A chalcone lignan glycoside compound isolated from sea buckthorn and its hepatoprotective uses.

Abstract

This invention discloses a novel chalcone gypsum glycoside compound with the structure shown in Formula I. The chalcone gypsum glycoside compound of this invention has a novel and unique structure, and for the first time, it is disclosed that this compound possesses significant hepatoprotective activity and can be used in the preparation of hepatoprotective drugs. This invention also discloses the use of the aforementioned novel chalcone gypsum glycoside compound in the preparation of hepatoprotective drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a novel chalcone lignan glycoside compound isolated from sea buckthorn, as well as the preparation method and hepatoprotective use of the compound. Background Technology

[0002] Sea buckthorn (Hippophae rhamnoides L.) is the fruit of a plant belonging to the genus Hippophae L. of the family Elaeagnaceae. Also known as vinegar willow, yellow sour thorn, sour thorn willow, black thorn, and sour thorn, it is widely distributed. As a common medicinal and edible herb, sea buckthorn fruit contains not only abundant nutrients but also various bioactive components. Therefore, sea buckthorn has extremely high development and utilization value. The 2020 edition of the Chinese Pharmacopoeia includes sea buckthorn fruit for medicinal use, possessing effects such as relieving cough and phlegm, strengthening the spleen and promoting digestion, and promoting blood circulation and removing blood stasis. Modern pharmacological research has found that sea buckthorn has various biological activities, including anti-inflammatory, antioxidant, anti-cancer, hypoglycemic, hypolipidemic, neuroprotective, and antibacterial properties. Therefore, it is necessary to further develop and utilize the potential medicinal value of sea buckthorn. Summary of the Invention

[0003] The purpose of this invention is to isolate and purify the hepatoprotective active components of sea buckthorn (a traditional Chinese medicine used for both medicinal and edible purposes) using a bioactivity-guided separation method, thereby obtaining a novel chalcone lignan glycoside compound with significant hepatoprotective activity. To date, no similar research results have been reported domestically or internationally.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] A novel chalcone lignan glycoside compound with the structure shown in Formula I:

[0006]

[0007] The molecular formula of the aforementioned neochalcone lignan glycoside compound is C2 43 H 50 O 15 Chemical name: 5-methoxyl-6-rutinoside-7-prenyl-chalcone-(3',4')-4”-methoxyl-7”,8”-phenylpropanollignan.

[0008] Another object of the present invention is to provide a method for preparing the aforementioned novel chalcone lignan glycoside compound, comprising the following steps:

[0009] Step (1): Crush the dried sea buckthorn fruit, soak it in 80-100% ethanol for 18-20 hours, heat and reflux to extract 3-5 times, 3-5 hours each time, combine the filtrates to obtain the total sea buckthorn extract;

[0010] Step (2): The total extract of sea buckthorn is concentrated and dried under vacuum at 45-55℃ to obtain the dried total extract of sea buckthorn.

[0011] Step (3): The total extract of sea buckthorn is uniformly suspended in distilled water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol to obtain the petroleum ether extract, ethyl acetate extract and n-butanol extract, respectively.

[0012] Step (4): The n-butanol extraction section is loaded onto an AB-8 macroporous adsorption resin column and eluted sequentially with 10% ethanol → 20% ethanol → 40% ethanol → 60% ethanol → 100% ethanol to obtain 5 main fractions: main fraction A, main fraction B, main fraction C, main fraction D, and main fraction E.

[0013] Step (5): The main fraction B is separated by medium-pressure liquid chromatography. The medium-pressure liquid chromatography uses a YMC-Pack ODS-A column (250×50mm, 5μm), mobile phase: 15-45% methanol, absorption wavelength: 200-350nm, flow rate: 10-15mL / min, to obtain 4 secondary fractions: secondary fraction B-1, secondary fraction B-2, secondary fraction B-3, and secondary fraction B-4.

[0014] Step (6): Subfraction B-3 is loaded onto a Toyopearl HW-40C gel resin column and eluted with dichloromethane-methanol = 1:1-1:3 (V / V) to obtain subfractions B-3-1, B-3-2, and B-3-3.

