Preparation methods and applications of biphenyl compounds in hawthorn leaves

Abstract

This invention discloses a method for preparing biphenyl compounds from hawthorn leaves and their applications, belonging to the field of pharmaceutical technology. The biphenyl compounds from hawthorn leaves, as shown in the structural formula below, are obtained by separating the 70% ethanol extract from the dried leaves of hawthorn (Crataegus pinnatifida Bge.), a plant of the Rosaceae family, using various column chromatography and HPLC methods. DPPH free radical scavenging activity tests show that compounds 1 and 2 have good DPPH free radical scavenging activity, and their activity is stronger than that of the positive control drug VC. ABTS free radical scavenging activity tests show that compounds 1 and 2 have good ABTS free radical scavenging activity, and their activity is stronger than that of the positive control drug VC. Compounds 1 and 2 provided by this invention exhibit superior antioxidant activity; therefore, the biphenyl compounds described in this invention have the potential to be used in the preparation of clinical antioxidant drugs.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and relates to a preparation method and use of biphenyl compounds, specifically to two biphenyl compounds isolated from the medicinal plant Hawthorn Leaf, and the application of the compounds in the preparation of antioxidant drugs. Background Art

[0002] Hawthorn Leaf is the dried leaf of the Rosaceae plant Crataegus pinnatifida Bge. var. major N.E. Br. or Crataegus pinnatifida Bge., which is mainly distributed in Northeast and North China. As early as in the "Elbow后方" during the Eastern Jin Dynasty, there was a record of using Hawthorn Leaf juice to treat lacquer sores. Hawthorn Leaf is usually harvested in summer and autumn, dried, and its powder is used medicinally or brewed as tea, which has the effects of promoting digestion and strengthening the stomach, promoting qi circulation and removing blood stasis, and reducing turbidity and lipid. Hawthorn Leaf and its preparations have been widely used in clinical treatment. For example, Shanmei Capsule uses the 50% ethanol extract of Hawthorn Leaf and the 70% ethanol extract of Rosa davurica Pall. as raw materials to treat coronary heart disease, cerebral arteriosclerosis and other diseases, and has been included in the "Chinese Pharmacopoeia" of 2020 edition, which is a model of both medicine and food.

[0003] Hawthorn Leaf contains rich chemical components, such as flavonoids, terpenoids, phenolic acids, biphenyls and their glycosides, glycosides and some trace organic compounds. The research on new compounds in Hawthorn Leaf is still ongoing. In this invention, 70% ethanol extraction and various column chromatography methods are used to separate and purify the chemical components in Hawthorn Leaf, and DPPH and ABTS free radical scavenging experiments are used to screen the antioxidant activities of two new compounds isolated from Hawthorn Leaf, and it is found that both of the two new compounds have good antioxidant activities. Summary of the Invention

[0004] The primary object of the present invention is to provide biphenyl compounds in Hawthorn Leaf, which are any one of the following structural formulas:

[0005]

[0006] The biphenyl compounds in the Hawthorn Leaf described above are isolated from the 70% ethanol extract of the dried leaf of the Rosaceae plant Crataegus pinnatifida Bge. by using various column chromatography and HPLC and other separation methods.

[0007] The preparation method of the biphenyl compounds in Hawthorn Leaf described in the present invention includes the following steps:

[0008] (1) Extract Hawthorn Leaf with 70% ethanol by reflux extraction;

[0009] (2) Concentrate the extract under reduced pressure to obtain a concentrated solution;

[0010] (3) The concentrate is adsorbed by macroporous adsorption resin;

[0011] (4) After dissolving the extract, it was separated into four crude fractions, Fr.AD, by vacuum polyamide chromatography.

[0012] (5) Fr.C was separated into five fractions, Fr.C1-C5, by gel column chromatography;

[0013] (6) Fr.C4 was separated into six fractions, Fr.4-1-Fr.4-6, by reversed-phase ODS column chromatography;

[0014] (7) Fr.4-2 was separated into four fractions, Fr.4-2-1-Fr.4-2-4, by gel column chromatography;

[0015] (8) Compound 2 was obtained by silica gel H column chromatography after separating Fr.4-2-2;

[0016] (9) Compound 1 was obtained by silica gel column chromatography after separating Fr.4-2-3;

[0017] The above preparation method: wherein

[0018] The volume fraction of ethanol in step (1) is 70%; the extraction is performed 2-3 times; and each extraction takes 2-3 hours.

