KR102525843B1 - Anti-inflammatory composition comprising Liriodendron tulipifera extracts or alkamide isolated therefrom - Google Patents

Abstract

The present invention is a tulip tree ( Liriodendron tulipifera ) Relates to an anti-inflammatory composition containing an extract or a compound isolated therefrom, and specifically, inhibits NO (Nitric Oxide) formation, inhibits the expression of iNOS, COX-2 and IL-1β, and affects IRAK1 degradation. It was confirmed that it has anti-inflammatory activity by inhibiting IKK phosphorylation and blocking the NF-κB signaling pathway without giving it, and it is expected to contribute to the prevention or treatment of inflammation using this.

Description

Anti-inflammatory composition comprising Liriodendron tulipifera extracts or alkamide isolated therefrom {Anti-inflammatory composition comprising Liriodendron tulipifera extracts or alkamide isolated therefrom}

The present invention is a tulip tree ( Liriodendron tulipifera ) extract or an anti-inflammatory composition containing Alkamide isolated therefrom.

Inflammatory response is a mechanism to restore and regenerate the damaged area when a living organism or tissue is subjected to invasion that causes any organic change, such as physical action, chemical substance, or bacterial infection. Once stimulation is applied, histamine, serotonin, Vasoactive substances such as bradykinin, prostaglandin, HETE (hydroxyeicosatetraenoic acid), and leukotrienes are released to increase vascular permeability, and inflammatory cytokines are increased by NF-κB to induce inflammation. This inflammation damages even normal cells, awakens harmful genes such as aging genes and cancer genes, and chronic inflammation can cause various diseases such as stroke, cancer, obesity, arthritis, Alzheimer's, heart disease, and depression.

Treatment of these inflammatory diseases has classically included steroidal anti-inflammatory drugs such as prednisolone, nonsteroidal anti-inflammatory drugs (NSAIDs) such as naproxen, and calcium- and calmodulin-dependent protein kinases such as cyclosporine or FK506. An immunosuppressive agent that binds to calcineurin and suppresses its action has been most commonly used. However, these steroids and other immunosuppressants have side effects such as renal toxicity, infection, lymphoma, diabetes, tremor, headache, diarrhea, high blood pressure, nausea, and renal dysfunction, so research on safer natural anti-inflammatory drugs is being actively conducted. there is.

Tulip tree ( Liriodendron) tulipifera ) is a broad-leaved arboreous tree belonging to the Magnoliaceae family. It is native to North America and is distributed in the mixed broad-leaved forests of eastern North America. The flowers that grow tall and bloom at the ends of branches are very similar to tulips. It was imported into Korea in the 32nd year of King Gojong (1895) as a street tree to be planted around Sinjak-ro, along with plane trees, sheep willows, and poplar trees. The scientific name means 'a tree with lily flowers', and the species name at the end also means 'a large tulip flower runs'.

The tulip tree is native to the northern United States, and its bark was used as a medicinal herb by Native Americans to treat irregular fever associated with malaria (Graziosea et al., 2011). Sesquiterpene lactones, aporphine alkaloids, phenylpropanoids, and lignans have been isolated from tulip trees (Muhammad et al., 1989; Lee et al., 2013), among which sesquiterpenes including costunolide and epitulipinolide have been isolated. Aporphin alkaloids, including quinterpene lactone, liriodenine, and tuliperolin, have been reported as major components exhibiting antioxidant and anticancer activities in this plant (Eliza et al., 2010; Nordin et al., 2015). However, research on the biological activity of the tulip tree, which has been used as a medicinal herb for a long time, and the isolation and structural analysis of chemicals having this activity are still insignificant.

Korea Patent Publication No. 1020140147430, which is a prior art, describes an antioxidant and anti-inflammatory composition containing essential oil obtained from tulip leaves, but is different from the anti-inflammatory composition containing alkamide isolated from tulip trees according to the present invention. and there is a difference in effect. Korean Patent No. 1619710 describes an anti-aging composition for external application for skin containing an extract of placenta cell culture of the tulip tree, but the anti-inflammatory composition containing the extract of Liriodendron tulipifera or Alkamide isolated therefrom not listed

Korean Patent Publication No. 1020140147430, antioxidant and anti-inflammatory composition containing essential oil obtained from tulip tree leaves, 2014.12.30. open. Korean Patent No. 1619710, Composition for external application for anti-aging skin containing tulip placenta cell culture extract, 2016.05.02. registration.

Graziosea, R.; Rathinasabapathy, T.; Lategan, C.; Poulev, A.; Smith, P. J.; Grace, M.; Lila, M. A.; Raskin, I., Antiplasmodial activity of aporphine alkaloids and sesquiterpene lactones from Liriodendron tulipifera L., J. Ethnopharmacol. 2011, 133, 26-30. Muhammad, I.; Hufford, C. D., Phenylpropanoids, Sesquiterpenes, and Alkaloids from the Seeds of Liriodendron tulipifera, J. Nat. Prod. 1989, 52, 1177-1179. Lee, C.-H.; Chen, H.-L.; Hong, Z.-L.; Hsieh, C.-W.; Juan, S.-W.; Huang, J.-C.; Wang, H.-M.; Chen, C.-Y., Chemical Constituents of Liriodendron tulipifera, Chem. Nat. Compd. 2013, 49, 398-400. Eliza, J.; Daisy, P.; Ignacimuthu, S., Antioxidant activity of costunolide and eremanthin isolated from Costus speciosus (Koen ex. Retz) Sm., Chem. Biol. Interact. 2010, 188, 467-472. Nordin, N.; Majid, N.A.; Hashim, N. M.; Rahman, M. A.; Hassan, Z.; Ali, H. M., Liriodenine, an aporphine alkaloid from Enicosanthellum pulchrum, inhibits proliferation of human ovarian cancer cells through induction of apoptosis via the mitochondrial signaling pathway and blocking cell cycle progression., Drug Des. Devel. Ther. 2015, 9, 1437-1448. Demir, S.; Karaalp, C.; Bedir, E., Specialized metabolites from the aerial parts of Centaurea polyclada DC., Phytochemistry 2017, 143, 12-18.

An object of the present invention is a tulip tree ( Liriodendron tulipifera ) to provide an anti-inflammatory composition comprising an extract or a compound isolated therefrom.

In order to solve the above problems, the present invention is a tulip tree ( Liriodendron tulipifera ) An anti-inflammatory composition comprising an extract or a compound isolated therefrom is provided.

The tulip tree extract is water, C1-4 lower alcohol, acetone, ethyl acetate, diethyl acetate, diethyl ether, benzene ), it may be an extract extracted with one or a mixed solvent selected from the group consisting of chloroform and hexane, preferably an extract extracted with water and C1-4 lower alcohol, more preferably is an extract extracted with methanol.

