Chalcone-lignan glycoside compound separated from sea-buckthorn and liver protection application of chalcone-lignan glycoside compound

Abstract

The invention discloses a novel chalcone-lignan glycoside compound with a structure as shown in a formula I which is described in the specification. The chalcone-lignan glycoside compound is novel and unique in structure, the fact that the compound has remarkable liver protection activity is disclosed for the first time, and the chalcone-lignan glycoside compound can be used for preparing liver protection drugs. The invention further discloses the application of the neochalcono lignan glycoside compound in preparation of a liver protection medicine, and further discloses the application of the neochalcono lignan glycoside compound in preparation of the liver protection medicine. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and relates to a new chalcone lignan glycoside compound separated from seabuckthorn, as well as a preparation method and liver-protecting use of the compound. Background Art

[0002] Seabuckthorn (Hippophae rhamnoides L.) is the fruit of the plant Hippophae L. of the Elaeagnaceae Juss. family. It is also known as vinegar willow, yellow sour thorn, sour thorn willow, black thorn, and sour thorn. The resources are widely distributed. As a common Chinese medicine for both medicinal and edible purposes, seabuckthorn contains not only rich nutrients but also a variety of biologically active ingredients in its fruit. Therefore, seabuckthorn has extremely high development and utilization value. The 2020 edition of the "Chinese Pharmacopoeia" includes seabuckthorn fruit as a medicine, which has the effects of relieving cough and expectoration, strengthening the spleen and digestion, and promoting blood circulation and removing blood stasis. Modern pharmacological studies have found that seabuckthorn has multiple biological activities such as anti-inflammatory, antioxidant, anti-cancer, hypoglycemic, hypolipidemic, neuroprotective, and antibacterial. Therefore, it is necessary to further develop and utilize the potential medicinal value of seabuckthorn. Summary of the invention

[0003] The present invention aims to use the Chinese medicine for both medicinal and edible purposes - seabuckthorn as the research object, and to separate and purify the liver-protecting active part of seabuckthorn by using the biological activity-guided separation method, thereby obtaining a new chalcone lignan glycoside compound, which has significant liver-protecting activity. So far, there has been no relevant report on this research result at home and abroad.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] A new chalcone lignan glycoside compound having a structure as shown in Formula I:

[0006]

[0007] The molecular formula of the new chalcone lignan glycoside compound is C 43 H 50 O 15 , chemical name: 5-methoxy-6-rutinoside-7-prenyl-chalcone-(3',4')-4"methoxy-7",8"-phenylpropanollignan.

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

[0009] Step (1), crushing the dried seabuckthorn fruit, soaking it in 80-100% ethanol for 18-20 hours, heating and refluxing for extraction 3-5 times, each time for 3-5 hours, combining the filtrate to obtain a total seabuckthorn extract;

[0010] Step (2), concentrating and drying the total seabuckthorn extract at 45-55° C. under vacuum conditions to obtain a dry total seabuckthorn extract;

[0011] Step (3), uniformly suspending the total extract of seabuckthorn in distilled water, and extracting with petroleum ether, ethyl acetate, and n-butanol in sequence to obtain a petroleum ether extraction fraction, an ethyl acetate extraction fraction, and an n-butanol extraction fraction, respectively;

[0012] Step (4), the n-butanol extraction portion is subjected to a AB-8 macroporous adsorption resin column, and gradient elution is performed in sequence using 10% ethanol → 20% ethanol → 40% ethanol → 60% ethanol → 100% ethanol to obtain five 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 adopts YMC-Pack ODS-A chromatographic column (250×50mm, 5μm), the mobile phase is 15-45% methanol, the absorption wavelength is 200-350nm, the flow rate is 10-15mL / min, and four sub-fractions are obtained: sub-fraction B-1, sub-fraction B-2, sub-fraction B-3, and sub-fraction B-4;

[0014] Step (6), the subfraction B-3 was 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), subfraction B-3-2 is applied to 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), the fine fraction B-3-2-2 is separated by preparative liquid chromatography, the preparative liquid chromatography uses a YMC-PackODS-A column (250×20 mm, 5 μm), the mobile phase is 15-45% methanol, the absorption wavelength is 200-350 nm, the flow rate is 3-5 mL / min, and the preparative liquid chromatography is repeated multiple times to purify compound I.

[0017] In step (1), the dried seabuckthorn is crushed into 50-70 meshes.

[0018] Each time the heating reflux extraction is performed, the solid-liquid ratio (ie, the mass volume ratio of the dried seabuckthorn to 80-100% ethanol) is 0.3:1-0.4:1 kg / L.

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

[0020] Based on the bioactivity-guided separation method, the different extraction fractions obtained were screened for activity preservation, and the n-butanol extraction fraction was determined to be the liver-protective 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 new chalcone lignan glycosides in the preparation of liver protection drugs.

[0023] Another object of the present invention is to provide the use of the new chalcone lignan glycosides in the preparation of drugs for treating and / or preventing liver damage.

