An antiviral composition containing extract of caesalpinia sappan leaf, and preparation method and application thereof

Abstract

The application provides an anti-virus composition containing Caesalpinia minssima leaf extract and a preparation method and application thereof. The composition contains the Caesalpinia minssima leaf extract, the Sophora japonica extract and a pharmaceutically acceptable carrier; the weight ratio of the Caesalpinia minssima leaf extract to the Sophora japonica extract is 1-20:1. The application improves the solubility and bioavailability of caesalpinin by using flavonoid glycosides in the Caesalpinia minssima leaf extract, realizes quality standardization by using the Sophora japonica extract to supplement caesalpinin, and synergistically enhances the anti-HCV activity of the two extracts. The anti-virus composition has significantly better anti-HCV activity than caesalpinin monomer, and maintains excellent inhibitory activity on DAAs-resistant HCV strains, and can be used for preparing an anti-hepatitis C virus drug.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an antiviral composition containing pigeon pea leaf extract, its preparation method, and its application. Background Technology

[0002] Pigeon pea (also known as genus *Pistacia*) is an erect, low-growing shrub belonging to the legume family (Fabaceae). It grows to a height of 1-3 meters and is currently cultivated on approximately 5.2 million hectares worldwide, primarily in Asia, Africa, and South America. It is also used in traditional medicine. Pigeon pea is primarily used for its cooling, heat-clearing, detoxifying, analgesic, hemostatic, antibacterial, and anti-inflammatory properties. Modern research indicates that pigeon pea leaves contain various stilbene compounds, including lignans, lignan A, and lignan C, as well as flavonoids such as pinocembrin, vitexin, and isovitexin. Lignan, also known as lignanstilbene acid, belongs to the stilbene class of compounds.

[0003] Sophora japonica fruit, the dried, ripe fruit of the Sophora japonica plant (family Fabaceae), was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and is known for its effects of clearing heat and purging fire, cooling the blood and stopping bleeding. Recent research has discovered that the ethyl acetate fraction of Sophora japonica fruit contains lignans, marking the first time lignans has been isolated from a plant in the *Sophora* genus.

[0004] Hepatitis C virus (HCV) causing acute and chronic hepatitis C has become a serious global threat to human health. Past treatment regimens relying on ribavirin and interferon had drawbacks such as long treatment cycles, significant side effects, and low sustained viral response rates. With the approval and marketing of direct-acting anti-HCV drugs (DAAs), the treatment of hepatitis C has undergone a revolutionary improvement. However, clinical treatment still faces many challenges, such as viral resistance, treatment of genotype 3 patients, and high treatment costs.

[0005] Professor Li Zhuorong's research group at the Institute of Pharmaceutical Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, discovered that fibrocin has strong inhibitory activity against HCV virus replication. However, the following technical problems still exist in the existing technology: 1) The water solubility of fibrocin monomer is extremely poor (AlogP=5.0), and its oral bioavailability is low, making it difficult to develop into an ideal drug formulation on its own; 2) The content of active ingredients in existing pigeon pea leaf extracts is unstable, affected by factors such as the origin of raw materials and the harvesting season, resulting in large batch-to-batch differences, making it difficult to achieve standardized quality control; 3) Existing DAA drugs have decreased efficacy against drug-resistant HCV strains, and there is an urgent need for anti-HCV drugs with new mechanisms of action; 4) The fibrocin structural analogs protected by existing patents CN103172512B and CN105030750B are artificially synthesized compounds, with complex preparation processes and high costs, and are fundamentally different from natural pigeon pea leaf extracts. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an antiviral composition containing pigeon pea leaf extract, its preparation method, and its application. This invention involves compounding pigeon pea leaf extract and sophora japonica extract in a specific ratio. The flavonoid glycosides in the pigeon pea leaf extract improve the solubility and bioavailability of lignans, while the sophora japonica extract supplements lignans. The two extracts synergistically enhance anti-HCV activity, thereby obtaining a pharmaceutical composition with excellent anti-HCV activity, effectiveness against DAA-resistant strains, and controllable quality.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: An antiviral composition containing pigeon pea leaf extract, comprising pigeon pea leaf extract, sophora japonica extract and a pharmaceutically acceptable carrier; wherein the weight ratio of pigeon pea leaf extract to sophora japonica extract is 1-20:1.

