Anti-esophageal cancer metastasis paris plant active component, active ingredient, composition and application

Abstract

This invention belongs to the field of phytochemistry and pharmaceutical technology, and provides active components, active ingredients, compositions, and applications of *Paris L.* plants that inhibit esophageal cancer metastasis. The active components mainly contain saponin monomeric compounds. The active components and active ingredients of *Paris L.* plants of this invention are obtained by extraction from *Paris L.* plant materials. Experiments show that the active components and active ingredients of *Paris L.* plants have significant anti-esophageal cancer metastasis effects and can be used as drugs for anti-esophageal cancer metastasis.

Description

Technical Field

[0001] This invention belongs to the fields of phytochemistry and pharmaceutical technology, and in particular relates to active components, active ingredients, and compositions of Paris polyphylla plants that have anti-esophageal cancer metastasis properties, as well as the application of these active components and active ingredients in the preparation of anti-esophageal cancer metastasis drugs. Background Technology

[0002] Esophageal cancer is one of the most common malignant tumors of the digestive system worldwide, ranking eighth among the most frequently diagnosed cancers globally and the sixth leading cause of cancer death worldwide. Asia is a high-incidence region for esophageal cancer; in 2020, nearly 79.7% of new esophageal cancer cases occurred in Asia, which accounts for 59.5% of the global population. China has the highest incidence rate, accounting for half of the global new cases. Esophageal cancer is extremely malignant; alarmingly, up to 50% of patients already have distant lymph node or organ metastases at the time of initial diagnosis. Because metastasis is a significant factor in postoperative recurrence, the five-year survival rate for esophageal cancer patients is extremely low, less than 5%. Therefore, the search for drugs to treat metastatic esophageal cancer is urgently needed.

[0003] *Rhizoma paridis* is the dried rhizome of a perennial herbaceous plant belonging to the genus *Rhizoma* L., family Melanthiaceae. There are 26 species worldwide, 22 of which are found in my country. In East Asia and Europe, *Rhizoma* plants are commonly used in traditional medicine to treat bleeding, diarrhea, gastritis, mumps, abscesses, and snake and insect bites. In China, however, they are primarily used as a hemostatic agent, in antibacterial and antitumor drugs, and can be used to treat lung cancer, osteoma, brain tumors, and other cancers. Previous studies have shown that *Rhizoma paridis* can inhibit the invasion and migration of adenomyosis cells, breast cancer cells, and lung cancer cells by suppressing epithelial-mesenchymal transition (EMT). Only three reports exist regarding the application of Paris polyphylla in esophageal cancer: a 95% ethanol extract of Paris polyphylla can inhibit the growth of EC109 cells by inducing apoptosis and upregulating the expression of connectin 26; a 75% ethanol extract of Paris polyphylla can inhibit the growth of esophageal cancer cells by inducing apoptosis and inhibiting the COX-2 pathway; and Paris polyphylla saponin VI can induce apoptosis in esophageal cancer cells by activating the JNK pathway. However, there are no reports on the application of Paris polyphylla, its active components, or active ingredients in the preparation of drugs for treating esophageal cancer metastasis. Summary of the Invention

[0004] The purpose of this invention is to provide anti-esophageal cancer metastasis active components, active ingredients, pharmaceutical compositions, and their use in the manufacture of anti-esophageal cancer metastasis drugs from plants of the genus Paris, especially P. polyphyllavar.Yunanensis and P. fargesii.

[0005] The objective of this invention is achieved through the following technical solutions.

[0006] On one hand, the present invention provides an active component and active ingredient from Paris polyphylla plants that inhibit esophageal cancer metastasis. The active component comprises PPY-3 or PF-3, which is obtained by extraction from Paris polyphylla plant material. The active ingredient is obtained from the active component PPY-3 or PF-3 and is one or more of Paris polyphylla saponin V, diosgenin, Paris polyphylla saponin I, fibrous diosgenin, Paris polyphylla saponin II, Paris polyphylla saponin VI, 17-hydroxydiosgenin, Paris polyphylla saponin H, 17-hydroxyfibrous diosgenin, and Paris polyphylla saponin VII. PPY-3 or PF-3 and the active ingredient are prepared by a method comprising the following steps:

[0007] 1) Add an aqueous ethanol solution to the Paris genus plant material, heat under reflux to extract, obtain an extract, and then concentrate the extract to obtain a paste.