The tulip tree extract is represented by the following Chemical Formula [1]: tulipiferamide A (Compound 1), tulipiferamide B (Compound 2), tulipiferamide C (Compound 3) and dehydrotemisin (Compound 4), N -acetylanonaine (Compound 5), N -acetylnornuciferin (Compound 6), tuliferoline (Compound 7), N -acetyl-3-methoxynornantenine (Compound 8), lysicamine (Compound 9), liriodenine (Compound 10), atherospermidine (Compound 11), liridine (Compound 12), oxoglaucine (Compound 13), oxophoebine (Compound 14), (±)-virolongin A (Compound 15), (±)-virolongin B (Compound 16), (-)-virolongin C (Compound 17), (+)-syringaresinol ( Compound 18), (-)(7 'S , 8R , 8'R )-4,4'-dihydroxy-3,3',5,5'-tetramethoxy-7',9-epoxylignan-9'-ol -7-one (Compound 19), (7 'S , 8R , 8'R )-lariciresinol (Compound 20), ( 2S , 3R )-dihydrodehydrodiconiferyl alcohol (Compound 21), costunolide (Compound 22), eupatolide (Compound 23), epi -tulipinolide (Compound 24), trans - N -feruloyl tyramine (Compound 25), trans - N -feruloyl-3-methoxytyramine (Compound 26), sakuranetin (Compound 27), 5,6,7- selected from the group consisting of trimethoxycoumarin (Compound 28), methoxyeugenol (Compound 29), N -phenethylbenzamide (Compound 30), N -phenethyl- N -methylacetamide (Compound 31), vanillin (Compound 32) and 4-hydroxybenzaldehyde (33) It provides an anti-inflammatory composition comprising one or more.

formula [1]

The tulip extract, tulipiferamide A (Compound 1), dehydrotemisin (Compound 4), tuliferoline (Compound 7), lysicamine (Compound 9), liriodenine (Compound 10), oxophoebine (Compound 14), eupatolide (Compound 23), epi -tulipinolide (Compound 24) and sakuranetin (Compound 27) may include one or more selected from the group consisting of.

As the extraction method of the tulip tree extract, any one of methods such as hot water extraction, cold brew extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression may be selected and used, and the desired extract is In addition, a conventional fractionation process may be performed, and it may be purified using a conventional purification method. The prepared tulip tree extract may be used as it is while being stored in a refrigerating chamber after centrifugation, filtration, and concentration followed by freeze-drying. In addition, the extract is further purified by using various chromatography methods such as silica gel column chromatography, thin layer chromatography, and high performance liquid chromatography to obtain a further purified fraction. , It can be purified to obtain the above compound.

The anti-inflammatory composition may inhibit the production of nitric oxide (NO) and inhibit the expression of iNOS, COX-2 and IL-1β. The anti-inflammatory composition may specifically inhibit NF-κB by inhibiting IKK phosphorylation without affecting degradation of IRAK1, an IKK upstream kinase of the TLR4 signaling pathway.

The compound isolated from the tulip tree extract of the present invention may be an alkamide. Alkamide isolated from the tulip extract of the present invention consists of tulipiferamide A (compound 1), tulipiferamide B (compound 2) and tulipiferamide C (compound 3) represented by the above formula [1]. It may include one or more selected from the group.

An anti-inflammatory composition comprising the tulip tree extract or a compound isolated therefrom of the present invention can be prepared as a pharmaceutical composition. The pharmaceutical composition of the anti-inflammatory composition is preferably 0.001 to 50% by weight, more preferably 0.001 to 40% by weight, most preferably 0.001 to 30% by weight based on the total weight of the total pharmaceutical composition. there is.

The pharmaceutical composition is formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, liquids, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. can Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, sweeteners, and acidulants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient, For example, it is prepared by mixing starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, preservatives, and acidulants are used. can be included Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. As a base for the suppository, witepsol, macrogol, tween-61, cacao butter, laurin paper, glycerogeratin, and the like may be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the prescriber's judgment. Determination of dosage based on these factors is within the level of those skilled in the art, and generally dosages range from 0.01 mg/kg/day to approximately 500 mg/kg/day. A preferred dose is 0.1 mg/kg/day to 200 mg/kg/day, and a more preferred dose is 1 mg/kg/day to 200 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, eg oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebrovascular injection and dermal application. The anti-inflammatory composition containing the tulip tree extract or a compound isolated therefrom of the present invention has little toxicity and side effects, so it is a drug that can be safely used even when taken for a long period of time for preventive purposes.

The present invention is a tulip tree ( Liriodendron tulipifera ) Provides an anti-inflammatory health functional food containing an extract or a compound isolated therefrom. The compounds isolated from the tulip extract are tulipiferamide A (Compound 1), tulipiferamide B (Compound 2), tulipiferamide C (Compound 3) and dehydrotemisin (Compound 4), N -acetylanonaine (Compound 5) represented by Formula [1]. ), N -acetylnornuciferin (Compound 6), tuliferoline (Compound 7), N -acetyl-3-methoxynornantenine (Compound 8), lysicamine (Compound 9), liriodenine (Compound 10), atherospermidine (Compound 11), liridine (Compound 12) ), oxoglaucine (Compound 13), oxophoebine (Compound 14), (±)-virolongin A (Compound 15), (±)-virolongin B (Compound 16), (-)-virolongin C (Compound 17), (+) -syringaresinol (Compound 18), (-)(7 'S , 8R , 8'R )-4,4'-dihydroxy-3,3',5,5'-tetramethoxy-7',9-epoxylignan-9 '-ol-7-one (Compound 19), (7' S ,8 R ,8' R )-lariciresinol (Compound 20), (2 S ,3 R )-dihydrodehydrodiconiferyl alcohol (Compound 21), costunolide (Compound 22 ), eupatolide (Compound 23), epi -tulipinolide (Compound 24), trans - N -feruloyl tyramine (Compound 25), trans - N -feruloyl-3-methoxytyramine (Compound 26), sakuranetin (Compound 27), 5,6 A group consisting of 7-trimethoxycoumarin (Compound 28), methoxyeugenol (Compound 29), N -phenethylbenzamide (Compound 30), N -phenethyl- N -methylacetamide (Compound 31), vanillin (Compound 32) and 4-hydroxybenzaldehyde (33) It may be one or more selected from.

The health functional food is preferably 0.001 to 50% by weight, more preferably 0.001 to 30% by weight, most preferably 0.001 to 30% by weight of the anti-inflammatory composition containing the tulip tree extract or a compound isolated therefrom, based on the total weight of the total food. It may be added as 0.001 to 10% by weight.

The health functional food includes the form of tablets, capsules, pills or liquids, and foods to which the extract of the present invention can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, health functional foods, etc.

The present invention is a tulip tree ( Liriodendron tulipifera ) Provides an anti-inflammatory cosmetic composition comprising an extract or a compound isolated therefrom. The compound isolated from the tulip tree extract of the present invention may be one or more selected from the group consisting of the above compounds 1 to 33. The compound isolated from the tulip tree extract of the present invention may be an alkamide, and may be an alkamide represented by Chemical Formula [1].

The cosmetic composition is preferably 0.001 to 50% by weight, more preferably 0.001 to 30% by weight, most preferably 0.001% by weight of the anti-inflammatory composition containing the tulip tree extract or a compound isolated therefrom, based on the total weight of the total food. ~10% by weight can be added.

The cosmetic composition contains a cosmetically or dermatologically acceptable medium or base. These are all formulations suitable for topical application, for example solutions, gels, solids, pasty anhydrous products, emulsions obtained by dispersing an oil phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules, or ionic forms (liposomes) and It may be provided in the form of a non-ionic follicular dispersant, or in the form of a cream, toner, lotion, powder, ointment, spray or conceal stick. These compositions can be prepared according to conventional methods in the art. The composition according to the invention can also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The cosmetic composition of the present invention contains a fatty substance, an organic solvent, a solubilizing agent, a thickening agent, a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, water, ionic or nonionic Cosmetics such as emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or any other ingredient commonly used in cosmetics or adjuvants commonly used in the field of dermatology. The adjuvant is added in an amount generally used in the field of cosmetology or dermatology.