[0008] Preferably, the content of lignan in the Sophora japonica extract is not less than 2.0%; the lignan is 3-hydroxy-4-isopentenyl-5-methoxystilbene-2-carboxylic acid, with the molecular formula C2. 21 H 22 O4 has a molecular weight of 338.4.

[0009] Preferably, the weight ratio of the pigeon pea leaf extract to the sophora japonica fruit extract is 10:1.

[0010] Preferably, the antiviral composition comprises the following raw materials in parts by weight: 5-40 parts of pigeon pea leaf extract, 1-20 parts of sophora japonica fruit extract, and 40-94 parts of a pharmaceutically acceptable carrier.

[0011] Preferably, the pigeon pea leaf extract is rich in vitexin, isovitexin and sennain.

[0012] Preferably, the dosage form of the pharmaceutical composition is a tablet, capsule, granule, oral liquid, or injection.

[0013] The present invention also provides a method for preparing the above-mentioned antiviral composition containing pigeon pea leaf extract, comprising the following steps: S1. Preparation of Pigeon Bean Leaf Extract Pigeon pea leaves were dried, pulverized, and passed through a 40-60 mesh sieve to obtain pigeon pea leaf powder. The pigeon pea leaf powder was then mixed with 60%–80% ethanol (volume concentration) at a material-to-liquid ratio of 1:15–25 and extracted at 40–55°C for 1–3 hours. The mixture was filtered, and the filtrate was collected. The filtrate was concentrated under reduced pressure until no alcohol odor remained, yielding a crude extract. The crude extract was enriched using a macroporous adsorption resin and eluted sequentially with water, 30% ethanol, 60% ethanol, and 80% ethanol. The 60%–80% ethanol eluent was collected. The collected eluent was dried to obtain the pigeon pea leaf extract. S2. Preparation of Sophora japonica extract The Sophora japonica fruit is dried, pulverized, and passed through a 40-60 mesh sieve to obtain Sophora japonica fruit powder. The Sophora japonica fruit powder is taken and ethanol with a volume concentration of 60%-80% is added. The mixture is refluxed at 60-80℃ for 2-4 hours, filtered, and the filtrate is collected. The filtrate is concentrated under reduced pressure until there is no alcohol odor to obtain a crude extract. The crude extract is dispersed in water and extracted with ethyl acetate. The ethyl acetate fraction is collected. The ethyl acetate fraction is dried to obtain Sophora japonica fruit extract. S3. Preparation of antiviral compositions The pigeon pea leaf extract obtained in step S1 and the sophora japonica fruit extract obtained in step S2 are mixed with a pharmaceutically acceptable carrier to prepare a formulation.

[0014] Preferably, the extraction method in step S1 is ultrasonic-assisted extraction or reflux extraction; the macroporous adsorption resin is type D101 or type AB-8.

[0015] Preferably, the mass ratio of ethanol to Sophora japonica powder in step S2 is 6-10:1.

[0016] The present invention also provides the use of the above-mentioned antiviral composition in the preparation of an anti-hepatitis C virus drug.

[0017] Preferably, the hepatitis C virus includes viral strains that have developed resistance to direct-acting antiviral drugs.