[0015] Step (7): Sub-fraction B-3-2 is loaded onto a Sephadex LH-20 gel column and eluted with 80-100% methanol to obtain fine fractions B-3-2-1, B-3-2-2, and B-3-2-3;

[0016] Step (8): Fine fraction B-3-2-2 was separated by preparative liquid chromatography. The preparative liquid chromatography used a YMC-PackODS-A column (250×20mm, 5μm), mobile phase: 15-45% methanol, absorption wavelength: 200-350nm, flow rate: 3-5mL / min. The preparative liquid chromatography was repeated multiple times to purify compound I.

[0017] In step (1), the dried sea buckthorn is crushed to 50-70 mesh.

[0018] For each heating and reflux extraction, the material-to-liquid ratio (i.e., the mass-to-volume ratio of dried sea buckthorn to 80-100% ethanol) is 0.3:1-0.4:1 kg / L.

[0019] In step (3), the mass-to-volume ratio of the total sea buckthorn extract to distilled water is 1:1.0-1:1.3 kg / L.

[0020] Based on the bioactivity-guided separation method, the different extract fractions were screened for activity protection, and the n-butanol extract fraction was determined to be the hepatoprotective active fraction based on the pharmacological activity results.

[0021] In step (4), the five fractions obtained were screened for hepatoprotective activity, and the results showed that the main fraction B had significant hepatoprotective activity.

[0022] Another object of the present invention is to provide the use of the aforementioned new chalcone lignan glycosides in the preparation of hepatoprotective drugs.

[0023] Another object of the present invention is to provide the use of the aforementioned new chalcone lignan glycoside compounds in the preparation of medicaments for treating and / or preventing liver injury.

[0024] Preferably, the liver injury is drug-induced liver injury.

[0025] Another object of the present invention is to provide a pharmaceutical composition having the aforementioned neochalcone lignan glycosides as the main active ingredient, formulated into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

[0026] The dosage form can be any pharmaceutically acceptable dosage form; preferably, the dosage form is a tablet, capsule, granule, pill, oral liquid, or suspension.

[0027] The beneficial effects of this invention are:

[0028] This invention selects sea buckthorn, a traditional Chinese medicine that can be used as both food and medicine, as the research object. This traditional Chinese medicine is abundant and the raw materials are readily available.

[0029] This invention uses 80-100% ethanol to extract sea buckthorn by heating and reflux. Based on the bioactivity-guided separation method, the hepatoprotective active components of sea buckthorn are separated and purified using the bioactivity-guided separation method, from which a new chalcone lignan glycoside compound is obtained. The preparation method is simple and easy to implement.

[0030] The present invention discloses a novel and unique structure of chalcone lignan glycoside compounds, and for the first time discloses that the compounds have significant hepatoprotective activity and can be used to prepare hepatoprotective drugs. Attached Figure Description

[0031] Figure 1This is a flowchart of the preparation process of compound I.

[0032] Figure 2 This is a coupling correlation diagram of compound I.

[0033] Figure 3 This is the NOESY correlation diagram for compound I. Detailed Implementation

[0034] The following specific embodiments further illustrate the substantive content of the present invention, but do not limit the scope of protection of the present invention.

[0035] Example 1

[0036] like Figure 1 A novel chalcone lignan glycoside compound was isolated and purified from sea buckthorn. The preparation method is as follows:

[0037] Step (1): Crush 10.0 kg of dried sea buckthorn fruit into 60 mesh, soak in 90% ethanol for 19 hours, and extract by reflux 4 times. Each time, use 30 L of 90% ethanol to extract by reflux for 4 hours. Combine the filtrates to obtain the total extract of sea buckthorn.

[0038] Step (2): Using a rotary evaporator, the total extract of sea buckthorn was concentrated and dried under vacuum at 50°C to obtain dried total extract of sea buckthorn (0.85 kg);

[0039] Step (3): The total sea buckthorn extract obtained in step (2) was uniformly suspended in distilled water at a mass-to-volume ratio of 1:1.3 kg / L to distilled water, and then extracted with petroleum ether, ethyl acetate and n-butanol in sequence to obtain petroleum ether extract (52.4 g), ethyl acetate extract (224.5 g) and n-butanol extract (417.2 g) respectively.

[0040] Based on the bioactivity-guided separation method, different extraction fractions were screened for activity protection, and the n-butanol extraction fraction was determined to be the hepatoprotective active fraction based on the pharmacological activity results.