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

[0025] Another object of the present invention is to provide a pharmaceutical composition, wherein the pharmaceutical composition uses the novel chalcone lignan glycosides as the main active ingredient and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

[0026] The dosage form is any pharmaceutically recognized dosage form; preferably, the dosage form is tablets, capsules, granules, pills, oral liquids, and suspensions.

[0027] Beneficial effects of the present invention:

[0028] The present invention selects seabuckthorn, a Chinese medicine for both medicinal and edible purposes, as a research object. The Chinese medicine has abundant resources and its raw materials are easily available.

[0029] The invention adopts 80-100% ethanol to heat and reflux extract the seabuckthorn, and based on the biological activity-guided separation method, the liver-protecting active part of the seabuckthorn is separated and purified by the biological activity-guided separation method, thereby separating a new chalcone lignan glycoside compound, and the preparation method is simple and easy.

[0030] The chalcone lignan glycoside compound of the present invention has a novel and unique structure, discloses for the first time that the compound has significant liver protection activity, and can be used for preparing liver protection drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1is a flow chart for the preparation of compound I.

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

[0033] Figure 3 It is the NOESY correlation diagram of compound I. DETAILED DESCRIPTION

[0034] The essential contents of the present invention are further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0035] Example 1

[0036] like Figure 1 , a new chalcone lignan glycoside compound is separated and purified from seabuckthorn, and the preparation method is as follows:

[0037] Step (1), crushing dried seabuckthorn fruit (10.0 kg) into 60 mesh, soaking in 90% ethanol for 19 hours, heating and refluxing extraction 4 times, using 30L 90% ethanol for 4 hours each time, combining the filtrates to obtain a total seabuckthorn extract;

[0038] Step (2), using a rotary evaporator to concentrate and dry the total seabuckthorn extract at 50° C. under vacuum conditions to obtain a dry total seabuckthorn extract (0.85 kg);

[0039] Step (3), according to the mass volume ratio of the total seabuckthorn extract to distilled water being 1:1.3 kg / L, the total seabuckthorn extract obtained in step (2) is uniformly suspended in distilled water, and extracted with petroleum ether, ethyl acetate, and n-butanol in sequence to obtain a petroleum ether extraction fraction (52.4 g), an ethyl acetate extraction fraction (224.5 g), and an n-butanol extraction fraction (417.2 g), respectively;

[0040] Based on the method of bioactivity-guided separation, different extraction sites were screened for activity preservation, and the n-butanol extraction site was determined to be the liver-protective active site based on the pharmacological activity results;

[0041] Step (4), applying the hepatoprotective active fraction, the n-butanol extraction fraction, to an AB-8 macroporous adsorption resin column, and gradient eluting with 10% ethanol → 20% ethanol → 40% ethanol → 60% ethanol → 100% ethanol in sequence, to obtain five 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] The 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, the medium pressure liquid chromatography used a YMC-Pack ODS-A column (250×50mm, 5μm); mobile phase: 19% methanol, absorption wavelength: 205nm, flow rate: 12mL / min; to obtain sub-fraction B-1 (8.0g), sub-fraction B-2 (10.6g), sub-fraction B-3 (12.4g), sub-fraction B-4 (7.6g);

[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: subfraction B-3-1 (3.1 g), B-3-2 (4.2 g), and B-3-3 (2.9 g);

[0045] Step (7), subfraction B-3-2 was subjected to a Sephadex LH-20 gel column and eluted with 90% methanol to obtain subfractions 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), the fine fraction B-3-2-2 was separated by preparative liquid chromatography, the preparative liquid chromatography used YMC-Pack (ODS-A column: 250×20 mm, 5 μm), mobile phase: 18% methanol, absorption wavelength: 205 nm, flow rate: 4 mL / min, and the preparative liquid chromatography was repeated several times to purify the new compound I (9.65 mg).

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

[0048] Compound I 1 H NMR (DMSO-d6, 600 MHz) and 13 C NMR (DMSO-d6, 150 MHz) data are shown in Table 1, HMBC, 1 H- 1 For information on H COSY coupling, see Figure 2 , NOESY related diagram see Figure 3 In compound I 1 H NMR and 13 In the C NMR spectrum, there is a typical trans double bond signal: δ H 7.98 (1H, d, J = 15.9 Hz, H-1), 7.59 (1H, d, J = 15.9 Hz, H-2) and δc 143.1 (C-1), 122.0 (C-2), a typical ABX system: δ H 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'), 125.4 (C-6') indicate that compound I is a chalcone compound. In addition, in the 1 H NMR and 13 There is a typical prenyl group signal on the C NMR spectrum: 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). According to the correlation between H-10 and C-7, H-9 and C-6, and H-8 and C-9 in the HMBC spectrum, it was determined that the prenyl group was connected to the C-7 position of glucose. 1 H NMR and 13 A 4"-methoxy-phenylpropenol structural fragment was found in the C 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). According to the HMBC spectrum, H-6" is related to C-7", H-2' is related to C-7", and H-9" is related to C7", it is determined that the structural fragment is connected at the C-3' / 4' position. 1 HNMR and 13 In the midfield 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), according to the correlation between H-1"' and C-6 in the HMBC spectrum, it shows that the rutinose unit is connected to the C-6 position. In summary, according to the spectral data of compound I and HMBC, 1 H- 1 The coupling related information such as H COSY and NOESY was searched by SciFinder, and it was determined that compound I was a new chalcone lignan glycoside compound.

