An anti-tumor effective part extracted from root of ramulus pruni mume and its preparation method and application

Abstract

The application discloses an anti-tumor effective part extracted from the root of Amur maotao and a preparation method and application thereof. The effective part is obtained by water extraction on the medicinal material of the root of Amur maotao, and then removing polysaccharide and other impurities by using an ethanol precipitation method, and collecting the water extraction alcohol precipitation part after refining. The composition group eluted after 60%-100% ethanol elutes a C18 solid-phase extraction column contains eugenol, rutin and quercetin, and the content of eugenol, rutin and quercetin is not less than 50 mg / g, 30 ug / g and 50 ug / g respectively. In-vivo and in-vitro pharmacodynamic experiments prove that the effective part of the root of Amur maotao provided by the application can significantly inhibit the tumor growth of a liver cancer H22 tumor-bearing mouse, reduce the serum alpha-fetal protein level, improve the oxidation stress index, and effectively inhibit the migration and proliferation capacity of human liver cancer cells HepG2 and Huh7, and simultaneously down-regulate the expression of PD-L1 protein and mRNA. The effective part composition is relatively definite, and can be used for preparing an auxiliary medicine for preventing and treating malignant tumors such as liver cancer with high PD-L1 expression, and a health product raw material.

Description

Technical Field

[0001] This invention relates to the field of Chinese herbal medicine extraction and separation technology, specifically to an effective anti-tumor component extracted from the root of the She ethnic group's traditional medicinal herb, the white peach root, its preparation method, and the application of this effective component in the preparation of anti-tumor drugs. Background Technology

[0002] Liver cancer is one of the most common malignant tumors worldwide, with extremely high incidence and mortality rates. It is characterized by its insidious onset, rapid progression, and tendency to develop resistance to traditional radiotherapy and chemotherapy, resulting in poor prognosis for patients. Currently, finding highly effective and low-toxicity anti-liver cancer drugs remains a major challenge in the scientific research field.

[0003] Traditional Chinese medicine has accumulated rich experience in the prevention and treatment of tumors, demonstrating unique advantages in multi-component, multi-target, and holistic regulation. Baishan Maotao root, scientifically known as *Actinidia pubescens* root, is a traditional anti-tumor medicine used by the She ethnic group. It is recorded in ancient books such as the Tang Dynasty's *Compendium of Materia Medica* and the Ming Dynasty's *Compendium of Materia Medica*. Modern research indicates that it has effects such as anti-gastric cancer and immune regulation. However, existing research mainly focuses on its crude extract, which suffers from complex composition, unclear pharmacodynamic material basis, and difficulties in quality control, limiting its in-depth development and clinical application.

[0004] Therefore, further isolation and purification from the root of Prunus cerasifera to obtain effective components with relatively clear components and significant medicinal effects is of great significance for elucidating its anti-tumor mechanism, developing new Chinese medicine anti-tumor drugs or health products with independent intellectual property rights, and adjuvant drugs for tumors. Summary of the Invention

[0005] Objective of this invention: The objective of this invention is to overcome the shortcomings of existing technologies and provide an effective anti-tumor component extracted from the root of *Prunus serrulata*, which has relatively well-defined components and significant anti-tumor effects. Another objective of this invention is to provide a method for preparing this effective component. A third objective of this invention is to provide the application of this effective component in the preparation of anti-tumor drugs.

[0006] Technical solution: To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing an effective anti-tumor component extracted from the root of *Prunus cerasifera*, comprising the following steps: (1) Take dried white peach root, crush it, add water and heat and reflux extract 1-3 times, combine the extracts, filter and obtain water extract; (2) The aqueous extract obtained in step (1) is concentrated under reduced pressure at 50-70℃ to a suitable density to obtain a concentrated solution; (3) Add ethanol to the concentrate in step (2), stir slowly to make the final concentration of ethanol reach 60%-85%, preferably 70%-80%, and let it stand at 4-10℃ for 8-24 hours to allow polysaccharide and other macromolecular impurities to fully precipitate. (4) Centrifuge or filter the alcohol precipitate from step (3) to separate and collect the supernatant; (5) Pass the supernatant obtained in step (4) through a C18 solid phase extraction column, elute with 60-100% ethanol, collect the eluent and recover the ethanol under reduced pressure, and dry (such as vacuum drying or spray drying) to obtain the effective anti-tumor component of the root of Prunus cerasifera.

