Phthaloisoquinoline alkaloids and pharmaceutically acceptable salts thereof and applications thereof, pharmaceutical compositions and applications thereof

Abstract

The present invention provides phthalide isoquinoline alkaloids and pharmaceutically acceptable salts thereof and applications thereof, pharmaceutical compositions and applications thereof, and relate to the field of biomedical technology. The phthalide isoquinoline alkaloids and pharmaceutically acceptable salts thereof provided by the present invention have the same chirality at C-1, C-9 and C-7', can significantly promote the occurrence of autophagic flux, are low-toxic and low-side-effect autophagic activators with development potential, are of great significance for the development of drugs for the treatment of neurodegenerative diseases, and have good application prospects in the application of drugs for the treatment of diseases related to autophagic flux and in the preparation of AMPK agonists. The preparation method of the phthalide isoquinoline alkaloids provided by the present invention does not require chemical synthesis, and is obtained by extracting the summer corms as a traditional bulk medicinal material as a raw material. The raw material source is wide, the acquisition is convenient, the cost is low, it conforms to the concept of inheritance and development of traditional Chinese medicine, the operation is simple, the production cost is low, and it is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a phthalide isoquinoline alkaloid and a preparation method and application thereof, a pharmaceutically acceptable salt of the phthalide isoquinoline alkaloid and an application thereof, and a phthalide isoquinoline alkaloid pharmaceutical composition and an application thereof. Background Art

[0002] Autophagy is the process by which cells degrade proteins or organelles through lysosomes, which not only helps to provide nutrients, but also removes harmful substances, such as misfolded proteins and invading microorganisms, helping to support cell homeostasis and survival. Therefore, autophagy defects are associated with a variety of human diseases. Studies have found that autophagy plays an important role in the development of nerve cells and the function of synapses. Abnormal levels of autophagy can cause damage to the nervous system, including autophagosome aggregation, increased neuronal sodium, mitochondrial depletion, and axonal and dendritic atrophy. Neurodegeneration is the chronic progressive degeneration and loss of neurons in the brain and spinal cord, which can lead to Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and Lewy body dementia (DLB). Abnormal proteins accumulate in the brains of patients with neurodegenerative diseases. As a protein quality control mechanism, autophagy is essential for removing aggregation-prone proteins (such as huntingtin and tau) that cause neurodegeneration, while impaired autophagy triggers their accumulation and promotes their toxicity. It can be seen that autophagy plays an important role in neurodegenerative diseases. Therefore, regulating autophagy as a means to treat these diseases is a feasible therapeutic strategy.

[0003] A large number of studies have shown that mTOR is a key regulator of autophagy, which can regulate different steps in the autophagy process (such as nucleation, autophagosome extension, autophagosome maturation and termination). Therefore, mTOR regulation of autophagy is a very promising target. At present, autophagy activators are mainly rapamycin (inducing cell autophagy in an mTOR-dependent manner) and trehalose (inducing cell autophagy in an mTOR-independent manner), but they have strong side effects. At this stage, there is an urgent need to find autophagy activators with low toxicity and low side effects to meet the development of clinical drugs for the treatment of neurodegenerative diseases.

[0004] The traditional Chinese medicine summer nectar is the dried tuber of Corydalis decumbens (Thunb.) pers. of the genus Corydalis of the family Papaveraceae, which has the effects of promoting qi and relieving pain, promoting blood circulation and unblocking collaterals. Summer nectar is often used to treat rheumatoid arthritis, sciatica, lumbar disc herniation, cervical spondylosis and other neuroinflammatory diseases. Chinese patent CN112940001A discloses a phthalide isoquinoline alkaloid, the structural formula of which is as follows:

[0005]

[0006] The phthalide isoquinoline alkaloid can significantly inhibit neuronal excitability and improve neuropathic pain, and can be used as a lead compound for the preparation of drugs that inhibit neuronal excitability and the development of drugs for treating neuropathic pain. However, there are currently no reports of phthalide isoquinoline alkaloids that promote autophagy activity. Summary of the invention

[0007] In view of this, the object of the present invention is to provide phthalide isoquinoline alkaloids and pharmaceutically acceptable salts thereof and their applications, pharmaceutical compositions and their applications. The phthalide isoquinoline alkaloids and pharmaceutically acceptable salts thereof provided by the present invention have a significant promoting effect on the occurrence of autophagic flux.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a phthalide isoquinoline alkaloid, including one or more of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7 and compound 8:

[0010]

[0011] The present invention provides a method for preparing the phthalide isoquinoline alkaloids described in the above technical solution, comprising the following steps:

[0012] The summer bulbless corms are mixed with an extractant, extracted and concentrated to obtain an extract; the extractant includes one or more of an alcohol solvent, a ketone solvent, an ester solvent, an ether solvent and a halogenated alkane solvent; the carbon number of the extractant is ≤6;

[0013] The pH value of the extract is adjusted to acidic before a first extraction is performed, and the pH value of the first extracted organic phase is adjusted to alkaline before a second extraction is performed to obtain a total alkaloid extract; the extractants of the first extraction and the second extraction independently include one or more of an ester solvent, an ether solvent and a halogenated alkane solvent, and the carbon number of the extractant is ≤6;

[0014] The total alkaloid extract is subjected to a first silica gel column chromatography separation to obtain five groups of components, which are sequentially recorded as Fr.A, Fr.B, Fr.C, Fr.D and Fr.E; the mobile phase used in the first silica gel column chromatography separation includes dichloromethane-methanol, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 100:1 to 1:1;

[0015] The preparation method of compound 3 and / or compound 4 comprises the following steps:

[0016] The Fr.A is subjected to a second silica gel column chromatography separation to obtain four components, which are sequentially recorded as Fr.A1, Fr.A2, Fr.A3 and Fr.A4; the mobile phase used in the second silica gel column chromatography separation includes petroleum ether-acetone, and the volume ratio of petroleum ether to acetone in the petroleum ether-acetone is 9:1 to 0:1;

[0017] The Fr.A1 is subjected to a first high performance liquid chromatography separation to obtain four groups of components, which are sequentially recorded as Fr.A1-1, Fr.A1-2, Fr.A1-3 and Fr.A1-4; the mobile phase system used in the first high performance liquid chromatography separation includes methanol-water, and the volume ratio of methanol to water in the methanol-water is 30:70 to 100:0;

[0018] The Fr.A1-3 is subjected to a third silica gel column chromatography separation to obtain compound 3 and compound 4, respectively; the mobile phase used in the third silica gel column chromatography separation includes petroleum ether-ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate is 6:1 to 1:1;

[0019] The preparation method of compound 6 and / or compound 8 comprises the following steps:

[0020] The Fr.D is separated by a reverse phase chromatography column to obtain five groups of components, which are sequentially recorded as Fr.D1, Fr.D2, Fr.D3, Fr.D4 and Fr.D5; the mobile phase used for the reverse phase chromatography column separation includes a methanol aqueous solution, and the volume ratio of methanol to water in the methanol aqueous solution is 30:70 to 100:0;

