Coumarin compounds isolated from gerbera hookeriana and preparation method and application thereof

Abstract

This invention relates to the field of natural product separation technology, specifically to coumarin compounds isolated from *Hypericum perforatum*, their preparation methods, and applications. The preparation method of these coumarin compounds includes alcohol extraction from *Hypericum perforatum*, followed by macroporous resin adsorption separation and column separation. The coumarin compounds of this invention can be used for anti-inflammatory purposes, exhibiting inhibitory activity against RAW264.7 cells and the expression of TNF-α, IL-1β, and IL-6 proteins.

Description

Technical Field

[0001] This invention relates to the field of natural product separation technology, and more specifically, to coumarin compounds isolated from Uncaria rhynchophylla, their preparation methods, and applications. Background Technology

[0002] *Hylocereus undatus* is a perennial herb belonging to the genus *Hylocereus* in the family Asteraceae. It is also known as *Hylocereus flavomarginata*, *Hylocereus nigra*, and *Hylocereus datus*. It is mainly distributed in Yunnan and Sichuan provinces of China. It has a pungent and slightly bitter taste, and is neutral in nature. It possesses the effects of clearing heat and detoxifying, promoting diuresis and reducing swelling, and resolving blood stasis and stopping bleeding. *Hylocereus undatus* contains numerous and complex chemical components. Research has found that the main chemical components are sugars, glycosides, flavonoids, and coumarins. These compounds in *Hylocereus undatus* contribute to the design and development of effective drugs for treating various diseases. Currently, many drugs based on natural compounds are used clinically or are being evaluated in clinical trials.

[0003] Inflammation is a protective response of the body to tissue damage, infection, and abnormal states. It plays a role in clearing harmful substances, repairing damaged tissues, and regulating immune function. However, excessive inflammatory responses can lead to tissue damage and functional disorders. RAW264.7 macrophages play a complex and crucial role in the occurrence, development, and homeostasis of inflammation by secreting pro-inflammatory cytokines and regulating polarization. When macrophages are activated, they produce large amounts of pro-inflammatory cytokines such as interleukin-12 (IL-12), TNF-α, interleukin-6 (IL-6), and interleukin-1β (IL-1β), triggering a positive feedback loop of immune activation and exacerbating tissue damage. Under the stimulation of different pathogens and cytokines, the phenotype and function of resting macrophages change, and they can be reprogrammed into classic (M1) and alternative (M2) types. In certain inflammatory diseases, M1 macrophage polarization exacerbates the inflammatory response, while M2 macrophages have anti-inflammatory effects; the balance between the two is crucial for maintaining homeostasis. Therefore, regulating the expression of macrophage inflammatory factors and modulating polarization are key to treating inflammatory diseases.

[0004] The herb *Hypericum esculentum* has great potential in the clinical treatment of various diseases, such as cough due to lung heat, urinary tract infections due to heat, diarrhea due to damp heat, redness and swelling of the lower limbs, traumatic bleeding, and anti-inflammation. These potential effects of *Hypericum esculentum* have attracted widespread attention. Therefore, further development of the uses of *Hypericum esculentum* is of great significance. Summary of the Invention

[0005] One object of the present invention is to provide a coumarin compound isolated from Uncaria rhynchophylla, and another object of the present invention is to provide a method for obtaining an effective chemical component from Uncaria rhynchophylla, and to provide the application of the coumarin compound.

[0006] According to a first aspect of the invention, the invention provides a coumarin compound isolated from *Hylocereus undatus*, said compound having a structure as shown in Formula 1:

[0007] .

[0008] According to a second aspect of the invention, the invention provides a coumarin compound isolated from *Hylocereus undatus*, said compound having a structure as shown in Formula 2:

[0009] .

[0010] According to a third aspect of the present invention, the present invention provides a method for preparing coumarin compounds isolated from Uncaria rhynchophylla, the method comprising: extracting Uncaria rhynchophylla with alcohol, adsorbing with macroporous resin and column separation to obtain compounds 1-2;

[0011] The compounds 1-2 have structures as shown in Formula 1 to Formula 2:

[0012] ;

[0013] Compound 1 and Compound 2 are an enantiomer pair; specifically, Compound 1 and Compound 2 are 5-methylcoumarin compounds.