[0007] The effective anti-tumor fraction is the group of components after elution with 60%-100% ethanol from a C18 solid-phase extraction column.

[0008] The effective fraction contains syringic acid, rutin, and quercetin at no less than 50 mg / g, 30 ug / g, and 50 ug / g, respectively.

[0009] Application of effective antitumor components in the preparation of antitumor drugs.

[0010] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. This invention is the first to identify and prepare the effective component of the root of Prunus cerasifera that plays a core role in anti-liver cancer. Through a refining process, ineffective or low-efficacy polysaccharide components and highly polar components are removed, thereby enriching the active ingredients.

[0011] 2. The effective components provided by this invention have clear anti-tumor effects and diverse mechanisms: in vivo experiments show that they can significantly inhibit tumor growth in H22 tumor-bearing mice; in vitro experiments demonstrate that they can effectively inhibit the migration, proliferation and colony formation of liver cancer cells; at the same time, they can also downregulate the expression of the immune checkpoint PD-L1, suggesting that they have immunomodulatory potential.

[0012] 3. The preparation process of this effective component is simple, the cost is low, it is easy to realize industrial production, and the quality is controllable, which lays a solid foundation for its development into a new traditional Chinese medicine.

[0013] 4. This effective component is derived from traditional Chinese medicine, and is expected to have fewer toxic side effects, showing good prospects for clinical application. Attached Figure Description

[0014] Figure 1 Effects of *Prunus cerasifera* root extract on body weight, tumor volume, and body weight in tumor-bearing mice (A: Mouse body weight over time; B: Mouse tumor volume over time; C: Tumor images for each group; D: Mouse tumor weight, *p<0.05). Model: Model group; MTGC: Alcohol extract group; MTGS: Water extract group; MTGD: Crude polysaccharide group; Figure 2 Changes in mouse serum (A: serum AFP; B: serum AST; C: serum ALT; D: serum IFN-γ; E: serum SOD; F: serum MDA; G: serum HDL; H: serum UA, * p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates no significant difference). Figure 3 Changes in the expression of PD-L1, P-JAK1, JAK1, P-STAT3, and STAT3 proteins and PD-L1 mRNA in tumor tissues of tumor-bearing mice (A: PD-L1 protein expression in tumor tissues of H22 tumor-bearing mice detected by Western blotting; B: PD-L1 mRNA expression in tumor tissues of H22 tumor-bearing mice detected by RT-PCR; C: Image showing changes in PD-L1 protein expression in tumor tissues of H22 tumor-bearing mice; CE: P-JAK1 and JAK1 protein expression in tumor tissues of H22 tumor-bearing mice detected by Western blotting; FH: P-STAT3 and STAT3 protein expression in tumor tissues of H22 tumor-bearing mice detected by Western blotting; IK: Graph showing changes in the expression of PD-L1, P-JAK1, JAK1, P-STAT3, and STAT3 proteins in tumor tissues of H22 tumor-bearing mice, * p < 0.05, ** p < 0.01, *** p<0.001, ns indicates no significant difference); Figure 4 The water extract of *Prunus cerasifera* root was passed through a C18 column, and the changes in PD-L1 protein expression in HepG2 and Huh7 cells were analyzed using 40%, 80%, and 100% ethanol eluents (AC: changes in PD-L1 protein expression in HepG2 cells and corresponding PD-L1 protein expression images with 40%, 80%, and 100% ethanol eluents; DF: changes in PD-L1 protein expression in Huh7 cells and corresponding PD-L1 protein expression images with 40%, 80%, and 100% ethanol eluents; * p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates no significant difference). Figure 5The effects of water extracts from the roots of *Prunus cerasifera* on PD-L1 mRNA expression in HepG2 and Huh7 cells were investigated using C18 columns and eluents of 40%, 80%, and 100% ethanol (AB: effect of 80% and 100% ethanol eluents on PD-L1 mRNA expression in HepG2 cells; CD: effect of 80% and 100% ethanol eluents on PD-L1 mRNA expression in Huh7 cells; * p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates no significant difference). Figure 6 Chromatograms of three standards: eugenol, rutin, and quercetin; Figure 7 Fingerprint of the water extract of Prunus cerasifera root eluent on a C18 column with 80-100% ethanol. Detailed Implementation

[0015] The present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0016] This invention discloses a method for preparing an effective anti-tumor component extracted from the root of *Prunus cerasifera*, which is prepared by the following method: (1) Take the root of the white mountain peach, grind it into powder, add water and heat and reflux to extract, and collect the extract; the heating and reflux extraction is performed 1-3 times, each time the amount of water added is 8-15 times the weight of the medicinal material, and the extraction time is 1-3 hours.