[0008] 2) The extract was completely dissolved and purified by column chromatography to obtain the active components PPY-3 and PF-3 from Paris polyphylla plants;

[0009] 3) The active components obtained in step 2) are subjected to column chromatography and semi-preparative high performance liquid chromatography in sequence to obtain the active ingredients.

[0010] In an embodiment of the present invention, the Paris species material is the rhizome of a Paris species. In a specific embodiment, Paris species refers to plants that are taxonomically classified as belonging to the Paris genus.

[0011] In an embodiment of the present invention, the active component PPY-3 contains pinotore-type steroidal saponins and diosgenin-type steroidal saponins, wherein the total saponin content is 50-60%.

[0012] In an embodiment of the present invention, the active ingredients obtained from the active component PPY-3 are Paris polyphylla saponin VII, Paris polyphylla saponin H, Paris polyphylla saponin II, Paris polyphylla saponin I, Paris polyphylla saponin V, diosgenin, fibrous diosgenin, 17-hydroxy diosgenin, and 17-hydroxy fibrous diosgenin.

[0013] In an embodiment of the present invention, the active component PF-3 contains phenotype steroidal saponins, and the total saponin content is 50-70%.

[0014] In an embodiment of the present invention, the active ingredients obtained from the active component PF-3 are Paris polyphylla saponin VII, Paris polyphylla saponin H, Paris polyphylla saponin VI, 17-hydroxydiosgenin, or 17-hydroxydiosgenin.

[0015] In a second aspect, the present invention provides a pharmaceutical composition for preventing esophageal cancer metastasis, comprising the above-mentioned active components or active ingredients of Paris polyphylla plants.

[0016] In a third aspect, the present invention provides the use of the above-mentioned active components or active ingredients of Paris species in the preparation of a medicament for treating esophageal cancer metastasis.

[0017] This invention uses Paris polyphylla plant materials as raw materials, and obtains the active components of Paris polyphylla plants that inhibit esophageal cancer metastasis through steps such as ethanol solution reflux extraction and macroporous resin separation and purification. The active components of Paris polyphylla plants are then separated by column chromatography and semi-preparative high-performance liquid chromatography to obtain active ingredients mainly composed of saponin monomers. Pharmacological experiments show that the active components or active ingredients of Paris polyphylla plants significantly inhibit the invasion and migration of esophageal cancer cells in vitro and inhibit tumor metastasis in liver and lung tissues in vivo, and can be used as drugs against esophageal cancer metastasis. Attached Figure Description

[0018] Figure 1 This is a high-performance liquid chromatogram of the active components of *Paris polyphylla* and *Paris sclerotium* from the present invention, wherein 1: Paris saponin V; 2: diosgenin; 3: Paris saponin I; 4: fine diosgenin; 5: Paris saponin II; 6: Paris saponin VI; 7: 17-hydroxydiosgenin; 8: Paris saponin H; 9: 17-hydroxyfine diosgenin; 10: Paris saponin VII.

[0019] Figure 2 The present invention relates to the active components of Paris polyphylla and Paris polyphylla sphericalis to inhibit the invasion of esophageal cancer cells.

[0020] Figure 3 The present invention relates to the active components of Paris polyphylla and Paris polyphylla spheroides to inhibit the migration of esophageal cancer cells.

[0021] Figure 4 The results show that the active components of Paris polyphylla and Paris polyphylla var. yunnanensis inhibit the expression of related proteins in the TGF-βRI / Smad and TGF-βRI / RhoA / ROCK1 pathways.

[0022] Figure 5 This invention presents the results of the active components of Paris polyphylla and Paris polyphylla var. yunnanensis inhibiting the expression of EMT-related proteins.

[0023] Figure 6 This invention relates to the effects of the active components of Paris polyphylla and Paris polyphylla spheroides on the growth of esophageal cancer tumors and the metastasis of tumors in liver and lung tissues in mice.

[0024] Figure 7 The active ingredient of this invention, Paris saponin H, inhibits the invasion and migration of esophageal cancer cells.