[0021] The Fr.D2 is subjected to a first Sephadex LH-20 column separation and a second high performance liquid chromatography separation in sequence to obtain compound 6 and compound 8, respectively; the mobile phase used in the first Sephadex LH-20 column separation includes methanol; the mobile phase system used in the second high performance liquid chromatography separation includes an acetonitrile aqueous solution, and the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 10:90 to 100:0;

[0022] The preparation method of one or more of the compound 1, compound 2 and compound 5 comprises the following steps:

[0023] The Fr.E is separated by a fourth silica gel column to obtain 6 groups of components, which are sequentially recorded as Fr.E1, Fr.E5, Fr.E3, Fr.E4, Fr.E5 and Fr.E6; the mobile phase used in the fourth silica gel column separation includes dichloromethane-methanol, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 40:1 to 1:1;

[0024] Sequentially subjecting the Fr.E1 to a second Sephadex LH-20 column separation and a third high performance liquid chromatography separation to obtain compound 1 and compound 2, respectively; the mobile phase used in the second Sephadex LH-20 column separation includes methanol; the mobile phase system used in the third high performance liquid chromatography separation includes an acetonitrile aqueous solution, wherein the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 10:90 to 100:0;

[0025] The Fr.E5 was subjected to a third Sephadex LH-20 column separation to obtain 8 groups of components, which were sequentially recorded as Fr.E5-1, Fr.E5-2, Fr.E5-3, Fr.E5-4, Fr.E5-5, Fr.E5-6, Fr.E5-7 and Fr.E5-8; the Fr.E5-3 was subjected to a fourth high performance liquid chromatography separation to obtain compound 5; the mobile phase used in the third Sephadex LH-20 column separation included methanol; the mobile phase system used in the fourth high performance liquid chromatography separation included a methanol-water solution, and the volume ratio of methanol to water in the methanol-water solution was 10:90 to 100:0.

[0026] Preferably, the solid-liquid ratio of the summer bulbless corms to the extractant is 1 kg: 3-5 L, and the water content of the summer bulbless corms is ≤5%.

[0027] Preferably, the extraction is performed 3 to 4 times;

[0028] The extraction includes cold soaking and hot extraction; the time of a single cold soaking is 18 to 24 hours; the temperature of the hot extraction is 45 to 65° C., and the time of a single hot extraction is 3 to 4 hours.

[0029] Preferably, the acidic pH value is 2-3.

[0030] Preferably, the alkaline pH value is 8-9.

[0031] The present invention provides a pharmaceutically acceptable salt of a phthalide isoquinoline alkaloid. The phthalide isoquinoline alkaloid is the phthalide isoquinoline alkaloid described in the above technical solution or the phthalide isoquinoline alkaloid prepared by the preparation method described in the above technical solution.

[0032] The present invention provides a phthalide isoquinoline alkaloid pharmaceutical composition, comprising an active component and a pharmaceutically acceptable excipient; the active component comprises a phthalide isoquinoline alkaloid and / or a pharmaceutically acceptable salt of the phthalide isoquinoline alkaloid described in the above technical solution; the phthalide isoquinoline alkaloid is the phthalide isoquinoline alkaloid described in the above technical solution or the phthalide isoquinoline alkaloid prepared by the preparation method described in the above technical solution.

[0033] The present invention also provides the use of the phthalide isoquinoline alkaloids described in the above technical scheme, the phthalide isoquinoline alkaloids prepared by the preparation method described in the above technical scheme, the pharmaceutically acceptable salts of the phthalide isoquinoline alkaloids described in the above technical scheme, or the phthalide isoquinoline alkaloid pharmaceutical composition described in the above technical scheme in the preparation of drugs for treating neurodegenerative diseases or AMPK agonists.

[0034] Preferably, the neurodegenerative disease includes a neurodegenerative disease associated with autophagic flux.

[0035] The phthalide isoquinoline alkaloids and pharmaceutically acceptable salts thereof provided by the present invention have the same chirality at C-1, C-9 and C-7', can significantly promote the occurrence of autophagic flux, are low-toxic and low-side-effect autophagic activators with development potential, are of great significance for the development of drugs for treating neurodegenerative diseases, and have good application prospects in the application of drugs for treating diseases related to autophagic flux and in the preparation of AMPK agonists.

[0036] The preparation method of the phthalide isoquinoline alkaloids of the present invention does not require chemical synthesis, and is extracted from the Chinese medicine summer corm as a raw material. The summer corm is a traditional bulk medicinal material with a wide source, convenient acquisition, and low cost, which conforms to the concept of inheritance and development of traditional Chinese medicine. The preparation method has simple process, simple operation, low production cost, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a graph showing the effects of compounds 1 to 8 on HM mCherry-GFP-LC3 cell viability as determined by the CCK8 kit;

[0038] Figure 2 Figure 3. Effects of compounds 1 to 8 on autophagic flux in HM mCherry-GFP-LC3 cells evaluated by flow cytometry. Rapamycin (Rapa) was used as a positive control.

[0039] Figure 3 Confocal was used to evaluate the effects of compound 2 and compound 6 on the autophagic flux process of HM mCherry-GFP-LC3 cells, and rapamycin was used as a positive control. DETAILED DESCRIPTION

[0040] The present invention provides a phthalide isoquinoline alkaloid, including one or more of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7 and compound 8:

[0041]

[0042] The present invention provides a method for preparing the phthalide isoquinoline alkaloids described in the above technical solution, comprising the following steps:

[0043] The summer bulbless corms are mixed with an extractant, extracted and concentrated to obtain an extract; the extractant includes one or more of an alcohol solvent, a ketone solvent, an ester solvent, an ether solvent and a halogenated alkane solvent; the carbon number of the extractant is ≤6;

[0044] The pH value of the extract is adjusted to acidic before a first extraction is performed, and the pH value of the first extracted organic phase is adjusted to alkaline before a second extraction is performed to obtain a total alkaloid extract; the extractants of the first extraction and the second extraction independently include one or more of an ester solvent, an ether solvent and a halogenated alkane solvent, and the carbon number of the extractant is ≤6;

[0045] The total alkaloid extract is subjected to a first silica gel column chromatography separation to obtain five groups of components, which are sequentially recorded as Fr.A, Fr.B, Fr.C, Fr.D and Fr.E; the mobile phase used in the first silica gel column chromatography separation includes dichloromethane-methanol, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 100:1 to 1:1;

[0046] The preparation method of compound 3 and / or compound 4 comprises the following steps:

[0047] The Fr.A is subjected to a second silica gel column chromatography separation to obtain four components, which are sequentially recorded as Fr.A1, Fr.A2, Fr.A3 and Fr.A4; the mobile phase used in the second silica gel column chromatography separation includes petroleum ether-acetone, and the volume ratio of petroleum ether to acetone in the petroleum ether-acetone is 9:1 to 0:1;

[0048] The Fr.A1 is subjected to a first high performance liquid chromatography separation to obtain four groups of components, which are sequentially recorded as Fr.A1-1, Fr.A1-2, Fr.A1-3 and Fr.A1-4; the mobile phase system used in the first high performance liquid chromatography separation includes methanol-water, and the volume ratio of methanol to water in the methanol-water is 30:70 to 100:0;

[0049] The Fr.A1-3 is subjected to a third silica gel column chromatography separation to obtain compound 3 and compound 4, respectively; the mobile phase used in the third silica gel column chromatography separation includes petroleum ether-ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate is 6:1 to 1:1;