Tulip tree of the present invention ( Liriodendron tulipifera ) The anti-inflammatory cosmetic composition containing an extract or a compound isolated therefrom is not particularly limited in its formulation, for example, softening lotion, astringent lotion, nutrient lotion, nutrient cream, massage cream, essence, eye cream , Eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil and body essence can be formulated into cosmetics.

The present invention is a tulip tree ( Liriodendron tulipifera ) Relates to an anti-inflammatory composition containing an extract or a compound isolated therefrom, and specifically, inhibits NO (Nitric Oxide) formation, inhibits the expression of iNOS, COX-2 and IL-1β, and affects IRAK1 degradation. It was confirmed that it has anti-inflammatory activity by inhibiting IKK phosphorylation and blocking the NF-κB signaling pathway without giving it, and it is expected to contribute to the prevention or treatment of inflammation using this.

1 shows the chemical structures of compounds 1 to 3 according to the present invention.
Figure 2 shows the main HMBC and COSY relationship of compounds 1 to 3 according to the present invention (A) and the chemical structure of compound 4 and the main HMBC, COSY relationship.
Figure 3 shows the cytotoxicity results for RAW 264.7 cells of compounds 1 to 33 according to the present invention.
Figure 4 shows the effect of tulipiferamide A (LT-1, Compound 1 ) on the LPS-induced inflammatory response in RAW264.7 macrophages. (A) RAW 264.7 macrophages were treated (None) or pre-treated (LT-1) with tulipiferamide A (30 µM) for 30 minutes and then treated with 1 µM LPS for the indicated times (0, 3, 6, 12 hours). (B) RT of iNOS, COX-2, IL-1β, TNFα, IL-6, and GAPDH in total RNA of each sample in (A) -PCR result
Figure 5 shows the mechanism of action of Tulipiferamide A (LT-1, 1) on the LPS-induced inflammatory response in RAW264.7 macrophages. (A) RAW 264.7 macrophages were treated with 1 μM LPS for the indicated times (0, 5, 15, 30, 60 min) in the absence (None) or presence (LT-1) of tulipiferamide A (30 μM) for 30 min. Immunoblotting results of IRAK1, IKKα/β, p65, ERK, JNK, p38, and each phosphorylated protein of each sample obtained (B) HEK293 cells transfected with a plasmid expressing flag-tagged IKKβ were treated with tulipperamide A ( Immunoblotting results after treatment for 24 hours in the absence or presence of LT-1, compound 1 ) (30 μM)

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the disclosure herein is provided so that it will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

< Example 1. Tulip tree ( Liriodendron tulipifera ) extracts and compounds isolated therefrom>

Tulip tree ( Liriodendron) tulipifera ) was collected at Chungnam National University in August 2016, and the voucher specimen (CNU201608) was deposited at the Pharmacology Research Institute, College of Pharmacy, Chungnam National University, Daejeon.

The tulip tree ( Liriodendron tulipifera ) roots (15.0 kg) were extracted twice (7 days × 100 L) with ethanol. The extract was concentrated under reduced pressure by rotary evaporation to obtain 887.3 g of a crude extract of tulip tree, of which 239.3 g was used in the experiment.

The crude extract was suspended and applied to silica gel VLC, and n-hexane-ethylacetate (8:2, 5:5, 3:7, 1:9; 6 L each step) and CHCl ₃ -MeOH (9:1, Seven fractions (LT1_LT7) were prepared by elution with a stepwise gradient of 7:3, 5:5, 2:8; 6 L each step; final wash with 10 L 100% MeOH). Fraction LT2 (24.1 g) was purified by reverse _- phase MPLC (Biotage SNAP cartridge, KP-C18-HS, 400 g) to obtain 11 small fractions (LT2-1 to LT2-11). Compounds 32 (11.4 mg, t _R = 37.2 min) and 33 (3.6 mg, t _R = 35.5 min) were obtained by preparing LT2-1 (115.5 mg) with a solvent composition of MeOH-H ₂ O (40:60 → 50:50) It was obtained by performing HPLC (Hector C ₁₈ column). Compound 28 (5.1 mg, t _R = 31.4 min) was obtained by performing reverse-phase HPLC (Hector C ₁₈ column) on fraction LT2-2 (106.2 mg) as a mobile phase of MeCN-H ₂ O (50:50 → 65:35) obtained. Compounds 4 (49.3 mg, t _R = 50.1 min), 23 (24.2 mg, t _R = 51.9 min), 29 (74.0 mg, t _R = 47.9 min), and 30 (38.9 mg, t _R = 49.5 min) Fraction LT2-3 (481.5 mg) was separated by MeCN-H ₂ O (40:60 → 100:30) linear gradient HPLC (Hector C ₁₈ column). Fractions LT2-4-1 (5.41 g) and LT2-5-1 (1.4 g) were obtained from methanol-soluble fractions LT2-4 (8.5 g) and LT2-5 (2.5 g), respectively. Fraction LT2-4-1 was purified by MPLC (Biotage SNAP Ultra cartridge, KP-Sil, 100 g) with a step gradient system of n -hexane-EtOAc (8:2, 7:3, 5:5, MeOH 100%). It was separated into 8 small fractions (LT2-4-1-1 to LT2-4-1-8). Among them, LT2-4-1-5 (3.1 g) was purified by HPLC (Phenomenex C ₁₈ column) eluting with gradient mixing of MeCN-H ₂ O (50:50 → 60:40) to obtain compound 24 (10.5 mg) , t _R = 42.8 min).

LT2-5-1 was analyzed by MPLC (Biotage SNAP Ultra cartridge, KP-Sil, 100 g) to obtain 6 small fractions (LT2-5-1-1 to LT2-5-1-6), of which fractions LT2-5-1-3 (732.6 mg) and LT2-5 -1-4 (321.4 mg) was analyzed by reverse phase HPLC [Phenomenex, MeCN-H ₂ O (60:40 → 70:30)] to give compounds 22 (235.4 mg, t _R = 34.3 min) and 27 (3.1 mg, t _R = 19.3 min). Compounds 15 (19.3 mg, t _R = 52.5 min) and 16 (17.8 mg, t _R = 50.3 min) were obtained by HPLC [Hector C ₁₈ column, MeCN-H ₂ O (70:30 → 80:20)]. In fractions LT2-8 (264.6 mg), MeCN-H ₂ O (80:20 → 90:10) through reverse-phase HPLC [Hector C ₁₈ column, compound 1 (17.1 mg, t _R = 30.5 min), 2 (4.7 mg , t _R = 35.6 min), 3 (5.1 mg, t _R = 32.3 min), and 17 (69.0 mg, t _R = 36.8 min) were isolated.

