Dioscorea panthaica extract, preparation method and application thereof

Abstract

The present application belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to a Polygonatum involucratum extract, a preparation method and application thereof. The present application takes dry Polygonatum involucratum powder as a raw material, removes the sugar-containing part through ethanol solution extraction and gradient elution of a polystyrene macroporous resin column, achieves the purpose of enriching small molecule substances such as flavonoids, saponins and amides, and obtains a first extract which has a good recovery effect on the decrease in liver cell activity caused by lysozyme-induced amyloidosis and the decrease in liver cell activity caused by alcohol. Then, the first extract is subjected to normal phase silica gel column chromatography elution and layering, and a second extract containing high isoflavone compounds has a significant recovery effect on the decrease in liver cell activity caused by lysozyme and the decrease in liver cell activity caused by alcohol, proving that the Polygonatum involucratum extract can be applied to the preparation of drugs for reversing liver amyloidosis and alcoholic liver injury, and has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine extraction technology, specifically relating to the extract of Polygonatum yunnanense, its preparation method, and its application. Background Technology

[0002] Hepatic amyloidosis is part of systemic amyloidosis. It occurs when amyloid substances deposit in the liver, leading to pathological changes and related clinical manifestations. Hepatic amyloidosis is a relatively rare clinical condition. It can be the hepatic manifestation of systemic amyloidosis or secondary to chronic infections, tuberculosis, rheumatoid arthritis, and other diseases. Common clinical manifestations include hepatomegaly, ascites, massive proteinuria, and hypoalbuminemia. It is easily misdiagnosed as fatty liver, Burch syndrome, etc., leading to prolonged misdiagnosis. Currently, there is no effective specific treatment for this disease, but early diagnosis, early intervention, and timely management of related complications can improve the patient's prognosis. Treatment for hepatic amyloidosis is primarily drug-based and is generally not curable; however, with active treatment, related symptoms can improve.

[0003] Alcoholic liver injury is a common liver disease caused by long-term heavy drinking. Early symptoms typically include alcoholic fatty liver. If left untreated, it can progress to alcoholic hepatitis, liver fibrosis, cirrhosis, and even hepatocellular carcinoma. Due to the complexity of its pathogenesis, there are currently no specific drugs for prevention or treatment. Treatment primarily involves abstinence from alcohol, nutritional therapy, drug therapy, and combination therapy. Currently, most liver-protecting drugs on the market are Western medicines, which are highly prone to causing toxic side effects during long-term use.

[0004] *Polygonatum kingianum* Coll. et Hemsl., a perennial herb belonging to the genus *Polygonatum* in the family Asparagaceae, is also known as *jiejiegao*, *maweigen*, *niuweibashu*, and *xianrenfan*. *Polygonatum kingianum* contains beneficial active compounds such as polysaccharides, alkaloids, saponins, flavonoids, and sterol amino acids. It possesses yin-nourishing, spleen-strengthening, lung-moistening, and kidney-tonifying effects, and is used to improve immunity, provide antioxidant benefits, improve cardiovascular health, and treat qi deficiency, dry cough due to lung deficiency, and insufficient essence and blood. It is also an important component of dozens of tonic traditional Chinese medicine formulas and prepared medicines, and has been valued by folk medicine practitioners and pharmacists since ancient times. *Polygonatum kingianum* is also one of the three *Polygonatum* source plants specified in the *Chinese Pharmacopoeia* (2020 edition), and is also commonly known as "big-headed *Polygonatum*".

[0005] Currently, there are no reports on the application of Polygonatum odoratum extract in reversing hepatic amyloidosis and alcoholic liver injury. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide an extract of Polygonatum yunnanense, its preparation method, and its application. The Polygonatum yunnanense extract of this invention has a restorative effect on the decreased hepatocyte viability caused by lysozyme-induced amyloidosis and the decreased hepatocyte viability caused by alcohol.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for preparing an extract of Polygonatum yunnanense, wherein the extract comprises a first extract and / or a second extract, the first extract containing flavonoids, amides, and saponins, and the second extract containing high levels of isoflavones; characterized in that the method for preparing the first extract comprises the following steps:

[0009] The powder of Polygonatum yunnanense was mixed with an ethanol solution for extraction, and the resulting extract was concentrated to obtain an extract paste.

