Monobenzylisoquinoline alkaloids, methods of making and using the same, and pharmaceutical compositions

Abstract

The present application relates to a kind of from Bauhinia championii single benzyl isoquinoline alkaloid and its preparation method and application and pharmaceutical composition, the compound is tested to show certain pharmacological activity by opioid delta receptor target point, can be used in the drug development for preventing and / or treating pain and other diseases.The compound structure

Description

Technical Field

[0001] The present invention belongs to the field of natural medicine chemistry, and relates to a preparation method of a monobenzylisoquinoline alkaloid and its application in the prevention or treatment of diseases such as pain, anxiety, and depression. Background Art

[0002] Menispermum dauricum DC. is a plant of the genus Menispermum in the family Menispermaceae. Its rhizome is a traditional Chinese medicine, also known as Beidougen. Clinically, it is commonly used to treat tonsillitis, rheumatoid arthritis, diarrhea, dysentery, gastroenteritis, cardiovascular diseases, and thrombosis diseases, etc. The main components of Menispermum dauricum roots are isoquinoline alkaloids, and they exhibit a wide range of biological activities, such as anti-arrhythmia, antibacterial, anti-inflammatory, and anti-tumor, etc. The isoquinoline alkaloids in Menispermum dauricum roots mainly include various subclasses such as bisbenzylisoquinoline, benzylisoquinoline, aporphine, protoberberine, and morphinan. Among them, the content of bisbenzylisoquinoline alkaloids is relatively high, accounting for more than 85% of the lipophilic alkaloids, represented by dauricine and daurisoline.

[0003] Based on the optical sensor's label-free cellular integrative pharmacology (CLIP) technology is a new type of phenotypic screening technology. Its principle is that when cells are stimulated by the external environment (such as the binding of drug small molecules to receptors), many physiological behaviors generated by the cells will bring about a redistribution of the internal mass of the cells. This change in mass distribution is detected by a resonant waveguide grating (RWG) biosensor, and the output signal is the change in the reflection wavelength, thus forming a visual dynamic mass redistribution (DMR) signal. Its experimental operation process can be carried out on the Epic system. The CLIP technology has the characteristics of label-free (receptors and compounds do not need to be labeled), unbiased (phenotypic screening), high-throughput, non-invasive, and high spatiotemporal resolution (the change in reflection wavelength within the entire measurement time range), so it has been widely used in many fields, including the screening and discovery of active molecules of natural products.

[0004] Menispermum dauricum roots are rich in isoquinoline alkaloids. In view of the important pharmacological activities of isoquinoline alkaloids widely reported at present, it is very meaningful to discover novel active isoquinoline alkaloids from Menispermum dauricum roots in combination with CLIP technology. Summary of the Invention

[0005] The present invention provides a preparation method of a monobenzylisoquinoline alkaloid and its application in the prevention or treatment of diseases such as pain, anxiety, and depression. The structural general formula of the compound is shown as follows:

[0006] .

[0007] in:

[0008] R1 to R6 are each independently selected from hydrogen, halogen, hydroxyl, carboxyl, C1 to C6 alkoxy, glycosyl, C1 to C6 alkenyl, etc.

[0009] Furthermore, R1 is selected from hydrogen or methyl, and R2 to R6 are selected from hydrogen, hydroxyl or methoxy.

[0010] Furthermore, R1 is selected from methyl, R2 and R3 are selected from methoxy, and R4 is selected from hydrogen, and the compound of formula (A) is shown in (B).

[0011] .

[0012] Furthermore, R5 and R6 are selected from hydroxyl groups, and the compound of formula (B) is shown in formula (C).

[0013] .

[0014] Furthermore, the stereoconfiguration of the compound of formula (C) is preferably selected from 1. R。

[0015] .

[0016] The present invention also provides a method for preparing a compound of formula (I), characterized by comprising the following steps:

[0017] (1) Extraction of medicinal materials: 1 kg to 100 kg of dried Sophora flavescens medicinal materials, add 6 to 10 L of 50% to 90% ethanol per kg of medicinal materials and soak for 1 to 24 hours. Heat to 50℃ to 90℃ and reflux for 1 to 3 hours. Filter to obtain the extract. Reflux extraction is performed 1 to 5 times in total. Combine the extracts and concentrate the obtained extract to 0.3 to 0.6 L per kg of medicinal materials to obtain Sophora flavescens extract.

