Benzyl isoquinoline alkaloid glycoside compound as well as preparation method and application thereof

Abstract

The invention discloses a benzylisoquinoline alkaloid glycoside compound as well as a preparation method and application thereof. The preparation method comprises the following steps: soaking tubers of rhizoma corydalis in a 6% acetic acid solution, crushing, soaking in water, and carrying out ultrasonic extraction to obtain a plant extract; after water dispersion, carrying out HPD100 type macroporous resin column chromatography separation, carrying out gradient elution by using ethanol aqueous solutions with different concentrations, and collecting a 50% ethanol elution part to obtain an elution part YH-D; carrying out MCI column chromatography separation, and carrying out gradient elution by using a 0-100% methanol-water solution to obtain four parts of eluents; the preparation method comprises the following steps: dissolving YH-D-2 in water, filtering, and carrying out medium-pressure ODS column chromatography separation, Sephadex LH-20 column chromatography separation, medium-pressure ODS column chromatography separation and semi-preparative C18 column HPLC (High Performance Liquid Chromatography) purification on the filtrate to obtain six new compounds 1-6. In-vitro experiments prove that the elution part YH-D-2 containing benzylisoquinoline alkaloid glycoside of the benzylisoquinoline alkaloid glycoside compounds 1-6 has NO generation inhibition activity, and has a good development prospect as an anti-inflammatory drug.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a benzyl isoquinoline alkaloid glycoside compound, its preparation method, and its application. Background Technology

[0002] Alkaloid glycosides are an important class of water-soluble natural products formed by the linkage of alkaloids to monosaccharides, disaccharides, or polysaccharides (glucose, rhamnose, arabinose, etc.) via CO, C-C, or NC bonds. Currently, natural product researchers have discovered approximately 230 alkaloid glycosides from plants. Based on the alkaloid aglycone, they are mainly classified into steroidal alkaloid glycosides (approximately 120), indole alkaloid glycosides (approximately 70), and other types of alkaloid glycosides (approximately 40). Reported alkaloid glycosides exhibit a range of biological activities, including antibacterial, antiviral, antitumor, analgesic, antipyretic, cholesterol-lowering, and anti-inflammatory effects, making them an important component of bioactive natural products. However, given the diversity of alkaloid structures (including steroidal alkaloids, indole alkaloids, phenanthridine alkaloids, isoquinoline alkaloids, diterpenoid alkaloids, etc.), the reported glycosylated alkaloids appear to be only the tip of the iceberg. A large number of unknown alkaloid glycosides exist in traditional Chinese medicine, warranting further exploration.

[0003] Benzylisoquinoline alkaloid glycosides are secondary metabolites formed from benzylisoquinoline and monosaccharides, disaccharides, or polysaccharides. Currently, very few benzylisoquinoline alkaloid glycosides have been discovered, with only six reported, and only one of them is a glucosinolate, while the others are glucosinolates. Therefore, novel benzylisoquinoline alkaloid glycosides warrant systematic exploration and pharmacological activity studies.

[0004] Corydalis yanhusuo, a traditional Chinese medicine, is the dried tuber of Corydalis yanhusuo WT Wang, a plant belonging to the genus Corydalis in the Papaveraceae family. It possesses properties such as promoting blood circulation, removing blood stasis, regulating qi, and relieving pain. The main chemical components of Corydalis yanhusuo are isoquinoline alkaloids, particularly corydaline, dehydrocorydaline, berberine, palmatine, and corydaline B, which are considered the main active ingredients due to their analgesic, sedative, antihypertensive, antimalarial, and antitumor effects. However, current research on the chemical components and pharmacological effects of Corydalis yanhusuo mainly focuses on its lipid-soluble fractions, with limited research on its water-soluble components, such as those with anti-inflammatory properties. Therefore, discovering novel benzyl isoquinoline alkaloid glycosides with potential anti-inflammatory effects from Corydalis yanhusuo is feasible. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the main objective of this invention is to provide a benzyl isoquinoline alkaloid glycoside compound, its preparation method, and its application.