[0010] The extract was mixed with water and then purified by passing it through a polystyrene macroporous resin column to obtain the first extract of Polygonatum yunnanense.

[0011] The preparation method of the second Polygonatum odoratum extract includes the following steps:

[0012] The first extract of Polygonatum yunnanense was separated on a silica gel column and eluted sequentially with petroleum ether and acetone in a first, second, and third volume ratio. The fraction obtained by elution with petroleum ether and acetone in a third volume ratio was collected to obtain the second extract of Polygonatum yunnanense. The first, second, and third volume ratios of petroleum ether and acetone were 90-100:1, 40-50:1, and 10-20:1, respectively.

[0013] Preferably, the volume fraction of ethanol in the ethanol solution is 70-95%.

[0014] Preferably, the extraction temperature is 10-30°C, the extraction is performed 2-5 times, and each extraction lasts for 2-3 days.

[0015] Preferably, the impurity removal process of the polystyrene macroporous resin column includes the following steps:

[0016] After wet loading, water, low-concentration ethanol solution, and high-concentration ethanol solution are used as eluents in sequence for elution, and the eluent obtained by elution with high-concentration ethanol solution is collected; the volume fraction of the low-concentration ethanol solution is 20%, and the volume fraction of the high-concentration ethanol solution is 85-90%.

[0017] Preferably, the high-concentration ethanol solution has a volume fraction of 90%.

[0018] Preferably, the polystyrene macroporous resin column is a D101 polystyrene macroporous resin column.

[0019] Preferably, the silica gel column separation uses 70-80 mesh silica gel for mixing the sample and 100-200 mesh silica gel for packing the column.

[0020] The present invention also provides a preparation method for the preparation of Polygonatum yunnanense obtained by the above technical solution, wherein the first Polygonatum yunnanense extract contains 3-5 wt% flavonoids, 2-4 wt% amides, and 1-3 wt% saponins, and the second Polygonatum yunnanense extract contains 2-4 wt% isoflavones.

[0021] The present invention also provides the application of the above-described Polygonatum odoratum extract in the preparation of health products or medicines for the prevention and / or treatment of liver diseases.

[0022] Preferably, the liver disease includes hepatic amyloidosis or alcoholic liver injury.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention provides a method for preparing an extract of Polygonatum yunnanense, comprising a first extract and / or a second extract. The first extract contains flavonoids, amides, and saponins, while the second extract contains high levels of isoflavones. The method includes the following steps: mixing Polygonatum yunnanense powder with an ethanol solution for extraction; concentrating the resulting extract to obtain an extract paste; mixing the extract paste with water and passing it through a polystyrene macroporous resin column to remove impurities, obtaining the first extract; separating the first extract onto a silica gel column and eluting it sequentially with petroleum ether and acetone at first, second, and third volume ratios, collecting the fraction eluted with the third volume ratio of petroleum ether and acetone to obtain the second extract; wherein the first, second, and third volume ratios of petroleum ether and acetone are 90–100:1, 40–50:1, and 10–20:1, respectively.

[0025] This invention employs cold extraction with ethanol solution, a mild extraction method that avoids alteration of chemical components while fully extracting total saponins, flavonoids, and polyphenols from *Polygonatum yunnanense*. A polystyrene macroporous resin column is used to effectively remove large molecular impurities from the extract, ensuring its purity and safety, while simultaneously enriching small molecules such as flavonoids, saponins, and amides. The resulting first *Polygonatum yunnanense* extract exhibits good restorative effects on hepatocyte viability reduction induced by lysozyme and alcohol. The first extract is then subjected to normal-phase silica gel column chromatography for elution and separation to obtain a second extract containing high levels of isoflavones, which also shows significant restorative effects on lysozyme-induced and alcohol-induced hepatocyte viability reduction.