[0014] Optionally, the extract of *Hedyotis diffusa* obtained by alcohol extraction is adsorbed by macroporous adsorption resin, eluted and concentrated with ethanol to obtain an ethanol fraction. The ethanol fraction is then subjected to a normal-phase column and eluted with a dichloromethane-methanol gradient to obtain eight fractions Fr.1-8. Fr.4 is separated by column chromatography to obtain compounds 1-2.

[0015] Optionally, the column separation of Fr.4 to obtain compounds 1-4 includes: eluting Fr.4 on a normal-phase column with petroleum ether-ethyl acetate to obtain 9 fractions Fr.4.1-4.9; eluting Fr.4.7 on a normal-phase column with a petroleum ether-ethyl acetate gradient to obtain 12 fractions Fr.4.7.1-4.7.12; eluting Fr.4.7.8 on a gel column and a normal-phase column gradient to obtain 2 fractions Fr.4.7.8.1-4.7.8.2; and separating Fr.4.7.8.1 on a gel column, a reverse-phase column, and a chiral column to obtain compounds 1-2.

[0016] According to a fourth aspect of the present invention, the present invention provides a pharmaceutical composition comprising the above-described coumarin compounds or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier or excipient.

[0017] According to a fifth aspect of the present invention, the present invention provides an inhibitor containing the above-described coumarin compounds.

[0018] According to a sixth aspect of the present invention, the present invention provides the use of the above-described coumarin compounds in the preparation of anti-inflammatory inhibitors.

[0019] According to a seventh aspect of the invention, the present invention provides the use of the above-described compound in the preparation of a RAW264.7 cell inhibitor.

[0020] According to an eighth aspect of the present invention, the present invention provides the use of the above-described compounds in the preparation of inhibitors of TNF-α, IL-1β, and IL-6 protein expression.

[0021] Optionally, the inhibitor may be in the form of tablets, pills, powders, granules, capsules, oral liquids, infusions, lyophilized powder injections, ointments, gels, or sprays.

[0022] The present invention has the following beneficial effects:

[0023] This invention isolates and extracts 5-methylcoumarin enantiomers of formula 1-2 from *Hedyotis diffusa*. Experimental testing shows that compounds 1-2 exhibit inhibitory activity against RAW264.7 cells and the expression of TNF-α, IL-1β, and IL-6 proteins, making them suitable for use as anti-inflammatory agents and in the preparation of related drugs. The preparation method of this invention is simple and low-cost, promoting the development of *Hedyotis diffusa* in medicine, health products, and food, and playing a significant role in promoting the sustainable development of the industry. Attached Figure Description

[0024] Figure 1 HR-ESI-MS of compounds 1 and 2 of the present invention are shown;

[0025] Figure 2 Compound 1 and Compound 2 of the present invention are shown. 1 H-NMR spectrum;

[0026] Figure 3 Compound 1 and Compound 2 of the present invention are shown. 13 C-NMR spectrum;

[0027] Figure 4 The nuclear magnetic resonance (HMQC) spectra of compounds 1 and 2 of this invention are shown.

[0028] Figure 5 The nuclear magnetic resonance HMBC spectra of compounds 1 and 2 of the present invention are shown.

[0029] Figure 6 The nuclear magnetic resonance (NMR) values ​​of compounds 1 and 2 of the present invention are shown. 1 H- 1 H COSY spectrum. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention; that is, the described embodiments are only a part of the embodiments of the invention, and not all of them.

[0031] Example 1

[0032] Step 1: Take 20kg of the herbal extract of *Houttuynia cordata* and extract it by reflux with 50% ethanol. Repeat the extraction 3 times, each time for 1.5 hours. Use 8 times the amount of 50% ethanol for the first extraction, and add 6 times the amount of 50% ethanol for the second and third extractions respectively. Filter the ethanol extract, combine the filtrates and recover the ethanol under reduced pressure. Heat and concentrate the extract while adding water to evaporate the ethanol until there is no obvious ethanol smell.

[0033] Step 2: After mixing the extract, it is coarsely separated by D101 macroporous adsorption resin. After adsorption by macroporous adsorption resin, it is eluted and concentrated successively with water and 50% ethanol to obtain different component fractions (water fraction and 50% ethanol fraction).