[0050] The preparation method of compound 6 and / or compound 8 comprises the following steps:

[0051] The Fr.D is separated by a reverse phase chromatography column to obtain five groups of components, which are sequentially recorded as Fr.D1, Fr.D2, Fr.D3, Fr.D4 and Fr.D5; the mobile phase used for the reverse phase chromatography column separation includes a methanol aqueous solution, and the volume ratio of methanol to water in the methanol aqueous solution is 30:70 to 100:0;

[0052] The Fr.D2 is subjected to a first Sephadex LH-20 column separation and a second high performance liquid chromatography separation in sequence to obtain compound 6 and compound 8, respectively; the mobile phase used in the first Sephadex LH-20 column separation includes methanol; the mobile phase system used in the second high performance liquid chromatography separation includes an acetonitrile aqueous solution, and the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 10:90 to 100:0;

[0053] The preparation method of one or more of the compound 1, compound 2 and compound 5 comprises the following steps:

[0054] The Fr.E is separated by a fourth silica gel column to obtain 6 groups of components, which are sequentially recorded as Fr.E1, Fr.E5, Fr.E3, Fr.E4, Fr.E5 and Fr.E6; the mobile phase used in the fourth silica gel column separation includes dichloromethane-methanol, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 40:1 to 1:1;

[0055] Sequentially subjecting the Fr.E1 to a second Sephadex LH-20 column separation and a third high performance liquid chromatography separation to obtain compound 1 and compound 2, respectively; the mobile phase used in the second Sephadex LH-20 column separation includes methanol; the mobile phase system used in the third high performance liquid chromatography separation includes an acetonitrile aqueous solution, wherein the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 10:90 to 100:0;

[0056] The Fr.E5 was subjected to a third Sephadex LH-20 column separation to obtain 8 groups of components, which were sequentially recorded as Fr.E5-1, Fr.E5-2, Fr.E5-3, Fr.E5-4, Fr.E5-5, Fr.E5-6, Fr.E5-7 and Fr.E5-8; the Fr.E5-3 was subjected to a fourth high performance liquid chromatography separation to obtain compound 5; the mobile phase used in the third Sephadex LH-20 column separation included methanol; the mobile phase system used in the fourth high performance liquid chromatography separation included a methanol-water solution, and the volume ratio of methanol to water in the methanol-water solution was 10:90 to 100:0.

[0057] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0058] The invention mixes the summer bulbless corms with an extractant, extracts and then concentrates to obtain an extract; the extractant comprises one or more of an alcohol solvent, a ketone solvent, an ester solvent, an ether solvent and a halogenated alkane solvent; and the carbon number of the extractant is ≤6.

[0059] In the present invention, the moisture content of the summer bulbless corms is preferably ≤5%. In the present invention, the summer bulbless corms are preferably crushed before use to obtain summer bulbless corm powder, and the particle size of the summer bulbless corm powder is preferably ≤100mm, more preferably 10 to 100mm.

[0060] In the present invention, the alcohol solvent is preferably a C1-C6 alcohol solvent, more preferably including one or more of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, n-pentanol, isopentanol, cyclopentanol, n-hexanol and cyclohexanol, more preferably methanol or ethanol. In the present invention, the ketone solvent is preferably a C3-C6 ketone solvent, more preferably including one or more of acetone, methyl ethyl ketone and methyl isobutyl ketone. In the present invention, the ester solvent preferably includes a C3-C6 ester solvent, more preferably including one or more of ethyl formate, ethyl acetate and ethyl propionate. In the present invention, the ether solvent preferably includes C2-C6 ether, more preferably including methyl ether and / or ethyl ether. In the present invention, the halogenated alkane solvent preferably includes a C1-C6 halogenated alkane solvent, more preferably including one or more of dichloromethane, chloroform and tetrachloroethane.

[0061] In the present invention, the solid-liquid ratio of the summer bulbless corms to the extractant is preferably 1 kg: 3 to 5 L, more preferably 1 kg: 3 to 4 L, and further preferably 1 kg: 3.5 to 4 L.

[0062] In the present invention, the number of extractions is preferably 3 to 4 times. In the present invention, the extraction preferably includes cold soaking and hot extraction; the temperature of the cold soaking is preferably room temperature, and the time of a single cold soaking is preferably 18 to 24 hours, more preferably 20 to 24 hours; the temperature of the hot extraction is preferably 45 to 65°C, more preferably 50 to 60°C; the time of a single hot extraction is preferably 3 to 4 hours, more preferably 3 to 3.5 hours.

[0063] After the extraction, the present invention preferably further comprises concentrating the obtained extract to recover the extractant to obtain an extract. The present invention has no particular limitation on the concentration, and the concentration can be performed by a concentration method well known to those skilled in the art until no solvent flows out, such as vacuum distillation. In the present invention, the recovered extractant can be reused.

[0064] After obtaining the extract, the present invention adjusts the pH value of the extract to acidic and then performs a first extraction, adjusts the pH value of the first extracted organic phase to alkaline and then performs a second extraction to obtain a total alkaloid extract; the extractants of the first extraction and the second extraction independently include one or more of an ester solvent, an ether solvent and a halogenated alkane solvent, and the carbon number of the extractant is ≤6.

[0065] The present invention preferably disperses the extract in water first and then separates the solid and liquid, and then adjusts the pH value of the obtained liquid component to acidic. The present invention does not specifically limit the amount of water used to disperse the extract, as long as the extract can be evenly dispersed in water. The present invention does not specifically limit the method of solid-liquid separation, and a solid-liquid separation method well known to those skilled in the art can be used, such as filtration, suction filtration or centrifugal separation.

[0066] In the present invention, the acidic pH value is preferably 2 to 3, more preferably 2.5 to 3. The present invention has no particular limitation on the acid used to adjust the pH value to acidity, and any acid well known to those skilled in the art can be used, such as hydrochloric acid, sulfuric acid or nitric acid; the concentration of the acid is preferably 2 to 10 wt%, more preferably 2 to 5 wt%.

[0067] In the present invention, the first extraction extractant (first extractant) includes one or more of an ester solvent, an ether solvent and a halogenated alkane solvent, and the carbon number of the first extractant is ≤6. In the present invention, the ester solvent preferably includes a C3-C6 ester solvent, and more preferably includes one or more of ethyl formate, ethyl acetate and ethyl propionate. In the present invention, the ether solvent preferably includes a C2-C6 ether, and more preferably includes methyl ether and / or ethyl ether. In the present invention, the halogenated alkane solvent preferably includes a C1-C6 halogenated alkane solvent, and more preferably includes one or more of dichloromethane, chloroform and tetrachloroethane. In a specific embodiment of the present invention, the first extraction preferably includes petroleum ether extraction and ethyl acetate extraction in sequence. In the present invention, the solid-liquid ratio of the extract to the first extractant is preferably 1kg:20-40L, more preferably 1kg:25-35L, and further preferably 1kg:25-30L.