[0025] Figure 8The results show that the active ingredient of this invention, Paris saponin H, inhibits the expression of TGF-βRI / Smad and TGF-βRI / RhoA / ROCK1 pathways and EMT-related proteins.

[0026] Figure 9 This invention relates to the effects of the active ingredient Paris saponin H on the growth of esophageal cancer tumors and the metastasis of tumors in liver and lung tissues in mice. Detailed Implementation

[0027] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above-mentioned and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0028] On one hand, the present invention provides an active component or active ingredient of Paris polyphylla plants that resists esophageal cancer metastasis. The active component includes active component PPY-3 or PF-3 of Paris polyphylla plants. The active component PPY-3 or PF-3 is obtained by extraction of Paris polyphylla plant material. The active ingredient is obtained from active component PPY-3 or PF-3, specifically one or more of Paris saponin V, diosgenin, Paris saponin I, fibrous diosgenin, Paris saponin II, Paris saponin VI, 17-hydroxydiosgenin, Paris saponin H, 17-hydroxyfibrous diosgenin and Paris saponin VII.

[0029] On the other hand, the present invention provides a method for preparing the above-mentioned active components PPY-3 or PF-3 and active ingredients from Paris species, comprising the following steps:

[0030] 1) Add an aqueous ethanol solution to the Paris genus plant material, heat and reflux to extract, obtain an extract, and then heat and concentrate the extract to obtain a paste;

[0031] 2) The extract was completely dissolved and purified by column chromatography to obtain the active components PPY-3 and PF-3 from Paris polyphylla plants;

[0032] 3) The active components obtained in step 2) are subjected to column chromatography and semi-preparative high performance liquid chromatography in sequence to obtain the active ingredients.

[0033] In the embodiments of this aspect, the Paris species material is the dried rhizome of a Paris species.

[0034] In this embodiment, *Paris* species refer to plants that taxonomically belong to the genus *Paris*. In a specific embodiment, *Paris* species may be *P. polyphylla* var. *Yunanensis*, *P. polyphylla*, etc. The species include *Paris var. chinensis*, *Paris polyphylla*, *Paris cronquistii*, *Paris yanchii*, *Paris luquanensis*, *Paris marmorata*, *Paris lancifolia*, *Paris delavayi*, *Paris dunniana*, *Paris fargesii*, *Paris vaniotii*, *Paris qiliangiana*, *Paris caobangensis*, *Paris vietnamensis*, *Paris verticillata*, *Paris maairei*, *Paris thibetica*, *Paris forrestii*, and *Paris liiana*. In a preferred embodiment, the *Paris* species are *Paris yunnanensis* and / or *Paris fargesii*.

[0035] In an embodiment of the present invention, in step 1), the mass-to-volume ratio of the Paris polyphylla plant material to the solvent, i.e., an aqueous ethanol solution, is 1:3 to 1:10, with units of g / mL. The mass-to-volume ratio of the plant material to the solvent refers to the ratio of the mass of the material to the volume of the solvent. For example, 3 to 10 mL of solvent is used to extract each gram of the material.

[0036] In an embodiment of the present invention, in step 1), the heating reflux time is 2 to 4 hours and the number of times is 2 to 4.

[0037] In an embodiment of the present invention, in step 1), the concentration of ethanol is 70% to 95%.

[0038] In an embodiment of the present invention, in step 2), the extract can be dissolved in water.

[0039] In an embodiment of the present invention, in step 2), the column chromatography can be macroporous adsorption column chromatography, and the mass ratio of plant material to macroporous adsorption resin packing is 1:1 to 1:3, for example 1:1, 1:1.5, 1:2, 1:2.5, 1:3, etc.

[0040] In an embodiment of the present invention, in step 2), the elution solvent used for column chromatography is any one or a combination of two or more of water, methanol, ethanol, acetonitrile, etc., preferably an ethanol-water system as the eluent, with an ethanol concentration of 0%, 40%, 75%, or 95%. In a specific embodiment, in step 2), the elution volume is 5 to 10 times the mass of the plant material. The eluent containing 75% ethanol is collected, concentrated, and the active components of the Paris genus are obtained.

[0041] In an embodiment of the present invention, in step 3), column chromatography includes normal-phase silica gel column chromatography, reversed-phase silica gel column chromatography, gel column chromatography, and semi-preparative high-performance liquid chromatography (HPLC); preferably, column chromatography includes normal-phase silica gel column chromatography, reversed-phase RP-18 column chromatography, and gel column chromatography.