[0018] (2) Preparation of total alkali: Add sulfuric acid with a volume concentration of 0.1%~3% to the above Sophora flavescens extract to adjust the pH to 1~4, then add ethyl acetate with a volume of 1~3 times the volume of the acid-adjusted extract to extract, separate the layers to obtain an ethyl acetate extract layer and an acidic water layer, and extract 1~5 times in total. Then add ammonia with a mass concentration of 25%~28% to the acidic water layer to adjust the pH to 8~10, then add n-butanol with a volume of 1~3 times the volume of the alkali-adjusted sample to extract, separate the layers to obtain an n-butanol layer and an alkaline water layer, and extract 1~5 times in total. Combine the organic layers, concentrate, and obtain crude alkali from Sophora flavescens. Then pass it through an ion exchange chromatography column to decolorize and remove impurities to obtain purified total alkali.

[0019] (3) The total alkali obtained in step (2) was separated and purified using a reverse-phase column packed with C18HCE packing material (particle size 5~60 μm). The elution was carried out using a volume ratio of 0:100~100:0 (volume concentration 0.01%~5%) formic acid-methanol / (volume concentration 0.01%~5%) formic acid-water solution, with an elution gradient of 0~80 minutes and 0%A~95%A. Eight fractions were collected according to time, namely F1 (RT: 0~17 min), F2 (RT: 17~24 min), F3 (RT: 24~28 min), F4 (RT: 28~32.5 min), F5 (RT: 32.5~38.5 min), F6 (RT: 38.5~52 min), F7 (RT: 52~62 min), and F8 (RT: 62~74 min).

[0020] (4) The fraction F6 obtained in step (3) was prepared by reversed-phase preparative HPLC with a C8CE stationary phase (5~60µm, 20×250~100×250). The mobile phase A was ammonia-methanol (volume fraction 0.01%~10%), and the mobile phase B was ammonia-water (volume fraction 0.01%~10%) (mass concentration 25%~28%). The elution gradient was 0~80 min, 0%A~95%A, resulting in 8 sub-fractions. The sub-fractions were collected over time and identified as F6-1 to F6-8, specifically F6-1 (RT: 2~7 min), F6-2 (RT: 7~11 min), F6-3 (RT: 11~13 min), F6-4 (RT: 13~17 min), F6-5 (RT: 17~22 min), F6-6 (RT: 22~27 min), F6-7 (RT: 27~31 min), and F6-8 (RT: 31~35 min).

[0021] (5) The fraction F6-6 obtained in step (4) was prepared by preparative HPLC. The chromatographic column used was a C18CE stationary phase (5~60µm, 4.6×250~20×250 mm), and the mobile phase was methanol (A) and water (B) (each containing 0.01~10% triethylamine acetate (the volume ratio of acetic acid to triethylamine was 1:1~1:5). The elution gradient was 0~60 min, 0%A~90%A. The resulting fraction F6-6-4 (RT: the peak corresponding to 22.5 min) was then passed through a C18HCE stationary phase (5~60µm, 4.6×250~20×250 mm). mm), mobile phase A is formic acid-methanol (volume fraction 0.01~1%), B is formic acid-water (volume fraction 0.01~1%), elution gradient is 0~40 min, 0%A~90%A, to obtain compound of formula (I) (RT: peak corresponding to 15 min);

[0022] In this invention: the glycosyl group refers to, but is not limited to, glucuronic acid, mannose, galactosyl, allosugar, fructose, sorbitol, fructose, rhamnose, cinnarizine, arabinose, lythose, xylose, ribosyl, and various disaccharides and polysaccharides formed from the above monosaccharides; the C1-C6 alkyl group refers to C1, C2, C3, C4, C5, and C6 alkyl groups, that is, straight-chain or branched alkyl groups having 1 to 6 carbon atoms; the C1-C6 alkenyl group refers to C1, C2, C3, C4, C5, and C6 alkyl groups, that is, straight-chain or branched alkenyl groups having 1 to 6 carbon atoms, and straight-chain or branched alkenyl groups having 1 to 6 double bonds.