[0034] Step 3: Load the 50% ethanol extract onto a normal-phase silica gel column and elute with a dichloromethane-methanol gradient of 10:0-5:5. Detect the eluent by thin-layer chromatography, develop the color, and combine the colored eluent fractions. Detect and combine the eluents by TLC to obtain 8 fractions Fr.1-8. Concentrate the combined eluent fractions to dryness under reduced pressure for later use.

[0035] Step 4: Fr.4 from Step 3 was subjected to normal-phase silica gel column chromatography with a petroleum ether-ethyl acetate gradient elution (10:0-5:5). After TLC analysis and concentration, the fractions were combined to obtain 9 fractions Fr.4.1-4.9. Among them, Fr.4.7 was subjected to normal-phase silica gel column chromatography with a petroleum ether-ethyl acetate gradient elution (10:0-5:5). After TLC analysis and concentration, the fractions were combined to obtain 12 fractions Fr.4.7.1-4.7.12. Among them, Fr.4.7.8 was subjected to repeated Toyopearl HW-40F and Sephadex LH-20 gel column chromatography (dichloromethane:methanol 1:1), followed by normal-phase silica gel column chromatography with a petroleum ether-ethyl acetate gradient elution (10:0-5:5). After TLC analysis and concentration, the fractions were combined to obtain 2 fractions Fr.4.7.8.1-4.7.8.2. Among them, Fr.4.7.8.1 was subjected to Toyopearl HW-40F gel column (methanol) and ODS reversed-phase column (20%-100% methanol-water) to obtain other compounds and white solids. The white solids were separated by Chiral MX(2)-RH (250mm×4.6mm, 5µm) chiral column (95% acetonitrile full gradient) to obtain compound 1 and compound 2.

[0036] In the above example, petroleum ether-ethyl acetate (10:0-5:5) means that the ratio of petroleum ether to ethyl acetate is 10:0-5:5.

[0037] The structural identification performed in this invention: using 1 H NMR, 13 C10 NMR spectroscopy, two-dimensional NMR spectroscopy, and high-resolution mass spectrometry are used to identify the structure of isolated monomeric compounds, such as... Figure 1-6 As shown.

[0038] Compound 1 and Compound 2 are an enantiomer pair.

[0039] Compound 1, appearance (white powder), molecular formula: C 20 H 24 O4; HR-ESI-MS m / z: 329.1756 [M+H] + The calculated value is 329.1747; its NMR data are shown in Table 1.

[0040] Compound 2, appearance (white powder), molecular formula: C 20 H 24 O4; HR-ESI-MS m / z: 351.1576 [M+Na] + The calculated value is 351.1567; its NMR data is in Table 1, and the corresponding chemical formulas with labels are shown in Formulas 1-2 below.

[0041] .

[0042] Table 1. Compounds 1 and 2 1 H (400 MHz) and 13 C(100 MHz) NMR

[0043]

[0044] Experimental Example 1

[0045] To achieve the above-mentioned objectives of this invention, the anti-inflammatory activity of 5-methylcoumarin enantiomers isolated from *Hedyotis diffusa* was studied. LPS (lipopolysaccharide)-induced mouse macrophages RAW264.7 were used as an inflammation model. The effect of the compounds on the survival rate of RAW264.7 cells was tested using the CCK-8 assay to determine the safe dosage concentration. The effects of the compounds on the formation of TNF-α, IL-1β, and IL-6 in the supernatant were measured using ELISA. This study is of profound significance in preliminarily revealing the significant anti-inflammatory effects of 5-methylcoumarin enantiomers isolated from *Hedyotis diffusa* and lays a solid foundation for subsequent research on the drug's mechanism of action. The specific steps are as follows:

[0046] Step 1, Cell Thawing: Quickly remove the frozen cells from the -80℃ freezer and thaw them rapidly in a 37℃ water bath. Spray the outside of the cryovials with medical alcohol for sterilization. Open the lid in a clean bench, aspirate the cell suspension using a pipette, and transfer it to a centrifuge tube. Slowly add 4-5 mL of culture medium dropwise, resuspending the cells by pipetting. Centrifuge at 1000 rpm for 3 minutes at room temperature. Aspirate the supernatant, add 1-2 mL of culture medium, resuspend, and transfer to a culture dish containing 3-4 mL of culture medium. Observe cell growth regularly.