[0026] Meanwhile, the preparation method of the extract of Polygonatum yunnanense in this invention is simple, the extraction solvent can be recycled and reused, it is green and environmentally friendly, and the obtained extract of Polygonatum yunnanense shows no change in TLC after standing, indicating good stability.

[0027] The extract of Polygonatum yunnanense obtained by this invention has a restorative effect on the decrease in hepatocyte viability caused by LYSO-6 and alcohol. The extract of Polygonatum yunnanense has the function of reversing hepatic amyloidosis and alcoholic liver injury. Therefore, it can be used for the preparation and development of products that reverse hepatic amyloidosis and alcoholic liver injury, which has important clinical application value and opens up a new research field for Polygonatum yunnanense extract. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a flowchart illustrating the preparation method of the Polygonatum odoratum extract in the examples;

[0030] Figure 2 The effects of different concentrations of crude extract of Rhizoma Scutellariae Radix (TE) and its small molecule product (Fr.C) on the viability of NCTC1469 hepatocytes stimulated with LYSO-6 for 24 h were investigated. A represents 25 μg / mL of crude extract of Rhizoma Scutellariae Radix (TE) and its small molecule product; B represents 50 μg / mL of crude extract of Rhizoma Scutellariae Radix (TE) and its small molecule product; and C represents 100 μg / mL of crude extract of Rhizoma Scutellariae Radix (TE) and its small molecule product (Note: ***P<0.001, **P<0.01, *P<0.05).

[0031] Figure 3The effects of different concentrations of crude extract of Rhizoma Scutellariae Radix (TE) and its small molecule product (Fr.C) on the viability of NCTC1469 hepatocytes stimulated with 100 mM alcohol for 24 h were investigated. A represents 25 μg / mL of crude extract of Rhizoma Scutellariae Radix (TE) and its small molecule product; B represents 50 μg / mL of crude extract of Rhizoma Scutellariae Radix (TE) and its small molecule product; C represents 100 μg / mL of crude extract of Rhizoma Scutellariae Radix (TE) and its small molecule product (Note: ***P<0.001, **P<0.01, *P<0.05).

[0032] Figure 4 Effects of different layered products of 50 μg / mL Polygonatum odoratum on the viability of NCTC1469 hepatocytes stimulated with LYSO-6 for 24 h (Note: n≧3, ***P<0.001, **P<0.01, *P<0.05);

[0033] Figure 5 The effect of different concentrations of different layered products of small molecules of Polygonatum yunnanense on the viability of NCTC1469 hepatocytes stimulated with LYSO-6 for 24 h was investigated. Among them, A represents different layered products of small molecules of Polygonatum yunnanense at 25 μg / mL; B represents different layered products of small molecules of Polygonatum yunnanense at 50 μg / mL; and C represents different layered products of small molecules of Polygonatum yunnanense at 100 μg / mL (Note: n≧3, ***P<0.001, **P<0.01, *P<0.05).

[0034] Figure 6 The effect of different concentrations of different layered products of small molecules of Polygonatum yunnanense on the viability of NCTC1469 hepatocytes stimulated with 100 mM alcohol for 24 h was investigated. Among them, A represents different layered products of 25 μg / mL Polygonatum yunnanense; B represents different layered products of 50 μg / mL Polygonatum yunnanense; and C represents different layered products of 100 μg / mL Polygonatum yunnanense (Note: n≧3, ***P<0.001, **P<0.01, *P<0.05). Detailed Implementation

[0035] This invention provides a method for preparing a Polygonatum yunnanense extract, wherein the Polygonatum yunnanense extract comprises a first Polygonatum yunnanense extract and / or a second Polygonatum yunnanense extract, wherein the first Polygonatum yunnanense extract contains flavonoids, amides and saponins, and the second Polygonatum yunnanense extract contains high levels of isoflavones;

[0036] The preparation method of the first Polygonatum yunnanense extract includes the following steps:

[0037] The powder of Polygonatum yunnanense was mixed with an ethanol solution for extraction, and the resulting extract was concentrated to obtain an extract paste.