[0023] Another object of the present invention is to provide a novel monobenzylisoquinoline alkaloid, or its crystal form, or its isomer, or its pharmaceutically acceptable salt, or its solvate, or its prodrug, or its metabolite, as an active ingredient, or any one or more of the above as a Delta receptor agonist, for use in the development of drugs for the prevention and / or treatment of pain, anxiety, and depression.

[0024] The pain conditions mentioned include, but are not limited to, traumatic pain, neuropathic pain, cancer pain, and psychosomatic pain, with inflammatory pain and cancer pain being preferred.

[0025] The pharmaceutical composition refers to one or more compounds of the present invention that can be used in combination with each other, or optionally in combination with any other active agent. If a group of compounds is used, these compounds can be administered to the test subject simultaneously, separately, or sequentially. The amount of the active ingredient (i.e., the compound of the present invention) in the pharmaceutical composition of the present invention can be specifically applied according to the patient's condition and the doctor's diagnosis. The dose or concentration of the active compound can be adjusted within a wide range, and the content of the active compound ranges from 1% to 90% of the pharmaceutical composition. Attached Figure Description

[0026] Figure 1 Flowchart of the preparation of compound I.

[0027] Figure 2 ESI of compound I + Primary mass spectrum.

[0028] Figure 3 Compound I 1 H NMR spectrum.

[0029] Figure 4 Compound I 13 C10 NMR spectrum.

[0030] Figure 5 Compound I1 H, 1 H-COSY is related to HMBC.

[0031] Figure 6 The dose-response curves of compound I in HEK-293-Delta cells for activating, desensitizing, and antagonizing effects are shown. ● represents the concentration-response curve of compound I, ▲ represents the DMR signal induced by SNC162 after 1 h of pretreatment with different concentrations of MDX, and ▼ represents the DMR signal induced by SNC162 after 1 h of pretreatment with different concentrations of SDM25N. Detailed Implementation

[0032] The following examples are intended to illustrate the invention and not to further limit it. The invention can be implemented in any of the ways described in the invention description.

[0033] Compound preparation

[0034] 100 kg of *Radix Sophorae Flavescentis* (a type of medicinal herb).

[0035] (1) Extraction of medicinal materials: Weigh 100kg of Sophora flavescens medicinal materials, add 1000L of 70% ethanol, soak for 24h, heat and reflux at 60℃ for 2h, filter to obtain extract 1, add 1000L of 70% ethanol to the filtered residue, heat and reflux at 60℃ for 2h, filter to obtain extract 2, add 1000L of 70% ethanol to the filtered residue, heat and reflux at 60℃ for 2h, filter to obtain extract 3, combine extracts 1~3, concentrate to 50L to obtain Sophora flavescens extract.