[0042] In an embodiment of the present invention, in step 3), the elution solvent used for normal-phase silica gel column chromatography is any one or a combination of two or more of water, chloroform, ethyl acetate, and methanol, preferably a chloroform-methanol-water system (10:1:0.1 → 1:1:0.5, v / v). The elution solvent used for reverse-phase column chromatography is any one or a combination of two or more of water, methanol, ethanol, and acetonitrile, preferably a methanol-water system as the eluent, with a methanol concentration of 40% to 90%. The mobile phase used for semi-preparative HPLC is any one or two of methanol, acetonitrile, and water, preferably an acetonitrile-water mobile phase, wherein the acetonitrile gradient is 20% to 80%. Gel column chromatography uses chloroform and methanol in a volume ratio of 1:1 for elution.

[0043] In an embodiment of the present invention, step 3) specifically includes the following sub-steps:

[0044] 3.1) The active components PPY-3 and PF-3 were mixed in equal amounts and then subjected to normal phase silica gel column chromatography. The elution was carried out with a chloroform-methanol-water system (10:1:0.1→1:1:0.5, v / v). The fractions were combined under thin-layer chromatography (TLC) to obtain five fractions: Fr.A-Fr.E. The mass ratio of silica gel to active component was 5:1, and each gradient was 3 to 5 column volumes.

[0045] 3.2) The Fr.C fraction was repeatedly subjected to reversed-phase RP-18 column chromatography (methanol-water: 4:6, 5:5, 6:4, 7:3, 1:0, v / v), eluted multiple times, and subjected to semi-preparative HPLC and recrystallization to obtain saponin monomers Paris saponin V, diosgenin, Paris saponin I, fibrous diosgenin, Paris saponin II, Paris saponin VI, 17-hydroxydiosgenin, Paris saponin H, 17-hydroxyfibrous diosgenin and Paris saponin VII, which are the active ingredients.

[0046] In an embodiment of the present invention, in step 3.2), the mobile phase used in the semi-preparative HPLC is acetonitrile and water, wherein the acetonitrile gradient is 20-80%.

[0047] In embodiments of the present invention, the active component PPY-3 mainly contains phenotype steroidal saponins and diosgenin steroidal saponins as active ingredients, with a total saponin content of 50-60%. In a specific embodiment, in the active component PPY-3, the content of Paris polyphylla saponin V is 0.1-0.4%; the content of diosgenin is 5%-9%; the content of Paris polyphylla saponin I is 14%-22%; the content of diosgenin fibrous diosgenin is 0.5%-2%; the content of Paris polyphylla saponin II is 14%-19%; the content of Paris polyphylla saponin VI is 0-0.1%; the content of both 17-hydroxydiosgenin and 17-hydroxydiosgenin fibrous diosgenin is 0.7%-2%; the content of Paris polyphylla saponin H is 2%-6%; and the content of Paris polyphylla saponin VII is 3%-11%.

[0048] In embodiments of the present invention, the active component PF-3 mainly contains phenotype steroidal saponins as active ingredients, with a total saponin content of 50-70%. In a specific embodiment, in the active component PF-3, the content of Paris polyphylla saponin VI is 8%-22.1%; the content of 17-hydroxydiosgenin or 17-hydroxydiosgenin is 2%-5%; the content of Paris polyphylla saponin H is 22%-35.1%; and the content of Paris polyphylla saponin VII is 14%-18%.

[0049] In embodiments of the present invention, the active ingredient is any one or more of the above-mentioned saponin monomer compounds, namely, any one or more of Paris saponin V, diosgenin, Paris saponin I, fibrous diosgenin, Paris saponin II, Paris saponin VI, 17-hydroxydiosgenin, Paris saponin H, 17-hydroxyfibrous diosgenin and Paris saponin VII.

[0050] In embodiments of the present invention, the above-mentioned saponin monomer compound has the following structural formula I:

[0051]

[0052] In a third aspect, the present invention provides a composition for preventing esophageal cancer metastasis, comprising the above-mentioned active components of Paris species or any one or more of the above-mentioned active ingredients, and a pharmaceutically acceptable carrier, adjuvant, and excipient.