[0038] The extract was mixed with water and then purified by passing it through a polystyrene macroporous resin column to obtain the first extract of Polygonatum yunnanense.

[0039] The preparation method of the second Polygonatum odoratum extract includes:

[0040] The first extract of Polygonatum yunnanense was separated on a silica gel column and eluted sequentially with petroleum ether and acetone in a first, second, and third volume ratio. The fraction obtained by elution with petroleum ether and acetone in a third volume ratio was collected to obtain the second extract of Polygonatum yunnanense. The first, second, and third volume ratios of petroleum ether and acetone were 90-100:1, 40-50:1, and 10-20:1, respectively.

[0041] Unless otherwise specified, all materials and equipment used in this invention are commercially available products in the field.

[0042] This invention involves mixing Polygonatum yunnanense powder with an ethanol solution for extraction, concentrating the resulting extract to obtain an extract paste.

[0043] In this invention, the powder of Polygonatum yunnanense is preferably powder of Polygonatum yunnanense rhizome.

[0044] In this invention, the volume fraction of ethanol in the ethanol solution is preferably 70-95%, more preferably 75-90%, and even more preferably 80-85%.

[0045] In this invention, the preferred ratio of the amount of Polygonatum odoratum powder to the ethanol solution is 1 kg: 6 L; the preferred mass ratio of the amount of Polygonatum odoratum powder to the ethanol solution is 1:4.5 to 5.5, more preferably 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5.0, 1:5.1, 1:5.2, 1:5.3, 1:5.4 or 1:5.5.

[0046] In this invention, the extraction temperature is preferably room temperature, preferably 10-30°C; the number of extractions is preferably 2-5 times, more preferably 3-4 times; and the extraction time for each extraction is preferably 2-3 days.

[0047] In this invention, the concentration conditions preferably include: a temperature of 50°C and a pressure of 300 bar, and the concentration is preferably up to an ethanol volume fraction of no more than 5%.

[0048] After obtaining the extract, the present invention mixes the extract with water and removes impurities through a polystyrene macroporous resin column to obtain the first extract of Polygonatum yunnanense.

[0049] In this invention, the water is preferably distilled water, and the mass fraction of the extract in the mixture obtained by mixing the extract and water is preferably 20-50%, more preferably 30%.

[0050] In this invention, the polystyrene macroporous resin column is preferably a D101 polystyrene macroporous resin column, and the mass ratio of the extract paste and water mixture to the macroporous resin in the polystyrene macroporous resin column is preferably 1:1.5 to 2.5, more preferably 1:1.5, 1:1.7, 1:2, 1:2.2 or 1:2.5.

[0051] In this invention, the impurity removal of the polystyrene macroporous resin column preferably includes the following steps:

[0052] After wet loading, water, low-concentration ethanol solution, and high-concentration ethanol solution are used as eluents in sequence for elution, and the eluent obtained by elution with high-concentration ethanol solution is collected; the volume fraction of the low-concentration ethanol solution is 20%, and the volume fraction of the high-concentration ethanol solution is 85-90%.

[0053] In this invention, the water and low-concentration ethanol solution eluents the sugar-containing portion. The sugar-containing portion can be removed by impurity removal using a polystyrene macroporous resin column, thereby achieving the purpose of enriching small molecule substances and obtaining small molecule substances. The small molecule substances preferably include flavonoids, amides, and saponins, and the saponins are preferably triterpenoid saponins.

[0054] In this invention, the volume fraction of the high-concentration ethanol solution is preferably 90%.

[0055] In this invention, the high-concentration ethanol solution is preferably followed by elution using 100% ethanol as the eluent.

