Psidium guajave acid derivative as well as preparation method and application thereof

Abstract

The invention discloses a psidium guajave acid derivative as well as a preparation method and application thereof. The preparation method comprises the following steps: crushing radix sophorae flavescentis root bark, heating and extracting with an ethanol solution, concentrating under reduced pressure to obtain a total extract, dispersing the total extract in water, filtering, eluting filtrate with 732 type cation exchange resin and distilled water to obtain a non-alkaloid part, and extracting with petroleum ether and ethyl acetate to obtain an ethyl acetate extraction part; carrying out silica gel column chromatography, polyamide column chromatography, ODS column chromatography, Sephadex LH-20 column chromatography and preparative HPLC (High Performance Liquid Chromatography) separation and purification on the ethyl acetate extraction part to obtain seven guava acid derivatives, wherein the compounds 1-6 are new compounds. According to the invention, alcohol-induced AML-12 cytotoxicity is adopted to evaluate the liver protection effect of monomeric compounds, and the compounds 1-7 are proved to have obvious inhibition effect on cytotoxicity. A rat alcoholic liver injury model is adopted to evaluate the in-vivo liver protection effect of the psidium guajave acid, it is proved that the psidium guajave acid has an obvious protection effect on alcohol-induced liver injury, and the new application of the psidium guajave acid in preparation of the liver protection medicine is proved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a guava acid derivative, its preparation method, and its application. Background Technology

[0002] Piscidic acid (PA) is derived from Cornus officinalis, a plant belonging to the genus Cornus of the legume family (Fabaceae). Piscidia erythrina Natural organic acids found in the dried root bark of *Linn.*. The main source plants include cacti (…). Opuntia Ficus-Indica L. Mill.), soapberry tree ( Quillaja saponaria Molina), Sophora flavescens ( Sophora flavescens Guava (Ait.) and other similar substances have main effects related to antioxidation and anti-inflammation. Studies have shown that guava acid has been proven to have antioxidant, anti-inflammatory, endocrine-regulating, and anti-diabetic effects.

[0003] Sophora flavescens is a plant belonging to the genus Sophora in the legume family (Fabaceae). S. flavescens The dried root of *Sophora flavescens* is produced throughout my country, both north and south. It has a bitter taste and cold nature, and enters the heart, liver, stomach, large intestine, and bladder meridians. It possesses the effects of clearing heat and drying dampness, killing parasites, and promoting diuresis. In traditional Chinese medicine, *Sophora flavescens* is commonly used to treat dysentery, hematochezia, jaundice, and urinary retention. For example, Compound *Sophora flavescens* Enteritis Tablets are used to treat damp-heat diarrhea or acute gastroenteritis, and Compound *Sophora flavescens* Injection is effective in treating liver cancer, breast cancer, and other cancers. Literature reports that the chemical components of *Sophora flavescens* mainly include alkaloids, flavonoids, and phenolic acids. Among them, the phenolic acids are mainly guava acid and its derivatives, exhibiting broad biological activity.

[0004] As the most metabolically active organ in the human body, the liver is the primary site of metabolism and detoxification. This crucial function makes it susceptible to damage from various factors, such as alcohol, chemical toxins, and pathogenic microorganisms, leading to alcoholic liver disease, liver fibrosis, and even hepatocellular carcinoma. Studies have shown that certain traditional Chinese medicine extracts possess unique advantages in treating liver diseases due to their antioxidant, anti-inflammatory, and hepatocyte membrane-stabilizing effects.

[0005] Ethanol is first oxidized to acetaldehyde by alcohol dehydrogenase within cells. Acetaldehyde is highly cytotoxic and can bind to intracellular proteins, nucleic acids, and other biomolecules, disrupting their structure and function and causing hepatocyte damage. Long-term or high-concentration ethanol stimulation induces upregulation of cytochrome P450 2E1 expression. This enzyme produces a large amount of reactive oxygen species (ROS) during ethanol metabolism, activating oxidative stress-related pathways (such as the MAPK and NF-κB pathways), inducing the release of inflammatory factors, and amplifying hepatocyte inflammatory damage. Ethanol and its metabolites interfere with the synthesis, transport, and breakdown of lipids within hepatocytes. Continuous oxidative stress and lipid accumulation activate hepatocyte apoptosis pathways. The ethanol-induced AML-12 cell (normal mouse hepatocyte line) damage model is a classic model for simulating in vitro studies of alcoholic liver disease (ALD). Its core principle is to induce hepatocyte damage, inflammation, and even apoptosis through the direct toxicity of ethanol and the oxidative stress and lipid metabolism disorders mediated by its metabolites, which highly matches the pathological mechanism of alcoholic liver disease in vivo.