In fraction LT4 (20.7 g), MPLC (Biotage SNAP cartridge, KP-C18-HS) using step gradient mixing of MeOH-H ₂ O (5:5, 7:3, 9:1, 10:0, MeCN 100%) , 400 g) to obtain compound 6 (300.4 mg) and 6 small fractions (LT4-1 to LT4-6). Fraction LT4-2 (2.9 g) was obtained by MPLC [Biotage SNAP cartridge, KP-C18-HS, 120 g, MeOH-H ₂ O (3:7 → 5:5, 5:5 → 10:0, MeOH 100%) ] to obtain 8 small fractions (LT4-2-1 ~ LT4-2-8), of which fraction LT4-2-3 (716.6 mg) is an isocratic solvent MeOH-H ₂ O (40:60) It was separated into four small fractions (LT4-2-3-1 to LT4-2-3-4) by HPLC (Phenomenex C ₁₈ column) using . Fractions LT4-2-3-2 (241.7 mg) and LT4-2-3-4 (101.5 mg) were prepared under MeOH-H ₂ O (40:60) isocratic conditions. Compounds 19 (57.7 mg, t _R = 44.2 min) and 20 (15.0 mg, t _R = 51.6 min) were obtained by HPLC (Phenomenex C ₁₈ column). Fraction LT4-2-4 (610.1 mg) was compound 18 (222.8 mg, t _R = 42.3 min) through HPLC (Hector C ₁₈ column) of a repetitive MeOH-H ₂ O (50:50 → 55:45) linear gradient ), 31 (12.1 mg, t _R = 59.8 min), and five subfractions (LT4-2-4-1 to LT4-2-4-3, LT4-2-4-5, LT4-2-4- 6), of which, compound ₂₁ ( _15.2 mg, t _R = 58.3 min). Compounds 25 (16.8 mg, t _R = 44.8 min) and 26 (25.6 mg, t _R = 46.0 min) were analyzed by HPLC [Hector phenyl column, MeOH-H ₂ O (50:50 → 60:40) as fraction LT4-2 -5 (236.2 mg). In fractions LT4-4 (0.7 g), compound 13 (75.4 mg, t _R = 39.6 min) was applied to prep HPLC (Hector phenyl column) using a linear gradient of MeOH-H ₂ O (80:20 → 90:10) was separated. Fraction LT4-5-1 (5.5 g) was obtained by stepwise gradient elution with MeOH-H ₂ O (7:3, 8:2, 9:1, 10:0) MPLC (Biotage SNAP cartridge, KP-C18-HS, 400 g) gave 7 small fractions (LT4-5-1-1 ~ LT4-5-1-7), of which fraction LT4-5-1-5 (461.0 mg) was repetitive MeOH-H ₂ O (80:20 → 90:10) Gradient mixing HPLC (Hector C ₁₈ column) gave compounds 10 (62.1 mg, t _R = 26.7 min), 12 (3.5 mg, t _R = 39.9 min), and 3 small fractions ( LT4-5-1-5-2 to LT4-5-1-5-4). Fractions LT4-5-1-5-2 (31.1 mg), LT4-5-1-5-3 (41.0 mg), and LT4-5-1-5-4 (207.1 mg) were analyzed by HPLC [Hector phenyl column, MeOH-H ₂ O (80:20 → 90:10)] via compounds 9 (6.5 mg, t _R = 36.4 min), 8 (30.5 mg, t _R = 40.1 min), and 7 (63.4 mg, t _R = 41.3 min) was obtained. Compounds 5 (6.1 mg, t _R = 40.8 min), 11 (2.6 mg, t _R = 65.6 min), and 14 (5.7 mg, t _R = 60.9 min) are equivalent to MeOH-H ₂ O (90:10). It was separated from fraction LT4-5-1-6 (907.4 mg) by HPLC (Phenomenex biphenyl column) eluting with every solvent.

< Example 2. Analysis of compounds isolated from tulip trees>

Compounds 1 to 33 isolated in Example 1 were analyzed. Specific rotations were measured on a JASCO DIP-370 polarimeter (Tokyo, Japan). The melting point was measured with an MPA100 melting point apparatus (Stanford Research Systems, U.S.A.). UV and IR spectra were recorded using a Shimadazu SPD-M20A PDA detector and a Thermo Scientific Nicolet iS10 spectrometer, respectively, and 1D and 2D NMR spectra were recorded using Bruker DMX 300 (1H-300 MHz, 13C-75 MHz) and Bruker DMX 600 (1H-600 MHz). , 13C-150 MHz) were recorded on a spectrometer. HRESIMS data were obtained on a SYNAPT G2 mass spectrometer (Waters, U.K.) and a Q Exactive mass spectrometer (Thermo Scientific, U.S.A.).

As a result, the compounds 1 to 33 contained 10 aporphine alkaloids (5-14), 7 lignans (15-21), and 4 sesquiterpene lactones (4, 22-24), 3 phenyl propanoids (25-26, 29), 1 flavanone (27), 1 coumarin (28), phenethylamine Two (30-31), two phenolic aldehydes (32-33) and three new alkamides (1-3) were identified, and they are shown in Table 1. Among them, compound 4 was first isolated from natural products.

No. Name No. Name One tulipiferamide A alkamide 18 (+)-syringaresinol lignans 2 tulipiferamide B alkamide 19 (-)(7 'S , 8R , 8'R )-4,4'-dihydroxy-3,3',5,5'-tetramethoxy-7',9-epoxylignan-9'-ol-7-one lignans 3 tulipiferamide C alkamide 20 (7 'S , 8R , 8'R )-lariciresinol lignans 4 dehydrotemisin sesquiterperne lactones 21 (2 S ,3 R )-dihydrodehydrodiconiferyl alcohol lignans 5 N -acetylanonaine aporphine alkaloids 22 costunolide sesquiterperne lactones 6 N -acetylnornuciferin aporphine alkaloids 23 eupatolide sesquiterperne lactones 7 Tuliferoline aporphine alkaloids 24 epi -tulipinolide sesquiterperne lactones 8 N -acetyl-3-methoxynornantenine aporphine alkaloids 25 trans - N -feruloyl tyramine phenyl propanoids 9 lysicamine aporphine alkaloids 26 trans - N -feruloyl-3-methoxytyramine phenyl propanoids 10 liriodenine aporphine alkaloids 27 sakuranetin flavanone 11 atherospermidine aporphine alkaloids 28 5,6,7-trimethoxycoumarin coumarin 12 liridine aporphine alkaloids 29 methoxyeugenol phenyl propanoids 13 oxoglaucine aporphine alkaloids 30 N -phenethylbenzamide phenethylamine 14 oxophoebine aporphine alkaloids 31 N -phenethyl- N -methylacetamide phenethylamine 15 (±)-virolongin A lignans 32 vanillin phenolic aldehydes 16 (±)-virolongin B lignans 33 4-hydroxybenzaldehyde phenolic aldehydes 17 (-)-virolongin C lignans

The compounds isolated from the tulip tree of the present invention are tulipiferamide A (Compound 1), tulipiferamide B (Compound 2), tulipiferamide C (Compound 3), dehydrotemisin (Compound 4), N -acetylanonaine (Compound 5), N -acetylnornuciferin (Compound 5). 6), tuliferoline (Compound 7), N -acetyl-3-methoxynornantenine (Compound 8), lysicamine (Compound 9), liriodenine (Compound 10), atherospermidine (Compound 11), liridine (Compound 12), oxoglaucine (Compound 13) , oxophoebine (Compound 14), (±)-virolongin A (Compound 15), (±)-virolongin B (Compound 16), (-)-virolongin C (Compound 17), (+)-syringaresinol (Compound 18), (-)(7 'S , 8R , 8'R )-4,4'-dihydroxy-3,3',5,5'-tetramethoxy-7',9-epoxylignan-9'-ol-7-one (Compound 19), (7 'S , 8R , 8'R )-lariciresinol (Compound 20), ( 2S , 3R )-dihydrodehydrodiconiferyl alcohol (Compound 21), costunolide (Compound 22), eupatolide (Compound 23) , epi -tulipinolide (Compound 24), trans - N -feruloyl tyramine (Compound 25), trans - N -feruloyl-3-methoxytyramine (Compound 26), sakuranetin (Compound 27), 5,6,7-trimethoxycoumarin (Compound 28 ), methoxyeugenol (Compound 29), N -phenethylbenzamide (Compound 30), N -phenethyl- N -methylacetamide (Compound 31), vanillin (Compound 32), and at least one selected from the group consisting of 4-hydroxybenzaldehyde (33) include

The chemical structures and main HMBC and COZY relationships of the new alkamide compounds 1 to 3 isolated from the tulip tree of the present invention and the compound 4 first isolated from natural products are shown in FIGS. 1 to 3.