[0068] In the present invention, the alkaline pH value is preferably 8 to 9, more preferably 8.5 to 9. The present invention has no particular limitation on the base used for adjusting the pH value to alkalinity, and a base well known to those skilled in the art can be used, such as one or more of sodium carbonate, potassium carbonate, sodium hydroxide and potassium hydroxide; the base is preferably a solid base and / or an alkaline aqueous solution, and the concentration of the alkaline aqueous solution is preferably 2 to 10 wt%, more preferably 5 to 10 wt%.

[0069] In the present invention, the second extraction extractant (second extractant) includes one or more of an ester solvent, an ether solvent and a halogenated alkane solvent, and the carbon number of the second extractant is ≤6. In the present invention, the ester solvent preferably includes a C3-C6 ester solvent, and more preferably includes one or more of ethyl formate, ethyl acetate and ethyl propionate. In the present invention, the ether solvent preferably includes a C2-C6 ether, and more preferably includes methyl ether and / or ethyl ether. In the present invention, the halogenated alkane solvent preferably includes a C1-C6 halogenated alkane solvent, and more preferably includes one or more of dichloromethane, chloroform and tetrachloroethane. In a specific embodiment of the present invention, the second extraction is preferably a dichloromethane extraction. In the present invention, the solid-liquid ratio of the extract to the second extractant is preferably 1kg:3-5L, more preferably 1kg:3-4L, and further preferably 1kg:3.5-4L.

[0070] After the second extraction, the present invention preferably further comprises concentrating the obtained second extraction organic phase to recover the extractant to obtain a total alkaloid extract. The present invention has no particular limitation on the concentration, and the concentration can be performed by a concentration method well known to those skilled in the art until no solvent flows out, such as vacuum distillation. In the present invention, the recovered extractant can be reused.

[0071] After obtaining the total alkaloid extract, the present invention performs a first silica gel column chromatography separation on the total alkaloid extract to obtain 5 groups of components, which are sequentially recorded as Fr.A, Fr.B, Fr.C, Fr.D and Fr.E; the mobile phase used in the first silica gel column chromatography separation includes dichloromethane-methanol, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 100:1 to 1:1. In the present invention, the elution mode of the first silica gel column chromatography is preferably gradient elution, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol during the gradient elution is 100:1, 50:1, 30:1, 10:1, 5:1 and 1:1, respectively.

[0072] The preparation method of compound 3 and / or compound 4 is described in detail below.

[0073] After obtaining Fr.A, the present invention performs a second silica gel column chromatography separation on the Fr.A to obtain four components, which are sequentially recorded as Fr.A1, Fr.A2, Fr.A3 and Fr.A4; the mobile phase used in the second silica gel column chromatography separation includes petroleum ether-acetone, and the volume ratio of petroleum ether to acetone in the petroleum ether-acetone is 9:1 to 0:1; the elution mode of the second silica gel column chromatography separation is preferably gradient elution, and the volume ratio of petroleum ether to acetone in the gradient elution process is 9:1, 5:1, 1:1 and 0:1, respectively.

[0074] After obtaining Fr.A1, the present invention performs a first high performance liquid chromatography separation on the Fr.A1 to obtain 4 groups of components, which are sequentially recorded as Fr.A1-1, Fr.A1-2, Fr.A1-3 and Fr.A1-4; the mobile phase system used in the first high performance liquid chromatography separation includes methanol-water, and the volume ratio of methanol to water in the methanol-water is 30:70 to 100:0; the chromatographic column used in the first high performance liquid chromatography separation preferably includes an MPLC column, and the elution mode of the first high performance liquid chromatography separation is gradient elution, and the volume ratio of methanol to water during the gradient elution process is 30:70, 50:50, 70:30, 90:10 and 100:0 respectively; the flow rate of the mobile phase of the first high performance liquid chromatography separation is preferably 2 to 4 mL / min, more preferably 3 mL / min; the column temperature is preferably 25 to 35°C, more preferably 30°C.

[0075] After obtaining Fr.A1-3, the present invention performs a third silica gel column chromatography separation on the Fr.A1-3 to obtain compound 3 and compound 4, respectively; the mobile phase used in the third silica gel column chromatography separation includes petroleum ether-ethyl acetate, and the volume ratio of petroleum ether and ethyl acetate in the petroleum ether-ethyl acetate is 6:1 to 1:1; the elution mode of the third silica gel column chromatography separation is preferably gradient elution, and the volume ratios of petroleum ether and ethyl acetate in the gradient elution process are 6:1, 3:1 and 1:1, respectively.

[0076] The preparation method of compound 6 and / or compound 8 is described in detail below.

[0077] After obtaining Fr.D, the Fr.D of the present invention is subjected to reverse phase chromatography column separation to obtain 5 groups of components, which are sequentially recorded as Fr.D1, Fr.D2, Fr.D3, Fr.D4 and Fr.D5; the mobile phase used for the reverse phase chromatography column separation includes methanol aqueous solution, and the volume ratio of methanol to water in the methanol aqueous solution is 30:70 to 100:0; the chromatographic column used for the reverse phase chromatography column separation preferably includes an RP-C18 column; the elution mode of the reverse phase chromatography column separation is preferably gradient elution, and the volume ratio of methanol to water during the gradient elution process is 30:70, 50:50, 70:30, 90:10 and 100:0 respectively; the flow rate of the mobile phase for the reverse phase chromatography column separation is preferably 2 to 4 mL / min, more preferably 3 mL / min.

[0078] After obtaining Fr.D2, the present invention sequentially performs a first Sephadex LH-20 column separation and a second high performance liquid chromatography separation on the Fr.D2 to obtain compound 6 and compound 8, respectively; the mobile phase used in the first Sephadex LH-20 column separation includes methanol; the mobile phase system used in the second high performance liquid chromatography separation includes an acetonitrile aqueous solution, and the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 10:90 to 100:0, preferably 15:85 to 50:50, and more preferably 20:80; the second high performance liquid chromatography separation is preferably semi-preparative HPLC separation, and the chromatographic column used preferably includes Waters X-bridge; the elution mode of the second high performance liquid chromatography separation is preferably isocratic elution; the flow rate of the mobile phase of the second high performance liquid chromatography separation is preferably 2 to 4 mL / min, and more preferably 3 mL / min; the column temperature is preferably 25 to 35°C, and more preferably 30°C.

[0079] The preparation methods of one or more of Compound 1, Compound 2 and Compound 5 are described in detail below.

[0080] After obtaining Fr.E, the present invention performs a fourth silica gel column separation on the Fr.E to obtain 6 groups of components, which are sequentially recorded as Fr.E1, Fr.E5, Fr.E3, Fr.E4, Fr.E5 and Fr.E6; the mobile phase used for the fourth silica gel column separation includes dichloromethane-methanol, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 40:1 to 1:1; the elution mode of the fourth silica gel column separation is preferably gradient elution, and the volume ratio of dichloromethane to methanol during the gradient elution process is 40:1, 30:1, 10:1, 5:1 and 1:1, respectively.

