Separation and application of 1-phenyl-1,3,3-propanetriamine, a compound with uric acid lowering function from coix seed

Abstract

The invention discloses the separation and application of 1-phenyl-1,3,3-propanetriamine, a compound with uric acid lowering function in coix seed, and belongs to the field of medical technology. After the coix seed is crushed, the present invention is placed in an extraction tank, ethanol is added, stirred and extracted, the filtrate is filtered and taken, the filter residue is repeatedly extracted once according to the above method, the two filtrates are combined, and the coix seed alcohol extract is obtained by reduced pressure concentration; the coix seed alcohol extract is first preliminarily separated by an MCI column, and then further separated by an ODS column to obtain a monomer compound, and the monomer compound is identified as 1-phenyl-1,3,3-propanetriamine by mass spectrometry and nuclear magnetic resonance analysis. The obtained compound has a significant uric acid lowering effect and can be used for the treatment of hyperuricemia.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to the separation and application of 1-phenyl-1,3,3-propanetriamine, a compound with uric acid lowering function in coix seeds. Background Art

[0002] Coix lacrma-jobi L. var. mayuen (Roman.) Stapf is the dried mature seed of the grass plant Coix lachryma-jobi. It has been used as a medicinal and edible material for thousands of years and is used for diuresis, treatment of rheumatism, warts and chronic ulcers. Modern pharmacological studies have shown that coix lachryma-jobi has multiple biological activities such as anti-inflammatory analgesia, immunomodulation, and lowering of serum uric acid. These physiological activities of coix lachryma-jobi extracts are attributed to their unique phytochemical components, such as polysaccharides, phenolic compounds, flavonoids and phytosterols. However, there are few studies on the effective components of coix lachryma-jobi in lowering uric acid. At present, some studies have found that coix lachryma-jobi polyphenol extracts can effectively reduce the serum uric acid level in rats with hyperuricemia, but there are few studies on the separation and chemical structure identification of the active components of coix lachryma-jobi in lowering uric acid. Summary of the invention

[0003] In view of the above situation, the present invention crushes coix seed, places it in an extraction tank, adds ethanol, stirs and extracts, filters and obtains the filtrate, and extracts the residue once again according to the above method, and combines the two filtrates. The coix seed alcohol extract is concentrated under reduced pressure to obtain the coix seed alcohol extract. The coix seed alcohol extract is firstly separated by an MCI column, and then further separated by an ODS column to obtain a monomer compound. The monomer compound is identified as 1-phenyl-1,3,3-propanetriamine by mass spectrometry and nuclear magnetic resonance analysis, and its structural formula is as follows:

[0004]

[0005] One object of the present invention is to provide a compound having a structure represented by formula (1) or a pharmaceutically acceptable salt thereof.

[0006]

[0007] In one embodiment of the present invention, the pharmaceutically acceptable salt includes an inorganic salt or an organic salt; the inorganic salt includes hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, phosphate, acid phosphate; the organic salt is selected from formates, acetates, trifluoroacetates, propionates, pyruvates, glycolates, oxalates, malonates, succinates, glutarates, fumarates, maleates, lactates, malates, citrates, tartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, salicylates, p-toluenesulfonates, and ascorbates.

[0008] The present invention also provides a method for preparing the above compound, comprising the following steps:

[0009] (1) crushing coix seed and placing it in an extraction tank, adding ethanol with a volume fraction of 30 to 70%, stirring and extracting at 30 to 60° C., filtering and collecting the filtrate after standing, repeatedly extracting the residue according to the same process, combining the filtrate; concentrating the filtrate to obtain coix seed alcohol extract;

[0010] (2) The above-mentioned coix seed alcohol extract is initially separated by MCI column, and water eluent, 30% ethanol eluent, 50% ethanol eluent, and 95% ethanol eluent are used in sequence, and the 50% ethanol eluate is collected; the 50% ethanol eluate is further separated by ODS column, and water, 10%, 30% and 50% ethanol are used in sequence for gradient elution, and the 30% ethanol eluate is collected; the 30% ethanol eluate is further separated by ODS column, and 20% and 30% ethanol are used in sequence for gradient elution, and the 30% ethanol eluate is collected, thereby obtaining the compound with the structure shown in formula (1).

[0011] The present invention also provides a uric acid-lowering drug containing a compound having a structure represented by the above formula (1) or a pharmaceutically acceptable salt thereof.

[0012] The present invention also provides a use of the compound having a structure represented by the above formula (1) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating hyperuricemia.