[0006] The present invention relates to guava acid or its novel derivatives derived from Sophora flavescens (… Sophora flavescens Guavaic acid was extracted and isolated from the dried roots of *Ait.*, and its hepatoprotective effect was evaluated by measuring the cytotoxicity of ethanol-induced AML-12 mouse hepatocytes. The in vivo hepatoprotective effect of guavaic acid was verified using a rat alcoholic liver injury model. The applications of the hepatoprotective effects of compounds 1-6 and compounds 1-7 involved in this invention have not been reported. Summary of the Invention

[0007] The main objective of this invention is to provide a guava acid derivative, its preparation method, and its application. A novel guava acid derivative is extracted and isolated from the dried root of Sophora flavescens, and the hepatoprotective effect of guava acid and its derivative is verified, solving the problem of the lack of stable and efficient hepatoprotective drugs in the prior art.

[0008] To achieve the aforementioned objectives, the technical solution adopted by this invention includes: A method for preparing a guava acid derivative includes the following steps: S1: After the root bark of Sophora flavescens is crushed, it is extracted with ethanol solution by heating. The extract is concentrated under reduced pressure to obtain the total extract. The total extract is dispersed in water, the pH is adjusted to 4-5, filtered, and the filtrate is passed through a 732 type cation exchange resin and eluted with distilled water to obtain the non-alkaloid fraction. The non-alkaloid fraction is dispersed in water to obtain the non-alkaloid fraction aqueous solution, which is extracted with petroleum ether and ethyl acetate respectively. The extract is concentrated under reduced pressure to obtain the ethyl acetate extract. S2: The ethyl acetate extract was separated by silica gel column chromatography and eluted sequentially with a dichloromethane-methanol gradient of 100:0 to 0:100 (v / v). The eluents obtained were collected and concentrated under reduced pressure and named Fr.A to Fr.L. S3: Fr.G was eluted by a polyamide column with an ethanol-water gradient of 0:100 to 100:0 (v / v), and the eluents were collected in 7 fractions. The fractions were concentrated under reduced pressure and named Fr.G1 to Fr.G7. S4: Fr.G2 was eluted on an ODS column using a first methanol-water gradient with a volume ratio of 0:100 to 100:0, and nine eluent fractions were collected. These fractions were concentrated under reduced pressure and named Fr.G21 to Fr.G29, respectively. Fr.G23 was eluted on an ODS column using a second methanol-water gradient with a volume ratio of 30:70 to 100:0, and four eluent fractions were collected. These fractions were concentrated under reduced pressure and named Fr.G231 to Fr.G234, respectively. Fr.G231 was prepared by preparative HPLC to obtain compound 7. S5: Fr.G4 was eluted on an ODS column with a methanol-water gradient of 0:100 to 100:0 (v / v), and nine eluent fractions were collected. These fractions were concentrated under reduced pressure and named Fr.G41 to Fr.G49, respectively. Fr.G42 was prepared by preparative HPLC to obtain compound 1. S6: Fr.G43 was eluted with methanol on a Sephadex LH-20 column, concentrated under reduced pressure, and prepared by preparative HPLC to obtain compound 3; S7: Fr.G45 was prepared by preparative HPLC to obtain compound 2; S8: Fr.I was separated by silica gel column chromatography, eluted sequentially with a first dichloromethane-methanol gradient at a volume ratio of 100:0 to 0:100, and nine eluent fractions were collected. After concentration under reduced pressure, they were named Fr.I1 to Fr.I9. Fr.I7 was eluted by Sephadex LH-20 column with a second dichloromethane-methanol solution as the eluent, and nine eluent fractions were obtained, named Fr.I71 to Fr.I79. S9: Compound 4 was prepared by preparative HPLC from Fr.I76; S10:Fr.I78 was prepared by preparative HPLC to obtain compounds 5 and 6.

[0009] The ethanol solution mentioned in step S1 is a 70% ethanol solution by volume, and the amount used is 3 times the weight of the Sophora flavescens medicinal material. The extraction is carried out 5 times and the time is 1.5 h each time. The petroleum ether and ethyl acetate extractions are carried out 3 times each, and the amount used is equal to the volume of the non-alkaloid aqueous solution.

[0010] In step S2, the mass ratio of the ethyl acetate extract to silica gel is 1:3. The silica gel column is 200-300 mesh. The volume ratio of dichloromethane to methanol is 100:0, 100:1, 50:1, 30:1, 15:1, 8:1, 4:1, 1:1, 0:100. The column volume is denoted as BV. Each ratio elutes 10 BV.