[0036] (2) Preparation of total alkali: Add 1% dilute sulfuric acid to the extract of Sophora flavescens to adjust the pH to between 2 and 3. Then add the same volume of ethyl acetate as the extract after acid adjustment and extract for the first time. Allow to stand and separate into layers to obtain ethyl acetate extract layer 1 and acid water layer 1. Then add 0.2% dilute sulfuric acid to the acid water layer 1 to adjust the pH to between 2 and 3. Then add the same volume of ethyl acetate as the extract after acid adjustment and extract for the second time to obtain ethyl acetate layer 2 and acid water layer 2. Then add 1% dilute sulfuric acid to the acid water layer 2 to adjust the pH to between 2 and 3. Then add the same volume of ethyl acetate as the extract after acid adjustment and extract for the third time to obtain ethyl acetate layer 3 and acid water layer 3. In the acidic aqueous layer 3, ammonia solution with a mass concentration of 25%–28% was added to adjust the pH to between 9 and 10. Then, the same volume of n-butanol as the alkali-adjusted sample was added for extraction. After standing and separation, n-butanol layer 1 and alkali aqueous layer 1 were obtained. Then, ammonia solution with a mass concentration of 25%–28% was added to the alkali aqueous layer 1 to adjust the pH to between 9 and 10. Then, the same volume of n-butanol as the alkali-adjusted extract was added for a second extraction, resulting in n-butanol layer 2 and alkali aqueous layer 2. Then, ammonia solution with a mass concentration of 25%–28% was added to the alkali aqueous layer 2 to adjust the pH to between 9 and 10. Then, the same volume of n-butanol as the alkali-adjusted extract was added for a third extraction, resulting in n-butanol layer 3 and alkali aqueous layer 3. The n-butanol layers 1–3 were combined and concentrated into an extract. The extract was redissolved in methanol to 12 L to obtain a crude alkali sample. 10 ml of the concentrated extract was tested, and the solid content was approximately 500 g / L. Therefore, approximately 6.0 kg of crude alkali from *Radix Sophorae Flavescentis* was obtained, accounting for 6.0% of the medicinal material's weight. Based on this calculation, take about 1L of crude alkali sample redissolved in methanol, add 1L of pure water to dilute and dissolve, centrifuge and filter to obtain supernatant, and then pass the supernatant through an ion exchange Q column with agarose gel matrix for decolorization and impurity removal to obtain about 500g of purified total alkali, with a recovery rate of about 97%.

[0037] (3) The total alkali obtained in step (2) was prepared by reversed-phase preparative HPLC. The stationary phase of the chromatographic column was a C18HCE reversed-phase column (particle size 10 μm, diameter and height 100×250 mm). The flow rate was 300 mL / min, and the mobile phase was methanol (A) and water (B) (each containing 0.1% formic acid by volume). The elution gradient was 0-10 min, 0% B (volume ratio); 10-20 min, 10% B; 20-35 min, 20% B; 35-45 min, 25% B; 45-60 min, 30% B. B; elute with 90% B for 60-75 min to obtain 8 fractions: F1 (RT: 0-17 min), F2 (RT: 17-24 min), F3 (RT: 24-28 min), F4 (RT: 28-32.5 min), F5 (RT: 32.5-38.5 min), F6 (RT: 38.5-52 min), F7 (RT: 52-62 min), and F8 (RT: 62-74 min).

[0038] (4) The fraction F6 obtained in step (3) was prepared by reversed-phase preparative HPLC. The chromatographic column was a C8CE stationary phase (particle size 10 μm, diameter and height 100 × 250 mm), and the flow rate was 300 m / s. The flow rate was mL / min, and the mobile phase was methanol (A) and water (B, containing 0.03% ammonia solution with a mass concentration of 25%–28%). The elution gradient was 0–30 min, 10%–95%A (linear gradient), 30–40 min, 95%A (volume ratio), resulting in 8 sub-fractions: F6-1 (RT: 2–7 min), F6-2 (RT: 7–11 min), F6-3 (RT: 11–13 min), F6-4 (RT: 13–17 min), F6-5 (RT: 17–22 min), F6-6 (RT: 22–27 min), F6-7 (RT: 27–31 min), and F6-8 (RT: 31–35 min).

[0039] (5) The fraction F6-6 obtained in step (4) was prepared by preparative HPLC. The chromatographic column used C18CE stationary phase (particle size 7 μm, diameter and height 10 × 250 mm), the flow rate was 3 mL / min, the mobile phase was methanol (A) and water (B) (each containing 0.05% triethylamine acetate (the volume ratio of acetic acid to triethylamine is 1:3)), the elution gradient was 0-10 min, 20%-60%A (linear gradient), 10-25 min, 60%A, 25 min-40 min, 90%A (volume ratio), and fractions F6-6-1 (RT: peak corresponding to 18 min), F6-6-2 (RT: peak corresponding to 19.5 min), F6-6-3 (RT: peak corresponding to 21 min), and F6-6-4 (RT: peak corresponding to 22.5 min) were obtained. The obtained fraction F6-6-4 was then passed through a C18HCE stationary phase (particle size 7 μm, diameter and height 10 × 250 mm), with mobile phases A and B being methanol and water (each containing 0.2% formic acid by volume). The elution gradient was 0–40 min, 0% A–90% A (linear gradient). Compound I was collected according to the peak (RT: the peak corresponding to 15 min).