[0056] In this invention, the water elution volume is preferably 4-5 BV; the low-concentration ethanol solution elution volume is preferably 2-3 BV; the high-concentration ethanol solution elution volume is preferably 4-5 BV; and the 100% ethanol elution volume is preferably 4-5 BV.

[0057] After obtaining the first extract of Polygonatum yunnanense, the present invention separates the first extract of Polygonatum yunnanense onto a silica gel column and elutes it sequentially with petroleum ether and acetone in a first volume ratio, a second volume ratio, and a third volume ratio. The fraction obtained by elution with petroleum ether and acetone in a third volume ratio is collected to obtain the second extract of Polygonatum yunnanense. The first volume ratio, the second volume ratio, and the third volume ratio of petroleum ether and acetone are 90-100:1, 40-50:1, and 10-20:1, respectively.

[0058] In this invention, the silica gel column separation preferably uses 70-80 mesh silica gel for mixing the sample and 100-200 mesh silica gel for packing the column.

[0059] In this invention, the first volume ratio, the second volume ratio, and the third volume ratio of petroleum ether and acetone are preferably 100:1, 50:1, and 20:1, respectively.

[0060] In this invention, after elution with a petroleum ether and acetone in a third volume ratio, it is preferable to further perform gradient elution with petroleum ether and acetone in volume ratios of 10:1, 5:1, and 1:1. The eluent obtained by the gradient elution is preferably subjected to TLC detection, and fractions of the same type are combined based on the Rf value under the same developing solvent conditions.

[0061] The present invention also provides a preparation method for Polygonatum yunnanense extract obtained by the above-described technical solution, wherein the first Polygonatum yunnanense extract contains 3-5 wt% flavonoids, 2-4 wt% amides, and 1-3 wt% saponins, and the second Polygonatum yunnanense extract contains 2-4 wt% isoflavones (polygonatone H).

[0062] The first and second extracts of Polygonatum yunnanense of the present invention have significant restorative effects on the reduction of NCTC1469 hepatocyte viability after 24 hours of LYSO-6 stimulation and the reduction of NCTC1469 hepatocyte viability after 24 hours of alcohol stimulation.

[0063] The present invention also provides the application of the above-described Polygonatum odoratum extract in the preparation of health products or medicines for the prevention and / or treatment of liver diseases.

[0064] In this invention, the liver disease preferably includes hepatic amyloidosis or alcoholic liver injury.

[0065] This invention provides an extract of Polygonatum yunnanense that reverses hepatic amyloidosis and alcoholic liver injury, and offers a new application for the extract in biomedicine, which is expected to lead to new applications and breakthroughs in the treatment of hepatic amyloidosis and alcoholic liver injury.

[0066] To further illustrate the present invention, the following detailed description of the extract of Polygonatum yunnanense, its preparation method, and its application, in conjunction with the accompanying drawings and embodiments, is provided but should not be construed as limiting the scope of protection of the present invention.

[0067] Figure 1 The flowchart of the preparation method of Polygonatum yunnanense extract in the example is as follows: Polygonatum yunnanense powder and ethanol solution are mixed and extracted. The resulting extract is concentrated to obtain an extract paste (total extract, TE). The extract paste is then mixed with water and passed through a polystyrene macroporous resin column to remove sugar, obtaining Polygonatum yunnanense extract Fr.C (first Polygonatum yunnanense extract). Then, Polygonatum yunnanense extract Fr.C is separated by silica gel column chromatography and eluted with a gradient of petroleum ether and acetone to obtain Polygonatum yunnanense extracts Fr.C1, Fr.C2, Fr.C3 (second Polygonatum yunnanense extract), Fr.C4, Fr.C5 and Fr.C6, respectively.

[0068] Example 1

[0069] The preparation method of the extract of Polygonatum yunnanense that reverses hepatic amyloidosis and alcoholic liver injury includes the following steps:

[0070] (1) Take 15 kg of dried powder of Polygonatum yunnanense and soak it in 80% ethanol (80 L) 4 times for 3 days each time. Combine the extracts and concentrate under reduced pressure to obtain the extract paste (Crude extract of Polygonatum yunnanense, TE).