Tulipiperamide A (Compound 1) : Red oil; UV (MeOH) λ _max (log ε) 277 (4.26), 251 (4.52), 210 (4.19); IR (KBr) vmax 3280, 2929, 2858, 1655, 1621, 1548, 1497, 1275 cm ^-1 ; ¹ H and ¹³ C NMR data, see Table 2; HRESIMS m/z 272.2014 [M + H] ⁺ (C ₁₈ H ₂₆ NO ⁺ , 272.2009), 294.1835 [M + Na] ⁺ (C ₁₈ H ₂ 5NONa ⁺ , 294.1828).

Compound 1 has a molecular formula of C ₁₈ H ₂₅ NO showing molecular ion peaks at m/z 272.2014 [M + H] ⁺ (calculated 272.2009) and 294.1835 [M + Na] ⁺ (calculated 294.1828) in the HRESIMS spectrum corresponding to the molecule. was obtained as a pale reddish yellow oil. δH _7.31 (2H, t, J = 7.4 Hz, H-3" and H-5"), 7.23 (1H, t, J = 7.4 Hz, H-4") in the ^1H NMR spectrum (Table 2), 7.20 (2H, d, J = 7.4 Hz, H-2" and H-6"), 3.61 (2H, q, J = 6.7 Hz, H ₂ -2') and 2.86 (2H, t, J = 6.7 Hz _. _ _{_} _ _ 4), olefin proton signals at 5.79 (1H, m, H-5) and 5.75 (1H, d, J = 14.8 Hz, H-2) confirmed the presence of two double bonds, also at _δH 2.29 ( 4 methylenes and δ at 2H, q, J = 7.5 Hz, H ₂ -6), 1.40 (2H, m, H ₂ -7), 1.30 (4H, m, H ₂ -8 and H ₂ -9) One methyl group was observed at _H 0.89 (3H, t, J = 6.8 Hz, H ₃ -10) In the ¹³ C NMR data (Table 2), the 16 carbon signals correspond to one amidecarbonyl carbon (δ _C 166.4) , one benzene ring ( δ _C 139.1, 128.9, 128.8, 126.7), four olefins ( δ _C 140.6, 136.3, 126.4, 123.7), six methylenes ( δ _C 40.8, 35.8, 31.6, 29.3, 28.3, 22.7) and one methyl group ( δ _C 14.2). This confirmed the location of the two double bonds at -4 (Fig. 2), which also coincided with the two fragment ion peaks at m/z 105 and 151 in the ESI-MS/MS spectrum of compound 1. are found to be 2 E and 4 Z deduced from the coupling constants J _{H -2/H-3} at 14.8 Hz and J _{H -4/H-5} at 11.5 Hz, respectively. Accordingly, Compound 1 was named deca-2 E ,4 Z -dienoic acid 2-phenylethylamide and Tulipiferamide A.

Position One 2 3 δ _H ( J in Hz) δC _{, type} δ _H ( J in Hz) δC _{, type} δ _H ( J in Hz) δC _{, type} One 166.4, C. 166.6, C. 172.6, C. 2 5.75,d
(14.8) 123.7, CH 5.38,d
(11.3) 118.3, CH 2.16, t
(7.4) 36.9 CH ₂ 3 7.54, dd
(11.5, 14.8) 136.3, CH 6.36, t
(11.3) 142.0, CH 2.34, q
(7.4) 23.6 CH ₂ 4 6.05, t
(11.5) 126.4, CH 7.42, d.d.
(11.3, 15.0) 127.0, CH 5.29 m 127.8, CH 5 5.79 m 140.6, CH 5.96 m 144.3, CH 5.40, overlapping 131.8, CH 6 2.29, q
(7.5) 28.3 CH ₂ 2.17, q
(7.2) 33.0 CH ₂ 2.01, q
(7.2) 27.3 CH ₂ 7 1.40 m 29.3 CH ₂ 1.42 m 28.8 CH ₂ 1.29, overlapping 29.4 CH ₂ 8 1.30, overlapping 31.6 CH ₂ 1.29, overlapping 31.6 CH ₂ 1.29, overlapping 31.6 CH ₂ 9 1.30, overlapping 22.7 CH ₂ 1.29, overlapping 22.6 CH ₂ 1.29, overlapping 22.7 CH ₂ 10 0.89, t
(6.8) 14.2 CH ₃ 0.88, t
(7.0) 14.2 CH ₃ 0.88, t
(7.1) 14.2 CH ₃ 2' 3.61, q
(6.7) 40.8 CH ₂ 3.58, q
(7.0) 40.6 CH ₂ 3.52, q
(6.9) 40.7 CH ₂ 3' 2.86, t
(6.7) 35.8 CH ₂ 2.85, t
(7.0) 35.9 CH ₂ 2.81, t
(6.9) 35.9 CH ₂ One" 139.1, C. 139.1, C. 139.1, C. 2", 6" 7.20,d
(7.4) 128.9, CH 7.21, d.d.
(1.4, 7.7) 128.9, CH 7.19,d
(7.3) 128.9, CH 3", 5" 7.31, t
(7.4) 128.8, CH 7.31, t
(7.7) 128.8, CH 7.31, t
(7.3) 128.8, CH 4" 7.23, t
(7.4) 126.7, CH 7.24 m 126.6, CH 7.23, t(7.3) 126.7, CH NH 5.52, brs 5.47, brs 5.40, overlapping

Tuliperamide B (Compound 2) : yellow oil; UV (MeOH) λ _max (log ε) 275 (4.24), 247 (4.45), 210 (4.26); IR (KBr) v _max 3338, 2929, 2858, 1669, 1541, 1454, 1369, 1238 cm ^-1 ; ¹ H and ¹³ C NMR data, see Table 2; HRESIMS m/z 272.2015 [M + H] ⁺ (C ₁₈ H ₂₆ NO ⁺ , 272.2009), 294.1836 [M + Na] ₊ (C ₁₈ H ₂ 5NONa ⁺ , 294.1828).

Compound 2 is a yellow oil with the same molecular formula C ₁₈ H ₂₆ NO as compound 1 based on the HRESIMS ion at m/z 272.2015 [M + H] ⁺ (calculated for C ₁₈ H ₂₆ NO ⁺ , 272.2009). ^{The 1H} and ^13C NMR resonances are _δH 7.42 (1H, dd, J = 11.3, 15.0 Hz, H-4), 6.36 (1H, t, J = 11.3 Hz, H-3), 5.96 (1H, m, H-5), 5.38 (1H, d, J = 11.3 Hz, H-2) were similar to those of Compound 1 except for the resonance of the olefin proton, indicating that Compound 2 is a geometric isomer of Compound 1. The MS/MS fragmentation pattern of compound 2 was identical to that of compound 1. The configurations of 2 Z and 4 E were inferred from the coupling constants J _{H2/ H3} of 11.3 Hz and J _{H4/ H5} of 15.0 Hz, respectively. Therefore, the structure of compound 2 was assigned as deca-2 Z ,4 E -dienoic acid 2-phenylethylamide and named as tulipiferamide B.