[0006] To achieve the aforementioned objectives, the technical solution adopted by this invention includes: A benzylisoquinoline alkaloid glycoside compound, namely compounds 1-6, specifically selected from compounds shown in the following formulas: .

[0007] A method for preparing benzyl isoquinoline alkaloid glycoside compounds, the method comprising the following steps: 1) The tubers of the Chinese herb Corydalis Rhizome were soaked in a 6% acetic acid solution, dried at 40°C, pulverized, soaked in water, and then extracted by ultrasound. The extracts were combined and concentrated under reduced pressure to obtain the plant extract. 2) The plant extract obtained in step 1) was dispersed in water and separated by HPD100 macroporous resin column chromatography. The column was eluted with water, 50% ethanol and 95% ethanol in a gradient. The 50% ethanol eluent was collected and concentrated under reduced pressure to obtain the eluent YH-D. 3) Take YH-D) and separate it by MCI column chromatography. Elute it with a gradient of 0%~100% methanol-water solution. Recover the solvent under reduced pressure to obtain four eluents, which are named YH-D-1~YH-D-4 respectively. 4) Dissolve YH-D-2 in water and filter. The filtrate is subjected to medium-pressure ODS column chromatography and eluted sequentially with 1% acetic acid-water, 5%~60% methanol-acid-water, and 100% pure methanol. The solvent is recovered under reduced pressure to obtain five eluent fractions, which are named A-E respectively. 5) The filtrate after dissolving and filtering the elution fraction A in water was subjected to Sephadex LH-20 column chromatography, eluted with pure water, and concentrated under reduced pressure to obtain six eluent fractions, named A1-A6 respectively. 6) Elution fraction A4 was eluted using a 0-30% methanol-water gradient on a medium-pressure ODS column to obtain seven eluent fractions, named A4-1 to A4-7; A4-5 was eluted with pure methanol on a Sephadex LH-20 column, concentrated under reduced pressure, and separated into four eluent fractions, named A4-5-1 to A4-5-4; A4-5-3 was further separated using a semi-preparative C... 18 Compound 1 was obtained by column HPLC purification. 7) Elution fraction A5 was eluted by medium-pressure ODS column chromatography with a methanol-water gradient of 0-30%, and concentrated under reduced pressure to obtain 9 eluent fractions, which were named A5-1 to A5-9 respectively. 8) The A5-4 obtained in step 7) was subjected to Sephadex LH-20 column chromatography, eluted with pure methanol, to obtain two eluent fractions, named A5-4-1 and A5-4-2, respectively; A5-4-2 was subjected to semi-preparative C 18 Column HPLC purification yielded compounds 2, 3, 4, and 6. 9) The A5-5 obtained in step 7) is semi-prepared into C 18 Compound 5 was obtained by column HPLC purification.

[0008] Step 1) involves ultrasonic extraction three times, with a water-to-medicinal-material mass-to-volume ratio of 1:1 each time, and an ultrasonic time of 1 hour.

[0009] In step 2), each gradient elutes 3 times the volume of the macroporous resin column, and in step 3), the volume ratio of methanol-water solution is 0%, 30%, 60%, and 100%, with each gradient eluting 2 times the volume of the MCI column.

[0010] In step 4), the elution time for 1% acetic acid-water is 30 min, and the flow rate is 45 ml / min. The 5%~60% methanol-acid water containing 1% acetic acid has an elution time of 180 min, and the flow rate is 45 ml / min. The elution time for 100% methanol is 20 min, and the flow rate is 45 ml / min.

[0011] In step 6), the elution time of the 0-30% methanol-water gradient is 60 min, and the flow rate is 35 ml / min; the semi-preparative C 18 The column HPLC conditions were as follows: mobile phase 6% acetonitrile-water containing 0.1% trifluoroacetic acid, flow rate 3 ml / min, detection wavelength 254 nm, and column temperature 25℃.

[0012] In step 7), the elution time of the 0-30% methanol-water gradient is 60 min, and the flow rate is 35 ml / min.