[0018] Fibromin has a strong inhibitory activity against HCV virus replication (EC). 50 =3.17 μM). Brucea oleiferin significantly reduced the level of chondroitin sulfate N-acetylgalactosyltransferase 1 (CSGalNAcT-1) protein in host cells. Brucea oleiferin did not affect the expression of the CSGalNAcT-1 gene, indicating that brucea oleiferin inhibits HCV replication by accelerating the degradation of the CSGalNAcT-1 protein. Since the target of brucea oleiferin is the host cell protein CSGalNAcT-1, rather than the viral protein, the virus is unlikely to evade the drug's action through gene mutation. Therefore, brucea oleiferin still has inhibitory activity against HCV strains that have developed resistance to DAAs. In addition to lignans, pigeon pea leaf extract contains various stilbene components such as lignan A and lignan C, as well as flavonoids such as vitexin, isovitexin, and fenestrant. Among them, the flavonoid glycosides (such as isovitexin and vitexin) have hydrophilic groups, which can form intermolecular hydrogen bonds or micellar-like structures with the fat-soluble lignans, increasing the dispersibility and solubility of lignans in aqueous media and improving its oral bioavailability.

[0019] Sophora japonica fruit extract contains fibroin, which can serve as a supplementary source of exogenous fibroin, compensating for the insufficient fibroin content in fibroin leaf extract due to batch variations in raw materials. At the same time, Sophora japonica fruit extract also contains various isoflavones such as chickpea sprout A and red clover extract. These components work synergistically at different stages of the HCV life cycle through multiple targets, enhancing the overall antiviral effect.

[0020] Through the above technical solution, the combination of pigeon pea leaf extract and sophora japonica fruit extract of the present invention improves the solubility and bioavailability of fibroin by the flavonoid glycosides in pigeon pea leaf extract, enabling more fibroin to reach target cells to exert antiviral effects; the sophora japonica fruit extract supplements exogenous fibroin, ensuring the stability of the total fibroin content in the composition; multiple active ingredients in the two extracts act on different stages or different targets of HCV life cycle with fibroin, forming multi-target synergistic inhibition; the synergistic effect of multiple components makes it more difficult for the virus to develop drug resistance through single-target mutation, further enhancing the ability of the composition to resist drug-resistant viral strains.

[0021] The beneficial effects of this invention are: This invention combines pigeon pea leaf extract with sophora japonica fruit extract. The flavonoids and various stilbene components in pigeon pea leaf extract work synergistically with the pachycarpus tinctoria and other isoflavones in sophora japonica fruit extract, resulting in significantly better anti-HCV activity than when an equal amount of pachycarpus tinctoria monomer is used alone.

[0022] The naturally occurring flavonoid glycosides (such as isovitexin) in pigeon pea leaf extract have hydrophilic groups that can form intermolecular hydrogen bonds or micellar-like structures with pigeon pea extract, increasing the dispersibility and solubility of pigeon pea extract in aqueous media and improving its oral bioavailability.

[0023] By supplementing exogenous fibroin with Sophora japonica extract, the total content of fibroin in the antiviral composition can be controlled within the target range, solving the problem of large batch-to-batch differences in natural extracts and achieving standardization and repeatability of product quality.

[0024] Fibromin inhibits HCV replication by downregulating the level of CSGalNAcT-1 protein in host cells. Its target is the host cell, which is completely different from the mechanism of action of DAAs. Therefore, the antiviral composition of the present invention still maintains inhibitory activity against HCV strains that have developed resistance to DAAs and can effectively inhibit the replication of DAA-resistant HCV strains. Attached Figure Description

[0025] Figure 1 This is the chemical structural formula of fibroin. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] Pharmaceutically acceptable carriers include, but are not limited to, diluents, excipients, fillers, binders, disintegrants, lubricants, solvents, encapsulating materials, and other pharmaceutical excipients known in the art.

[0028] Preparation Example 1: Preparation of Pigeon Bean Leaf Extract 10 kg of dried pigeon pea leaves were crushed and passed through a 40-mesh sieve to obtain pigeon pea leaf powder. 70% ethanol (volume concentration) was added at a material-to-liquid ratio of 1:20, and extraction was performed at 50℃ with ultrasonic assistance for 2 hours. The mixture was filtered, and the filtrate was collected. The filtrate was concentrated under reduced pressure until no alcohol odor remained, yielding a crude extract. The crude extract was enriched using a D101 macroporous adsorption resin, followed by gradient elution with water, 30% ethanol (volume concentration), 60% ethanol, and 80% ethanol. The 60%–80% ethanol eluent was collected. The collected eluent was dried under reduced pressure to obtain approximately 0.85 kg of refined pigeon pea leaf extract.