[0040] The preparation process of compound I is as follows: Figure 1 As shown.

[0041] (6) The planar structure of compound I was determined by ultraviolet, mass spectrometry and nuclear magnetic resonance characterization, and its stereoconfiguration was determined by electron circular dichroism (ECD). Its structural formula is shown in formula (I), and the identification information is as follows:

[0042] .

[0043] Compound I: 4 mg, C 26 H 29 NO5, MW: 435.2046, white powder, soluble in methanol.

[0044] 1 H NMR (CD3OD, 600 MHz): δ 4.38 (1H, dd, J = 10.0, 4.5 Hz, H-1), 3.31 (1H,m, H-3 α ), 3.68 (1H, m, H-3 β ), 3.13 (1H, ddd, J = 17.6, 10.8, 7.0 Hz H-4 α ), 3.01(1H, m, H-4 β), 6.79 (1H, s, H-5), 5.92 (1H, s, H-8), 3.37 (1H, dd, J = 13.2, 4.5 Hz, H-9 α ), 2.99 (1H, dd, J = 13.2, 10.0 Hz, H-9 β ), 7.07 (1H, br d, J = 8.5Hz, H-10), 6.88 (1H, br d, J = 8.5 Hz, H-11), 6.88 (1H, br d, J = 8.5 Hz, H-13),7.07(1H, br d, J = 8.5 Hz, H-14), 6.93 (1H, d, J = 8.3 Hz, H-3′), 6.95 (1H, d, J =2.0 Hz, H-4′), 4.48 (2H, s, H-7′), 2.89 (3H, s, N -Me), 3.80 (3H, s, CH3O-6), 3.49 (3H, s, CH3O-7).

[0045] ECD(MeOH) λ max (D ε ) 211 (‒33.62), 233 (‒16.96), 287 (‒4.52) nm; ESI of compound I + First-order mass spectrum 1 H NMR and 13 C NMR spectrum and 1 H, 1 H-COSY and HMBC related information Figures 2-5 As shown.

[0046] Activity test examples:

[0047] The sample was a newly prepared compound (compound I); HEK293-delta cells were derived from HEK293 cells overexpressing the delta receptor, used by the Dalian Institute of Chemical Physics, Chinese Academy of Sciences, and were constructed through laboratory transfection. (Reference: Xu, FF; Zhou, H.; Liu, XM; Zhang, XL; Wang, Z. W.; Hou, T.; Wang, JX; Qu, LL; Zhang, PY; Piao, HL; Liang, XM, Label-free cellphenotypic study of FFA4 and FFA1 and discovery of novel agonists of FFA4 from natural products. Rsc Advances 2019, 9 (26)). 15073-15083; SNC162 (catalog number: 1529) and SDM25N (catalog number: 1410) were purchased from Tocris; DMEM high-glucose culture medium (catalog number: C11995500BT) was purchased from Thermo Fisher Scientific; fetal bovine serum (catalog number: 04000101A) was purchased from Shenyang Huibai Biotechnology Co., Ltd.; balanced salt solutions HBSS (catalog number: 14065-056) and HEPES (catalog number: 15630-080) were purchased from Gibco. The detection platform was a Corning Epic® third-generation imager, and the signal detected was the wavelength shift caused by dynamic mass resetting (DMR).

[0048] HEK293-delta cells in logarithmic growth phase were seeded into Epic® 384-well (total 384 wells) biosensor microplates. The volume of cell suspension seeded per well was 40 µL, and the number of cells seeded per well was 2.5 × 10⁶. 4 Each 384-well plate was incubated in a cell culture incubator (air containing 5% CO2, 37°C) for 22-24 h. When the cell confluence reached approximately 95%, the culture medium in each well of the 384-well microplate inoculated with HEK293-delta cells was aspirated, and 30 µL of HBSS buffer (containing 20 mM HEPES) was added to each well. The plate was then equilibrated on an EpicR high-throughput high-content activity screening system before the experiment was performed.