[0013] In step 8), C is prepared 18 The column HPLC conditions were: mobile phase 19% acetonitrile-water containing 0.1% trifluoroacetic acid, flow rate 3 ml / min, detection wavelength 254 nm, and column temperature 25 °C; the preparation of C in step 9) 18 The column HPLC conditions were as follows: mobile phase 17% acetonitrile-water containing 0.1% trifluoroacetic acid, flow rate 3 ml / min, detection wavelength 254 nm, and column temperature 25℃.

[0014] A pharmaceutical composition comprising at least one of the benzyl isoquinoline alkaloid glycoside compounds and its pharmaceutically acceptable salt or pharmaceutically acceptable carrier or excipient, or the elution site YH-D-2 and its pharmaceutically acceptable carrier or excipient.

[0015] The pharmaceutical composition is used in the preparation of anti-inflammatory drugs.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention is the first to isolate six novel benzyl isoquinoline alkaloid glycoside compounds from the traditional Chinese medicine Corydalis yanhusuo.

[0017] 2. In vitro experiments confirmed that the benzyl isoquinoline alkaloid glycosides 1-6 of the present invention, and the elution fraction YH-D-2 containing benzyl isoquinoline alkaloid glycosides, possess NO generation inhibitory activity. Benzyl isoquinoline alkaloid glycosides 1-6 and the elution fraction YH-D-2 show promising development potential as anti-inflammatory drugs. Attached Figure Description

[0018] Figure 1 ECD and calculated ECD spectra of compounds 1-6; Figure 2 HRMS spectra of compounds 1-6. Detailed Implementation

[0019] The embodiments described below are exemplary descriptions of key experimental evidence and are not intended to limit the core content and application scope of this invention due to the amount of evidence. It should be noted that all the accompanying drawings and corresponding descriptions merely illustrate the concept, principles, and representative experimental evidence of the disclosed embodiments of this invention. Where the chain of evidence is complete, it is unnecessary to show all the specific details and extended details of the various embodiments listed in this invention.

[0020] Unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0021] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0022] Experimental apparatus and packing materials: Ultraviolet spectrometer: JASCO V-650 (JASCO Corporation, Japan); Infrared spectrometer: Nicolet 5700 Fourier transform infrared spectrometer (FT-IR Microscope Transmission). Optical polarimeter: JASCO P-2000 polarimeter (JASCO Corporation, Japan); Nuclear magnetic resonance spectrometers: AV-Ⅲ-600 (Bruker, Germany) and Inova-600 (Varian, USA). Mass spectrometers: Micromass Auto spec-Ultima ETFO; AccuTo FCS IMS-J100CS; Agilent 1000 Series LC-MSD-Trap-SL (Agilent Technologies, USA). Circular dichroism (ECD) spectrometer: JASCO J-815 circular dichroism spectrometer (JASCO Corporation, Japan); Medium-pressure liquid chromatograph: Reveleris medium-pressure liquid chromatograph (Grace Corporation, USA); High Performance Liquid Chromatograph (HPLC): Agilent 1260 HPLC (Agilent Technologies, USA). Shimadzu SPD-20A UV detector, LC-6AD high-pressure infusion pump and CBM-20A connector (SHIMADZU Corporation, Japan); Electronic analytical balance: AL104 electronic balance [Mettler-Toledo Instruments (Shanghai) Co., Ltd.]; Rotary evaporator: EYELA SB-2000 (Tokyo Rika Kiki Co., Ltd.); Diaphragm pump: MPC-301Z diaphragm pump (Welch GmbH, Germany); Cooling water circulation device: EYELA CCA-1112A (Tokyo Rikka Equipment Co., Ltd.); Macroporous resin column chromatography: HPD100 macroporous resin (Hebei Cangzhou Baoen Chemical Co., Ltd.); Gel column chromatography: hydroxypropyl dextran gel Sephadex LH-20 (Amersham Bioscience, Sweden); Silica gel thin-layer chromatography: Silica gel GF 254 (Qingdao Marine Chemical Plant); MCI column chromatography: CHP20P (Mitsubishi Chemical Inc., Tokyo, Japan); Reversed-phase column: MGII-C 18 Semi-preparative chromatographic column (250 × 10 mm, 5 μm); Ultimate XB-CN semi-preparative chromatographic column (250 × 10 mm, 5 μm) [Yuexu Technology (Shanghai) Co., Ltd.]; Color development: Spray with 10% sulfuric acid ethanol solution followed by heating or develop color with potassium bismuth iodide solution; Reagents and solvents: Purified water: Produced by Hangzhou Wahaha Company; Chromatographic acetonitrile: Honeywell Pharmaceuticals, USA, chromatographic grade; Chromatographic methanol: Honeywell Pharmaceuticals, USA, chromatographic grade; DMSO: Beijing Bailingwei Technology Co., Ltd., for biochemical use; Deuterated methanol: Cambridge Isotope Laboratories, Inc., USA; D-rhamnose, L-rhamnose, D-glucose, L-glucose, D-arabinose and L-arabinose: Beijing Bailingwei Technology Co., Ltd., all of which were analytical grade; DMEM cell culture medium: Gibco; Lipopolysaccharide (LPS): Shanghai Yuanye Biotechnology Co., Ltd.