[0053] Suitable pharmaceutical excipients are well known to those skilled in the art. Pharmaceutical carriers or excipients are one or more solid, semi-solid, and liquid diluents, fillers, and pharmaceutical excipients, including but not limited to fillers (diluents), lubricants (flow aids or anti-adhesion agents), dispersants, wetting agents, binders, solubilizers, antioxidants, antibacterial agents, emulsifiers, disintegrants, etc. Binders include syrups, gum arabic, gelatin, sorbitol, astragalus gum, cellulose and its derivatives (such as microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose, or hydroxypropyl methyl cellulose, etc.), gelatin paste, syrup, starch paste, or polyvinylpyrrolidone, etc.; fillers include lactose, powdered sugar, dextrin, starch and its derivatives, cellulose and its derivatives, inorganic calcium salts (such as calcium sulfate, calcium phosphate, calcium hydrogen phosphate, precipitated calcium carbonate, etc.), sorbitol, or glycine, etc.; lubricants include micronized silica gel, magnesium stearate, talc, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol, etc. Disintegrants include starch and its derivatives (such as sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, corn starch, etc.), polyvinylpyrrolidone, or microcrystalline cellulose; humectants include sodium dodecyl sulfate, water, or alcohol; antioxidants include sodium sulfite, sodium bisulfite, sodium metabisulfite, dibutylbenzoic acid, etc.; antibacterial agents include 0.5% phenol, 0.3% cresol, 0.5% chlorobutanol, etc.; emulsifiers include polysorbate-80, sorbitan, lecithin, soybean lecithin, etc.; and solubilizers include Tween-80, bile, glycerin, etc.

[0054] In four aspects, the present invention provides the use of the above-mentioned active components of Paris species or any one or more active ingredients in the manufacture of a medicament for treating esophageal cancer metastasis.

[0055] When used as a medicine, the compounds of the present invention can be applied directly or in the form of a pharmaceutical composition. In the pharmaceutical compositions of the present invention, the composition may contain 0.1-99%, preferably 0.5-90%, of an active ingredient or active component, such as Paris saponin H, based on the total weight of the pharmaceutical composition.

[0056] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. It should be understood that the following embodiments are given for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and intent, and all such modifications and substitutions fall within the scope of protection claimed in the present invention.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0058] Example 1: Preparation method of PPY-3, an active component of Paris polyphylla.

[0059] In this embodiment, the active components of Paris polyphylla are prepared using the following steps:

[0060] (1) The rhizome sample (5kg) of Paris polyphylla was dried and pulverized to obtain Paris polyphylla plant material;

[0061] (2) The plant material of Paris polyphylla was extracted by heating and reflux with ethanol aqueous solution. The mass-volume ratio of plant material to solvent was 1:5, and the unit was g / mL. The extraction was performed 3 times in total, with extraction times of 3h, 2h and 2h. The extracts were combined, filtered, and the filtrate was concentrated under reduced pressure to obtain a paste extract PPY-1 (1.1kg).

[0062] (3) The paste extract was dissolved in water and subjected to D101 macroporous adsorption resin column chromatography (the mass ratio of resin to plant material was 2:1). Gradient elution was performed using ethanol-water as the eluent, with a volume-to-mass ratio of plant material to eluent of 1:5 (in g / mL). The 40% ethanol eluent was PPY-2 (55.5 g), and the 75% ethanol eluent was the active component of Paris polyphylla, PPY-3 (125 g).

[0063] (4) The paste extract was dissolved in water and subjected to D101 macroporous adsorption resin column chromatography (the mass ratio of resin to plant material was 2:1). Gradient elution was performed using ethanol-water as the eluent, with a volume mass ratio of plant material to eluent of 1:5 (in g / mL). Elution was performed using 50% and 75% ethanol and 35% and 80% ethanol, respectively, to obtain 75% ethanol eluent fraction PPY-4 and 80% ethanol eluent fraction PPY-5.