[0041] Step (4): The hepatoprotective active fraction—the n-butanol extract fraction—was loaded onto an AB-8 macroporous adsorption resin column and eluted sequentially with 10% ethanol → 20% ethanol → 40% ethanol → 60% ethanol → 100% ethanol to obtain 5 main fractions: main fraction A (30.5 g), main fraction B (45.5 g), main fraction C (58.6 g), main fraction D (60.9 g), and main fraction E (41.6 g).

[0042] Five main fractions were screened for hepatoprotective activity, and the results showed that main fraction B had significant hepatoprotective activity.

[0043] Step (5): The main fraction B was separated by medium-pressure liquid chromatography using a YMC-Pack ODS-A column (250×50mm, 5μm); mobile phase: 19% methanol, absorption wavelength: 205nm, flow rate: 12mL / min; secondary fractions B-1 (8.0g), B-2 (10.6g), B-3 (12.4g), and B-4 (7.6g) were obtained.

[0044] Step (6): Subfraction B-3 was loaded onto a Toyopearl HW-40C gel resin column and eluted with dichloromethane-methanol = 1 / 3 (V / V) to obtain three subfractions: subfractions B-3-1 (3.1 g), B-3-2 (4.2 g), and B-3-3 (2.9 g);

[0045] Step (7): Sub-fraction B-3-2 was loaded onto a Sephadex LH-20 gel column and eluted with 90% methanol to obtain fine fractions B-3-2-1 (1.1 g), B-3-2-2 (2.3 g), and B-3-2-3 (0.9 g);

[0046] Step (8): Fine fraction B-3-2-2 was separated by preparative liquid chromatography. The preparative liquid chromatography used YMC-Pack (ODS-A column: 250×20mm, 5μm), mobile phase: 18% methanol, absorption wavelength: 205nm, flow rate: 4mL / min. The preparative liquid chromatography was repeated multiple times to purify and obtain new compound I (9.65 mg).

[0047] Compound I is a pale yellow solid, HR-ESI-MS m / z 829.3044 [M+Na] + Its molecular formula is C 43 H 50 O 15 (calcd.for C 43 H 50 O 15 Na, 829.3047). IRν of compound I max 3286, 1678, 1612, 1362cm -1 ;UV(MeOH)λ max 205, 286, 340nm.

[0048] Compound I 1 H NMR (DMSO-d6, 600MHz) and 13 C NMR (DMSO-d6, 150MHz) data are shown in Table 1, HMBC, 1 H- 1 For information related to H COSY and other couplings, please see [link / reference]. Figure 2 See related images for NOESY. Figure 3 In compound I 1 H NMR and 13 In the 1C NMR spectrum, there is a typical trans double bond signal: δ H 7.98 (1H,d,J=15.9Hz,H⁻¹), 7.59 (1H,d,J=15.9Hz,H⁻²) and δc 143.1 (C⁻¹), 122.0 (C⁻²), a typical ABX system: δ H The values ​​of 7.69 (1H, d, J = 2.0 Hz, H-2'), 7.45 (1H, d, J = 8.0 Hz, H-5'), 7.51 (1H, dd, J = 8.0, 2.0 Hz, H-6'), and δc 129.3 (C-1'), 119.2 (C-2'), 127.3 (C-3'), 155.1 (C-4'), 111.5 (C-5'), and 125.4 (C-6') indicate that compound I is a chalcone compound. Furthermore, in compound I... 1 H NMR and 13 The C NMR spectrum shows a typical prenyl (isopreneyl) group signal: δ H 3.29 (2H, d, J = 6.5 Hz, H-9), 5.77 (1H, t, J = 6.5, 1.5 Hz, H-10), 1.70 (3H, s, 12-CH3), 1.83 (3H, s, 13-CH3) and δ C 23.1 (C-9), 120.6 (C-10), 133.0 (C-11), 19.1 (12-CH3), 24.3 (13-CH3). Based on the correlation between H-10 and C-7, H-9 and C-6, and H-8 and C-9 in the HMBC spectrum, the prenyl group is determined to be attached to the C-7 position of glucose. In compound I... 1 H NMR and 13 A 4”-methoxy-phenylenepropenol structural fragment was found in the C1 NMR spectrum: δ Hand δc 128.5(C-1”), 129.8(C-2” / 6”), 115.1(C-3” / 5”), 161.1(C-4”), 112.6(C-7”), 151.9(C-8”), 58.1(C-9”), 56.5(4”-OCH3). Based on the correlations between H-6” and C-7”, H-2” and C-7”, and H-9” and C-7” in the HMBC spectrum, the structural fragment is determined to be attached at the C-3’ / 4’ position. In compound I... 1 HNMR and 13 In the mid-field and high-field regions of the C NMR spectrum, there is a typical rutin signal: δ H 5.02(1H,d,J=7.8Hz,H-1"'), 3.53(1H,m,H-2"'), 3.81(1H,m,H-3"'), 3.39(1H,m, H-4"'), 3.78(1H,m,H-5"'),3.66(1H,dd,J=14.1,6.5Hz,H-6"'a),3.94(1H,dd,J= 6.5,3.3Hz,H-6”'b),4.66(1H,d,J=1.8Hz,H-1””), 3.75(1H,m,H-2””), 3.60(1H,m ,H-3””),3.44(1H,m,H-4””)3.59(1H,m,H-5””),1.21(3H,d,J=7.2Hz,6””-CH3) and δc 103.0 (C-1”'), 72.3 (C-2”'), 73.2 (C-3”'), 71.5 (C-4”'), 72.6 (C-5”'), 65.0 (C-6”'), 100.1 (C-1”'), 75.3 (C-2”'), 76.8 (C-3”'), 73.5 (C-4”'), 76.5 (C-5”'), 16.9 (6”'-CH3). Based on the correlation between H-1”' and C-6 in the HMBC spectrum, it indicates that the rutin unit is connected at the C-6 position. In summary, based on the spectral data of compound I and HMBC, 1 H- 1 H COSY, NOESY and other coupling-related information were used to search for SciFinder and compound I was identified as a new chalcone lignan glycoside.