Tulipiperamide C (Compound 3) : yellow oil; UV (MeOH) λ _max (log ε) 259 (2.58), 255 (2.53), 200 (4.03); IR (KBr) v _max 3330, 2928, 2856, 1645, 1550, 1454, 1228, 1030 cm ^-1 ; ¹ H and ¹³ C NMR data, see Table 2; HRESIMS m/z 274.2172 [M + H] ⁺ (calculated for C ₁₈ H ₂₈ NO ^{+ ,} 274.2165), 296.1991 [M + Na] ⁺ (calculated for C ₁₈ H ₂₇ NONa ⁺ , 296.1985).

Compound 3 was identified as an alkamide based on MS and NMR spectroscopic data. The molecular formula C ₁₈ H ₂₇ NO of compound 3 was deduced from the protonated molecule [M + H] ⁺ at m/z 274.2172 by HRESIMS. The difference of the two mass units compared to compound 1 and compound 2 (C ₁₈ H ₂₅ NO) indicates that one of the double bonds must be saturated. The location of the double bond was clearly located by HMBC and COZY correlations (Fig. 2). The HMBC cross peaks from H ₂ -3 to C-1 and C-5 and H ₂ -2 and H ₂ -6 to C-4 confirmed the location of the double bond at C-4 (Fig. 2). Two MS fragment ions (m/z 91 and 168) corresponding to the phenyl methane and deca-4-enamide backbones were detected in the ESI-MS/MS spectrum. The Z configuration was confirmed by chemical shifts of _{the allyl methylene carbons δC} 27.3 (C-6) and 23.6 (C-3). For the Z configuration, the general chemical shift of the allyl carbon is close to δC _27–30 and that of the E configuration is close to δC _32–35.7 , so the structure of compound 3 is deca-4 Z -enoic acid phenylethylamide. It was determined and named as Tulipiferamide C.

Dihydrothemycin (Compound 4): Colorless crystalline needles; mp 141.8 °C; [α] ²² _D +147.7 ( c 0.10, MeOH); ¹ H NMR (CDCl ₃ , 600 MHz) δ _H 6.18 (1H, d, J = 3.0 Hz, H ₂ -13a), 5.98 (1H, J = 3.0 Hz, H ₂ -13b), 5.80 (1H, m, H -1), 5.06 (1H, s, H ₂ -3a), 5.03 (1H, d, J = 10.8 Hz, H ₂ -2a), 4.98 (1H, d, J = 17.4 Hz, H ₂ -2b), 4.71 (1H, brs, H ₂ -3b), 4.12 (1H, d, J = 11.3 Hz, H-6), 4.08 (1H, dd, J = 3.7, 10.9 Hz, H-8), 2.61 (1H, tt, J = 3.0, 10.8 Hz, H-7), 2.37 (1H, d, J = 11.7 Hz, H-5), 1.84 (1H, dd, J = 4.1, 13.0 Hz, H ₂ -9a), 1.79 (3H, s, H ₃ - 15), 1.66 (1H, m, H ₂ -9b), 1.09 (3H, s, H ₃ -14); ¹³ C NMR (CDCl ₃ , 150 MHz) δ _C 170.2 (C-12), 146.9 (C-1), 140.0 (C-4), 137.7 (C-11), 120.4 (C-13), 116.2 (C- 3), 112.1 (C-2), 78.8 (C-6), 67.8 (C-8), 55.6 (C-5), 55.0 (C-7), 50.0 (C-9), 42.4 (C-10) ), 23.9 (C-15), 19.8 (C-14); HRESIMS m/z 247.1333 [M - H] ^- (calcd for C ₁₅ H ₁₉ O ₃ ^- , 247.1340).

Compound 4 is an Elemanse sesqui terpene lactone, which was first isolated from a natural product. Examination of the ¹ H and ¹³ C NMR spectra of compound 4 showed characteristic signals for an elemane-type sesquiterpene lactone. The presence of α-methylene γ-lactone groups is indicated by the ^1H NMR resonances at δH _6.18 (1H, d, J = 3.0 Hz, H ₂ -13a), 5.98 (1H, d, J = 3.0 Hz, H ₂ -13b) and δ _C 170.2 (C-12), 137.7 (C-11), 120.4 (C-13) by ¹³ C NMR resonance. In ^{the 1} H NMR spectrum, δ _H 5.80 (1H, m, H-1), 5.03 (1H, d, J = 10.8 Hz, H ₂ -2a), 4.98 (1H, d, J = 17.4 Hz, H ₂ -2b ) indicated the terminal vinyl group. Another olefin proton cluster at δ _H 5.06 (1H, s, H ₂ -3a), 4.71 (1H, brs, H ₂ -3b) and methyl at δ _H 1.79 (3H, s, H ₃ -15) is The propenyl skeleton is shown. Downfield shifted proton signals at δH _4.12 (1H, d, J = 11.3 Hz, H-6) and 4.08 (1H, dd, J = 3.7, 10.9 Hz, H-8) indicate the presence of two oxygen substituents. indicate Additionally, two methine ( δ _H 2.37, 2.61), one methylene (δ _H 1.84, 1.66) and one methyl (δ _H 1.09) groups were observed. The HSQC and HMBC cross peaks were extensively used to complete the NMR assignment to the elemane backbone (Fig. 3). The relative composition of the five stereocenters in compound 4 was inferred by interpretation of NOESY correlations. The NOE correlation between H ₃ -14 and H-6/H-8 suggested a position on the same side, whereas between H ₂ -9b and H-1/H-7 it showed a position on the opposite side (FIG. 3). According to Demir et al. (2017), H-7 of all elemanolides was recorded in the α direction. Therefore, the experimental specific rotation value for compound 4 was +147.7 ( c 0.10, MeOH). As a result, the structure of compound 4 was established as dehydrotemisin, which was first isolated from a natural product.

< Example 3. Confirmation of the effect of compounds isolated from tulip trees on NO production>

3-1. Cell culture and viability check

RAW 264.7 cells were maintained at 5% in Dulbecco's modified Eagle's medium (DMEM, Wellgene, Gyeongsan-si, Korea) supplemented with 10% heat-inactivated fetal bovine serum (HyClone FBS, GE Healthcare, IL, USA) and 1% L-glutamine. Incubated at 37°C in CO2. To determine cell viability, RAW 264.7 cells were seeded in 96-well plates at a confluency of 75% cells and incubated overnight, then cells were treated with the indicated compounds or samples at various concentrations for 30 min followed by 1 μg/ml LPS stimulated for 24 hours. For the MTT assay, cell viability was determined by adding 5 mg/ml MTT solution (Sigma-Aldrich, Miamisburg, OH, USA) and the plates were incubated at 37°C for 1 hour. After removing the supernatant, 100 μl of DMSO was added to each well, and then measured at 590 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the results are shown in FIG. 3 . As shown in FIG. 3, compounds 10 and 24 showed strong cytotoxicity at a concentration of 20 μM, and the other compounds showed no cytotoxicity up to a concentration of 40 μM.