[0011] The ethanol-water volume ratio in step S3 is 0:100, 10:90, 30:70, 50:50, 70:30, 95:5, 100:0, and the column volume is denoted as BV. Each ratio elutes 8 BV.

[0012] In step S4, the volume ratio of the first methanol-water solution is 0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 90:10, 100:0, and the column volume is denoted as BV. Each ratio elutes 8 BV. The volume ratio of the second methanol-water solution is 30:70 and 100:0. The preparative HPLC detection wavelength is 225 nm, the mobile phase is methanol-water with a volume ratio of 20:80, and the flow rate is 3 mL / min.

[0013] In step S5, the methanol-water solution volume ratio is 0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 90:10, 100:0, and the column volume is denoted as BV. Each ratio elutes 8 BV. The preparative HPLC in step S5 uses a detection wavelength of 225 nm, a mobile phase of methanol-water with a volume ratio of 40:60, and a flow rate of 3 mL / min. In step S6, methanol is eluted with 3 times the volume of Sephadex LH-20 column. The preparative HPLC in step S6 is performed with a detection wavelength of 225 nm, a mobile phase of methanol-water with a volume ratio of 35:65, and a flow rate of 3 mL / min. In step S7, the preparative HPLC is performed with a detection wavelength of 225 nm, a mobile phase of methanol-water with a volume ratio of 40:60, and a flow rate of 3 mL / min.

[0014] In step S8, the mass ratio of Fr.I to silica gel is 1:10, the silica gel column is 200-300 mesh, and the volume ratio of the first dichloromethane-methanol solution is 100:0, 50:1, 30:1, 15:1, 8:1, 4:1, 1:1, 0:100, with the column volume denoted as BV, and 8 BV is eluted for each ratio; the volume ratio of the second dichloromethane-methanol solution is 1:1, and 3 times the volume of the Sephadex LH-20 column is eluted; in step S9, the preparative HPLC detection wavelength is 225 nm, the mobile phase is methanol-water with a volume ratio of 45:55, and the flow rate is 3 mL / min; in step S10, the preparative HPLC detection wavelength is 225 nm, the mobile phase is methanol-water with a volume ratio of 45:55, and the flow rate is 3 mL / min.

[0015] A guava acid derivative, wherein the guava acid derivative is compound 1 to 6, and the specific structural formula is shown below: .

[0017] A pharmaceutical composition comprising at least one of the guava acid derivative compounds 1 to 7 and a pharmaceutically acceptable salt or a pharmaceutically acceptable carrier or excipient thereof.

[0018] The pharmaceutical composition is used in the preparation of hepatoprotective active drugs.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1) This invention is the first to isolate and purify six new compounds 1-6 from Sophora flavescens.

[0020] 2) This invention uses alcohol-induced AML-12 cytotoxicity to evaluate the hepatoprotective effect of monomeric compounds, and confirms that compounds 1-7 have significant inhibitory effects on cytotoxicity.

[0021] 3) This invention uses a rat alcoholic liver injury model to evaluate the in vivo hepatoprotective effect of guava acid, confirming that guava acid has a significant protective effect against alcohol-induced liver injury, and for the first time discovering and confirming its new use in developing hepatoprotective drugs. Attached Figure Description

[0022] Figure 1 The 1H NMR spectrum of compound 1 in Example 1 of this invention; Figure 2 The carbon NMR spectrum of compound 1 in Example 1 of this invention; Figure 3 This is the high-resolution mass spectrum of compound 1 in Example 1 of the present invention; Figure 4 The 1H NMR spectrum of compound 2 in Example 1 of this invention; Figure 5 The carbon NMR spectrum of compound 2 in Example 1 of this invention; Figure 6 This is the high-resolution mass spectrum of compound 2 in Example 1 of the present invention; Figure 7 The 1H NMR spectrum of compound 3 in Example 1 of this invention; Figure 8 The carbon NMR spectrum of compound 3 in Example 1 of this invention; Figure 9 This is the high-resolution mass spectrum of compound 3 in Example 1 of the present invention; Figure 10 The 1H NMR spectrum of compound 4 in Example 1 of this invention; Figure 11 The carbon NMR spectrum of compound 4 in Example 1 of this invention; Figure 12 This is the high-resolution mass spectrum of compound 4 in Example 1 of the present invention; Figure 13 The 1H NMR spectrum of compound 5 in Example 1 of this invention; Figure 14 The carbon NMR spectrum of compound 5 in Example 1 of this invention; Figure 15 This is the high-resolution mass spectrum of compound 5 in Example 1 of the present invention; Figure 16 The 1H NMR spectrum of compound 6 in Example 1 of this invention; Figure 17 The carbon NMR spectrum of compound 6 in Example 1 of this invention; Figure 18 This is the high-resolution mass spectrum of compound 6 in Example 1 of the present invention; Figure 19 The 1H NMR spectrum of compound 7 in Example 1 of this invention; Figure 20 The carbon NMR spectrum of compound 7 in Example 1 of this invention; Figure 21 This is the high-resolution mass spectrum of compound 7 in Example 1 of the present invention; Figure 22 The effects of compounds 1-7 in Example 2 of this invention on ethanol-induced AML-12 cytotoxicity; Figure 23 A represents the H&E staining results of guava acid on the liver of rats with alcoholic liver injury in Example 3 of this invention; Figure 23 B represents the effect on serum alanine aminotransferase (ALT); Figure 23 C represents the effect on serum aspartate aminotransferase (AST); Figure 23D represents the effect on serum superoxide dismutase (SOD); Figure 23 E represents the effect on serum total cholesterol (T-CHO). Detailed Implementation