[0049] Table 1. Compound I 1 H NMR, 13 C NMR and HMBC related data

[0050]

[0051]

[0052] Example 2

[0053] Hepatoprotective activity assay of compound I

[0054] 1. Experimental cells, drugs, and equipment

[0055] HepG2 cells; paracetamol (APAP); compound I; bocyclol; dimethyl sulfoxide (DMSO); MTT solution; 96-well plate; microplate reader.

[0056] 2. Cytotoxicity of Compound I on HepG2 Cells

[0057] HepG2 cells were seeded in 96-well cell culture plates and cultured for 24 hours. The old culture medium was removed, and then culture medium containing the test compound I (final concentration of compound I: 9.5 mM) was added. A blank control group (containing culture medium without compound I) was also set up. Each treatment had three parallel wells. After 48 hours of treatment, the culture medium was removed, and 100 mL of 0.5 mg / mL MTT solution was added to each well. The cells were cultured for another 4 hours, and then the MTT solution was removed. 150 mL of dimethyl sulfoxide (DMSO) was added to each well, and the mixture was thoroughly vortexed. The absorbance was measured at 570 nm using a microplate reader, and cell viability was calculated.

[0058] Cell viability (%) = (average OD of treated cells / average OD of blank control cells) × 100%.

[0059] Experimental results: After treatment of HepG2 cells with compound I at a concentration of 9.5 mM for 48 h, the cell viability was greater than 93%, indicating that compound I had no significant toxicity to HepG2 cells at this concentration. Therefore, a concentration of 9.5 mM of compound I was selected for subsequent experiments.

[0060] 3. Protective effect of compound I against APAP-induced damage to HepG2 cells

[0061] HepG2 cells were seeded in 96-well cell culture plates and cultured for 24 h. After removing the old culture medium, four groups were set up: Compound I group, positive drug control group, model group, and blank control group. Compound I group: culture medium containing both test compound I and paracetamol (APAP) was added (final concentration of compound I was 9.5 mM and final concentration of APAP was 8 mM). Positive drug control group: culture medium containing both bocyclol and APAP was added (final concentration of bocyclol was 9.5 mM and final concentration of APAP was 8 mM). Blank control group: fresh culture medium was added. Model group: culture medium containing APAP was added (final concentration of APAP was 8 mM). After HepG2 cells were treated with the drug for 48 hours, the culture medium was removed, and 100 mL of MTT solution with a concentration of 0.5 mg / mL was added to each well. The cells were cultured for another 4 hours. Then, the MTT solution was removed, and 150 mL of dimethyl sulfoxide (DMSO) was added to each well. The cells were thoroughly shaken and mixed. The absorbance value was measured at 570 nm using an ELISA reader, and the cell viability was calculated.