[0081] The Fr.E1 is subjected to a second Sephadex LH-20 column separation and a third high performance liquid chromatography separation in sequence to obtain compound 1 and compound 2, respectively; the mobile phase used in the second Sephadex LH-20 column separation includes methanol; the mobile phase system used in the third high performance liquid chromatography separation includes an acetonitrile aqueous solution, and the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 10:90 to 100:0, preferably 30:70 to 50:50, and more preferably 38:62; the third high performance liquid chromatography separation is preferably a semi-preparative HPLC separation, and the chromatographic column used preferably includes a Waters X-bridge; the elution mode of the third high performance liquid chromatography separation is preferably isocratic elution; the flow rate of the mobile phase of the third high performance liquid chromatography separation is preferably 2 to 4 mL / min, and more preferably 3 mL / min; the column temperature is preferably 25 to 35°C, and more preferably 30°C.

[0082] After obtaining Fr.E5, the present invention performs a third Sephadex LH-20 column separation on the Fr.E5 to obtain 8 groups of components, which are sequentially recorded as Fr.E5-1, Fr.E5-2, Fr.E5-3, Fr.E5-4, Fr.E5-5, Fr.E5-6, Fr.E5-7 and Fr.E5-8; the Fr.E5-3 is subjected to a fourth high performance liquid chromatography separation to obtain compound 5; the third Sephadex The mobile phase used for LH-20 column separation includes methanol; the mobile phase system used for the fourth HPLC separation includes methanol-water solution, and the volume ratio of methanol to water in the methanol-water solution is 10:90-100:0, preferably 15:85-40:60, and more preferably 25:75; the fourth HPLC separation is preferably semi-preparative HPLC separation, and the chromatographic column used preferably includes Waters X-bridge; the elution mode of the fourth HPLC separation is preferably isocratic elution; the flow rate of the mobile phase of the fourth HPLC separation is preferably 2-4 mL / min, more preferably 3 mL / min; the column temperature is preferably 25-35°C, more preferably 30°C.

[0083] The present invention also provides a pharmaceutically acceptable salt of a phthalide isoquinoline alkaloid, wherein the phthalide isoquinoline alkaloid is the phthalide isoquinoline alkaloid described in the above technical solution or the phthalide isoquinoline alkaloid prepared by the preparation method described in the above technical solution. In the present invention, the pharmaceutically acceptable salt of the phthalide isoquinoline alkaloid preferably includes an organic acid salt of the phthalide isoquinoline alkaloid or an inorganic acid salt of the phthalide isoquinoline alkaloid; the organic acid salt of the phthalide isoquinoline alkaloid preferably includes a tartrate of the phthalide isoquinoline alkaloid, a citrate of the phthalide isoquinoline alkaloid, a formate of the phthalide isoquinoline alkaloid, an acetate of the phthalide isoquinoline alkaloid or an oxalate of the phthalide isoquinoline alkaloid; the inorganic acid salt of the phthalide isoquinoline alkaloid preferably includes a hydrochloride of the phthalide isoquinoline alkaloid, a hydrobromide of the phthalide isoquinoline alkaloid, a nitrate of the phthalide isoquinoline alkaloid or a sulfate of the phthalide isoquinoline alkaloid.

[0084] In the present invention, the method for preparing the pharmaceutically acceptable salt of the phthalide isoquinoline alkaloid preferably comprises the following steps: subjecting the phthalide isoquinoline alkaloid to a salt-forming reaction with an organic acid or an inorganic acid to obtain a pharmaceutically acceptable salt of the phthalide isoquinoline alkaloid. In the present invention, the organic acid preferably comprises tartaric acid, citric acid, formic acid, acetic acid or oxalic acid; the inorganic acid preferably comprises hydrochloric acid, hydrobromic acid, nitric acid or sulfuric acid. The present invention does not specifically limit the conditions of the salt-forming reaction, and the conditions of the salt-forming reaction well known to those skilled in the art may be used.

[0085] The present invention also provides a phthalide isoquinoline alkaloid pharmaceutical composition, comprising an active component and a pharmaceutically acceptable excipient; the active component comprises a phthalide isoquinoline alkaloid and / or a pharmaceutically acceptable salt of the phthalide isoquinoline alkaloid described in the above technical scheme; the phthalide isoquinoline alkaloid is the phthalide isoquinoline alkaloid described in the above technical scheme or the phthalide isoquinoline alkaloid prepared by the preparation method described in the above technical scheme.

[0086] In the present invention, the mass percentage of the active component in the phthalide isoquinoline alkaloid pharmaceutical composition is preferably 0.1 to 99%, more preferably 0.5 to 90%, and further preferably 1 to 50%.

[0087] In the present invention, the pharmaceutically acceptable excipient preferably includes a carrier and / or an excipient. The present invention has no particular limitation on the carrier and / or excipient, and any carrier and / or excipient well known to those skilled in the art may be used, such as one or more of a solid diluent, a semisolid diluent, a liquid diluent, a filler or a pharmaceutical product adjuvant.

[0088] In the present invention, the preparation types of the phthalide isoquinoline alkaloid pharmaceutical composition preferably include liquid preparations, solid preparations, sprays or mists; the liquid preparations preferably include injections, suspensions, emulsions, solutions or syrups; the solid preparations preferably include tablets, capsules, granules or granules. In the present invention, there is no special limitation on the preparation method of the phthalide isoquinoline alkaloid pharmaceutical composition, and the preparation method well known in the art can be used.

[0089] In the present invention, the administration method of the phthalide isoquinoline alkaloid pharmaceutical composition is preferably injection, oral administration, sublingual administration or mucosal dialysis; the injection preferably includes intravenous injection, intravenous drip, intramuscular injection, intraperitoneal injection or subcutaneous injection.

[0090] In the present invention, the phthalide isoquinoline alkaloid pharmaceutical composition is preferably used in the form of a dosage per unit body weight; the dosage per unit body weight is preferably 0.5 to 20 mg / kg, more preferably 1 to 15 mg / kg, and further preferably 5 to 10 mg / kg.

[0091] The present invention provides the use of the phthalide isoquinoline alkaloids described in the above technical solution, the phthalide isoquinoline alkaloids prepared by the preparation method described in the above technical solution, the pharmaceutically acceptable salts of the phthalide isoquinoline alkaloids described in the above technical solution, or the phthalide isoquinoline alkaloid pharmaceutical composition described in the above technical solution in the preparation of drugs or AMPK agonists for treating neurodegenerative diseases. In the present invention, the neurodegenerative diseases preferably include neurodegenerative diseases associated with autophagic flux, and more preferably include neurodegenerative diseases caused by autophagosome aggregation, increased neuronal sodium, mitochondrial depletion, and axonal and dendritic atrophy. The phthalide isoquinoline alkaloids or pharmaceutically acceptable salts thereof provided by the present invention can significantly promote the occurrence of autophagic flux, and have potential application value in the treatment of neurodegenerative diseases.

[0092] In order to further illustrate the present invention, the following detailed description of phthalide isoquinoline alkaloids and preparation methods and uses thereof, pharmaceutically acceptable salts of phthalide isoquinoline alkaloids and uses thereof, phthalide isoquinoline alkaloid pharmaceutical compositions and uses thereof are provided in conjunction with the following examples, but they should not be construed as limiting the scope of protection of the present invention.

[0093] Example 1

[0094] 50.0 kg of dried Corydalis decumbens corms (water content ≤ 5%) were crushed to a particle size of ≤ 100 mm, and cold-soaked in methanol for 3 times at room temperature, with a single cold-soaking time of 1 day. The combined extracts were concentrated under reduced pressure and the solvent was recovered to obtain a methanol total extract. The solid-liquid ratio of dried Corydalis decumbens corms to the methanol used for the single cold-soaking was 1 kg: 3 L.