3-2. Check the effect on nitric oxide (NO) production

The nitrite concentration in the cell supernatant was evaluated as an indicator of NO production using the Griess Reagent Kit (Sigma-Aldrich, Miamisburg, OH, USA). RAW 264.7 cells were seeded in a 96-well plate at a confluence of 75% cells and cultured overnight, and then the cells were treated with compounds 1 to 33 of Example 1 at 0, 1, 2.5, 5, 10, 20, 40, 80, They were treated for 30 minutes with 100 μM concentration and then stimulated with 1 μM LPS for 24 hours. Thereafter, 100 μl of the supernatant was mixed with 100 μl of Griess reagent in a 96-well plate and incubated for 30 minutes at room temperature. Absorbance was measured at 540 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The nitrite concentration was calculated by regressing from a standard solution of sodium nitrite, compared to the untreated control, and the IC ₅₀ was calculated and shown in Table 3.

No. IC ₅₀ ( μM ) No IC ₅₀ ( μM ) No IC ₅₀ ( μM ) One 13.3 ± 1.1 12 > 50.0 23 7.3±1.0 2 > 50.0 13 > 50.0 24 19.8 ^a ± 2.0 3 > 50.0 14 34.2 ± 2.2 25 > 50.0 4 12.0±1.5 15 > 50.0 26 > 50.0 5 > 50.0 16 > 50.0 27 21.6 ± 2.3 6 > 50.0 17 > 50.0 28 > 50.0 7 14.0 ± 1.2 18 > 50.0 29 > 50.0 8 > 50.0 19 > 50.0 30 > 50.0 9 6.4±1.5 20 > 50.0 31 > 50.0 10 10.5 ^a ± 1.1 21 > 50.0 32 > 50.0 11 > 50.0 22 > 50.0 33 > 50.0 L- NMMA ^b 35.5 ± 2.1

Each value represents the mean ± SEM of three determinations. a : Cytotoxic effect observed at 20 μM by MTT assay, b : Positive control compound, L-NMMA (nitric oxide synthase inhibitor).

As shown in Table 3, compounds 1, 4, 7, 9, 10, 14, 23, 24 and 27 effectively inhibited the production of nitric oxide in LPS-stimulated RAW 264.7 cells. However, compounds 10 and 24 showed strong cytotoxicity at a concentration of 20 μM. As a result, as a result of pre-treatment of RAW264.7 macrophages with 7 compounds 1, 4, 7, 9, 14, 23, and 27 among the test compounds, LPS-induced NO production was significant with an IC ₅₀ value of less than 50 μM without morphological signs. was confirmed to be suppressed. At this time, the positive control group, NG-monomethyl-L-arginine monoacetate and L-NMMA, had an IC ₅₀ value of 35.5 μM.

< Example 4. Confirmation of anti-inflammatory activity of compound 1>

It was confirmed that compounds 1, 4, 7, 9, 14, 23, and 27 isolated from tulip trees in Example 3 inhibit LPS-induced NO production, and among them, tulipiferamide A, a novel substance previously unknown. The anti-inflammatory activity of (Compound 1) was investigated.

The effect of compound 1 on the expression of iNOS, COX-2, and IL-1β, which are NF-κB target genes, was confirmed. RAW264.7 macrophages were maintained in the same manner as in Example 3-1, and RAW 264.7 macrophages were treated with or without treatment with 30 μM of compound 1 (tulipiferamide A) for 30 minutes, followed by 0, 3, 6, and 12 hours while treated with 1 μM LPS. Thereafter, the lysate of the whole cells of the sample was separated by 10% SDS-PAGE and immunoblotting was performed with iNOS, COX-2, IL-1β and β-actin antibodies, and the results are shown in FIG. 4A.

As shown in Figure 4A, LPS increased the expression of iNOS, COX-2 and IL-1β in RAW264.7 macrophages, and this increase was suppressed by Compound 1. In particular, the expression of iNOS and COX-2 continuously increased with the LPS treatment time, which was suppressed by Compound 1. The expression of IL-1β increased the most at 6 hours after LPS treatment, and the expression level gradually decreased, and compound 1 also suppressed this increase in IL-1β.

The expression of iNOS, COX-2, IL-1β, TNFα and IL-6, which are NF-κB target genes of Compound 1, was analyzed by RT-PCR analysis. RAW 264.7 macrophages were treated with or without 30 µM of compound 1 (tulipiferamide A) for 30 minutes, and then treated with 1 µM LPS for 0, 2, or 4 hours. Then, after preparing total RNA using the easy-BLUE total RNA extraction kit (iNtRON Biotechnology, Inc., Seongnam, Korea), oligo (dT)-primed cDNA was converted to RT-PCR kit (Promega, Madison, CA, USA) was synthesized using PCR was performed using the following primer pairs for each mouse gene (m iNOS F: 5'-CGAAACGCTTCACTTCCAA-3' R: 5'-TGAGCCTATATTGCTGTGGCT-3'; m Cox2 F: 5'-AACCGCATTGCCTCTGAAT- 3' R: 5'- CATGTTCCAGGAGGATGGAG-3'; m IL-1β F: 5'-ATGGCAACTGTTCCTGAACTCAACT-3' R: 5'-CAGGACAGGTATAGATTCTTTCCTTT-3'; m TNFα F: 5'-TTCTGTCTACTGAACTTCGGGGTGATCGGTCC-3' R: 5'-GTATGAGATAGCAAATCGGCTGACGGTGTGGG-3 '; m IL-6 F: 5'-ACATCCTCGACGGCATCT-3' R: 5'-GCCTCTTTGCTGCTTTCAC-3'; m GAPDH, 5'-GACCCCTTTCTTGACCTC-3' and 5'-GCCATCCACAGTCTTCTG-3'). After PCR amplification, the products were separated by agarose gel electrophoresis and visualized using ethidium bromide staining and are shown in Figure 4B.

As shown in Figure 4B, LPS increased the expression of iNOS, COX-2, IL-1β, TNFα and IL-6 in RAW264.7 macrophages, and this increase was suppressed by compound 1. Through this, it was confirmed that compound 1, which is a novel substance isolated from the tulip tree extract of the present invention, inhibits the NF-κB-mediated inflammatory pathway.

Expression of proteins involved in NF-κB signaling was systematically monitored to determine the molecular mechanisms involved in compound 1-mediated inhibition of the inflammatory response. RAW 264.7 macrophages were treated with or without 30 μM of Compound 1 (tulipiferamide A) and then treated with 1 μM LPS for 0, 5, 15, 30, or 60 minutes. Then, the harvested cells were lysed, separated by SDS-PAGE, and transferred to NC membranes, followed by JNK1, Phospho-p38, p38 and ERK (Santa Cruz, CA, USA); IKBα, Phospho-IKBα, Phospho-IKKα/β, Phospho-ERK, Phospho-JNK, Phospho-p65, p65, and IRAK1 (Cell Signaling Technology, Beverly, MA, USA); IKKβ (Upstate Biotech, Waltham, MA, USA); and β-actin (Sigma-Aldrich, Miamisburg, OH, USA) primary antibodies were maintained at 4°C for 24 hours, and then the membrane was incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Then, the band was detected using an enhanced chemiluminescence western blotting detection reagent (WestGlow; Biomax, Inc., Seoul, Korea) and is shown in FIG. 5A.