[0062] Cell viability (%) = 100 × mean OD of the drug-treated group / mean OD of the blank control group.

[0063] The experimental results are shown in Table 2. APAP (final concentration 8 mM) caused significant damage to HepG2 cells after 48 hours of treatment, with a cell survival rate of only 48.59%. The positive control drug bicyclol (concentration 9.5 mM) showed a significant protective effect against APAP-induced hepatocyte damage, with a cell survival rate of 60.20%. Compound I (concentration 9.5 mM) also showed a significant protective effect against APAP-induced HepG2 cell damage, with a cell survival rate as high as 77.56%. Therefore, compound I has a protective effect against APAP-induced HepG2 cell damage.

[0064] Table 2. Protective effect of compound I against APAP-induced HepG2 cell damage

[0065]

[0066] Note: *** P < 0.001, compared with the blank control group; # P < 0.05, compared with the model group.

Claims

1. Chalcone lignan glycosides with structures as shown in Formula I:

2. A method for preparing the chalcone lignan glycoside compound according to claim 1, characterized in that: Includes the following steps: Step (1): Crush the dried sea buckthorn fruit, soak it in 80-100% ethanol for 18-20 hours, heat and reflux to extract 3-5 times, 3-5 hours each time, combine the filtrates to obtain the total sea buckthorn extract; Step (2): The total extract of sea buckthorn is concentrated and dried under vacuum at 45-55℃ to obtain the dried total extract of sea buckthorn. Step (3): The total extract of sea buckthorn is uniformly suspended in distilled water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol to obtain the petroleum ether extract, ethyl acetate extract and n-butanol extract, respectively. Step (4): The n-butanol extraction section is loaded onto an AB-8 macroporous adsorption resin column and eluted sequentially with 10% ethanol → 20% ethanol → 40% ethanol → 60% ethanol → 100% ethanol to obtain 5 main fractions: main fraction A, main fraction B, main fraction C, main fraction D, and main fraction E. Step (5): The main fraction B is separated by medium-pressure liquid chromatography. The medium-pressure liquid chromatography uses a YMC-Pack ODS-A column, 250×50mm, 5μm, mobile phase: 15-45% methanol, absorption wavelength: 200-350nm, flow rate: 10-15mL / min, to obtain 4 secondary fractions: secondary fraction B-1, secondary fraction B-2, secondary fraction B-3, and secondary fraction B-4. Step (6): Subfraction B-3 is loaded onto a Toyopearl HW-40C gel resin column and eluted with dichloromethane-methanol = 1:1-1:3 (V / V) to obtain subfractions B-3-1, B-3-2, and B-3-3. Step (7): Sub-fraction B-3-2 is loaded onto a Sephadex LH-20 gel column and eluted with 80-100% methanol to obtain fine fractions B-3-2-1, B-3-2-2, and B-3-2-3; Step (8): Fine fraction B-3-2-2 was separated by preparative liquid chromatography. The preparative liquid chromatography used a YMC-Pack ODS-A column, 250×20mm, 5μm, with a mobile phase of 15-45% methanol, an absorption wavelength of 200-350nm, and a flow rate of 3-5mL / min to obtain compound I.

3. The method for preparing chalcone lignan glycosides according to claim 2, characterized in that: In step (1), the dried sea buckthorn is crushed to 50-70 mesh.

4. The method for preparing chalcone lignan glycosides according to claim 2, characterized in that: In step (1), the material-to-liquid ratio is 0.3:1-0.4:1 kg / L for each heating and reflux extraction.

5. The method for preparing chalcone lignan glycosides according to claim 2, characterized in that: In step (3), the mass-to-volume ratio of the total sea buckthorn extract to distilled water is 1:1.0-1:1.3 kg / L.

6. The use of the chalcone lignan glycoside compound of claim 1 in the preparation of hepatoprotective drugs.

7. Use of the chalcone lignan glycoside compound of claim 1 in the preparation of medicaments for treating and / or preventing liver injury.

8. The use according to claim 7, characterized in that: The liver injury described is drug-induced liver injury.

9. A pharmaceutical composition, characterized in that: The pharmaceutical composition uses the chalcone lignan glycoside compound of claim 1 as the main active ingredient, and is formulated into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 9, characterized in that: The dosage forms mentioned are tablets, capsules, granules, pills, oral liquids, and suspensions.

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