[0019] The temperature for vacuum concentration in step (2) is 50-55℃.

[0020] In step (3), the gradient elution conditions for macroporous adsorption of D101 lipid adsorption are EtOH / H2O = 0:100-95:5.

[0021] The reduced pressure polyamide chromatography gradient elution conditions in step (4) are as follows: CH2Cl2-MeOH = 5:1-0:1 gradient elution system.

[0022] The elution conditions for gel column chromatography in step (5) are MeOH:H2O = 70:30.

[0023] The gradient elution conditions for reversed-phase ODS column chromatography in step (6) are MeOH:H2O = 20:80-100:0.

[0024] The gradient elution conditions for gel column chromatography in step (7) are MeOH:H2O = 70:30.

[0025] In steps (8) and (9), the gradient elution conditions for silica gel H-column chromatography were CH2Cl2-MeOH = 5:1-0:1. The HPLC separation conditions for compound 2 were acetonitrile and water at a volume fraction of 20%. The HPLC separation conditions for compound 1 were methanol and water at a volume fraction of 33%.

[0026] A pharmaceutical composition comprising any one of the hawthorn leaf compounds 1-2 or any one of pharmaceutically acceptable salts of the compounds. It also includes pharmaceutically acceptable excipients.

[0027] The present invention also provides the use of the hawthorn leaf compounds 1-2 or pharmaceutical compositions containing the compounds in the preparation of antioxidant drugs.

[0028] The beneficial effects of this invention are:

[0029] Two compounds 1-2 from hawthorn leaves described in this invention were initially screened for DPPH and ABTS free radical scavenging activities at a concentration of 100 μM. DPPH free radical scavenging activity screening: at a concentration of 100 μM, the antioxidant activities of the positive control VC, compound 1, and compound 2 were 50.3%, 36.6%, and 61.4%, respectively. ABTS free radical scavenging activity screening: at a concentration of 100 μM, the antioxidant activities of the positive control VC, compound 1, and compound 2 were 76.3%, 74.3%, and 83.8%, respectively. The DPPH and ABTS free radical scavenging activities of compounds 1 and 2 at concentrations ranging from 12.5 to 200 μM were further tested. The DPPH free radical scavenging activity test results showed that compound 1 (IC50) exhibited strong DPPH and ABTS activity. 50 =149.3±0.65μM) and compound 2 (IC) 50 =54.9±0.95μM) exhibited good DPPH free radical scavenging activity, and its activity was stronger than that of the positive control drug VC (IC50). 50 =157.5±0.90μM). ABTS free radical scavenging activity test results showed that biphenyl compound 1 (IC50) 50 =16.5±1.42μM) and compound 2 (IC) 50 =11.0±0.03μM) exhibited good ABTS free radical scavenging activity, and its activity was stronger than that of the positive control drug VC (IC50). 50 =17.1±1.05 μM). The compounds exhibit superior antioxidant activity; therefore, the biphenyl compounds described in this invention show promise for the preparation of clinical antioxidant drugs. Attached Figure Description

[0030] Figure 1 (+)-HRESIMS spectrum of compound 1;

[0031] Figure 2 Compound 1 1 1H NMR spectrum (600MHz, DMSO-d6);

[0032] Figure 3 Compound 1 13 C NMR spectrum (150MHz, DMSO-d6);

[0033] Figure 4 (+)-HRESIMS spectrum of compound 2;

[0034] Figure 5 Compound 2 1 H NMR spectrum (600MHz, Methanol-d4);

[0035] Figure 6 Compound 2 13 C10 NMR spectrum (150 MHz, Methanol-d4);

[0036] Figure 7 In vitro antioxidant activity of compounds 1-2 and VC. Detailed Implementation

[0037] The present invention will be further described in conjunction with specific embodiments.

[0038] Example 1

[0039] The preparation method of compounds 1-2 from hawthorn leaves is as follows:

[0040] (1) Select 25 kg of hawthorn leaves and extract them three times with 70% (v:v) industrial ethanol by cold soaking and reflux, each time for 2 hours. The extract is then concentrated under reduced pressure to obtain a concentrated solution.