[0023] 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.

[0024] 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.

[0025] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0026] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0027] Example 1: Method for separating and purifying guava acid derivatives from Sophora flavescens 1) Sophora flavescens ( Sophora flavescens 8.0 kg of dried root bark of *Ait.* was pulverized and extracted five times with 3 times its volume of 70% ethanol solution under reflux for 1.5 h each time. The extracts were combined, and the solvent was recovered under reduced pressure at 35 °C. The extract was then concentrated to obtain a total extract. The total extract was dispersed in water, and the pH was adjusted to 4-5 with 5% dilute hydrochloric acid. The mixture was filtered, and the filtrate was passed through a 732 type cation exchange resin and eluted with distilled water to obtain a non-alkaloid fraction (1190 g). This non-alkaloid fraction was then eluted with 5% hydrochloric acid to obtain an alkaloid fraction. The non-alkaloid fraction was dispersed in 2 L of water and extracted three times with equal volumes of petroleum ether and three times with equal volumes of ethyl acetate. The extracts were combined and concentrated under reduced pressure to obtain the ethyl acetate extract.

[0028] 2) The ethyl acetate extract (216 g) was mixed with 1.5 times the amount of silica gel (100-200 mesh), with a mass ratio of ethyl acetate extract to silica gel of 1:3. Separation was performed by silica gel column chromatography (200-300 mesh), with column volume recorded as BV. Elution was carried out sequentially using a dichloromethane-methanol gradient (100:0, 100:1, 50:1, 30:1, 15:1, 8:1, 4:1, 1:1, 0:100, V / V), with 10 BV eluted at each ratio. Thin-layer chromatography was performed on a GF254 silica gel chromatograph with petroleum ether:ethyl acetate = 3:1, dichloromethane:methanol = 50:1, 15:1, 8:1, 5:1, and 3:1, respectively. Vanillin-concentrated sulfuric acid was used for color development. Similar components were combined, and 12 fractions of eluent were collected. The resulting eluents were concentrated under reduced pressure and named Fr.A~Fr.L.

[0029] 3) Fr.G (72 g) was eluted through a polyamide column with an ethanol-water gradient (0:100, 10:90, 30:70, 50:50, 70:30, 95:5, 100:0, V / V). The column volume was recorded as BV. Each ratio was eluted by 8 BV. The eluents were collected into 7 fractions, concentrated under reduced pressure, and named Fr.G1 to Fr.G7 respectively.

[0030] 4) Fr.G2 (3 g) was eluted on an ODS column with a methanol-water gradient (0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 90:10, 100:0, V / V). The column volume was recorded as BV. Each ratio was eluted by 8 BV. The eluents were collected in 9 fractions, concentrated under reduced pressure, and named Fr.G21~Fr.G29 respectively.

[0031] 5) Fr.G23 (122 mg) was eluted by an ODS column with a methanol-water gradient (30:70, 100:0, V / V). The column volume was recorded as BV. Each fraction was eluted by 5 BV. The eluents were collected into four fractions, concentrated under reduced pressure, and named Fr.G231~Fr.G234 respectively.

[0032] 6) Fr.G231 (37 mg) was prepared by preparative HPLC with a detection wavelength of 225 nm. The compound 7 was obtained by elution with methanol-water (20:80, V / V, 3 mL / min). 7) Fr.G4 (3 g) was eluted on an ODS column with a methanol-water gradient (0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 90:10, 100:0, V / V). The column volume was recorded as BV. Each ratio was eluted by 8 BV. The eluents were collected in 9 fractions, concentrated under reduced pressure, and named Fr.G41 to Fr.G49.