[0049] The experiment is conducted in the following three steps:

[0050] (1) Activation experiment: First, a baseline of 2 min was established. Then, different final concentrations (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.563 μM, 0.781 μM, 0.391 μM, 0.195 μM, 0.098 μM, 0.049 μM, 0.024 μM) of compound I (10 µL) were added to different wells. The experimental results were monitored for 1 h. Figure 6 As shown, compound I induced a dose-dependent DMR response in HEK293-delta cells. The dose-response curve of compound I was a monophasic "S"-shaped curve that reached a saturation response, corresponding to EC50. 50 The value is 2.5 ± 0.3 μM.

[0051] (2) Desensitization experiment: HEK293-delta cells were pretreated for 1 h with 10 µL of compound I (10 µL) at different final concentrations (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.563 μM, 0.781 μM, 0.391 μM, 0.195 μM, 0.098 μM, 0.049 μM, 0.024 μM) for 2 min. A baseline of 2 min was established. Then, 10 µL (fixed final concentration of 400 nM) of Delta receptor-specific agonist SNC162 was added to each well. The results after 1 h were monitored and are shown in Figure 6. Compound I completely desensitized the DMR response signal (Compound I_SNC162) of Delta receptor agonist SNC162, showing a dose-dependent effect. Its IC50 value was 10 µL. 50 The value was 45.3 ± 5.5 μM.

[0052] (3) Antagonism experiment: HEK293-delta cells were pretreated with 10 µL of the Delta receptor-specific antagonist SDM25N at different final concentrations (32 μM, 16 μM, 8 μM, 4 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM, 0.125 μM, 0.063 μM, 0.031 μM, 0.016 μM, 0.008 μM, 0.004 μM) for 1 h. A 2-min baseline was established, and then 10 µL (fixed final concentration of 50 μM) of compound I was added to each well. The results after 1 h were monitored, as shown in Figure 6. The Delta receptor selective antagonist SDM25N completely inhibited the DMR response signal of compound I on HEK293-delta cells (SDM25N_CompoundI) in a dose-dependent manner. Its IC50 value was 10 µL / min. 50 The value was 0.31 ± 0.09 μM, indicating that the compound is a Delta agonist.

[0053] Current research indicates that opioid Delta receptors are associated with conditions such as pain, anxiety, and depression. The compounds of this invention have a different core structure from existing Delta agonists, making them important for pharmacological research and clinical applications in diseases such as pain, anxiety, and depression.