[0047] Step 2, Cell Culture and Passaging: Observe cell growth daily. If cell growth is slow and has not yet reached 70-80% confluence, only medium change is needed. Remove the cell culture flask or dish from the incubator, spray it with 75% medical alcohol, and then transfer it to a laminar flow hood for further processing. In the laminar flow hood, gently aspirate the culture medium from the flask or dish and wash the cells with physiological saline twice. After washing twice, add approximately 3 mL of complete culture medium and allow the cells to continue culturing. When the cells reach 70-80% confluence, passaging is necessary. Similarly, remove the cell culture flask or dish from the incubator, spray it with 75% medical alcohol, and then transfer it to a laminar flow hood. Next, gently aspirate the culture medium from the flask or dish and wash the cells with physiological saline twice. After washing twice, add approximately 1 mL of trypsin to digest the cells. The entire digestion process takes about 2 minutes. After trypsin digestion, the digestion solution needs to be transferred to a centrifuge tube. Use culture medium to wash the cells adhering to the walls of the culture flask or dish, and transfer them to the centrifuge tube as well. Centrifuge at 1000 rpm for 3 minutes. After centrifugation, discard the supernatant to obtain the cells adhering to the tube wall. At this point, add complete culture medium to the cells and repeatedly pipette to evenly disperse them before aliquoting and culturing.

[0048] Step 3: Divide the experiment into a control group and groups with different concentrations of LPS. The control group was given complete DMEM medium, and the groups with different concentrations of LPS were given medium with final LPS concentrations of 0.25, 0.5, 1, 2 and 4 μg / mL, respectively.

[0049] Step 4: Collect cells in the logarithmic growth phase and adjust the cell concentration to approximately 3 × 10⁻⁶. 5 Approximately 100 μL / mL was inoculated into each well of a 96-well plate and incubated at 37°C with 5% CO2 for 24 h to allow adhesion. After 24 h of incubation, the plate was washed once with PBS, and the OD value was measured using the CCK-8 assay. Each concentration was replicated in 5 wells, and the experiment was repeated 3 times.

[0050] Step 5: Preliminary screening of compounds with anti-inflammatory activity from *Hedyotis diffusa*: RAW264.7 cells in the logarithmic growth phase were used, and the cell concentration was adjusted to 3 × 10⁻⁶. 5Cells were seeded at a density of approximately 100 μL / mL in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. Cells were then divided into Control, Model, Positive, and Drug-treated groups. The Control and Model groups were cultured in RAW264.7 cell-specific medium, the Positive group in medium containing DEX, and the Drug-treated group in medium containing the monomeric compound. The final concentrations of DEX and the compound were 25 μmol / L, 25 and 50 μmol / L, and 1 μg / mL, respectively. After 3 h of incubation, the Control group was cultured in complete RAW264.7 cell-specific medium, the Model group in medium containing LPS, the Positive group in a mixture of DEX and LPS, and the Drug-treated group in a mixture of the compound and LPS. After 24 h of incubation, the culture supernatant was collected, and the effects of the supernatant on the production of TNF-α, IL-1β, and IL-6 were determined by ELISA. Each concentration was tested in triplicate, and the experiment was repeated three times.

[0051] Compared with the normal group, the serum levels of TNF-α (P<0.05), IL-6 (P<0.01), and IL-1β (P<0.01) in the model group mice were significantly increased. Compared with the model group, the positive group significantly reduced the serum levels of TNF-α (P<0.01), IL-6 (P<0.001), and IL-1β (P<0.01) in mice. After intervention with Uncaria rhynchophylla, compared with the model group, the serum levels of TNF-α, IL-6, and IL-1β in mice were significantly reduced (P<0.05, P<0.01, P<0.001). The IC50 values ​​of compounds 1 and 2 for TNF-α, IL-1β, and IL-6 were significantly reduced. 50 The values ​​are shown in Table 2.