[0023] In the various embodiments, 50% ethanol is an abbreviation for a 50% ethanol aqueous solution by volume, 95% ethanol is an abbreviation for a 95% ethanol aqueous solution by volume, and so on.

[0024] Example 1 The preparation of Corydalis extract includes the following steps: (1) Acetic acid processing: 50 kg of the tuber of Corydalis yanhusuo WT Wang was soaked in 10 L of 6% acetic acid solution and then dried at 40℃.

[0025] (2) Preparation of extract: The medicinal material processed with acetic acid is pulverized and soaked in water, and then extracted by ultrasound 3 times, each time with 50L of water and ultrasound for 1h. The extracts are combined and concentrated under reduced pressure to obtain the plant extract.

[0026] (3) LC-MS detection: The obtained plant extract was detected by LC-MS and it was determined that it contained the main benzyl isoquinoline components such as corydaline.

[0027] Example 2 The preparation method of benzyl isoquinoline alkaloid glycoside includes the following steps: The Corydalis yanhusuo aqueous extract obtained in Example 1 was dispersed in water and then separated by HPD100 macroporous resin column chromatography. Eluent was obtained sequentially with water, 50% ethanol, and 95% ethanol, with each gradient eluting at three times the column volume (approximately 10 L). The eluent was concentrated under reduced pressure to obtain three fractions. The 50% ethanol eluent fraction was collected to obtain fraction YH-D.

[0028] YH-D was separated by MCI column chromatography and eluted with pure water, 30% methanol, 60% methanol and pure methanol respectively. The methanol-water solution ratio was used here. Each gradient elution was twice the volume of the MCI column, about 2 L. The solvent was recovered under reduced pressure to obtain four eluents, which were named YH-D-1 to YH-D-4 respectively.

[0029] After dissolving the YH-D-2 eluent (30% methanol eluent) in water, a precipitate was formed. The precipitate was filtered, and the filtrate was subjected to medium-pressure ODS column chromatography. The eluent was eluted with 1% acetic acid-water for 30 min at a flow rate of 45 ml / min, then eluted with 5%–60% methanol-water (containing 1% acetic acid) for 180 min at a flow rate of 45 ml / min, and finally eluted with pure methanol for 20 min at a flow rate of 45 ml / min. The eluent was analyzed by thin-layer chromatography with dichloromethanol = 3:1 as the developing solvent. The same components were combined, and the solvent was recovered under reduced pressure to obtain five eluent fractions, named A–E.

[0030] The filtrate from the dissolved water of eluent fraction A was subjected to Sephadex LH-20 column chromatography with pure water elution. The eluent was then analyzed by thin-layer chromatography with dichloromethanol = 3:1 as the developing solvent. Identical components were combined and concentrated under reduced pressure to obtain six eluent fractions, named A1 to A6.