Claims

1. A monobenzylisoquinoline alkaloid, characterized in that: Its structural formula is shown in equation (I): 。 2. A method for preparing the monobenzylisoquinoline alkaloid of claim 1, characterized in that: Includes the following steps: (1) Extraction of medicinal materials: 1 kg to 100 kg of dried Sophora flavescens medicinal materials, add 6 to 10 L of 50% to 90% ethanol per kg of medicinal materials and soak for 1 to 24 hours. Heat to 50℃ to 90℃ and reflux for 1 to 3 hours. Filter to obtain the extract. Reflux extraction is performed 1 to 5 times in total. Combine the extracts and concentrate the obtained extract to 0.3 to 0.6 L per kg of medicinal materials to obtain Sophora flavescens extract. (2) Preparation of total alkali: After adjusting the pH to 1-4 by adding sulfuric acid with a volume concentration of 0.1%-3% to the extract of Sophora flavescens in step (1), add ethyl acetate with a volume of 1-3 times the volume of the acid-adjusted extract for extraction, separate the layers to obtain an ethyl acetate extract layer and an acidic water layer, and extract for a total of 1-5 times. Then, add ammonia with a mass concentration of 25%-28% to the acidic water layer to adjust the pH to 8-10, and then add n-butanol with a volume of 1-3 times the volume of the alkali-adjusted sample for extraction, separate the layers to obtain an n-butanol layer and an alkaline water layer, and extract for a total of 1-5 times. Combine the n-butanol layers and concentrate to obtain crude alkali of Sophora flavescens. Then, pass it through an ion exchange chromatography column to decolorize and remove impurities to obtain purified total alkali. (3) The total alkali obtained in step (2) was separated and purified using a reverse-phase column packed with C18HCE packing material. The particle size of the packing material was 5~60 μm. The mobile phase A was methanol containing 0.01%~5% formic acid by volume, and the mobile phase B was an aqueous solution of 0.01%~5% formic acid by volume. The elution gradient was 0~80 minutes, 0%B~95%B. Eight fractions were collected according to time, namely F1 with RT: 0~17 min, F2 with RT: 17~24 min, F3 with RT: 24~28 min, F4 with RT: 28~32.5 min, F5 with RT: 32.5~38.5 min, F6 with RT: 38.5~52 min, F7 with RT: 52~62 min, and F8 with RT: 62~74 min. (4) The fraction F6 obtained in step (3) was prepared by reversed-phase preparative HPLC. The chromatographic column was a C8CE stationary phase with a particle size of 5~60µm and a column diameter and height of 20×250~100×250 mm. The mobile phase A was 0.01%~10% ammonia-methanol and the mobile phase B was 0.01%~10% ammonia-water. The mass concentration of ammonia was 25%~28%. The elution gradient was 0~80 minutes and 0%A~95%A. Eight fractions F6-1 to F6-8 were obtained by collecting the fractions over time. The fractions were F6-1 with a RT of 2~7 min, F6-2 with a RT of 7~11 min, and F6-2 with a RT of 11~13 min. F6-3, RT: 13~17min; F6-4, RT: 17~22min; F6-5, RT: 22~27min; F6-6, RT: 27~31min; F6-7, RT: 31~35min; F6-8. (5) The fraction F6-6 obtained in step (4) was prepared by preparative HPLC. The chromatographic column used was C18CE stationary phase with a packing particle size of 5~60µm and a column diameter and height of 4.6×250~20×250 mm. The mobile phase was methanol A and water B. Each of the mobile phases A and B contained 0.01~10% acetic acid and triethylamine by volume, with the volume ratio of acetic acid to triethylamine being 1:1~1:

5. The elution gradient was 0~60min, 0%A~90%A. The peak corresponding to the F6-6-4 fraction with RT: 22.5min was then passed through C18HCE stationary phase. The mobile phase A was 0.01~1% formic acid-methanol by volume, and the mobile phase B was 0.01~1% formic acid-water by volume. The elution gradient was 0~40min, 0%A~90%A. The peak corresponding to the compound of formula (I) with RT: 15min was obtained.

3. The method for preparing monobenzylisoquinoline alkaloids according to claim 2, characterized in that, In step (1), when reflux extraction is performed 2 to 5 times, the material from the previous reflux extraction process is filtered. The filtered residue is then refluxed with 6 to 10 L of 50% to 90% ethanol per kilogram of medicinal material for 1 to 3 hours at 50°C to 90°C. The extract is then filtered and the extracts are combined. In step (2), when ethyl acetate is extracted 2 to 5 times, the pH of the acidic water layer from the previous separation is first adjusted to 1 to 4 using sulfuric acid with a volume concentration of 0.1% to 3%. Then, ethyl acetate with a volume of 1 to 3 times its volume is added to the acidic water layer for extraction, and the layers are separated to obtain an ethyl acetate extraction layer and an acidic water layer. The weak base is ammonia water with a mass concentration of 10 to 25%. When extracting n-butanol 2 to 5 times, first use a weak alkali to adjust the pH of the previous alkaline water layer to 8 to 10, then add 1 to 3 times the volume of n-butanol to the alkaline water layer to extract the layers and obtain n-butanol and alkaline water layer.

4. The use of the monobenzylisoquinoline alkaloid of claim 1 in the preparation of a medicament for the prevention and / or treatment of pain, anxiety and depression.

5. A pharmaceutical composition, characterized in that: The preparation is a formulation made by adding a pharmaceutically acceptable carrier or excipient to the monobenzylisoquinoline alkaloid of claim 1.

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