[0071] (2) Dissolve the extract (8 kg) obtained in step (1) in purified water (8 L), pack the column with D101 polystyrene macroporous resin (id. 25 cm × 75 cm, diameter × length), load the sample by wet method, and elute in sequence with water, 20% volume fraction ethanol solution, 90% volume fraction ethanol solution and 100% ethanol as eluents, with the amounts being 30 L, 20 L, 30 L and 30 L respectively. Collect the elution fraction of 90% volume fraction ethanol solution, concentrate the eluent under reduced pressure to obtain Polygonatum extract (small molecule product, Fr.C).

[0072] (3) The 90% volume fraction ethanol solution eluent obtained in step (2) (Fr.C) was separated by normal phase chromatography on silica gel column. Gradient elution was performed sequentially with petroleum ether-acetone at volume ratios of 100:1, 50:1, 20:1, 10:1, 5:1 and 1:1. The eluents were detected by TLC. Fractions with the same Rf value under the same developing solvent conditions were combined. Fractions under different gradients were collected and concentrated to an ethanol volume fraction of no more than 5% and dried (at a temperature of 50°C and a negative pressure of 300 bar) to obtain Polygonatum extracts Fr.C1, Fr.C2, Fr.C3, Fr.C4, Fr.C5 and Fr.C6.

[0073] Example 2

[0074] The crude extract (TE) of Polygonatum yunnanense prepared in Example 1, and the restorative effects of Polygonatum yunnanense extracts Fr.C, Fr.C1, Fr.C2, Fr.C3, Fr.C4, Fr.C5 and Fr.C6 on LYSO-6-induced and alcohol-induced decreases in hepatocyte viability were determined.

[0075] (1) Preparation of lysozyme LYSO (LYSO-6)

[0076] The LYSO incubation solution was placed in a thermostatic shaker set at 65°C and 50 rpm. Incubation was continued for 6 days.

[0077] (2) Cell Culture

[0078] The NCTC1469 mouse normal hepatocyte line was cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 100 U / mL penicillin, 100 g / mL streptomycin and 0.2 mg / mL G-418. Cells in the logarithmic growth phase were used for experiments.

[0079] (3) Cell seeding plate

[0080] When the cell density reaches 80-90%, passage is performed, and the cells are diluted with high-glucose medium containing 3% fetal bovine serum at a concentration of 1×10⁻⁶. 5 Inoculate the 96-well plate at a density of 1 cell / well, with 5 duplicate wells per group, and allow it to adhere to the wall for 24 hours.

[0081] (4) Activity screening and results

[0082] ① Effects of different concentrations of crude extract (TE) and small molecule products (Fr.C) of *Scutellaria baicalensis* on the viability of NCTC1469 hepatocytes stimulated with LYSO-6 for 24 h.

[0083] Cells were seeded into plates, and a blank control group (control) and a LYSO-6 group (1×10⁻⁶) were set up. -5 The final concentrations of crude extract of *Dioscorea nipponica* and its small molecule product (Fr.C) were 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. After administration of the drug 2 h prior to treatment, the patient was stimulated with LYSO-6 for 24 h. After 24 h, the culture medium was removed, CCK-8 reagent was added, and the mixture was incubated at 37℃ for 2-3 h. The absorbance (OD value) at 450 nm was measured using a microplate reader.

[0084] Effects of different concentrations of crude extract (TE) and small molecule product (Fr.C) of Rhizoma Scutellariae Radix et Rhizoma on the viability of NCTC1469 hepatocytes stimulated with LYSO-6 for 24 h: The protective effects of different concentrations of crude extract (TE) and small molecule product of Rhizoma Scutellariae Radix et Rhizoma on NCTC1469 hepatocytes stimulated with LYSO-6 for 24 h are shown in the results. Figure 2 As shown, the crude extract of Polygonatum yunnanense at concentrations of 50 μg / mL and 100 μg / mL had a certain restorative effect on the decrease in hepatocyte viability induced by LYSO-6; the small molecule product at concentrations of 25 μg / mL, 50 μg / mL and 100 μg / mL all had a significant restorative effect on the decrease in hepatocyte viability induced by LYSO-6.