[0052] Table 2. IC50 of compounds 1-2 against TNF-α, IL-1β, and IL-6 50 value

[0053]

[0054] Table 2 shows that the changes in TNF-α, IL-1β, and IL-6 levels in the supernatant were detected by ELISA. This indicates that compounds 1-2 all exhibited inhibitory activity against RAW264.7 cells and the expression of TNF-α, IL-1β, and IL-6 proteins, with compound 2 showing significant inhibitory activity against IL-6 protein expression. These experimental results preliminarily reveal the anti-inflammatory effects of the 5-methylcoumarin enantiomers 1-2 isolated from *Uncaria rhynchophylla*, providing strong evidence for further in-depth research on their anti-inflammatory mechanisms. This suggests that the compounds of this invention can be used as a reference for the inhibitory use of inflammation and for the preparation of related drugs, laying the foundation for research on drug mechanisms of action.

[0055] Uses, preparations, administration:

[0056] Medical uses and indications:

[0057] The biological data provided by this invention indicate that the compounds of this invention are beneficial for the treatment or prevention of diseases caused by abnormalities in RAW264.7 (macrophages). Therefore, the compounds of this invention are beneficial for the treatment of autoimmune diseases, including but not limited to: psoriasis, vitiligo, dermatitis, alopecia areata, rheumatoid arthritis, colitis, multiple sclerosis, systemic lupus erythematosus, and Crohn's disease. The compounds of this invention can be used as monotherapy or in combination therapy, and can be used in combination with other drugs besides those of this invention.

[0058] Pharmaceutical methods:

[0059] The pharmaceutical method of the present invention includes determining a therapeutically effective dose for a subject requiring the compounds of the present invention. The "therapeutically effective dose" varies depending on the stage, progression, or severity of the disease. The daily dose of the compounds and compositions of the present invention will depend on various factors of the patient, including the condition being treated, the severity of the condition, the efficacy of the specific compound used, the specific composition, age, weight, general health status, sex and diet, route and schedule of administration, metabolism and / or the rate of excretion of the compound, duration of treatment, etc. Furthermore, the required dose of the compounds of the present invention, after being formulated into a pharmaceutically acceptable carrier, can be administered to humans and other animals. Routes of administration include oral, rectal, parenteral, intracisional, intravaginal, intraperitoneal, topical (e.g., via transdermal patches, powders, ointments, or drops), sublingual, buccal, or nasal spray, etc. The effective dose of the compounds of the present invention is generally measured in terms of the amount administered per kilogram of patient body weight, preferably 0.1 to 125 mg / kg body weight, and generally 0.01 to 500 mg / kg body weight. Dosing can be done once or multiple times, daily, weekly, every other day or every few days, or on an intermittent schedule. For example, the compound can be administered daily, weekly (e.g., every Monday), indefinitely, or for several weeks (e.g., 4-10 weeks). The effective dose of the compounds of the present invention will vary depending on the compound used, the dosing regimen, the severity of the disease, the conditions being treated, and various physical factors associated with the patient. In most cases, satisfactory therapeutic effects are achieved when the daily dose of the preferred compounds of the present invention is about 0.01 to 500 mg / kg. The preferred dose is 0.1 to 125 mg / kg, and the more preferred dose is 1 to 25 mg / kg. Parenteral doses are typically at about 10%-20% of the oral dose level. When the compounds of the present invention are used as part of a combination therapy regimen, each component of the composition will be administered during a desired treatment period. Whether as a single dosage unit or as a single dosage form containing two components, the components in the composition can be administered simultaneously during the treatment period, or at different times during the treatment period, or one can be administered as a pretreatment of the other.

[0060] Regarding compounds:

[0061] The compounds of this invention can be used for treatment in their free form or, where appropriate, in the form of pharmaceutically acceptable salts or other derivatives. As used herein, "pharmaceutically acceptable salt" refers to the organic and inorganic salts of the compounds of this invention, which are suitable for humans and lower animals, without excessive toxicity, irritation, allergic reactions, etc., and have a reasonable benefit / risk ratio. Pharmaceutically acceptable salts of amines, carboxylic acids, phosphonates, and other types of compounds are well known in the art. Such salts can be prepared by reacting the compounds isolated and purified in this invention with a suitable free base or acid.

[0062] Salts formed from pharmaceutically non-toxic acids, including but not limited to amino salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, malonic acid, or obtained by means well known in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentane, disglucuronate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptahydrate, glycerophosphate, gluconate, hemisulfate, heptahydrate, hydroiodate, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, per-3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Other pharmaceutically acceptable salts include suitable non-toxic ammonium, quaternary ammonium, and amino cations formed using ions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0063] Furthermore, the term "prodrug" as used herein refers to a compound that can be converted in vivo into a compound represented by Formula 1 or Formula 2 of this invention. This conversion is achieved through the hydrolysis of the prodrug in the blood or through enzymatic action in the blood or tissues to the parent compound.