[0041] (2) The concentrate was eluted with a gradient of EtOH / H2O = 0:100, 25:75, 55:45, 95:5 (v:v), adsorbed by macroporous adsorption resin, and dried with 55% EtOH component to obtain a brownish-red solid powder.

[0042] (3) After dissolving and filtering the brownish-red extract, it was eluted by a gradient of CH2Cl2-MeOH = 5:1-0:1 (v:v) and rapidly separated into four crude fractions Fr.AD by reduced pressure polyamide chromatography;

[0043] (4) Fr.C was separated into five fractions (Fr.C1-C5) by gel column chromatography with a concentration of MeOH:H2O = 70:30 (v:v);

[0044] (5) Fr.C4 was eluted by a gradient of MeOH:H2O = 20:80-100:0 (v:v) and separated by reversed-phase ODS column chromatography to obtain six fractions Fr.4-1-Fr.4-6;

[0045] (6) Fr.4-2 was separated into four fractions, Fr.4-2-1-Fr.4-2-4, by gel column chromatography with a concentration of MeOH:H2O = 70:30 (v:v);

[0046] (7) Fr.4-2-2 was eluted by a CH2Cl2-MeOH gradient of 5:1-0:1 (v:v) and separated by silica gel H column chromatography to obtain compound 2; elution conditions: acetonitrile:water (20:80) (v:v), and 14.7 mg of compound 2 was obtained by HPLC.

[0047] (8) Fr.4-2-3 was eluted with a CH2Cl2-MeOH gradient of 5:1-0:1 (v:v) and separated by silica gel column chromatography to obtain compound 1; elution conditions: methanol:water (33:67) (v:v); 7.6 mg of compound 1 was obtained by HPLC.

[0048] The obtained compounds 1-2 were systematically structurally identified, and the results are as follows (see attached). Figure 1-6 ):

[0049] Compound 1: Yellow powder (MeOH). It turns purple with 10% vanillin sulfate reagent. HR-ESI-MS gives a value of 431.0933 [M+Na]. + (calcd.431.0949), its molecular formula is determined to be C 19 H 20 O 10 The calculated unsaturation degree is 10.

[0050] 1 The H-NMR (600MHz, DMSO-d6) spectrum shows four aromatic proton signals δ H 7.65 (1H, d, J = 8.4 Hz), 7.46 (1H, s), 6.97 (1H, d, J = 2.0 Hz), 6.77 (1H, dd, J = 8.4, 2.0 Hz); a set of sugar signals, sugar-terminal proton signal δ H 4.69 (1H,d,J=7.6Hz), the δ signal of the proton at position 6′ on the sugar. H 3.77 (1H, dd, J = 11.6, 1.5 Hz), 3.51 (1H, dd, J = 11.6, 6.2 Hz), chemical shift at δ H Four proton signals on sugars between 3.36 and 3.18.1 The H-NMR spectrum also showed a methoxy signal δ. H 3.99 (3H,s). 13 The C-NMR (150MHz, DMSO-d6) spectrum showed δ signals from 12 benzene ring carbons. C The carbon signals δ from the proton NMR spectrum are 156.9, 156.7, 143.9, 143.5, 139.2, 133.0, 120.3, 116.0, 115.0, 111.5, 102.5, and 98.3. Based on the signals and unsaturation calculations from the proton NMR spectrum, the compound is presumably a biphenyl, with one benzene ring substituted with ABX and the other with penta-substituted carbons. A set of β-glucose carbon signals δ... C 103.8, 77.4, 76.0, 73.5, 69.9, 60.8; Additionally, there is a methoxy carbon signal δ. C With a binding unsaturation of 10, it is speculated that the compound also has a cyclic structure.