[0064] Example 2: Preparation method of PF-3, the active component of Paris polyphylla spheres

[0065] In this embodiment, the active components of the spherical drug *Paris polyphylla* are prepared using the following steps:

[0066] (1) The rhizome sample (5 kg) of Paris polyphylla was dried and pulverized to obtain Paris polyphylla plant material;

[0067] (2) The plant material of Paris polyphylla was extracted by heating and reflux with ethanol aqueous solution. The mass-volume ratio of plant material to solvent was 1:5, and the unit was g / mL. The extraction was performed 3 times in total, with extraction times of 3h, 2h and 2h. The extracts were combined, filtered, and the filtrate was concentrated under reduced pressure to obtain a paste extract PF-1 (1.2kg).

[0068] (3) The paste extract was dissolved in water and subjected to D101 macroporous adsorption resin column chromatography (the mass ratio of resin to plant material was 2:1). Gradient elution was performed using ethanol-water as the eluent, with a volume mass ratio of plant material to eluent of 1:5 (in g / mL). The 40% ethanol eluent was PF-2 (76.0 g), and the 75% ethanol eluent was the active component of Paris polyphylla, PF-3 (214.0 g).

[0069] (4) The paste extract was dissolved in water and subjected to D101 macroporous adsorption resin column chromatography (the mass ratio of resin to plant material was 2:1). Gradient elution was performed using ethanol-water as the eluent, with a volume mass ratio of plant material to eluent of 1:5 (in g / mL). The 75% ethanol eluent fractions PF-4 and PF-5 were obtained by elution with 50% and 75% ethanol and 35% and 75% ethanol, respectively.

[0070] Example 3: Evaluation of the anti-toxic activity of active components of Paris polyphylla and Paris polyphylla spherical granules against esophageal cancer cells.

[0071] Human esophageal squamous cell carcinoma EC109 cells were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection. KYSE520 squamous cell carcinoma cells were purchased from the DSMZ (German Center for Microbiology and Cell Culture) at the Leibniz Institute, Germany. Both cell types were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (PS).

[0072] The effects of active components of Paris polyphylla and Leonurus japonicus on the viability of esophageal cancer cell lines EC109 and KYSE520 were evaluated using the MTT assay (see LI L, YUE GG, LEE JK, et al. The adjuvant value of Andrographis paniculata in metastatic esophageal cancer treatment - from preclinical perspectives[J]. Sci Rep, 2017, 7(1):854). [1] 5×10 3EC109 and KYSE520 cells were seeded in 100 μL of RPMI 1640 medium (containing 10% phosphate-buffered saline (PBS) and 1% PS) in 96-well plates and cultured overnight in a cell culture incubator (37°C, 5% CO2). Then, 100 μL of the above RPMI 1640 medium containing PPY-1, PPY-2, PPY-3, PF-1, PF-2, and PF-3 were added, respectively. The final concentrations of PPY-1 were 0, 12.5, 25, 50, 100, and 200 μg / mL, respectively. The same concentrations of PPY-2, PPY-3, PF-1, PF-2, and PF-3 were set, with final concentrations of 0, 3.13, 6.25, 12.5, 25, and 50 μg / mL, respectively. Cisplatin was used as a positive control, with final concentrations of 0, 2.5, 5, 10, 20, and 40 μg / mL in EC109 cells and 0, 12.5, 25, 50, 100, and 200 μg / mL in KYSE520 cells. After 48 h of cell culture, 30 μL of LTT solution (5 μg / mL) was added, and incubation continued for another 4 h. Absorbance was then measured using a microplate reader, and viability was calculated. The results showed that after 48 h of treatment, components PPY-3 and PF-3 significantly reduced the proliferation of esophageal cancer cells and exhibited the strongest activity, representing the active components of *Paris polyphylla* and *Paris spheroides* (see Table 1).

[0073] Table 1. IC50 of active components of Paris polyphylla and Paris polyphylla sclerotium in inhibiting esophageal cancer cells. 50 value

[0074]

[0075] Example 4: Isolation and identification of active ingredients from the active components of Paris polyphylla and Paris polyphylla var. yunnanensis.

[0076] In this embodiment, the active ingredient is prepared using the following steps:

[0077] (1) The active components PPY-3 and PF-3 were mixed in equal amounts and then subjected to normal silica gel column chromatography (the mass ratio of silica gel to the mixed active components was 5:1). Gradient elution was performed using a chloroform-methanol-water (10:1:0.1→1:1:0.5, v / v) system (3 to 5 column volumes for each gradient). The fractions were combined under TLC detection to obtain five fractions: Fr.A-Fr.E.