[0064] Composition:

[0065] The compositions described in this patent consist of any of the compounds described herein (or prodrugs, or pharmaceutically acceptable salts thereof, or other pharmaceutically acceptable derivatives thereof), and one or more pharmaceutically acceptable carriers or excipients. These compositions may optionally further comprise one or more additional therapeutic agents. The compounds of the present invention can be administered to the desired patient in combination with one or more other treatment regimens (e.g., tofacitinib or other kinase inhibitors, interferon, bone marrow transplantation, farnesyltransferase inhibitors, bisphosphonates, thalidomide administration combinations, cancer vaccines, hormone therapy, antibodies, radiation, etc.). The pharmaceutical composition of the compounds may be another one or more anti-inflammatory or anticancer agents.

[0066] As described herein, the compositions of the present invention comprise the compounds of the present invention and pharmaceutically acceptable carriers, including any and all solvents, diluents or other carriers, dispersants or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., to suit a particular dosage form. Examples of pharmaceutically acceptable carrier materials include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; ethylene glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; and phosphate buffer solutions, as well as other non-toxic and compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and colorants, release agents, coating agents, sweeteners, flavoring agents and aromatizers, preservatives, and antioxidants may also be present in the composition.

[0067] formula:

[0068] This invention also covers a class of compositions (collectively referred to herein as “carrier” materials) using the active compounds of this invention in combination with one or more pharmaceutically acceptable carriers and / or diluents and / or adjuvants, and, if desired, other active ingredients. The active compounds of this invention can be administered via any suitable route, preferably in the form of a pharmaceutical composition suitable for such route of administration at an effective dose required for the intended treatment. The compounds and compositions of this invention can be administered orally, via mucosal, topical, rectal, pulmonary, such as by inhalation spray, or parenterally, including intravascular, intravenous, intraperitoneal, subcutaneous, intramuscular, intrasternal, and infusion techniques. Administration is in dosage form and contains a pharmaceutically acceptable carrier, adjuvant, and excipient. For oral administration, the pharmaceutical composition can be in the following forms, for example, tablets, capsules, suspensions, or liquids. Examples of dosage units are tablets or capsules. For example, they may contain an amount of active ingredient from 1 to 2000 mg, preferably from 1 to 500 mg, and more commonly from 5 to 200 mg. The appropriate daily dose for an individual or other mammal may vary depending on the patient and other factors, but can be determined again using conventional methods. As previously stated, the amount of compound in the administration and dosing regimens of the compounds and / or compositions involved in this invention depends on a variety of factors, including the subject's age, weight, sex, and medical condition, type of disease, severity of the disease, route and frequency of administration, and the specific compound used. Therefore, dosing regimens can vary considerably, but can be determined using standard methods. Typical daily doses are 0.01 to 500 mg / kg body weight, preferably 0.1 to 125 mg / kg body weight, and more preferably 1 to 25 mg / kg body weight.