As shown in FIG. 5A , compound 1 (30 μM) significantly inhibited NF-κB activation through phosphorylation of IKKα/β, IkBα and relA (p65), thereby exhibiting anti-inflammatory activity of compound 1. In addition, it was confirmed that LPS-induced phosphorylation of MAPKs (ERK, JNK and p38) was not affected by Compound 1, and that the inhibitory effect of Compound 1 was specific to NF-κB. More importantly, compound 1 inhibits IKK phosphorylation without affecting degradation of IRAK1, an IKK upstream kinase in the TLR4 signaling pathway. This means that compound 1 restricts the LPS-induced NF-κB signaling pathway by targeting IKK activity. That is, IKK phosphorylation caused by ectopic expression of IKKβ was significantly inhibited by compound 1, which means that compound 1 inhibits the level of IKKβ regardless of upstream kinases such as IRAK1.

As described above, the present inventors isolated 33 compounds from the roots of the tulip tree and identified a new alkamide component having a strong effect of inhibiting the inflammatory response induced by targeting the activity of IKKβ. From these results, it was confirmed that the novel compound tulipperamide A isolated from the tulip tree of the present invention can be used for the treatment of inflammatory diseases including abnormal overactivation of the IKK-NF-κB signaling cascade.

< formulation example 1. Pharmaceutical preparations>

1.1. manufacture of tablets

Tulip tree of the present invention ( Liriodendron tulipifera ) extract or 200 mg of tulipiferamide A isolated therefrom was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. After adding 10% gelatin solution to this mixture, it was pulverized and passed through a 14 mesh sieve. It was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of active agent, and 5 g of magnesium stearate was made into tablets.

1.2. Preparation of injection solution

Tulip tree of the present invention ( Liriodendron 100 mg of tulipifera ) extract or tulippiperamide A isolated therefrom, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was bottled and sterilized by heating at 20° C. for 30 minutes.

< formulation example 2. Manufacture of health functional food>

2.1. Manufacture of health functional food

Tulip tree of the present invention ( Liriodendron tulipifera ) extract or Tulipiferamide A 2g isolated therefrom, proper amount of vitamin mixture, vitamin A acetate 70μg, vitamin E 1.0mg, vitamin B1 0.13mg, vitamin B2 0.15mg, vitamin B6 0.5mg, vitamin B12 0.2μg, Vitamin C 10mg, Biotin 10μg, Nicotinamide 1.7mg, Folic Acid 50μg, Calcium Pantothenate 0.5mg, Mineral Mixture Appropriate amount, Ferrous Sulfate 1.75mg, Zinc Oxide 0.82mg, Magnesium Carbonate 25.3mg, Potassium Phosphate Monobasic 15mg , 55 mg of dibasic calcium phosphate, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride were mixed to form granules, but it can be prepared by transforming into various formulations depending on the use. In addition, the composition ratio of the vitamin and mineral mixture may be arbitrarily modified, and it may be prepared by mixing the above components according to a conventional health functional food manufacturing method.

2.2. Manufacture of health functional beverages

Tulip tree of the present invention ( Liriodendron A beverage was prepared by mixing 1 g of tulipifera ) extract or tulippiperamide A isolated therefrom, 0.1 g of citric acid, 100 g of fructooligosaccharide, and 900 g of purified water, followed by stirring, heating, filtering, sterilization, and refrigeration according to a conventional beverage manufacturing method.

< formulation example 3. cosmetic manufacturing>

2.1. Manufacture of skin lotion

Tulip tree of the present invention ( Liriodendron tulipifera ) extract or isolated therefrom, 0.1 g of tulippiperamide A, 2.0 g of butylene glycol, 2.0 g of propylene glycol, 0.1 g of carboxyvinyl polymer, PEG-12 nonylphenyl ether 0.2 g, 0.4 g of polysorbate 80, A skin lotion was prepared by mixing 10.0 g of ethanol, 0.1 g of triethanolamine, appropriate amounts of preservatives, pigments, fragrances, and the remaining amount of purified water.

2.2. Manufacture of massage cream

Tulip tree of the present invention ( Liriodendron tulipifera ) extract or Tulipiferamide A isolated therefrom 0.1 g, beeswax 10.0 g, polysorbate 60 1.5 g, PEG 60 hydrogenated castor oil 2.0 g, sorbitan sesquioleate 0.8 g, liquid paraffin 40.0 g, squalane A massage cream was prepared by mixing 5.0 g of glycerin, 5.0 g of glycerin, 3.0 g of butylene glycol, 3.0 g of propylene glycol, 0.2 g of triethanolamine, an appropriate amount of preservative, colorant, fragrance, and the remaining amount of purified water.

Claims (13)

Tulip tree ( Liriodendron tulipifera ) In the anti-inflammatory composition comprising a root extract or a compound isolated therefrom,
The tulipifera root extract or a compound isolated therefrom is represented by the following formula [1]: tulipiferamide A (Compound 1) and dehydrotemisin (Compound 4), tuliferoline (Compound 7), lysicamine (Compound 9), liriodenine (Compound 10) ), oxophoebine (Compound 14), eupatolide (Compound 23), epi-tulipinolide (Compound 24) and sakuranetin (Compound 27) Anti-inflammatory composition comprising at least one selected from the group consisting of.
Formula [1]

According to claim 1,
The tulip tree root extract is obtained from water, C1-4 lower alcohol, acetone, ethyl acetate, diethyl acetate, diethyl ether, benzene ( benzene), chloroform (chloroform) and hexane (hexane) selected from the group consisting of one or a mixed solvent of these extracts characterized in that the anti-inflammatory composition. delete delete According to claim 1 or 2,
The anti-inflammatory composition is an anti-inflammatory composition, characterized in that for suppressing the production of NO (nitric oxide). According to claim 1 or 2,
The anti-inflammatory composition is an anti-inflammatory composition, characterized in that inhibiting the expression of iNOS, COX-2 and IL-1β. According to claim 1,
The compound isolated from the tulip tree root extract consists of tulipiferamide A (compound 1), tulipiferamide B (compound 2) and tulipiferamide C (tulipiferamide C, compound 3). Anti-inflammatory composition comprising at least one selected from the group. In the anti-inflammatory health functional food containing a Liriodendron tulipifera root extract or a compound isolated therefrom,
The tulipifera root extract or a compound isolated therefrom is, tulipiferamide A (Compound 1), dehydrotemisin (Compound 4), tuliferoline (Compound 7), lysicamine (Compound 9), liriodenine (Compound 10), oxophoebine (Compound 14), An anti-inflammatory health functional food comprising at least one selected from the group consisting of eupatolide (Compound 23), epi-tulipinolide (Compound 24) and sakuranetin (Compound 27). delete Tulip tree ( Liriodendron tulipifera ) In the anti-inflammatory cosmetic composition comprising a root extract or a compound isolated therefrom,
The tulipifera root extract or a compound isolated therefrom is, tulipiferamide A (Compound 1), dehydrotemisin (Compound 4), tuliferoline (Compound 7), lysicamine (Compound 9), liriodenine (Compound 10), oxophoebine (Compound 14), An anti-inflammatory cosmetic composition comprising at least one selected from the group consisting of eupatolide (Compound 23), epi-tulipinolide (Compound 24) and sakuranetin (Compound 27). delete According to claim 10,
The anti-inflammatory cosmetic composition is a softening lotion, astringent lotion, nutrient lotion, nutrient cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil And an anti-inflammatory cosmetic composition characterized in that it has a formulation selected from the group consisting of body essence. From the group consisting of tulipiferamide A (compound 1), tulipiferamide B (compound 2) and tulipiferamide C (compound 3) represented by the following formula [1] One new compound of choice.
formula [1]

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