[0031] Elution fraction A4 was eluted by medium-pressure ODS column chromatography with a 0-30% methanol-water gradient for 60 min at a flow rate of 35 ml / min. The eluent was analyzed by thin-layer chromatography with dichloromethanol:methanol = 3:1 as the developing solvent. Identical fractions were combined to obtain seven eluents, named A4-1 to A4-7. A4-5 was eluted by Sephadex LH-20 column chromatography with pure methanol. The eluent was analyzed by thin-layer chromatography with dichloromethanol:methanol = 3:1 as the developing solvent. Identical fractions were combined, concentrated under reduced pressure, and separated to obtain four eluents, named A4-5-1 to A4-5-4. A4-5-3 was subjected to semi-preparative C... 18 HPLC purification was performed using the following conditions: mobile phase: 6% acetonitrile-water containing 0.1% trifluoroacetic acid; flow rate: 3 mL / min; detection wavelength: 254 nm; column temperature: 25 °C. Compound 1 (2 mg, t) was obtained. R =12.3min).

[0032] Elution fraction A5 was eluted by medium-pressure ODS column chromatography with a methanol-water gradient of 0-30% for 60 min at a flow rate of 35 ml / min. The eluent was detected by thin-layer chromatography with dichloromethanol = 3:1 as the developing solvent. Identical components were combined and concentrated under reduced pressure to obtain nine eluent fractions, which were named A5-1 to A5-9.

[0033] A5-4 was subjected to Sephadex LH-20 column chromatography, eluted with pure methanol. The eluent was analyzed by thin-layer chromatography with dichloromethanol as the developing solvent (3:1). Identical fractions were combined to obtain two eluents, named A5-4-1 and A5-4-2. Fraction A5-4-2 was subjected to semi-preparative C... 18HPLC purification was performed using the following conditions: mobile phase 19% acetonitrile-water containing 0.1% trifluoroacetic acid, flow rate 3 mL / min, detection wavelength 254 nm, and column temperature 25 °C, yielding compound 2 (t). R =15.6 min, 4.0mg), compound 3 (t R =16.2min, 2.0mg), compound 4 (t R =16.9 min, 2.0 mg) and compound 6 (t R = 17.5 min, 3.0 mg).

[0034] A5-5 was prepared by half-processing C 18 HPLC purification was performed using the following conditions: mobile phase: 17% acetonitrile-water containing 0.1% trifluoroacetic acid; flow rate: 3 mL / min; detection wavelength: 254 nm; column temperature: 25 °C. Compound 5 (t) was obtained. R =17.8 min, 3.0 mg).

[0035] Using physicochemical constants and modern spectroscopic techniques (HRMS, NMR, ORD, ECD), combined with relevant literature data, compounds 1-6 were identified as novel compounds, with the structures shown below:

[0036] Table 1. Compounds 1-3 1 H NMR data a

[0037] a The solvent was deuterated methanol; the NMR instrument was 600 MHz. Table 2. Compounds 4-6 1 H NMR data a

[0038] a The solvent was deuterated methanol; the NMR instrument was 600 MHz. Table 2. Compounds 1-6 13 C NMR data b

[0039] b The solvent was deuterated methanol; the NMR instrument was 150 MHz. Example 3 The anti-inflammatory activity of compounds 1-6 and the elution fraction YH-D-2 containing compounds 1-6 in inhibiting NO release was evaluated using the following steps: Raw cells were seeded in 96-well cell culture plates and cultured at 37°C for 24 hours. Different concentrations of test compounds 1-6, elution site YH-D-2, or positive control drug indomethacin were added to the treatment groups (three parallel wells for each drug concentration). At the same time, control groups (with added lipopolysaccharide) and blank control groups (without added lipopolysaccharide) were set up.

[0040] Take a sterile 24-well plate and divide it into groups of three consecutive wells. Add 1000 ml of lipopolysaccharide (LPS)-containing medium to the first well of each group, 450 ml of LPS-containing medium to the second well, and 450 ml of LPS-containing medium to the third well. Add 2 ml of each compound (10 µM) sequentially to the first well of each group and mix thoroughly. Then, add 50 ml of medium from the first well of each group to the second well and mix thoroughly. Finally, add 50 ml of medium from the second well to the third well and mix thoroughly. The resulting drug concentration in the first well of each group is 2 × 10⁻⁶. - 5 M, the drug concentration in the second well is 2×10 -6 M, the drug concentration in the third well is 2×10 -7 M.