[0095] Disperse in water in the methanol total extract, filter, adjust the pH value of the obtained filtrate to 2-3 with 2wt% hydrochloric acid aqueous solution, extract (degrease) 3 times with petroleum ether, extract 3 times with ethyl acetate respectively, collect all organic phases, adjust the pH value to 9-10 with 1wt0% sodium hydroxide aqueous solution, extract with dichloromethane, and obtain 800g of total alkaloid extract. Among them, the solid-liquid ratio of the methanol total extract and the dispersion water is 1kg:5L; the solid-liquid ratio of the methanol total extract and the petroleum ether for single extraction is 1kg:3L; the solid-liquid ratio of the methanol total extract and the ethyl acetate for single extraction is 1kg:3L.

[0096] The total alkaloid portion was separated by silica gel column chromatography (gradient elution, CH2Cl2 / MeOH, volume ratios of 100:1, 50:1, 30:1, 10:1, 5:1 and 1:1, respectively) to obtain 5 main components, which were respectively recorded as Fr.A (159.0 g), Fr.B, Fr.V, Fr.D (24.0 g) and Fr.E (120.0 g).

[0097] The Fr.A was separated by silica gel column chromatography (gradient elution, petroleum ether / acetone, 9:1, 5:1, 1:1 and 0:1, V / V) to obtain 4 subcomponents, which were successively recorded as Fr.A1 (12.5 g), Fr.A2, Fr.A3 and Fr.A4. The Fr.A1 was separated by preparative MPLC column (gradient elution, MeOH / H2O, volume ratios of 30:70, 50:50, 70:30, 90:10 and 100:0, the flow rate of the mobile phase was 3 mL / min, and the column temperature was 30°C) to obtain 4 subcomponents, which were successively recorded as Fr.A1-1, Fr.A1-2, Fr.A1-3 (5.5 g) and Fr.A1-4. The Fr.A1-3 was separated by silica gel column chromatography (gradient elution, petroleum ether / ethyl acetate, volume ratios of 6:1, 3:1 and 1:1 respectively) to obtain compound 3 (82.0 mg, yield 0.164%, purity 98%, yellow powder) and compound 4 (4.5 mg, yield 0.0084%, purity 98%, white powder), respectively.

[0098] The Fr.D was separated by RP-C18 column (MeOH / H2O, volume ratio of 30:70, 50:50, 70:30, 90:10 and 100:0, the flow rate of the mobile phase was 3 mL / min, and the column temperature was 30°C) to obtain 5 subcomponents, which were recorded as Fr.D1, Fr.D2 (3.0 g), Fr.D3, Fr.D4 and Fr.D5. The Fr.D2 was separated by Sephadex LH-20 column (methanol), and then separated by semi-preparative high performance liquid chromatography HPLC (chromatographic column was Waters X-bridge (5μm, 10mm×150mm), MeCN:H2O volume ratio = 20:80, the flow rate of the mobile phase was 3 mL / min, and the column temperature was 30°C), and compound 6 (2.3 mg, t R =12.0min, yield 0.0046%, purity 98%, yellow powder), compound 7 (25.3mg, t R =17.5min, yield 0.0506%, purity 98%, yellow powder) and compound 8 (7.5mg, t R =23.0 min, yield 0.015%, purity 98%, yellow powder).

[0099] The Fr.E was separated by silica gel column chromatography (gradient elution, CH2Cl2 / MeOH, volume ratios of 40:1, 30:1, 10:1, 5:1 and 1:1, respectively), and 6 subcomponents were obtained, which were recorded as Fr.E1 (5.8 g), Fr.E2, Fr.E3, Fr.E4, Fr.E5 and Fr.E6, respectively. The Fr.E1 was separated by Sephadex LH-20 column (methanol), and then separated by semi-preparative HPLC (chromatographic column Waters X-bridge (5μm, 10mm×150mm), MeCN / H2O volume ratio = 38:62, the flow rate of the mobile phase was 3mL / min, and the column temperature was 30°C), and compound 1 (7.6 mg, t R =13.5min, yield 0.0152%, purity 98%, colorless needle crystals) and compound 2 (8.4mg, t R =12.0min, yield 0.0168%, purity 98%, yellow powder). The Fr.E5 (6.3g) was separated by Sephadex LH-20 (methanol) to obtain 8 components, which were named Fr.E5-1, Fr.E5-2, Fr.E5-3, Fr.E5-4, Fr.E5-5, Fr.E5-6, Fr.E5-7 and Fr.E5-8. The Fr.E5-3 was separated by semi-preparative HPLC (chromatographic column Waters X-bridge (5μm, 10mm×150mm, flow rate of mobile phase was 3mL / min, column temperature was 30°C), MeOH / H2O volume ratio = 25:75) to obtain compound 5 (9.0mg, t R =17.5min, yield 0.018%, purity 98%, yellow powder).

[0100] Compound 1: Colorless needles; UV(MeOH)λmax(logε)200.0(4.30),262.0(2.78)nm; ECD(0.00049M,MeOH)λ max (Δε)200.0(+9.6),210.0(-14.8)nm; IR(KBr)V max 3439,2933,1612,1516,1470,1376,1248cm -1 ; HRESIMS m / z 400.1753[M+H] + (calcd for C 22 H 25 NO6+H,400.1755).

[0101] Compound 2: Yellowish powder; UV (MeOH) λmax (logε) 200.0 (4.43), 261.0 (2.73) nm; ECD (0.00035M, MeOH) λ max (Δε) 199.0 (+11.3), 210.0 (-12.7) nm; IR (KBr) V max 3436, 2924, 2853, 1611, 1516, 1470, 1247 cm -1 ; HRESIMS m / z 400.1758 [M+H] + (calcd for C 22 H 25 NO6+H, 400.1755).

[0102] Compound 3: Yellow powder; UV (MeOH) λmax (logε) 265.0 (2.77), 291.0 (3.33) nm; ECD (0.00039M, MeOH) λ max (Δε) 203.0 (-6.8), 215.0 (+2.4), 239.0 (-0.2) nm; IR (KBr) V max 3436, 2902, 2793, 1771, 1617, 1471, 1247 cm -1 ; HRESIMS m / z384.1447 [M+H] + (calcd for C 21 H 21 NO6+H, 384.1442).

[0103] Compound 4: Colorless crystals; UV (MeOH) λ max (logε) 202.0 (4.63), 261.0 (3.15) nm; ECD (0.00023M, MeOH) λmax (Δε) 203.0 (-6.1), 212.0 (+6.2), 235.0 (-1.7) nm nm; IR (KBr) ν max 3435, 2951, 2919, 2797, 1628, 1471, 1245 cm -1 ; HRESIMS m / z370.1290 [M+H] + (calcd for C 20 H 19 (calcd for C 20 H 19 N4O6+H, 370.1285).