[0017] (2) After concentrating the extract obtained in step (1), add ethanol for alcohol precipitation treatment to make the final ethanol concentration reach 60%-85%, let stand, and separate the precipitate; the final ethanol concentration is 70%-80%, and the standing time is 8-24 hours.

[0018] (3) Collect the supernatant from step (2), pass it through a C18 solid phase extraction column, and obtain the antitumor effective fraction after the eluent is concentrated and dried.

[0019] The effective antitumor fraction is a group of components obtained after elution with 60%-100% ethanol from a C18 solid-phase extraction column. This group of components can downregulate the expression of PD-L1 protein and mRNA. The effective fraction contains at least 50 mg / g of syringic acid, 30 μg / g of rutin, and 50 μg / g of quercetin.

[0020] The application of the effective antitumor component in the preparation of antitumor drugs. The tumor is liver cancer.

[0021] The effective antitumor components are formulated into capsules, tablets, granules, oral liquids, or injections using pharmaceutically acceptable carriers.

[0022] Example 1: Preparation of Aqueous Extract from Root of Prunus serrulata Dry the root of *Prunus cerasifera* at 60℃ for 4 hours, then pulverize it. Weigh 200g of the powder, add 20-50 times the amount of distilled water, and extract twice by reflux at 100℃, 1.5 hours each time. Combine the two extracts, filter through four layers of gauze, and concentrate the filtrate under reduced pressure at 60℃ to approximately 200mL (approximately 1g of raw herb / mL). Slowly add 4 times the volume of anhydrous ethanol to the concentrate (to make the ethanol concentration approximately 80%) while stirring, and let it stand overnight (approximately 12 hours) at 4℃. The next day, centrifuge at 4000 r / min for 15 minutes and carefully collect the supernatant. The precipitate is crude polysaccharide (save separately). Recover the ethanol from the supernatant under reduced pressure at 50℃, and further concentrate to a thick paste. Transfer to a vacuum drying oven and dry to constant weight at 60℃, then pulverize to obtain the water extract of *Prunus cerasifera* root, with an extraction rate of approximately 10-15%.

[0023] Example 2: Preparation of crude polysaccharide from the root of Prunus serrulata (comparative example) This embodiment is intended to compare with the effective components of the present invention. The precipitate obtained by centrifugation after alcohol precipitation in Example 1 was washed successively with anhydrous ethanol and acetone, and then dried in a vacuum drying oven to obtain the crude polysaccharide sample, with an extraction rate of approximately 5%.

[0024] Example 3: Preparation of alcoholic extract from the root of Prunus serrulata (comparative example) This embodiment is intended to compare with the effective components of the present invention. 100g of dried white peach root powder was soaked overnight in 10 times its volume of 95% ethanol, followed by ultrasonic extraction twice, 2 hours each time. The extracts were then filtered under reduced pressure, and the filtrates were combined. The residue was then ultrasonically extracted twice again with 5 times its volume of 95% ethanol, filtered, and all filtrates were combined. The ethanol solvent was recovered from the filtrate under reduced pressure at 50°C. The residue was washed with an appropriate amount of ethanol and then dried in a vacuum centrifuge to obtain the total ethanol extract, with an extraction rate of approximately 9%.

[0025] Example 4: C18 chromatographic column separation of water extract from white peach root Take a 3 mL C18 solid-phase extraction column and place it in a solid-phase extraction apparatus. Add 2 mL of anhydrous ethanol to the column, followed by an equal volume of 2 mL of pure water. After the liquid has naturally run off, add 1 mL of the aqueous extract (sample from Example 1), followed by 1 mL of pure water. Add 20%, 40%, 60%, 80%, and 100% ethanol solutions sequentially to the column. After the liquid has run off, collect the corresponding eluents sequentially using an EP tube. Add 1 mL of anhydrous ethanol to the column, followed by an equal volume of pure water. After the liquid has run off naturally, load the sample again and repeat the subsequent steps until the drug solution is completely eluted.