[0033] 8) Fr.G42 (289 mg) was prepared by preparative HPLC with a detection wavelength of 225 nm. It was eluted with methanol-water (40:60, V / V, 3 mL / min) to separate compound 1. 9) Fr.G43 (119 mg) was eluted with methanol on a Sephadex LH-20 column. The column volume was recorded as BV. After elution of 3 BV, the eluent was concentrated under reduced pressure and then prepared by preparative HPLC. The detection wavelength was 225 nm. The elution was carried out with methanol-water (35:65, V / V, 3 mL / min) to separate compound 3.

[0034] 10) Fr.G45 (67 mg) was prepared by preparative HPLC with a detection wavelength of 225 nm. It was eluted with methanol-water (40:60, V / V, 3 mL / min) to separate compound 2. 11) Fr.I (89 g) was mixed with an equal volume of silica gel (100-200 mesh), with a mass ratio of Fr.I to silica gel of (1:10). Separation was performed by silica gel column chromatography (200-300 mesh), with the column volume recorded as BV. Elution was carried out sequentially using a dichloromethane-methanol gradient (100:0, 50:1, 30:1, 15:1, 8:1, 4:1, 1:1, 0:100, V / V), with 8 BV eluted at each ratio. The eluents were developed using silica gel thin-layer chromatography on a GF254 chromatograph with dichloromethane:methanol ratios of 15:1, 8:1, 5:1, and 3:1, respectively. Color development was performed using vanillin-concentrated sulfuric acid. Similar components were combined, and nine fractions of eluent were collected. These eluents were concentrated under reduced pressure and named Fr.I1~Fr.I9.

[0035] 12) Fr.I7 (8 g) was eluted on a Sephadex LH-20 column with dichloromethane-methanol (1:1, V / V) as the eluent for 3 BV to obtain 9 fractions, which were named Fr.I71 to Fr.I79 respectively.

[0036] 13) Fr.I76 (135 mg) was prepared by preparative HPLC with a detection wavelength of 225 nm. It was eluted with methanol-water (45:55, V / V, 3 mL / min) to separate compound 4. 14) Fr.I78 (118 mg) was prepared by preparative HPLC with a detection wavelength of 225 nm and eluted with methanol-water (45:55, V / V, 3 mL / min) to separate compounds 5 and 6.

[0037] The proton NMR spectrum, carbon NMR spectrum, and high-resolution mass spectra of compound 1 are as follows: Figure 1 , 2 As shown in Figures 1 and 3; the proton NMR spectrum, carbon NMR spectrum, and high-resolution mass spectra of compound 2 are shown in Figures 3 and 4 respectively. Figure 4 , 5 As shown in Figures 6; the proton NMR spectrum, carbon NMR spectrum, and high-resolution mass spectra of compound 3 are shown in Figures 7 and 8, respectively. Figure 7 , 8 As shown in Figures 9; the proton NMR spectrum, carbon NMR spectrum, and high-resolution mass spectra of compound 4 are shown in Figures 1 and 9, respectively. Figure 10 , 11 As shown in Figures 1 and 12; the proton NMR spectrum, carbon NMR spectrum, and high-resolution mass spectra of compound 5 are shown in Figures 1 and 12, respectively. Figure 13 , 14 As shown in Figures 1 and 15; the proton NMR spectrum, carbon NMR spectrum, and high-resolution mass spectra of compound 6 are shown in Figures 1 and 15, respectively. Figure 16 , 17 As shown in Figures 1 and 18; the proton NMR spectrum, carbon NMR spectrum, and high-resolution mass spectra of compound 7 are shown in Figures 1 and 18, respectively. Figure 19 , 20 As shown in Figure 21, the structures of compounds 1-7 were determined using physicochemical constants and modern spectroscopic techniques. Compounds 1-6 are novel compounds not previously reported in the literature. Their structural formulas are shown below:

[0038] Compound 1: Sophora acid A Yellow amorphous powder (methanol); +21.4 ( c 0.1,MeOH);UV (MeOH) λ max (log ε )202(4.50),225 (4.10),291 (4.31) nm;IR (KBr) ν max :3238, 1738, 1664, 1512, 1269,1217, 1101, 1022 cm 1 HRESIMS m / z 377.0878 [M H] (calcd forC 18 H17 O9,377.0873), the molecular formula of compound 1 was determined to be C 18 H 18 O9; 1 H (600 MHz, DMSO- d 6) and 13 C-NMR (150 MHz, DMSO- d 6) The data are shown in Table 1; the structural formula is shown in Formula I, which contains a benzene ring and a furan ring.