[0104] Compound 5: Yellowish powder; UV (MeOH) λ max (logε) 201.0 (4.32), 261.0 (2.68) nm; ECD (0.00048 M, MeOH) λmax (Δε) 203.0 (+16.1), 212.0 (-16.9) nm; IR (KBr) ν max 3429, 2922, 1612, 1519, 1471, 1365, 1248 cm -1 ; HRESIMS m / z 416.1710 [M + H] + (calcd for C 22 H 25 NO7 + H, 416.1704).

[0105] Compound 6: Yellowish powder; UV (MeOH) λ max (logε) 202.0 (4.86), 264.0 (3.74) nm; ECD (0.00015 M, MeOH) λ max (Δε) 205.0 (+24.0), 225.0 (-8.3) nm; IR (KBr) V max 3439, 2920, 1767, 1626, 1503, 1749, 1255 cm -1 ; HRESIMS m / z 384.1081 [M + H] + (calcd for C 20 H 17 NO7 + H, 384.1078).

[0106] Compound 7: Yellow powder; UV (MeOH) λ max (logε) 204.0 (4.37), 261.0 (3.01) nm; ECD (0.00036 M, MeOH) λ max (Δε) 205.0 (+27.2), 224.0 (-12.1) nm; IR (KBr) ν max 3424, 2931, 1772, 1609, 1519, 1477, 1255 cm -1 ; HRESIMS m / z 400.1391 [M + H] + (calcd for C 21 H 21NO7+H,400.1391).

[0107] Compound 8: Yellow powder; UV(MeOH)λ max (logε)200.0(3.70),267.0(2.83),346.0(2.66)nm; ECD(0.00130M,MeOH)λ max (Δε)204.0(+1.9),225.0(-0.5)nm; IR(KBr)ν max 3463,2923,1764,1620,1513,1477,1255cm -1 ; HRESIMS m / z386.1237[M+H] + (calcd for C 20 H 19 NO7+H,386.1234).

[0108] Table 1 Compounds 1 to 5 1 H NMR data (deuterated chloroform, 500 MH)

[0109]

[0110] Note: a in Table 1 and Table 3 indicates signal overlap.

[0111] Table 2 Compounds 1 to 5 13 C NMR data (deuterated chloroform, 125 MHz)

[0112]

[0113]

[0114] Table 3 Compounds 6 to 8 1 H NMR data (deuterated chloroform, 500 MHz)

[0115]

[0116]

[0117] Table 4 Compounds 6 to 8 13 C NMR data (deuterated chloroform, 125 MHz)

[0118]

[0119]

[0120] It can be seen from Tables 1 to 4 that Example 1 successfully prepared phthalide isoquinoline alkaloids (Compounds 1 to 8).

[0121] Example 2

[0122] The phthalide isoquinoline alkaloids prepared in Example 1 and water for injection are uniformly mixed, and fine filtration and filling sterilization are performed in sequence to obtain a phthalide isoquinoline alkaloid injection, wherein the concentration of the phthalide isoquinoline alkaloids is 2-5%, specifically 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%.

[0123] Example 3

[0124] The phthalide isoquinoline alkaloids prepared in Example 1 are reacted with 4% mass concentration of tartaric acid aqueous solution, citric acid aqueous solution, formic acid aqueous solution, acetic acid aqueous solution, oxalic acid aqueous solution, hydrochloric acid aqueous solution, hydrobromic acid aqueous solution, nitric acid aqueous solution and sulfuric acid aqueous solution to obtain tartaric acid salt of phthalide isoquinoline alkaloids, citrate of phthalide isoquinoline alkaloids, formate of phthalide isoquinoline alkaloids, acetate of phthalide isoquinoline alkaloids, oxalate of phthalide isoquinoline alkaloids, hydrochloride of phthalide isoquinoline alkaloids, hydrobromide of phthalide isoquinoline alkaloids, nitrate of phthalide isoquinoline alkaloids and sulfate of phthalide isoquinoline alkaloids, respectively.

[0125] Example 4

[0126] The phthalide isoquinoline alkaloid prepared in Example 1 is dissolved in sterile water for injection, stirred until the phthalide isoquinoline alkaloid is dissolved, filtered through a sterile suction filtration funnel and sterile fine filtration, and then packaged into ampoules, and then freeze-dried at low temperature and aseptically sealed to obtain a phthalide isoquinoline alkaloid pharmaceutical composition powder injection, wherein the solid-liquid ratio of the phthalide isoquinoline alkaloid and the sterile water for injection is 1g:3-5mL, specifically 1g:3mL, 1g:3.5mL, 1g:4mL, 1g:4.5 or 1g:5mL.

[0127] Example 5

[0128] The phthalide isoquinoline alkaloid prepared in Example 1 and the excipient were uniformly mixed in a mass ratio of 9:1, and subjected to low-temperature freeze-drying and sterilization to obtain a phthalide isoquinoline alkaloid pharmaceutical composition powder.

[0129] Example 6

[0130] The phthalide isoquinoline alkaloids prepared in Example 1 and the excipients are uniformly mixed in a mass ratio of 1:5-10 (specifically 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5 or 1:10), granulated and tableted to obtain phthalide isoquinoline alkaloid pharmaceutical composition tablets.

[0131] Example 7

[0132] The phthalide isoquinoline alkaloids prepared in Example 1 were used in a conventional method for preparing oral liquids to prepare a phthalide isoquinoline alkaloids pharmaceutical composition oral liquid.

[0133] Example 8

[0134] The phthalide isoquinoline alkaloids prepared in Example 1 and the excipients were uniformly mixed in a mass ratio of 5:1, and conventional preparation methods of capsules, granules and granules were respectively used to prepare phthalide isoquinoline alkaloid pharmaceutical composition capsules, phthalide isoquinoline alkaloid pharmaceutical composition granules and phthalide isoquinoline alkaloid pharmaceutical composition granules.

[0135] Example 9

[0136] The phthalide isoquinoline alkaloids prepared in Example 1 and the excipients were uniformly mixed in a mass ratio of 3:1, and conventional preparation methods of capsules, granules and granules were respectively used to prepare phthalide isoquinoline alkaloid pharmaceutical composition capsules, phthalide isoquinoline alkaloid pharmaceutical composition granules and phthalide isoquinoline alkaloid pharmaceutical composition granules.

[0137] Test Example 1

[0138] Evaluation of the activity of phthalide isoquinoline alkaloids

[0139] (1) Cell culture and treatment

[0140] HM mCherry-GFP-LC3 cells were provided and cultured by the Kunming Institute of Zoology, Chinese Academy of Sciences. The cells were cultured in Dulbecco's modified medium (Gbico, 11140050) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 mg / mL streptomycin in a humidified incubator at 37°C, 5% CO2 and 95% humidity. Chemical agents were directly applied to the culture medium for treatment, and the cells were harvested 24 hours after treatment.

[0141] (2) CCK-8 kit to test the cytotoxicity of compounds

[0142] Cell Counting Kit-8, referred to as CCK-8 kit or CCK8 kit, is a WST-8-based kit widely used for rapid and highly sensitive detection of cell proliferation and cytotoxicity. In this experiment, according to the manufacturer's requirements (Beyotime Biotechnology, C0038), HM mCherry-GFP-LC3 cells were inoculated in 96-well plates, 100 microliters of 5000 cells were added to each well, and different concentrations (5μM and 20μM) of compounds 1 to 8 were added after 24 hours of culture. After 24 hours of compound treatment, the microplate reader was used for detection. The absorbance was measured at 450nm, and 630nm was used as the reference wavelength for dual-wavelength measurement.