[0069] The active compounds of the present invention are typically administered via a route of administration with one or more adjuvants, excipients, or carriers. If administered orally, the compounds may be mixed with lactose, sucrose, starch powder, cellulose esters, alkyl cellulose esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric acid and sulfuric acid, gelatin, farnesian gum, sodium alginate, polyvinylpyrrolidone / or polyvinyl alcohol, and then compressed into tablets or capsules for convenient administration. Such capsules or tablets may contain a controlled-release formulation that disperses the active compound in hydroxypropyl methylcellulose. Formulations suitable for topical administration include liquid or semi-liquid formulations (such as liniments, lotions, ointments, creams, or pastes) suitable for penetration through the skin and drops suitable for application to the eyes, ears, or nose. Suitable topical doses of the compounds of the present invention are 0.1 to 150 mg, once to four times daily, preferably once to twice daily. For topical administration, when using ointments, the active ingredient may be based on any paraffin or water-miscible ointment. Alternatively, the active ingredient can be formulated as a water-in-oil emulsion base cream. If desired, the aqueous phase of the cream base may include, for example, at least 30% by weight of, polyols such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerin, polyethylene glycol, and mixtures thereof. Topical formulations may include compounds that enhance the absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethyl sulfoxide and related analogues. The compound may also be administered via a transdermal device. Transdermal administration is preferably achieved using patches containing a reservoir and a porous membrane or solid base. The oil phase of the emulsion of the present invention may be composed of known ingredients in a known manner, comprising at least one emulsifier in a mixture of fat or oil, or a mixture of both fat and oil. Optionally, the hydrophilic emulsifier may be used in conjunction with a lipophilic emulsifier as a stabilizer; additionally, it is preferred that it may also be used in conjunction with oil and fat. Suitable emulsifiers and emulsion stabilizers for use in the formulations of this invention include Tween 60, Span 80, cetearyl alcohol, myristicin, glyceryl monostearate, sodium lauryl sulfate, glyceryl distearate alone, or mixtures thereof with emulsifying waxes, or other materials known in the art. Creams should preferably be non-greasy, non-staining, and washable, and have a suitable consistency to prevent leakage from tubes or other containers. Straight-chain or branched, mono- or dialkyl esters such as diisohexadiate, isohexadecanoyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate, or a mixture of branched esters may also be used. Alternatively, high-melting-point lipids such as white soft paraffin and / or liquid paraffin or other mineral oils may be used. Formulations suitable for topical administration to the eye also include eye drops, wherein the active ingredient is dissolved or suspended in a suitable carrier, particularly for aqueous solvents of the active ingredient.The active ingredient in these formulations is preferably 0.5% to 20% by weight, more advantageously 0.5% to 10%, and most preferably about 1.5% concentration. For parenteral administration, the formulations can be in the form of aqueous or non-aqueous isotonic sterile injectable solutions or suspensions. These solutions and suspensions can be prepared from one or more sterile powders or granules, using the formulations mentioned herein for oral administration or using other suitable dispersants or wetting agents and suspending agents, carriers, or diluents. The compounds can be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, tragacanth gum, and / or various buffer solutions. Other adjuvants and routes of administration are well known in the pharmaceutical industry.

[0070] The active ingredient can also be administered by injection, in combination with suitable carriers including saline, glucose, or water, or with cyclodextrin (Captisol), co-solvent solubilization (i.e., propylene glycol), or micellar solubilization (i.e., Tween 80). The formulation can also be a sterile injectable solution or a suspension in a non-toxic, parenteral acceptable diluent or solvent, such as 1,3-butanediol. Suitable solvents include water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile, non-volatile oils are commonly used as solvents or suspension media. Any mild fixed oil used for this purpose can be used, including synthetic mono- or diglycerides.

[0071] For pulmonary administration, the pharmaceutical composition can be administered as an aerosol or via inhaler, including dry powder aerosols. Suppositories for rectal administration can be prepared by combining the drug with suitable non-irritating excipients, such as cocoa butter and polyethylene glycol, which are solids at room temperature but liquids at rectal temperature, thus melting and releasing the drug in the rectum. The pharmaceutical composition can be incorporated into conventional pharmaceutical processes such as sterilization and / or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc. Tablets and pills can also be prepared with enteric coating. Such compositions may also contain adjuvants such as wetting agents, sweeteners, flavoring agents, and aromatizers.

[0072] The pharmaceutical compositions of the present invention comprise a compound of formula 1 or 2 as described herein, or a pharmaceutically acceptable salt thereof, a kinase inhibitor (small molecule, peptide, antibody, etc.), an immunosuppressant, an anticancer drug, an antiviral agent, an anti-inflammatory agent, an antifungal agent, an antibiotic, or an antiangiogenic compound, and any pharmaceutically acceptable carrier, adjuvant, or excipient. Alternative compositions of the present invention comprise compounds having formula 1 or 2 as described herein, or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable carriers, adjuvants, or excipients. Such compositions may optionally comprise one or more additional therapeutic agents, including, for example, kinase inhibitors (small molecule, peptide, antibody, etc.), immunosuppressants, anticancer agents, antiviral agents, anti-inflammatory agents, antifungal agents, antibiotics, or antiangiogenic compounds.