[0051] In the HMBC spectrum, δ H 7.65(H-9) and δ C A correlation exists between 98.3 (C-6), 156.9 (C-7), and 115.0 (C-8), δ H 6.97(H-6) and δ C A correlation exists between 156.9 (C-7), 115.0 (C-8), and 116.0 (C-9a), δ H 6.77(H-8) and δ C A correlation exists between 156.7 (C-5a), 98.3 (C-6), and 116.0 (C-9a), determining the carbon signals and OH- linkage positions of the ABX-substituted benzene ring. δ H 7.46(H-1) and δ C A correlation exists between 143.5 (C-2), 139.2 (C-3), and 116.0 (C-9a), δ H 3.99(4-OCH3) and δ C A correlation exists at 133.0(C-4), δ H 4.69(H-1′) and δ C A correlation was found at 143.5 (C-2), which determined the carbon signal and substituent connection positions of the pentasubstituted benzene ring. The accurate assignment of the C and H signals is detailed in Table 1. Based on the above analysis, the planar structure of compound 1 was determined.

[0052] After a systematic literature search, it was identified as a novel compound that had not been previously reported. It was systematically named 2,3,7-trihydroxy-4-methox-ydibenzofuran 3-O-β-D-glucopyranoside, and commonly known as crataegusnoside G.

[0053] Table 1. 1H NMR (600MHz) and 1C NMR (150MHz) data for compound 1 (deuterated dimethyl sulfoxide). 1 H-NMR (600MHz) and 13 C-NMR(150MHz)data of compound 1(DMSO-d6).

[0054]

[0055] Compound 2: Colorless oil (MeOH), readily soluble in solvents such as methanol. It turns dark orange with 10% vanillin sulfate reagent. HR-ESI-MS gives a value of 395.1340 [M+H]. + (calcd.395.1336), its molecular formula is determined to be C 19 H 22 O9, the calculated degree of unsaturation is 9.

[0056] 1 The H-NMR (600MHz, methanol-d4) spectrum shows two sets of proton signals on the benzene ring, one of which is a proton signal δ H 7.08 (1H,d,J=1.8Hz), 6.94 (1H,d,J=1.8Hz), indicating a 1,3,4,5-tetrasubstituted benzene ring. Another set of proton signals δ H 7.26 (1H, dd, J = 7.9, 1.6 Hz), 7.10 (1H, td, J = 7.9, 1.6 Hz), 6.89 (1H, dd, J = 8.0, 1.4 Hz), 6.86 (1H, dd, J = 8.0, 1.4 Hz) indicate a 1,2-disubstituted benzene ring. A set of monosaccharide signals, with the sugar-terminal proton signal δ... H 4.84 (1H,d,J=7.7Hz), hydrogen signal δ at C-6′ position on sugar. H 3.72 (1H, dd, J = 12.0, 5.1 Hz), 3.86 (1H, dd, J = 12.0, 2.4 Hz), chemical shift in δ H Four proton signals on sugars between 3.38 and 3.53. 1 The H-NMR spectrum also showed an oxymethyl signal δ. H3.87 (3H,s). 13 The C-NMR (150MHz, methanol-d4) spectrum showed δ signals from 12 aromatic carbons. C 155.2, 149.2, 146.6, 136.6, 131.5, 131.2, 129.6, 129.1, 121.0, 117.0, 112.7, 109.7, a set of carbon signals δ for β-glucose. C 104.2, 78.2, 77.6, 74.9, 71.2, 62.3; Additionally, there is one methoxy carbon signal δ. C Based on the signals given in the 1H NMR spectrum, the compound is likely a biphenyl compound with two -OH groups, one sugar group and one methoxy group.

[0057] In the HMBC spectrum, δ H 7.08(H-2) and δ C A correlation exists between 146.6(C-3), 136.6(C-4), 109.7(C-6), and 129.6(C-1′), δ H 6.94(H-6) and δ C A correlation exists between 112.7(C-2), 136.6(C-4), 149.2(C-5), and 129.6(C-1′), δ H 3.87 and δ C A correlation exists between 149.2 (C-5) and the terminal proton signal δ of the sugar. H 4.84(H-1″) and δ C A correlation exists at 146.6 (C-3), which determined the carbon signals and substituent linkage positions of the 1,3,4,5-tetrasubstituted benzene ring. δ H 7.10(H-4′) and δ C A correlation exists between 155.2(C-2′), 117.0(C-3′), 121.0(C-5′), and 131.5(C-6′), δ. H 6.86(H-3′) and δ C Correlation was found at 129.6 (C-1′), 155.2 (C-2′), and 121.0 (C-5′), which determined the carbon signals and substituent positions of the 1,2-disubstituted benzene ring. The precise assignments of the C and H signals are detailed in Table 2. Based on the above analysis, the planar structure of compound 2 was determined.