[0013] In one embodiment of the present invention, the drug further comprises pharmaceutical excipients, including: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, flocculants and deflocculating agents, filter aids and release retardants.

[0014] In one embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier selected from microcapsules, microspheres, nanoparticles and liposomes.

[0015] In one embodiment of the present invention, the dosage forms of the drug include: injection, lyophilized powder for injection, suspension, implant, embolic agent, capsule, tablet, pill and oral solution.

[0016] In one embodiment of the present invention, animal experiments show that the optimized composition has a significant uric acid-lowering effect. The inhibition of xanthine oxidase activity test shows that it has a strong inhibitory effect on XOD activity, with an inhibition rate of 49.52%.

[0017] Beneficial effects:

[0018] The present invention uses a model of hyperuricemia mice induced by potassium oxonate and hypoxanthine to evaluate the anti-hyperuricemia activity with indicators such as serum uric acid and liver function. A variety of chromatographic methods are used to obtain the uric acid-lowering effective components from the coix seed ethanol extract, providing a new therapeutic approach for lowering uric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The coix alkaloids obtained in Example 1 13 C NMR spectrum.

[0020] Figure 2 The coix alkaloids obtained in Example 1 135 DEPT NMR spectrum.

[0021] Figure 3 The mass spectrum of coix seed alkaloids obtained in Example 1 is shown in FIG. DETAILED DESCRIPTION

[0022] The present invention is described in detail by the following examples, but it is not intended that the present invention is limited thereto. The specific implementation scheme is as follows.

[0023] Example 1

[0024] Weigh 10 kg of coix seed, crush it, and place it in a 100L extraction tank. Add 70% ethanol by volume at a solid-liquid ratio of 1:10 (g:mL). Stir and extract for 3 hours at 60°C (stirring at 150 rpm). After standing, filter and take the filtrate. Repeat the extraction of the residue once according to the above method, and combine the two filtrates. Concentrate under reduced pressure at 60°C to obtain the coix seed alcohol extract. The coix seed alcohol extract first passes through an MCI column (10×150cm), and then uses 30% ethanol eluent, 50% ethanol eluent, and 95% ethanol eluent as four different elution components, and the elution volume is 5 liters each time. Take the 50% ethanol elution part and further separate it on an ODS (5×100 cm) column, use water, 10%, 30% and 50% ethanol for gradient elution in sequence, the elution volume is 3 liters each time, collect the 30% ethanol eluate and separate it on an ODS column, use 20% and 30% ethanol for gradient elution in sequence, collect the 30% ethanol eluate to obtain the monomer compound.

[0025] The compound is a yellow powder, soluble in water, easily soluble in methanol and ethanol. The mass spectrometry analysis of compound 1 shows that its quasi-molecular ion peak [MH] - m / z = 164.0395, from which its molecular formula is inferred to be C9H 15 N3 (calculated value is 165.2233).

[0026] 1H NMR δ7.25(4H,t,J=8.0Hz), δ7.19(1H,d,J=7.0Hz), δ4.10(1H,t,J=12.0Hz), 2.97(1H,d,J=12.0Hz), 2.78(1H,m) and 2.47(1H,m). 13 C NMR delta 138.7, 129.5, 129.1, 128.1, 127.9, 126.1, 71.5, 55.6, 40.3.

[0027] from 1 From the H NMR spectrum, the presence of hydrogen signals at δ7.25 (4H, t, J = 8.0 Hz) and δ7.19 (1H, d, J = 7.0 Hz) indicates the presence of a monosubstituted benzene ring. There are four hydrogen signals at δ4.10 (1H, t, J = 12.0 Hz), 2.97 (1H, d, J = 12.0 Hz), 2.78 (1H, m) and 2.47 (1H, m). 13 Six aromatic carbon signals can be observed in the C NMR spectrum, δ138.7, 129.5, 129.1, 128.1, 127.9, 126.1, combined with 135 The DEPT NMR spectrum shows that δ138.7 is a quaternary carbon signal, which also proves the existence of a monosubstituted benzene ring. The three carbon signals at δ71.5, 55.6 and 40.3 infer that δ40.3 is a carbon signal on the methylene group based on the chemical shift value, the carbon at δ55.6ppm should be connected to one nitrogen atom, and the carbon at δ71.5ppm should be connected to two nitrogen atoms. In summary, compound 1 is determined to be 1-phenyl-1,3,3-propanetriamine, and its molecular weight and molecular formula are consistent with the mass spectrometry information. This compound is a new alkaloid isolated from natural products for the first time, named coixolidine, and its structural formula is as follows:

[0028]