[0041] The dosing group consisted of 54 wells: First, 100 ml of culture medium was sequentially added from the third well of each group on the 24-well plate to three wells on the 96-well plate. Then, 100 ml of culture medium was sequentially added from the second well of each group on the 24-well plate to the corresponding three wells on the 96-well plate. Finally, 100 ml of culture medium was sequentially added from the third well of each group on the 24-well plate to the corresponding three wells on the 96-well plate. After adding 100 ml of culture medium to each well, the actual concentration of the compound became 10. -5 10 -6 and 10 -7 M, thus each compound has three concentration gradients. 100 ml of LPS-free medium was added to the blank control group, and 100 ml of LPS-containing medium was added to both the control and treatment groups. The 96-well plate was incubated at 37°C. After observing that the control group's medium turned yellow and the blank control group's color was darker than the control group, nitrite (NO) could be determined using the Griess method. 2- (This is used to reflect the concentration of NO.)

[0042] Evaluation of the anti-inflammatory effect of the compound: NO release inhibition rate (%) = (NO release amount in blank control group) NO release amount in the treatment group / control group / NO release amount in the blank control group × 100.

[0043] The results are shown in Table 4.

[0044] Table 4. Anti-inflammatory activity of compounds 1-6 and elution fraction YH-D-2 in inhibiting NO release.

[0045] Example 4 The anti-inflammatory activity of compound 6 (IC) 50 Value determination, the steps are as described in Example 3 above, IC 50 The value is obtained through calculation.

[0046] Table 5. Anti-inflammatory activity of compound 6 in inhibiting NO release (IC50) 50 value

[0047] The results showed that the elution sites YH-D-2 and compounds 1-6 could inhibit the anti-inflammatory activity of NO release, among which compound 6 had the most significant anti-inflammatory activity and had potential anti-inflammatory effects, and could be used in the preparation of anti-inflammatory drugs.

[0048] Compounds 1-6 and the elution fraction YH-D-2 of the present invention can be formulated into preparations suitable for oral or injectable applications by conventional techniques, together with pharmaceutically acceptable salts, carriers or excipients. For example, tablets, capsules, powders, syrups, injections, etc., can be prepared by adding pharmaceutically acceptable carriers and / or excipients according to conventional techniques.

[0049] The above embodiments only describe a portion of the specific implementation methods of the present invention in detail, and are not limited to the embodiments disclosed herein. Furthermore, the substantive content protected by the present invention is not limited thereto. Any other modifications, equivalent substitutions, improvements, etc., made based on the principles and techniques of the present invention without departing from its design scope are all within the protection scope of the present invention.