[0058] After a systematic literature search, it was identified as a novel compound that had not been reported before. It was systematically named 2',4-Dihydroxy-5-methoxy[1,1'-biphenyl]-3-ylβ-D-glucopyranoside, and commonly known as crataegusnoside H.

[0059] Table 2 shows the 1H NMR (600MHz) and 1C NMR (150MHz) spectra of compound 2 (deuterated methanol). 1 H-NMR (600MHz) and 13 C-NMR(150MHz)data of compound 2(Methanol-d4).

[0060]

[0061]

[0062] Example 2

[0063] Screening of compounds 1-2 for DPPH and ABTS radical scavenging activities.

[0064] Materials and instruments: 96-well plate (Nest Biotech, USA), microplate reader (Thermo, USA), UV spectrophotometer (UV-2600i, Shimadzu, Japan), electronic balance (ML-104, China).

[0065] Reagents: DMSO (AR grade, China), Vitamin C (China), DPPH (Tokyo Chemical Industry, Japan), ABTS + (Tokyo Chemical Industry, Japan).

[0066] Experimental methods

[0067] Antioxidant activity experiments included the ability to scavenge DPPH free radicals and scavenge ABTS. + Free radical capacity has two parts.

[0068] I. Ability to scavenge DPPH free radicals

[0069] (1) Preparation of experimental reagents

[0070] Accurately weigh the compound into an EP tube and add a quantitative amount of DMSO to prepare a 10 mM sample solution.

[0071] 1‰ DMSO: Take 10 μL of DMSO into a 10 mL EP tube and add 9990 mL of pure water.

[0072] 100μM sample solution: Take 10mM sample solution and prepare 100μM sample solution with 1‰ DMSO.

[0073] DPPH solution: Accurately weigh 7.9 mg of DPPH powder into a 15 mL EP tube, add 10 mL of anhydrous ethanol to obtain a 2 mM DPPH stock solution. Take 1 mL of the stock solution into a 15 mL EP tube, add 9 mL of anhydrous ethanol to obtain a 0.2 mM DPPH solution, ready for use. Store at 4℃, prepare and use immediately.

[0074] (2) Experiment Content

[0075] Accurately transfer 100 μL of a 100 μM sample into a microplate, add 100 μL of 0.2 mM DPPH solution, and react in the dark for 30 min. Measure the absorbance at 517 nm (A1). Simultaneously, accurately transfer 100 μL of the sample and 100 μL of anhydrous ethanol to form the background group (A2). The blank control (A0) consists of 100 μL of 0.2 mM DPPH and 100 μL of anhydrous ethanol. Perform three parallel experiments for each concentration. The scavenging rate R (R = DPPH free radical scavenging rate, %) is calculated using the formula: R = [A0 - (A1 - A2)] / A0 × 100%.

[0076] Table 3 shows the amount of DPPH free radical scavenging testreagent added.

[0077]

[0078] II. Clear ABTS + Free radical ability

[0079] (1) Preparation of experimental reagents

[0080] Accurately weigh the compound into an EP tube and add a quantitative amount of DMSO to prepare a 10 mM sample solution.

[0081] 1‰ DMSO: Take 10 μL of DMSO into a 10 mL EP tube and add 9990 mL of pure water.

[0082] 100μM sample solution: Take 10mM sample solution and prepare 100μM sample solution with 1‰ DMSO.

[0083] Five sample solutions of different concentrations: Take 10 mM sample solution and add 1‰ DMSO to prepare sample solutions of 200 μM, 100 μM, 50 μM, 25 μM and 12.5 μM concentrations.

[0084] ABTS + Working solution: A 1:1 volume ratio mixture (7mM ABTS) prepared by dark treatment for 24 hours. +ABTS was prepared by diluting ABTS 40-50 times with 2.5 mM K2S2O8 to achieve an absorbance of 0.7 ± 0.02 at 734 nm. + Working fluid.