[0029] Preparation Example 2: Preparation of Sophora japonica fruit extract 10 kg of dried Sophora japonica fruit was crushed and passed through a 40-mesh sieve to obtain Sophora japonica fruit powder. 70% ethanol (volume concentration) was added, with an ethanol-to-Sophora japonica fruit powder mass ratio of 8:1. The mixture was refluxed three times at 70℃, 3.5 hours each time. The extracts were filtered and combined. The filtrates were concentrated under reduced pressure until no alcohol odor remained, yielding a crude extract. The crude extract was dispersed in water and extracted three times with ethyl acetate. The ethyl acetate fractions were combined, the solvent was recovered under reduced pressure, and the extract was dried to obtain approximately 0.6 kg of Sophora japonica fruit extract. HPLC analysis showed that the lignan content in the obtained Sophora japonica fruit extract was 2.5%.

[0030] Example 1 A method for preparing an antiviral composition containing pigeon pea leaf extract, comprising the following steps: 100g of pigeon pea leaf extract and 10g of sophora japonica fruit extract (the weight ratio of pigeon pea leaf extract to sophora japonica fruit extract is 10:1) are mixed evenly with 110g of pharmaceutically acceptable carriers such as starch and microcrystalline cellulose. The mixture is then granulated, compressed, and made into 1000 tablets, each containing 100mg of pigeon pea leaf extract and 10mg of sophora japonica fruit extract.

[0031] Example 2 A method for preparing an antiviral composition containing pigeon pea leaf extract, comprising the following steps: 100g of pigeon pea leaf extract and 20g of sophora japonica fruit extract (the weight ratio of pigeon pea leaf extract to sophora japonica fruit extract is 5:1) are mixed evenly with 110g of pharmaceutically acceptable carriers such as starch and microcrystalline cellulose, and then filled into capsules to make 1000 capsules, each containing 100mg of pigeon pea leaf extract and 20mg of sophora japonica fruit extract.

[0032] Example 3 A method for preparing an antiviral composition containing pigeon pea leaf extract, comprising the following steps: 100g of pigeon pea leaf extract and 5g of sophora japonica fruit extract (the weight ratio of pigeon pea leaf extract to sophora japonica fruit extract is 20:1) are mixed evenly with 110g of pharmaceutically acceptable carriers such as starch and microcrystalline cellulose, granulated, and made into granules. Each bag contains 100mg of pigeon pea leaf extract and 5mg of sophora japonica fruit extract.

[0033] Comparative Example 1 A method for preparing an antiviral composition includes the following steps: 10g of pure genistein (purity ≥98%) is mixed evenly with 110g of pharmaceutically acceptable carriers such as starch and microcrystalline cellulose to form tablets, each containing 10mg of genistein.

[0034] Comparative Example 2 A method for preparing an antiviral composition containing pigeon pea leaf extract, comprising the following steps: Take 100g of pigeon pea leaf extract and mix it evenly with 110g of pharmaceutically acceptable carriers such as starch and microcrystalline cellulose to make tablets, each containing 100mg of pigeon pea leaf extract.

[0035] Comparative Example 3 A method for preparing an antiviral composition containing pigeon pea leaf extract, comprising the following steps: Take 10 kg of dried pigeon pea leaves, pulverize them through a 40-mesh sieve, add 70% ethanol (volume concentration) at a material-to-liquid ratio of 1:20, and extract with ultrasonic assistance at 50℃ for 2 hours. Filter the extract, concentrate the filtrate to dryness under reduced pressure to obtain crude pigeon pea leaf extract. Take 100 g of this crude extract, mix it evenly with 110 g of pharmaceutically acceptable carriers such as starch and microcrystalline cellulose, and prepare tablets.