[0085] ② Effects of different concentrations of crude extract and small molecule products of *Scutellaria baicalensis* on the viability of NCTC1469 hepatocytes stimulated with 100 mM alcohol for 24 h.

[0086] Cells were seeded into plates, and control and model groups were set up, using 100mM ethanol + TE and 100mM ethanol + Fr.C. The final concentrations of crude extract of *Scutellaria baicalensis* and small molecule products were 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. Two hours before administration, cells were stimulated with 100mM ethanol for 24 hours. After 24 hours, the culture medium was removed, CCK-8 reagent was added, and the cells were incubated at 37℃ for 2-3 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader.

[0087] Effects of different concentrations of crude extract of *Rhizoma Scutellariae Radix* and its small molecule products on the viability of NCTC1469 hepatocytes stimulated with 100 mM alcohol for 24 h: The protective effects of different concentrations of crude extract of *Rhizoma Scutellariae Radix* and its small molecule products on NCTC1469 hepatocytes stimulated with 100 mM alcohol for 24 h are shown in the results below. Figure 3 As shown, crude extract of Dianhuangjing and its small molecule products have significant restorative effects on the reduction of hepatocyte viability induced by 100mM alcohol at concentrations of 25μg / mL, 50μg / mL and 100μg / mL.

[0088] ③ Effects of different concentrations of small-molecule, layered products of Polygonatum yunnanense on the viability of NCTC1469 hepatocytes stimulated with LYSO-6 for 24 h

[0089] Cells were seeded into plates, and a control group and a LYSO-6 group (1×10⁻⁶) were set up. -5 The final concentrations of different small-molecule phases of crude extract of *Dioscorea nipponica* were 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. The products were LYSO-6+Fr.C1, LYSO-6+Fr.C2, LYSO-6+Fr.C3, LYSO-6+Fr.C4, LYSO-6+Fr.C5, and LYSO-6+Fr.C6. After administration 2 h prior to treatment, the product was stimulated with LYSO-6 for 24 h. After 24 h, the culture medium was removed, CCK-8 reagent was added, and the product was incubated at 37℃ for 2-3 h. The absorbance (OD value) at 450 nm was measured using a microplate reader.

[0090] Effects of different concentrations of different layered products of small molecules from *Polygonatum yunnanense* on the viability of NCTC1469 hepatocytes stimulated with LYSO-6 for 24 h: The protective effects of different concentrations of different layered products of small molecules from *Polygonatum yunnanense* on NCTC1469 hepatocytes stimulated with LYSO-6 for 24 h are shown in the results. Figure 4As shown, Fr.C1, Fr.C2, and Fr.C3 layers at a concentration of 50 μg / mL showed a restorative effect on LYSO-6-induced hepatocyte viability reduction, while Fr.C4, Fr.C5, and Fr.C6 layers did not. Therefore, the Fr.C1, Fr.C2, and Fr.C3 layers were subjected to activity tests at other concentrations to restore LYSO-6-induced hepatocyte viability reduction, and the results are as follows. Figure 5 The Fr.C3 layer showed significant restorative effects on LYSO-6-induced hepatocyte viability reduction at concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL. Fr.C1 and Fr.C2 also showed significant restorative effects on LYSO-6-induced hepatocyte viability reduction at concentrations of 50 μg / mL and 100 μg / mL, but their effects were less pronounced than those of the Fr.C3 layer. At a concentration of 25 μg / mL, they did not show any restorative effect on LYSO-6-induced hepatocyte viability reduction.