[0026] The collected eluent was placed in a vacuum drying centrifuge, dried and weighed, and each eluent was dissolved in DMSO to prepare a solution of 200 mg / mL.

[0027] Example 5: Pharmacodynamic experiment of white peach root extract against H22 tumor-bearing mice SPF-grade healthy male KM mice and H22 mouse hepatocellular carcinoma cell line were used. Mice, except for the normal control group, were subcutaneously inoculated with H22 ascites cells on their right back to establish the hepatocellular carcinoma model. After successful modeling, mice were randomly divided into four groups: Model group, Model group, MTGC (ethanol extract group), MTGS (water extract group), and MTGD (crude polysaccharide group). All groups were administered the drug at a dose of 0.5 g / kg / day via gavage for 14 consecutive days.

[0028] Before administration, all mice were in normal condition. On days 3, 5, 7, 9, and 11 after administration, there was no significant difference in mouse body weight compared to the normal group (p>0.05), indicating that the three extracts from *Prunus cerasifera* root had no significant effect on mouse body weight. Figure 1 The growth of subcutaneous tumors is as follows: Figure 1 As shown, the growth rate of the model group and the crude polysaccharide group was relatively fast, with little difference between the two groups. However, the growth rate of the water extract group was significantly slower than that of the model group (p<0.05). This indicates that the alcohol and water extracts of Prunus cerasifera root have an inhibitory effect on tumor growth, while the crude polysaccharide of Prunus cerasifera root has no significant effect on tumor growth.

[0029] Figure 2 The results showed that the alcohol and water extracts reduced the expression of alpha-fetoprotein (AFP) significantly compared to the model group (p<0.05). AST and ALT are biomarkers for assessing liver damage; both levels increased in the model group, while serum levels of both approached those of the normal group after gavage administration of *Prunus serrulata* root extract to tumor-bearing mice. SOD levels increased in both the alcohol and water extract groups compared to the model group (p<0.05), while no significant change was observed in the crude polysaccharide group. MDA (malondialdehyde), as an end product of lipid peroxidation, reflects worsening oxidative damage and is positively correlated with poor prognosis; MDA levels decreased significantly in both the alcohol and water extract groups compared to the model group (p<0.05), while no significant change was observed in the crude polysaccharide group.

[0030] Example 6: Effects of White Prunus serrulata root extract on PD-L1, P-JAK1, JAK1, P-STAT3, and STAT3 proteins and PD-L1 mRNA in tumor tissues of H22 tumor-bearing mice Figure 3As shown, the expression of PD-L1 protein and mRNA in both the ethanol and water extract groups decreased compared to the model group (p<0.05). This indicates that the ethanol and water extracts of *Prunus serrulata* root can inhibit PD-L1 protein synthesis at the transcriptional level. Previous studies have confirmed that JAK1 / STAT3 pathway activation is a key mechanism for inducing PD-L1 transcriptional upregulation. Therefore, the expression levels of upstream pathway regulators P-STAT3, STAT3, P-JAK1, and JAK1 proteins were detected, and their expression in tumor tissues showed a decreasing trend. These results suggest that the antitumor effect of *Prunus serrulata* root extract may be achieved by inhibiting the JAK / STAT3 pathway, thereby reducing PD-L1 protein expression and inhibiting tumor cell immune escape mechanisms.

[0031] Example 7: Effect of C18 column eluent from the aqueous extract of Prunus cerasifera root on PD-L1 expression The aqueous extract of Prunus cerasifera root, when eluented with 20% and 40% ethanol on a C18 column, showed no cytotoxicity to HepG2 cells. The IC50 values ​​for the 60% ethanol eluent were 642.5 mg / mL, 80% ethanol eluent was 407.8 mg / mL, and 100% ethanol eluent was 139.1 mg / mL. Furthermore, at a concentration of 200 mg / mL, the different eluents showed a clear distinction in their ability to kill liver cancer cells.