[0036] Comparative Example 4 A method for preparing an antiviral composition includes the following steps: Take 10g of Sophora japonica extract and mix it evenly with 110g of pharmaceutically acceptable carriers such as starch and microcrystalline cellulose to make tablets, each containing 10mg of Sophora japonica extract.

[0037] Effect Example 1. In vitro anti-HCV activity assay Cells: Huh7.5 cells Virus: HCV JFH1 (genotype 2a) and DAA-resistant strain (NS5A resistant mutant) Test drug: Antiviral compositions prepared in Examples 1-3 and Comparative Examples 1-4 Positive control: Sofosbuvir Huh7.5 cells were seeded in 96-well plates and cultured for 24 hours. HCV JFH1 virus solution was then added for infection for 2 hours. The virus solution was discarded, and different concentrations of the test drugs were added, followed by further culturing for 72 hours. Intracellular HCV RNA levels were detected using real-time quantitative PCR, and the half-maximal effective concentration (MCP) of each drug was calculated. 50 Simultaneously, cell controls and virus controls were set up.

[0038] Table 1

[0039] As shown in Table 1, the anti-HCV activity of the compositions in Examples 1-3 of this invention is superior to that of the pigeon pea extract monomer used alone in Comparative Example 1, the pigeon pea leaf extract used alone in Comparative Example 2, and the Sophora japonica extract used alone in Comparative Example 4. Among them, the pigeon pea leaf extract to Sophora japonica extract in Example 1, with a weight ratio of 10:1, exhibits the best anti-HCV activity; the crude pigeon pea leaf extract in Comparative Example 3, which was not purified by macroporous resin, has the worst activity, indicating that the purification and extraction process of this invention is crucial for maintaining and enhancing antiviral activity.

[0040] 2. Inhibitory activity experiment against DAA-resistant HCV strains Cells: Huh7.5 cells Virus: HCV NS5A drug-resistant mutant strain (carrying L31V or Y93H mutation) Test drugs: Example 1, Comparative Example 1 Positive control: Sofosbuvir Huh7.5 cells were seeded in 96-well plates and cultured for 24 hours. HCV NS5A virus solution was then added for infection for 2 hours. The virus solution was discarded, and different concentrations of the test drugs were added, followed by further culturing for 72 hours. Intracellular HCV RNA levels were detected using real-time quantitative PCR, and the half-maximal effective concentration (MCP) of each drug was calculated. 50 ).

[0041] Table 2

[0042] Table 2 shows that the EC50 of ligustin monomer against NS5A resistant mutants... 50Comparable to wild-type strains, this indicates that ligustin remains effective against DAA-resistant HCV strains. The composition of Example 1 of this invention also maintains excellent inhibitory activity against resistant mutants, significantly superior to ligustin monomer, while the positive control sofosbuvir showed a significant decrease in activity against resistant mutants.

[0043] 3. Ligustrazine dissolution test Examples 1, 1, 2, and 4 were taken respectively, and the cumulative dissolution percentage of fibroin was determined by HPLC according to the dissolution determination method (paddle method) in the Chinese Pharmacopoeia, using pH 6.8 phosphate buffer as the dissolution medium, rotating at 50 rpm, and at a temperature of 37±0.5℃.

[0044] Table 3

[0045] The results showed that the dissolution rate of fibroin monomer in Comparative Example 1 was only 29.5% after 60 minutes, indicating that fibroin has extremely poor water solubility. The dissolution rate of fibroin in the Sophora japonica extract in Comparative Example 4 was comparable to that of fibroin monomer (28.6%), indicating that the Sophora japonica extract itself had no significant promoting effect on the dissolution of fibroin. The antiviral composition in Example 1 of this invention achieved a dissolution rate of 78.6% after 60 minutes, indicating that the combination of fibroin leaf extract and Sophora japonica extract enhanced the effect on improving the dissolution rate of fibroin.