[0039] Compound 2: Sophora acid B Yellow amorphous powder (methanol); 25.5 ( c 0.1, MeOH); UV (MeOH) λ max (log ε ) 202(4.20), 226 (3.98), 290 (4.01) nm; IR (KBr) ν max : 3313, 1734, 1660, 1271, 1213,1105, 1018 cm 1 HRESIMS m / z 385.0896 [M + Na] + (calcd forC 18 H 18 O8Na, 385.0899), the molecular formula of compound 2 was determined to be C 18 H 18 O8; 1 H (600 MHz, DMSO- d 6) and 13 C-NMR (150 MHz, DMSO- d 6) The data are shown in Table 1; the structural formula is shown in Formula II, which contains a benzene ring and a furan ring.

[0040] Compound 3: Sophora acid C White amorphous powder (methanol); +68.6 ( c 0.1, MeOH); UV (MeOH) λ max (log ε) 201(4.21), 225 (3.98), 277 (4.08) nm; IR (KBr) ν max : 3413, 1738, 1672, 1510, 1234,1117, 1018 cm 1 HRESIMS m / z 401.0844 [M + Na] + (calcd forC 18 H 18 O8Na, 401.0849), the molecular formula of compound 3 was determined to be C 18 H 18 O8; 1 H (600 MHz, DMSO- d 6) and 13 C-NMR (150 MHz, DMSO- d 6) The data are shown in Table 1; the structural formula is shown in Formula III, which contains a benzene ring and a furan ring.

[0041] Compound 4: Sophora acid E White amorphous powder (methanol); 101.6 ( c 0.1, MeOH); UV (MeOH) λ max (log ε ) 201(4.63), 229 (4.42), 261 (4.40), 300 (3.95) nm; IR (KBr) ν max : 3330, 1734, 1624,1512, 1446, 1248, 1070 cm 1 HRESIMS m / z 669.1822 [M +H] + (calcd forC 33 H 33 O 15 (669.1819), the molecular formula of compound 4 was determined to be C 33 H 32 O 15 ; 1 H (600 MHz, DMSO- d 6) and 13 C-NMR (150MHz, DMSO-d 6) The data are shown in Table 2; the structural formula is shown in Formula IV, which contains a benzene ring and an isoflavone glycoside.

[0042] Compound 5: Sophora acid F White amorphous powder (methanol); 96.0 ( c 0.1, MeOH); UV (MeOH) λ max (log ε ) 203(4.83), 226 (4.24), 280 (3.64), 312 (3.80) nm; IR (KBr) ν max : 3388, 1736, 1616,1510, 1473, 1028 cm 1 HRESIMS m / z 685.1766 [M +H] + (calcd forC 33 H 33 O 16 (685.1769), the molecular formula of compound 5 was determined to be C 33 H 32 O 16 ; 1 H (600 MHz, DMSO- d 6) and 13 C-NMR (150MHz, DMSO- d 6), the data are shown in Table 2; the structural formula is shown in Formula V, which contains a benzene ring and an isoflavone glycoside.

[0043] Compound 6: Sophora acid G White amorphous powder (methanol); 100.0 ( c 0.1, MeOH); UV (MeOH) λ max (log ε ) 203(4.92), 226 (4.27), 283 (3.60), 312 (3.80) nm; IR (KBr) ν max : 3330, 1741, 1620,1504, 1475, 1030 cm 1HRESIMS m / z 699.1921 [M +H] + (calcd forC 34 H 35 O 16 (699.1925), the molecular formula of compound 6 was determined to be C 34 H 34 O 16 ; 1 H (600 MHz, DMSO- d 6) and 13 C-NMR (150MHz, DMSO- d 6), the data are shown in Table 2; the structural formula is shown in Formula VI, which contains a benzene ring and an isoflavone glycoside.

[0044] Compound 7: Guava acid Pale yellow needle-like crystals (methanol); +39.5 ( c 0.1, MeOH); HRESIMS m / z 279.0477 [M + Na] + (calcd forC 11 H 12 O7Na,279.0481), the molecular formula of compound 7 was determined to be C 11 H 12 O7; 1 H (600 MHz, CD3OD) and 13 C-NMR (150 MHz, CD3OD), data are shown in Table 3; the structural formula is shown in Formula VII, which contains a benzene ring.