[0073] The term (pharmaceutically acceptable carrier or adjuvant) refers to a carrier or adjuvant that can be administered to a patient together with the compound of the present invention, and which does not impair the drug activity and is non-toxic when the dose is sufficient to deliver a therapeutic dose. Pharmaceutically acceptable carriers, adjuvants, and excipients may be used in the pharmaceutical compositions of the present invention, including but not limited to ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-atocopHerol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tween or other similar polymer delivery matrices, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, surfactants used in mixtures of saturated vegetable fatty acids in the form of glycerides, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin. Cyclodextrins such as α-, β-, and γ-cyclodextrins, or chemically modified derivatives such as hydroxyalkyl, including 2- and 3-hydroxypropyl-cyclodextrins, or other soluble derivatives, may also be advantageously used to improve the delivery of compounds of the structural formula described herein. The pharmaceutical composition may be administered orally in any acceptable dosage form, including but not limited to capsules, tablets, emulsions, and aqueous suspensions, dispersions, and solutions. In the case of tablets for oral administration, commonly used carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When administered orally using aqueous suspensions and / or emulsions, the active ingredient may be suspended or dissolved in the oil phase with the emulsion and / or suspending agent. If desired, certain sweeteners, flavoring agents, and / or coloring agents may be added. The pharmaceutical composition may include the use of liposomes or microencapsulation techniques, various examples of which can be found in the literature. The pharmaceutical composition may be administered via nasal aerosol or inhalation. Such compositions are prepared according to known techniques in the field of pharmaceutical formulations and can be prepared into solutions in saline, using benzyl alcohol or other suitable preservatives, absorption enhancers to improve bioavailability, fluorocarbons, and / or other solubilizers or dispersants, examples of which are well known in the prior art.

[0074] Combination therapy:

[0075] The compounds of the present invention can be used alone or in combination with one or more other compounds of the present invention or with one or more other pharmaceutical agents. When administered in combination, the therapeutic agents can be formulated for simultaneous or sequential administration at different times, or the therapeutic agents can be administered as a single composition. The term "combination therapy" refers to the use of the compounds of the present invention in conjunction with another pharmaceutical agent, administered either simultaneously or sequentially, with the aim of achieving optimal drug efficacy. Co-administration includes simultaneous delivery formulations as well as separate formulations for each compound. Therefore, the administration of the compounds of the present invention can be used concurrently with other known therapies in the art, such as in cancer treatment using radiotherapy or adjunctive therapies such as cell growth inhibitors, cytotoxic agents, or other anticancer agents to improve cancer symptoms. The present invention is not limited to the order of administration; the compounds of the present invention can be administered prior to, concurrently with, or after other anticancer agents or cytotoxic agents.

[0076] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. A coumarin compound isolated from *Hylocereus undatus*, characterized in that, The compound has the structure shown in Formula 1: 。 2. A coumarin compound isolated from *Hylocereus undatus*, characterized in that, The compound has the structure shown in Formula 2: 。 3. A method for preparing coumarin compounds isolated from Uncaria rhynchophylla, characterized in that, The preparation method includes: extracting Uncaria rhynchophylla with alcohol, adsorbing with macroporous resin and separating with column to obtain compounds 1-2; The compounds 1-2 have structures as shown in Formula 1 to Formula 2: 。 4. The method for preparing coumarin compounds isolated from Uncaria rhynchophylla as described in claim 3, characterized in that, The extract of *Hedyotis diffusa* obtained by alcohol extraction is adsorbed onto a macroporous adsorption resin, eluted and concentrated with ethanol to obtain an ethanol fraction. The ethanol fraction is then passed through a normal-phase column and eluted with a dichloromethane-methanol gradient to obtain eight fractions Fr.1-8. Fr.4 is separated by column chromatography to obtain compounds 1-2.

5. A pharmaceutical composition comprising a coumarin compound as described in claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

6. An inhibitor containing a coumarin compound as described in claim 1.

7. An inhibitor containing a coumarin compound as described in claim 2.

8. The use of the coumarin compounds as described in claim 1 or claim 2 in the preparation of anti-inflammatory inhibitors.

9. The use of the compound of claim 1 or claim 2 in the preparation of RAW264.7 cell inhibitors.

10. The use of the compound of claim 1 or claim 2 in the preparation of inhibitors of TNF-α, IL-1β, and IL-6 protein expression.

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