[0046] This invention combines pigeon pea leaf extract and sophora japonica fruit extract in a specific ratio. The flavonoid glycosides in the pigeon pea leaf extract improve the solubility and bioavailability of pachycarpus, while the sophora japonica fruit extract supplements pachycarpus. The two extracts synergistically enhance anti-HCV activity, thereby obtaining a drug composition with excellent anti-HCV activity, effective against DAA-resistant strains, and controllable quality.

[0047] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An antiviral composition containing pigeon pea leaf extract, characterized in that, It comprises pigeon pea leaf extract, sophora japonica fruit extract and a pharmaceutically acceptable carrier; the weight ratio of pigeon pea leaf extract to sophora japonica fruit extract is 1-20:

1.

2. The antiviral composition according to claim 1, characterized in that, The content of lignan in the Sophora japonica extract is not less than 2.0%; the lignan is 3-hydroxy-4-isopentenyl-5-methoxystilbene-2-carboxylic acid, with the molecular formula C2. 21 H 22 O4 has a molecular weight of 338.

4.

3. The antiviral composition according to claim 1, characterized in that, The weight ratio of the pigeon pea leaf extract to the sophora japonica fruit extract is 10:

1.

4. The antiviral composition according to claim 1, characterized in that, The antiviral composition comprises the following raw materials in parts by weight: 5-40 parts of pigeon pea leaf extract, 1-20 parts of sophora japonica fruit extract, and 40-94 parts of a pharmaceutically acceptable carrier.

5. The antiviral composition according to claim 1, characterized in that, The pigeon pea leaf extract is rich in vitexin, isovitexin and sennain.

6. The antiviral composition according to claim 1, characterized in that, The dosage form of the pharmaceutical composition is tablets, capsules, granules, oral liquid, or injection.

7. A method for preparing an antiviral composition containing pigeon pea leaf extract as described in any one of claims 1-6, characterized in that, Includes the following steps: S1. Preparation of Pigeon Bean Leaf Extract Pigeon pea leaves were dried, pulverized, and passed through a 40-60 mesh sieve to obtain pigeon pea leaf powder. The pigeon pea leaf powder was then mixed with 60%–80% ethanol (volume concentration) at a material-to-liquid ratio of 1:15–25 and extracted at 40–55°C for 1–3 hours. The mixture was filtered, and the filtrate was collected. The filtrate was concentrated under reduced pressure until no alcohol odor remained, yielding a crude extract. The crude extract was enriched using a macroporous adsorption resin and eluted sequentially with water, 30% ethanol, 60% ethanol, and 80% ethanol. The 60%–80% ethanol eluent was collected. The collected eluent was dried to obtain the pigeon pea leaf extract. S2. Preparation of Sophora japonica extract The Sophora japonica fruit is dried, pulverized, and passed through a 40-60 mesh sieve to obtain Sophora japonica fruit powder. The Sophora japonica fruit powder is taken and ethanol with a volume concentration of 60%-80% is added. The mixture is refluxed at 60-80℃ for 2-4 hours, filtered, and the filtrate is collected. The filtrate is concentrated under reduced pressure until there is no alcohol odor to obtain a crude extract. The crude extract is dispersed in water and extracted with ethyl acetate. The ethyl acetate fraction is collected. The ethyl acetate fraction is dried to obtain Sophora japonica fruit extract. S3. Preparation of antiviral compositions The pigeon pea leaf extract obtained in step S1 and the sophora japonica fruit extract obtained in step S2 are mixed with a pharmaceutically acceptable carrier to prepare a formulation.

8. The preparation method according to claim 7, characterized in that, The extraction method described in step S1 is ultrasonic-assisted extraction or reflux extraction; the macroporous adsorption resin is type D101 or type AB-8.

9. The use of the antiviral composition according to any one of claims 1-6 in the preparation of an anti-hepatitis C virus medicament.

10. The application according to claim 9, characterized in that, The hepatitis C virus includes strains that have developed resistance to direct-acting antiviral drugs.

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