[0143] (3) Flow cytometric analysis

[0144] HM mCherry-GFP-LC3 cells stably express a triple fusion protein (red fluorescent protein (mCherry), green fluorescent protein (GFP), and autophagosome marker LC3), which can directly reflect the intensity of autophagic flux and is used to detect the biological activity of compounds. HM mCherry-GFP-LC3 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, in a 37°C incubator, 5% carbon dioxide, and 95% humidity. HM mCherry-GFP-LC3 cells were cultured in 12-well plates, and the compounds were directly added to the culture medium (5 μM and 20 μM). After 24 h, the cells were harvested, fixed with 4% PFA (paraformaldehyde), and then subjected to flow cytometry to check whether the autophagic flux was enhanced. Data were analyzed using FlowJo software (FLOWJO, LLC).

[0145] (4) mCherry-GFP-LC3 fluorescence confocal imaging analysis

[0146] To further evaluate the effects of compound 2 and compound 6 on autophagic flux, HM mCherry-GFP-LC3 cells were treated with the compounds for 24 h, fixed with 4% PFA (paraformaldehyde), and analyzed using an Olympus FluoView TM The cells were photographed using a 1000 confocal microscope (Olympus, USA).

[0147] The experimental results are as follows:

[0148] Figure 1 The CCK-8 kit was used to measure the effects of compounds 1 to 8 on the viability of HM mCherry-GFP-LC3 cells, and the results showed that these compounds had no obvious cytotoxicity.

[0149] Figure 2In order to evaluate the effect of compounds 1 to 8 on the autophagic flux of HM mCherry-GFP-LC3 cells by flow cytometry analysis, the autophagy inducer rapamycin (Rapa) was used as a positive control. The results showed that consistent with the effect of rapamycin, compounds 1, 2 and 6 could significantly promote the process of autophagic flux.

[0150] Figure 3 This is a diagram for further evaluating the effects of compound 2 and compound 6 on the autophagic flux process of HM mCherry-GFP-LC3 cells using Confocal. The results showed that the number of red dots in HM mCherry-GFP-LC3 cells treated with the positive control, autophagy inducer Rapa, increased, indicating an increase in the level of autophagic flux. The effects of compound 2 and compound 6 on the increase in autophagic flux were similar to those of Rapa.

[0151] Although the above-mentioned embodiments have made a detailed description of the present invention, they are only some embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on the embodiments of the present invention without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A phthalide isoquinoline alkaloid, characterized in that: Including one or more of compound 1, compound 2 and compound 6: 。 2. The method for preparing phthalide isoquinoline alkaloids according to claim 1, characterized in that: The following steps are involved: The summer bulbless corms are mixed with an extractant, extracted and concentrated to obtain an extract; the extractant includes one or more of an alcohol solvent, a ketone solvent, an ester solvent, an ether solvent and a halogenated alkane solvent; the carbon number of the extractant is ≤6; The pH value of the extract is adjusted to acidic before a first extraction is performed, and the pH value of the first extracted organic phase is adjusted to alkaline before a second extraction is performed to obtain a total alkaloid extract; the extractants of the first extraction and the second extraction independently include one or more of an ester solvent, an ether solvent and a halogenated alkane solvent, and the carbon number of the extractant is ≤6; The total alkaloid extract is subjected to a first silica gel column chromatography separation to obtain five groups of components, which are sequentially recorded as Fr.A, Fr.B, Fr.C, Fr.D and Fr.E; the mobile phase used in the first silica gel column chromatography separation includes dichloromethane-methanol, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 100:1~1:1; The preparation method of compound 6 comprises the following steps: The Fr.D is separated by a reverse phase chromatography column to obtain five groups of components, which are sequentially recorded as Fr.D1, Fr.D2, Fr.D3, Fr.D4 and Fr.D5; the mobile phase used for the reverse phase chromatography column separation includes a methanol aqueous solution, and the volume ratio of methanol to water in the methanol aqueous solution is 30:70-100:0; Sequentially subjecting the Fr.D2 to a first Sephadex LH-20 column separation and a second high performance liquid chromatography separation to obtain compound 6; the mobile phase used in the first Sephadex LH-20 column separation includes methanol; the mobile phase system used in the second high performance liquid chromatography separation includes an acetonitrile aqueous solution, wherein the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 10:90 to 100:0; The preparation method of one or more of the compound 1 and compound 2 comprises the following steps: The Fr.E is separated by a fourth silica gel column to obtain 6 groups of components, which are sequentially recorded as Fr.E1, Fr.E5, Fr.E3, Fr.E4, Fr.E5 and Fr.E6; the mobile phase used in the fourth silica gel column separation includes dichloromethane-methanol, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 40:1~1:1; The Fr.E1 is subjected to a second Sephadex LH-20 column separation and a third high performance liquid chromatography separation in sequence to obtain compound 1 and compound 2, respectively; the mobile phase used in the second Sephadex LH-20 column separation includes methanol; the mobile phase system used in the third high performance liquid chromatography separation includes an acetonitrile aqueous solution, and the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 10:90~100:

0.

3. The preparation method according to claim 2, characterized in that: The solid-liquid ratio of the summer bulbless corms and the extractant is 1kg:3-5L, and the water content of the summer bulbless corms is ≤5%.

4. The preparation method according to claim 2 or 3, characterized in that: The number of extractions is 3 to 4 times; The extraction includes cold soaking and hot extraction; the time of a single cold soaking is 18-24 hours; the temperature of the hot extraction is 45-65° C., and the time of a single hot extraction is 3-4 hours.

5. The preparation method according to claim 2, characterized in that: The acidic pH value is 2-3.

6. The preparation method according to claim 2, characterized in that: The alkaline pH value is 8-9.

7. A pharmaceutically acceptable salt of a phthalide isoquinoline alkaloid, characterized in that: The phthalide isoquinoline alkaloid is the phthalide isoquinoline alkaloid described in claim 1 or the phthalide isoquinoline alkaloid prepared by the preparation method described in any one of claims 2 to 6.

8. A phthalide isoquinoline alkaloid pharmaceutical composition, comprising an active ingredient and a pharmaceutically acceptable excipient; the active ingredient comprises a phthalide isoquinoline alkaloid and / or a pharmaceutically acceptable salt of the phthalide isoquinoline alkaloid according to claim 7; the phthalide isoquinoline alkaloid is the phthalide isoquinoline alkaloid according to claim 1 or the phthalide isoquinoline alkaloid prepared by the preparation method according to any one of claims 2 to 6.

9. Use of the phthalide isoquinoline alkaloid according to claim 1, the phthalide isoquinoline alkaloid prepared by the preparation method according to any one of claims 2 to 6, the pharmaceutically acceptable salt of the phthalide isoquinoline alkaloid according to claim 7, or the phthalide isoquinoline alkaloid pharmaceutical composition according to claim 8 in the preparation of drugs for treating neurodegenerative diseases.

10. The use according to claim 9, characterized in that: The neurodegenerative diseases include neurodegenerative diseases associated with autophagic flux.

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