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

[0050]

[0051]

[0052] Example 2

[0053] Determination of hepatoprotective activity 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 inoculated in a 96-well cell culture plate, cultured for 24 hours, the old culture medium was removed, and then the culture medium containing the test compound I was added (the final concentration of compound I was 9.5mM). At the same time, a blank control group was set up (the culture medium without compound I was added), and 3 parallel wells were set up for each treatment. After the HepG2 cells were treated with drugs for 48 hours, the culture medium was removed, and 100mL of MTT solution with a concentration of 0.5mg / mL was added to each well. The culture was continued for 4 hours, the MTT solution was removed, and 150mL of dimethyl sulfoxide (DMSO) was added to each well. The mixture was fully shaken and mixed, and the absorbance value was measured at a wavelength of 570nm using an enzyme marker to calculate the cell survival rate.

[0058] Cell survival rate (%) = (mean OD of administered cells / mean OD of blank control cells) × 100%.

[0059] Experimental results: After compound I was treated with HepG2 cells at a concentration of 9.5 mM for 48 hours, the cell survival rate was greater than 93%, indicating that compound I had no obvious 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 on HepG2 cell injury induced by APAP

[0061] HepG2 cells were seeded in a 96-well cell culture plate and cultured for 24 h. The old culture medium was removed and a compound I group, a positive drug control group, a model group and a blank control group (Control) were set up. Compound I group: a culture medium containing both the test compound I and paracetamol (APAP) was added (the final concentration of compound I was 9.5 mM, and the final concentration of APAP was 8 mM). Positive drug control group: a culture medium containing both bocyclol and APAP was added (the final concentration of bocyclol was 9.5 mM, and the final concentration of APAP was 8 mM). Blank control group: fresh culture medium was added. Model group: culture medium containing APAP was added (the final concentration of APAP was 8 mM). After HepG2 cells were exposed to drugs for 48 h, 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 culture was continued for 4 h, and then the MTT solution was removed, and 150 mL of dimethyl sulfoxide (DMSO) was added to each well. The mixture was thoroughly shaken and mixed, and the absorbance was measured at a wavelength of 570 nm using an enzyme reader to calculate the cell survival rate.

[0062] Cell survival rate (%) = 100 × average OD value of the drug-treated group / average OD value of the blank control group.

[0063] The experimental results are shown in Table 2, which show that APAP (final concentration 8mM) caused significant damage to HepG2 cells after 48 hours of action, and the cell survival rate was only 48.59%. The positive control drug bicyclol (concentration 9.5mM) had a significant protective effect on APAP-induced liver cell damage, and its cell survival rate was 60.20%. Compound I (concentration 9.5mM) had a significant protective effect on APAP-induced HepG2 cell damage, and its cell survival rate was as high as 77.56%. Therefore, Compound I has a protective effect on APAP-induced HepG2 cell damage.

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

[0065]

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

Claims

1. A chalcone lignan glycoside compound having a structure as shown in Formula I:

2. A method for preparing the chalcone lignan glycosides compound according to claim 1, characterized in that: The following steps are involved: Step (1), crushing the dried seabuckthorn fruit, soaking it in 80-100% ethanol for 18-20 hours, heating and refluxing for extraction 3-5 times, each time for 3-5 hours, combining the filtrate to obtain a total seabuckthorn extract; Step (2), concentrating and drying the total seabuckthorn extract at 45-55° C. under vacuum conditions to obtain a dry total seabuckthorn extract; Step (3), uniformly suspending the total extract of seabuckthorn in distilled water, and extracting with petroleum ether, ethyl acetate, and n-butanol in sequence to obtain a petroleum ether extraction fraction, an ethyl acetate extraction fraction, and an n-butanol extraction fraction, respectively; Step (4), the n-butanol extraction portion is subjected to a AB-8 macroporous adsorption resin column, and gradient elution is performed in sequence using 10% ethanol → 20% ethanol → 40% ethanol → 60% ethanol → 100% ethanol to obtain five 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, 250×50 mm, 5 μm, mobile phase: 15-45% methanol, absorption wavelength: 200-350 nm, flow rate: 10-15 mL / min, to obtain 4 sub-fractions: sub-fraction B-1, sub-fraction B-2, sub-fraction B-3, sub-fraction B-4; Step (6), the subfraction B-3 was 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), subfraction B-3-2 is applied to 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), the fine fraction B-3-2-2 was separated by preparative liquid chromatography, using a YMC-Pack ODS-A column, 250×20 mm, 5 μm, mobile phase: 15-45% methanol, absorption wavelength: 200-350 nm, flow rate: 3-5 mL / 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 seabuckthorn is crushed into 50-70 meshes.

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

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

6. Use of the chalcone lignan glycosides compound according to claim 1 in the preparation of liver protection drugs.

7. Use of the chalcone lignan glycoside compound according to claim 1 in the preparation of a drug for treating and / or preventing liver damage.

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

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

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

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