[0045] Table 1. 1H and 1C NMR spectra of compounds 1-3 ( δ (in ppm)

[0046] a Recorded in DMSO- d 6. b Recorded on CD3OD. Table 2. 1H and 1C NMR spectra of compounds 4–6 ( δ in ppm, DMSO- d 6)

[0047] Table 3. 1H and 1C NMR spectra of compound 7 ( δ (in ppm, CD3OD)

[0048] Example 2: Hepatoprotective activity of compounds 1-7 against ethanol-induced AML-12 cells Mouse AML-12 normal hepatocytes were cultured in DMEM / F12 medium (Wuhan Pronosai Life Sciences Co., Ltd.) and incubated at 37°C in a 5% CO2 cell culture incubator. AML-12 cells in the logarithmic growth phase were seeded into 96-well plates (1×10⁻⁶ cells / well). 4 The samples were cultured at 37°C and 5% CO2 in wells. Once approximately 70% confluence was reached, they were divided into a control group, a model group, and a drug-treated group. Except for the control group, ethanol was added to both the model and drug-treated groups to achieve a final ethanol concentration of 7%. After incubation at 37°C for 4 hours, the culture medium was discarded from each group, and fresh culture medium was added. In the drug-treated group, the test drug (compounds 1-7) and the positive control drug (silymarin) were dissolved in DMSO and added to the 96-well plates to achieve a final drug concentration of 10%. μ After incubating at 37°C for 24 hours, cell viability was determined using the MTT assay.

[0049] Experimental results are as follows Figure 22 As shown, the results indicate that compounds 1-7 of this invention have the activity of inhibiting ethanol-induced AML-12 hepatocyte damage, and have great potential for use as hepatoprotective drugs.

[0050] Compositions containing the compounds described in this invention can be prepared into hepatoprotective drugs suitable for oral or injectable applications using conventional preparation methods, for example, in tablet, capsule, injection, or powder form.

[0051] Example 3: Hepatoprotective effect of compound 7 on rats with alcoholic liver injury Thirty 8-week-old SD rats (180 ± 20 g) were housed in an environment with a temperature of 23 ± 2 °C, humidity of 50 ± 5%, and a 12-hour diurnal cycle. After 7 days of acclimatization, they were randomly divided into 6 groups (control group, model group, low-dose group, medium-dose group, high-dose group, and positive control group), with 5 rats in each group. Except for the control group, rats were administered 56% ethanol (7 mL / kg) by gavage daily. Two hours after each gavage, they were given physiological saline. The low-dose, medium-dose, and high-dose groups were given compound 7 at 30 mg / kg, 60 mg / kg, and 120 mg / kg, respectively. The positive control group was given silymarin at 50 mg / kg. After 7 days of gavage administration and 24 hours of fasting, blood was collected from the abdominal aorta and liver was harvested on the eighth day. Pathological changes in liver tissue were observed using H&E staining, and serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), superoxide dismutase (SOD), and total cholesterol (T-CHO) levels were detected by ELISA.

[0052] Experimental results are as follows Figure 23 As shown, the results indicate that compound 7 of the present invention can improve alcoholic liver injury in rats, reduce AST, ALT, and T-CHO levels, and increase SOD levels, and can be used to prepare hepatoprotective active drugs.

[0053] 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 method for preparing a guava acid derivative, characterized in that, Includes the following steps: S1: After the root bark of Sophora flavescens is crushed, it is extracted with ethanol solution by heating. The extract is concentrated under reduced pressure to obtain the total extract. The total extract is dispersed in water, the pH is adjusted to 4-5, filtered, and the filtrate is passed through a 732 type cation exchange resin and eluted with distilled water to obtain the non-alkaloid fraction. The non-alkaloid fraction is dispersed in water to obtain the non-alkaloid fraction aqueous solution, which is extracted with petroleum ether and ethyl acetate respectively. The extract is concentrated under reduced pressure to obtain the ethyl acetate extract. S2: The ethyl acetate extract was separated by silica gel column chromatography and eluted sequentially with a dichloromethane-methanol gradient of 100:0 to 0:100 (v / v). The eluents obtained were collected and concentrated under reduced pressure and named Fr.A to Fr.L. S3: Fr.G was eluted by a polyamide column with an ethanol-water gradient of 0:100 to 100:0 (v / v), and the eluents were collected in 7 fractions. The fractions were concentrated under reduced pressure and named Fr.G1 to Fr.G7. S4: Fr.G2 was eluted on an ODS column using a first methanol-water gradient with a volume ratio of 0:100 to 100:0, and nine eluent fractions were collected. These fractions were concentrated under reduced pressure and named Fr.G21 to Fr.G29, respectively. Fr.G23 was eluted on an ODS column using a second methanol-water gradient with a volume ratio of 30:70 to 100:0, and four eluent fractions were collected. These fractions were concentrated under reduced pressure and named Fr.G231 to Fr.G234, respectively. Fr.G231 was prepared by preparative HPLC to obtain compound 7. S5: Fr.G4 was eluted on an ODS column with a methanol-water gradient of 0:100 to 100:0 (v / v), and nine eluent fractions were collected. These fractions were concentrated under reduced pressure and named Fr.G41 to Fr.G49, respectively. Fr.G42 was prepared by preparative HPLC to obtain compound 1. S6: Fr.G43 was eluted with methanol on a Sephadex LH-20 column, concentrated under reduced pressure, and prepared by preparative HPLC to obtain compound 3; S7: Fr.G45 was prepared by preparative HPLC to obtain compound 2; S8: Fr.I was separated by silica gel column chromatography, and eluted sequentially with a first dichloromethane-methanol gradient at a volume ratio of 100:0 to 0:

100. Nine eluents were collected, concentrated under reduced pressure, and named Fr.I1 to Fr.I9. Fr.I7 was eluted by Sephadex LH-20 column with a second dichloromethane-methanol solution as the eluent, and nine eluents were obtained, named Fr.I71 to Fr.I79. S9: Compound 4 was prepared by preparative HPLC from Fr.I76; S10:Fr.I78 was prepared by preparative HPLC to obtain compounds 5 and 6.

2. The method for preparing the guava acid derivative according to claim 1, characterized in that, The ethanol solution mentioned in step S1 is a 70% ethanol solution by volume, and the amount used is 3 times the weight of the Sophora flavescens medicinal material. The extraction is carried out 5 times and the time is 1.5 hours each time. The petroleum ether and ethyl acetate extractions are carried out 3 times each, and the amount used is equal to the volume of the non-alkaloid aqueous solution.

3. The method for preparing the guava acid derivative according to claim 1, characterized in that, In step S2, the mass ratio of the ethyl acetate extract to silica gel is 1:

3. The silica gel column is 200-300 mesh. The volume ratio of dichloromethane to methanol is 100:0, 100:1, 50:1, 30:1, 15:1, 8:1, 4:1, 1:1, 0:

100. The column volume is denoted as BV. Each ratio elutes 10 BV.

4. The method for preparing the guava acid derivative according to claim 1, characterized in that, The ethanol-water volume ratio in step S3 is 0:100, 10:90, 30:70, 50:50, 70:30, 95:5, 100:0, and the column volume is denoted as BV. Each ratio elutes 8 BV.

5. The method for preparing the guava acid derivative according to claim 1, characterized in that, In step S4, the volume ratio of the first methanol-water solution is 0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 90:10, 100:0, and the column volume is denoted as BV. Each ratio elutes 8 BV. The volume ratio of the second methanol-water solution is 30:70 and 100:

0. The preparative HPLC detection wavelength is 225 nm, the mobile phase is methanol-water with a volume ratio of 20:80, and the flow rate is 3 mL / min.

6. The method for preparing the guava acid derivative according to claim 1, characterized in that, In step S5, the methanol-water solution volume ratio is 0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 90:10, 100:0, and the column volume is denoted as BV. Each ratio elutes 8 BV. In step S5, the preparative HPLC detection wavelength is 225 nm, the mobile phase is methanol-water with a volume ratio of 40:60, and the flow rate is 3 mL / min. In step S6, methanol is eluted with 3 times the Sephadex LH-20 column volume. In step S6, the preparative HPLC is performed with a detection wavelength of 225 nm, the mobile phase is methanol-water with a volume ratio of 35:65, and the flow rate is 3 mL / min. In step S7, the preparative HPLC is performed with a detection wavelength of 225 nm, the mobile phase is methanol-water with a volume ratio of 40:60, and the flow rate is 3 mL / min.

7. The method for preparing the guava acid derivative according to claim 1, characterized in that, In step S8, the mass ratio of Fr.I to silica gel is 1:10, the silica gel column is 200-300 mesh, and the volume ratio of the first dichloromethane-methanol solution is 100:0, 50:1, 30:1, 15:1, 8:1, 4:1, 1:1, 0:100, with the column volume denoted as BV, and 8 BV is eluted for each ratio; the volume ratio of the second dichloromethane-methanol solution is 1:1, and 3 times the volume of the Sephadex LH-20 column is eluted; in step S9, the preparative HPLC detection wavelength is 225 nm, the mobile phase is methanol-water with a volume ratio of 45:55, and the flow rate is 3 mL / min; in step S10, the preparative HPLC detection wavelength is 225 nm, the mobile phase is methanol-water with a volume ratio of 45:55, and the flow rate is 3 mL / min.

8. A guava acid derivative, characterized in that, The guava acid derivatives are compounds 1-6, and their specific structural formulas are shown below: 。 9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises at least one of the guava acid derivative compounds 1 to 7 of claim 1, and a pharmaceutically acceptable salt or a pharmaceutically acceptable carrier or excipient thereof.

10. The use of the pharmaceutical composition of claim 9 in the preparation of a hepatoprotective active pharmaceutical ingredient.

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