[0085] (2) Experiment Content

[0086] Accurately transfer 100 μL of samples with concentrations of 200 μM, 100 μM, 50 μM, 25 μM, and 12.5 μM into a microplate, and then add 100 μL of ABTS to each well. + The working solution was reacted in the dark for 30 min, and its absorbance was measured at 734 nm (A1). Simultaneously, 100 μL of the sample and 100 μL of anhydrous ethanol were accurately transferred to form the background sample group (A2). The blank control (A0) consisted of 100 μL of ABTS. + Working solution and 100 μL of anhydrous ethanol. Three parallel experiments were performed for each concentration. The clearance rate R (R = ABTS) was recorded. + The free radical scavenging rate (%) is calculated using the formula: R = [A0 - (A1 - A2)] / A0 × 100%.

[0087] Table 4 shows the amount of ABTS free radical scavenging testreagent added.

[0088]

[0089] III. Experimental Results and Discussion

[0090] Compounds 1-2 from hawthorn leaves described in this invention were subjected to preliminary screening for DPPH and ABTS free radical scavenging activities at a concentration of 100 μM. DPPH free radical scavenging activity screening: at a concentration of 100 μM, the antioxidant activities of the positive control VC, compound 1, and compound 2 were 50.3%, 36.6%, and 61.4%, respectively. ABTS free radical scavenging activity screening: at a concentration of 100 μM, the antioxidant activities of the positive control VC, compound 1, and compound 2 were 76.3%, 74.3%, and 83.8%, respectively. Figure 7 ).

[0091] The DPPH and ABTS free radical scavenging activities of compounds 1 and 2 were further tested at concentrations ranging from 12.5 to 200 μM. The DPPH free radical scavenging activity test results showed that compound 1 (IC50) exhibited strong free radical scavenging activity. 50 =149.3±0.65μM) and compound 2 (IC) 50 =54.9±0.95μM) exhibited good DPPH free radical scavenging activity, and its activity was stronger than that of the positive control drug VC (IC50).50 =157.5±0.90μM). ABTS free radical scavenging activity test results showed that biphenyl compound 1 (IC50) 50 =16.5±1.42μM) and compound 2 (IC) 50 =11.0±0.03μM) exhibited good ABTS free radical scavenging activity, and its activity was stronger than that of the positive control drug VC (IC50). 50 =17.1±1.05 μM). The compounds exhibit superior antioxidant activity; therefore, the biphenyl compounds described in this invention show promise for the preparation of clinical antioxidant drugs. Figure 7 ).

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that, The compound has the structural formula: 。 2. A process for the preparation of a compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The method comprises the following steps: (1) extracting hawthorn leaves by reflux extraction with 70% ethanol; (2) concentrating the extract under reduced pressure to obtain a concentrated solution; (3) adsorbing the concentrated solution on a D101 macroporous adsorption resin column and eluting with EtOH / H2O=0:100-95:5 gradient elution; (4) dissolving the extract and subjecting it to polyamide column chromatography under reduced pressure, and eluting with CH2Cl2-MeOH=5:1-0:1 gradient elution, so as to divide it into four crude fractions Fr. A-D; (5) separating Fr. C by gel column chromatography to obtain five parts Fr. C1-C5, and the elution condition is MeOH:H2O=70:30; (6) separating Fr. C4 by reverse-phase ODS column chromatography, and eluting with MeOH:H2O=20:80-100:0 gradient elution, so as to obtain six parts Fr. 4-1-Fr. 4-6; (7) separating Fr. 4-2 by gel column chromatography, and eluting with MeOH:H2O=70:30, so as to obtain four parts Fr. 4-2-1-Fr. 4-2-4; (8) separating Fr. 4-2-3 by silica gel column chromatography, and eluting with CH2Cl2-MeOH=5:1-0:1 gradient elution, and separating by HPLC, and the elution condition is 33% methanol and water, so as to obtain compound 1.

3. The preparation method according to claim 2, characterized in that, The volume fraction of ethanol in step (1) is 70%; the extraction times are 2-3 times; and the extraction time of each time is 2-3 hours.

4. A pharmaceutical composition, characterized by, Any one of the compounds or pharmaceutically acceptable salts thereof in claim 1.

5. Use of the compound in claim 1 or compound 2 or a pharmaceutically acceptable salt thereof in the preparation of an antioxidant drug. 。 6. Use of the pharmaceutical composition in claim 4 in the preparation of an antioxidant drug.

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