[0029] Example 2

[0030] The biological activity test of coix alkaloids obtained in Example 1, namely 1-phenyl-1,3,3-propanetriamine:

[0031] In vivo test: 40 SPF male ICR mice (weight 18-22g) were purchased and fed freely under standard environmental conditions (20-26℃, 40-70%). After seven days of adaptive feeding, the mice were randomly divided into 4 groups (n=10): (NC) normal control group, (M) hyperuricemia model group, (P) allopurine positive control group (10mg / kg), coixolidine group (100mg / kg). One hour before the last administration, except for the normal control group, the mice in the other groups were intraperitoneally injected with potassium oxonate and hypoxanthine to establish a hyperuricemia model. One hour after administration on the seventh day, blood samples of mice were collected and centrifuged at 3500r / min for 15min at 4℃ to obtain serum. The levels of uric acid and creatinine in serum were detected by an automatic biochemical analyzer. The results are shown in Table 1 below.

[0032] Table 1 Serum uric acid and creatinine levels

[0033]

[0034] The results show that the coix seed alkaloid group of the present invention has a significant uric acid lowering effect.

[0035] In vitro test: Coixine and xanthine oxidase were dissolved in phosphate buffer (0.2M, pH 7.5). 0.8mL of xanthine oxidase solution (0.1U / mL) was mixed with the same volume of coixine (200μg / mL), then incubated at 37°C for 5 minutes, xanthine was added and the absorbance of the mixture was measured at 295nm every 10 seconds for 5 minutes. Zero adjustment was made with phosphate buffer, and allopurinol (200μg / mL) was set as a positive control. The inhibition rate of xanthine oxidase was calculated according to the following equation: Inhibition rate % = (R0-R A ) / R0×100%; R0 is the reaction rate of the negative control group, R A is the reaction rate of the coixolidine group. The specific results are shown in Table 2.

[0036] Table 2 Effect of coixine on XOD activity

[0037]

[0038] Note: XOD control group refers to an equal amount of xanthine oxidase dissolved in phosphate buffer without adding other drugs. ** P<0.01.

[0039] Results The coixine group had a significant inhibitory effect on xanthine oxidase, thereby achieving the effect of lowering uric acid.

[0040] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A compound having a structure represented by formula (1) or a pharmaceutically acceptable salt thereof, 2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: Pharmaceutically acceptable salts are inorganic salts or organic salts; the inorganic salts are selected from hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, phosphate, acid phosphate; the organic salts are selected from formates, acetates, trifluoroacetates, propionates, pyruvates, glycolates, oxalates, malonates, succinates, glutarates, fumarates, maleates, lactates, malates, citrates, tartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, salicylates, p-toluenesulfonates, and ascorbates.

3. A method for preparing the compound according to claim 1, characterized in that: The process includes the following: (1) crushing coix seed and placing it in an extraction tank, adding ethanol with a volume fraction of 30 to 70%, stirring and extracting at 30 to 60° C., filtering and collecting the filtrate after standing, repeatedly extracting the residue according to the same process, combining the filtrate; concentrating the filtrate to obtain coix seed alcohol extract; (2) The above-mentioned coix seed alcohol extract is initially separated by MCI column, and water eluent, 30% ethanol eluent, 50% ethanol eluent, and 95% ethanol eluent are used in sequence, and the 50% ethanol eluate is collected; the 50% ethanol eluate is further separated by ODS column, and water, 10%, 30% and 50% ethanol are used in sequence for gradient elution, and the 30% ethanol eluate is collected; the 30% ethanol eluate is further separated by ODS column, and 20% and 30% ethanol are used in sequence for gradient elution, and the 30% ethanol eluate is collected, thereby obtaining the compound with the structure shown in formula (1).

4. A uric acid-lowering drug comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

5. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating hyperuricemia.

6. The uric acid lowering drug according to claim 4, characterized in that: The medicine also contains pharmaceutical excipients.

7. The uric acid lowering drug according to claim 6, characterized in that: Pharmaceutical excipients are selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, flocculants and deflocculating agents, filter aids and release retardants.

8. The uric acid lowering drug according to claim 4, characterized in that: The medicine also contains a pharmaceutically acceptable carrier.

9. The uric acid lowering drug according to claim 8, characterized in that: The pharmaceutical carrier is selected from microcapsules, microspheres, nanoparticles and liposomes.

10. The uric acid lowering drug according to claim 4, characterized in that: The dosage form of the drug is selected from the group consisting of injection, freeze-dried powder for injection, suspension, implant, embolic agent, capsule, tablet, pill and oral solution.

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