Claims

1. A benzyl isoquinoline alkaloid glycoside compound, characterized in that, Compounds 1-6 are specifically selected from compounds shown in the following formulas: 。 2. The method for preparing the benzyl isoquinoline alkaloid glycoside compound according to claim 1, characterized in that, The method includes the following steps: 1) The tubers of the Chinese herb Corydalis Rhizome were soaked in a 6% acetic acid solution, dried at 40°C, pulverized, soaked in water, and then extracted by ultrasound. The extracts were combined and concentrated under reduced pressure to obtain the plant extract. 2) The plant extract obtained in step 1) was dispersed in water and separated by HPD100 macroporous resin column chromatography. The column was eluted with water, 50% ethanol and 95% ethanol in a gradient. The 50% ethanol eluent was collected and concentrated under reduced pressure to obtain the eluent YH-D. 3) Take YH-D) and separate it by MCI column chromatography. Elute it with a gradient of 0%~100% methanol-water solution. Recover the solvent under reduced pressure to obtain four eluents, which are named YH-D-1~YH-D-4 respectively. 4) Dissolve YH-D-2 in water and filter. The filtrate is subjected to medium-pressure ODS column chromatography and eluted sequentially with 1% acetic acid-water, 5%~60% methanol-acid-water, and 100% pure methanol. The solvent is recovered under reduced pressure to obtain five eluent fractions, which are named A-E respectively. 5) The filtrate after dissolving and filtering the elution fraction A in water was subjected to Sephadex LH-20 column chromatography, eluted with pure water, and concentrated under reduced pressure to obtain six eluent fractions, named A1-A6 respectively. 6) Elution fraction A4 was eluted using a 0-30% methanol-water gradient on a medium-pressure ODS column to obtain seven eluent fractions, named A4-1 to A4-7; A4-5 was eluted with pure methanol on a Sephadex LH-20 column, concentrated under reduced pressure, and separated into four eluent fractions, named A4-5-1 to A4-5-4; A4-5-3 was further separated using a semi-preparative C... 18 Compound 1 was obtained by column HPLC purification. 7) Elution fraction A5 was eluted by medium-pressure ODS column chromatography with a methanol-water gradient of 0-30%, and concentrated under reduced pressure to obtain 9 eluent fractions, which were named A5-1 to A5-9 respectively. 8) The A5-4 obtained in step 7) was subjected to Sephadex LH-20 column chromatography, eluted with pure methanol, to obtain two eluent fractions, named A5-4-1 and A5-4-2, respectively; A5-4-2 was subjected to semi-preparative C 18 Column HPLC purification yielded compounds 2, 3, 4, and 6. 9) The A5-5 obtained in step 7) is semi-prepared into C 18 Compound 5 was obtained by column HPLC purification.

3. The method for preparing the benzyl isoquinoline alkaloid glycoside compound according to claim 2, characterized in that, Step 1) involves ultrasonic extraction three times, with a water-to-medicinal-material mass-to-volume ratio of 1:1 each time, and an ultrasonic time of 1 hour.

4. The method for preparing the benzyl isoquinoline alkaloid glycoside compound according to claim 2, characterized in that, In step 2), each gradient elutes 3 times the volume of the macroporous resin column, and in step 3), the volume ratio of methanol-water solution is 0%, 30%, 60%, and 100%, with each gradient eluting 2 times the volume of the MCI column.

5. The method for preparing the benzyl isoquinoline alkaloid glycoside compound according to claim 2, characterized in that, In step 4), the elution time for 1% acetic acid-water is 30 min, and the flow rate is 45 ml / min. The 5%~60% methanol-acid water containing 1% acetic acid has an elution time of 180 min, and the flow rate is 45 ml / min. The elution time for 100% methanol is 20 min, and the flow rate is 45 ml / min.

6. The method for preparing the benzyl isoquinoline alkaloid glycoside compound according to claim 2, characterized in that, In step 6), the elution time of the 0-30% methanol-water gradient is 60 min, and the flow rate is 35 ml / min; the semi-preparative C 18 The column HPLC conditions were as follows: mobile phase 6% acetonitrile-water containing 0.1% trifluoroacetic acid, flow rate 3 ml / min, detection wavelength 254 nm, and column temperature 25 °C.

7. The method for preparing the benzyl isoquinoline alkaloid glycoside compound according to claim 2, characterized in that, In step 7), the elution time of the 0-30% methanol-water gradient is 60 min, and the flow rate is 35 ml / min.

8. The method for preparing the benzyl isoquinoline alkaloid glycoside compound according to claim 2, characterized in that, In step 8), C is prepared 18 The column HPLC conditions were: mobile phase 19% acetonitrile-water containing 0.1% trifluoroacetic acid, flow rate 3 ml / min, detection wavelength 254 nm, and column temperature 25 °C; the preparation of C in step 9) 18 The column HPLC conditions were as follows: mobile phase 17% acetonitrile-water containing 0.1% trifluoroacetic acid, flow rate 3 ml / min, detection wavelength 254 nm, and column temperature 25 °C.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises at least one of the benzyl isoquinoline alkaloid glycoside compounds of claim 1 and its pharmaceutically acceptable salt or pharmaceutically acceptable carrier or excipient, or the elution site YH-D-2 of claim 2 and its pharmaceutically acceptable carrier or excipient.

10. Use of the pharmaceutical composition of claim 9 in the preparation of an anti-inflammatory drug.

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