[0032] For Huh7 cells, the 20% and 40% ethanol eluents showed no cytotoxicity. The IC50 values ​​for the 60% ethanol eluent were 607.2 mg / mL, 80% ethanol eluent was 406.5 mg / mL, and 100% ethanol eluent was 390.8 mg / mL. Furthermore, at a concentration of 200 mg / mL, the different eluents were able to clearly distinguish the relative killing effects of each on liver cancer cells.

[0033] For HepG2 and Huh7 cells, the 40% ethanol eluent had no effect on PD-L1 protein expression, while the 80% and 100% ethanol eluents reduced PD-L1 protein and mRNA expression in a dose-dependent manner.

[0034] Example 8: Fingerprint of water extract of Prunus cerasifera root eluent with 80-100% ethanol on C18 column and content of its active ingredients The eluent collected in Example 4 was placed in a vacuum drying centrifuge, dried, and weighed. Each eluent was dissolved in 50% methanol to prepare a 100 mg / mL mother liquid. After passing through a 0.22 μm membrane, HPLC-UV fingerprinting was performed. Syringic acid, rutin, and quercetin were prepared separately with 50% methanol to create working curves, and the corresponding compounds in the fingerprint were quantified. The chromatographic conditions were as follows: Phase A was 0.1% formic acid water, Phase B was chromatographic grade acetonitrile, column temperature was 40℃, flow rate was 1.0 ml / min, and UV detection wavelengths were 300 nm and 360 nm. Quantitative analysis showed that the roots of *Prunus persica* contained syringic acid, rutin, and quercetin at concentrations of 100.0 ± 40 mg / g (n=3), 60.0 ± 20.3 ug / g (n=3), and 101.2 ± 40.3 ug / g (n=3), respectively.

[0035] Example 9: Preparation of effective fraction granules Take 10g of the PD-L1 inhibitory active fraction obtained by eluting the root extract of *Prunus cerasifera* prepared in Example 4 through a C18 column, add 1-2.5 times the amount of dextrin, and mix thoroughly. Use an appropriate amount of 95% ethanol as a wetting agent to prepare a soft mass. Granulate the soft mass through a 12-mesh galvanized wire sieve using a granulator. Dry the wet granules at approximately 60°C. After drying, granulate the granules through a suitable sieve to remove excessively large and small particles to obtain granules. The moisture content of the granules is less than 6%, and the granules contain [missing information - likely a specific ingredient or ingredient].

[0036] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing an effective antitumor component extracted from the root of *Prunus cerasifera*, characterized in that, It is prepared by the following method: (1) Take the root of the white mountain peach, grind it into powder, add water and heat and reflux to extract, and collect the extract; (2) After concentrating the extract obtained in step (1), add ethanol for alcohol precipitation treatment to make the final concentration of ethanol reach 60%-85%, let stand, and separate the precipitate. (3) Collect the supernatant from step (2), pass it through a C18 solid phase extraction column, and obtain the antitumor effective fraction after the eluent is concentrated and dried.

2. The method for preparing the effective antitumor fraction according to claim 1, characterized in that, In step (1), the heating and reflux extraction is performed 1-3 times, with the amount of water added each time being 8-15 times the weight of the medicinal material, and the extraction time being 1-3 hours.

3. The method for preparing the effective antitumor fraction according to claim 1, characterized in that, In step (2), the final concentration of ethanol is 70%-80%, and the standing time is 8-24 hours.

4. The effective antitumor fraction according to claim 1, characterized in that, The effective components are the group of components after elution with 60%-100% ethanol from a C18 solid-phase extraction column.

5. The effective antitumor fraction according to claim 4, characterized in that, The effective fraction contains syringic acid, rutin, and quercetin at no less than 50 mg / g, 30 ug / g, and 50 ug / g, respectively.

6. The use of the antitumor effective fraction according to any one of claims 4-5 in the preparation of antitumor drugs.

7. The application according to claim 6, characterized in that, The tumor is liver cancer.

8. The application according to claim 6, characterized in that... The component group can downregulate the expression of PD-L1 protein and mRNA.

9. The application according to claim 6, 7, or 8, characterized in that, The effective antitumor components are formulated into capsules, tablets, granules, oral liquids, or injections using pharmaceutically acceptable carriers.

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