[0091] ④ Effects of different concentrations of small-molecule, layered products of Polygonatum yunnanense on the viability of NCTC1469 hepatocytes stimulated with 100mM alcohol for 24h

[0092] Cells were seeded into plates, and control and model groups were set up. The following groups were prepared using 100mM ethanol + Fr.C1, 100mM ethanol + Fr.C2, 100mM ethanol + Fr.C3, 100mM ethanol + Fr.C4, 100mM ethanol + Fr.C5, and 100mM ethanol + Fr.C6. The final concentrations of different small-molecule phases of the crude extract of *Scutellaria baicalensis* were 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. Two hours before administration, the cells were stimulated with 100mM ethanol for 24 hours. After 24 hours, the culture medium was removed, CCK-8 reagent was added, and the cells were incubated at 37℃ for 2-3 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader.

[0093] Effects of different concentrations of different layered products of small molecules from *Polygonatum yunnanense* on the viability of NCTC1469 hepatocytes stimulated with 100 mM alcohol for 24 h: The protective effects of different concentrations of different layered products of small molecules from *Polygonatum yunnanense* on NCTC1469 hepatocytes stimulated with 100 mM alcohol for 24 h are shown in the results. Figure 6 As shown, layers Fr.C1, Fr.C2, and Fr.C3 had a restorative effect on the decrease in hepatocyte viability induced by 100 mM alcohol, with Fr.C3 showing the best effect. The CellViability results were: Fr.C1 = 88.74%, Fr.C2 = 99.56%, and Fr.C3 = 126.53%, respectively. Layers Fr.C4, Fr.C5, and Fr.C6 had no restorative effect on the decrease in hepatocyte viability induced by 100 mM alcohol.

[0094] The results of Fr.C and Fr.C3 on alcoholic liver injury and hepatic amyloidosis are shown in Table 1. For alcoholic liver injury, Fr.C3 showed better protective activity at a concentration of 100 μg / mL; for hepatic amyloidosis, Fr.C showed better protective activity at concentrations of 25, 50 and 100 μg / mL.

[0095] Table 1. Results of Fr.C and Fr.C3 on alcoholic liver injury and hepatic amyloidosis.

[0096]

[0097] In summary, the Fr.C and Fr.C3 fractions of the Polygonatum odoratum extract provided by this invention exhibit significantly superior activity compared to other fractions, and have a restorative effect on the decreased hepatocyte viability caused by LYSO-6 and alcohol, making them suitable for the preparation of drugs or health products that reverse hepatic amyloidosis and alcoholic liver injury.

[0098] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a Polygonatum odoratum extract for the prevention and / or treatment of alcoholic liver injury, wherein the Polygonatum odoratum extract contains high levels of isoflavones; characterized in that, Includes the following steps: The powder of Polygonatum yunnanense and an ethanol solution were mixed and extracted. The resulting extract was concentrated to obtain an extract paste. The ratio of Polygonatum yunnanense powder to ethanol solution was 1 kg: 6 L. The volume fraction of ethanol in the ethanol solution was 80%. The extraction temperature was 10~30℃. The extraction was performed 4 times, and each extraction lasted for 3 days. The extract was mixed with water and then wet-loaded onto a D101 polystyrene macroporous resin column. The column was eluted sequentially with water, 20% ethanol solution, and 90% ethanol solution. The eluent obtained from the 90% ethanol solution was collected and concentrated under reduced pressure to obtain the extract Fr.C. The extract was separated on a silica gel column using Fr.C., and then eluted sequentially with petroleum ether and acetone in volume ratios of 100:1, 50:1, and 20:

1. The fraction obtained by elution with petroleum ether and acetone in a volume ratio of 20:1 was collected, concentrated to a volume fraction of ethanol of no more than 5%, and dried to obtain the Polygonatum yunnanense extract.

2. The Polygonatum odoratum extract obtained by the preparation method according to claim 1, characterized in that, The content of isoflavones in the extract of Polygonatum yunnanense is 2-4 wt%.

3. The use of the Polygonatum odoratum extract according to claim 2 in the preparation of a medicine for the prevention and / or treatment of alcoholic liver injury.

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