[0078] (2) The collected Fr.C fraction was a brown oily substance. It was subjected to reversed-phase RP-18 column chromatography (methanol-water: 4:6, 5:5, 6:4, 7:3, 1:0, v / v), eluted multiple times, and semi-preparative HPLC was used to obtain Paris saponin V, diosgenin, Paris saponin I, fibrous diosgenin, Paris saponin II, Paris saponin VI, 17-hydroxydiosgenin, Paris saponin H, 17-hydroxyfibrous diosgenin and Paris saponin VII. The purity of the saponin was determined to be 98% by HPLC.

[0079] Example 5: Determination of the content of active ingredients in *Paris polyphylla* and *Paris stolonifera* var. *yunnanensis* (PPY-3 and PF-3).

[0080] (1) Weigh 50 mg of the active components PPY-3, PF-3, and PPY-4, PPY-5, PF-4, and PF-5 of Paris polyphylla that have been dried overnight in a vacuum dryer, add them to a 10 mL volumetric flask, dilute to volume with chromatographic grade methanol, and store at 4 °C for later use.

[0081] (2) Weigh 5 mg of the active ingredients that have been dried overnight in a vacuum dryer, namely Paris saponin V, diosgenin, Paris saponin I, fibrous diosgenin, Paris saponin II, Paris saponin VI, 17-hydroxy diosgenin, Paris saponin H, 17-hydroxy fibrous diosgenin and Paris saponin VII, and add them to a 10 mL volumetric flask. Dilute to volume with chromatographic grade methanol to prepare a standard stock solution. Store at 4 °C for later use.

[0082] (3) The main components of the active ingredients of *Paris polyphylla* and *Paris stolonifera* were analyzed using a high-performance liquid chromatograph (Agilent 1260HPLC) with a ZORBAX SB-C18 column (150×4.6mm, 5μm, Agilent). The detection wavelength was set at 203nm, and the injection volume was 5–10μL. Acetonitrile-water was used as the mobile phase, and the gradient elution conditions were: 0–30.0 min, 36.0%–50.3% acetonitrile; 30–50 min, 50.3%–50.3% acetonitrile. Standard solutions were injected at volumes of 0.5μL, 1μL, 3μL, 5μL, 10μL, and 15μL, and the peak areas were recorded. A standard curve was plotted with peak area as the ordinate (Y) and injection volume (μg) as the abscissa (X).

[0083] The content analysis of PPY-3 and PF-3 is shown in the figure. Figure 1 The composition and content of the active components PPY-3, PF-3, and PPY-4, PPY-5, PF-4, and PF-5 of Paris polyphylla are shown in Table 2.

[0084] exist Figure 1In Table 2, 1: Paris saponin V; 2: Diosgenin; 3: Paris saponin I; 4: Slender diosgenin; 5: Paris saponin II; 6: Paris saponin VI; 7: 17-hydroxy diosgenin; 8: Paris saponin H; 9: 17-hydroxy slender diosgenin; 10: Paris saponin VII.

[0085] Table 2. Composition and content of active components of Paris polyphylla and active components of Paris polyphylla spheroidae.

[0086]

[0087] Example 6: Evaluation of the anti-esophageal cancer activity of active components in the active components of Paris polyphylla and Paris polyphylla var. yunnanensis.

[0088] The culture methods for esophageal cancer cell lines EC109 and KYSE520 were the same as in Example 3. The cytotoxic activity of the active ingredients of Paris polyphylla and Leonurus japonicus against esophageal cancer cell lines EC109 and KYSE520 was evaluated using the MTT assay, following the same method as in Example 3.

[0089] The results showed that after 48 hours of treatment, the active ingredients Paris polyphylla saponin V, diosgenin, Paris polyphylla saponin I, sclerodiclofenac saponin, Paris polyphylla saponin II, Paris polyphylla saponin VI, 17-hydroxydiosgenin, Paris polyphylla saponin H, 17-hydroxysclerodiclofenac saponin, and Paris polyphylla saponin VII all significantly inhibited the proliferation of esophageal cancer cells. 50 The values ​​are shown in Table 3, among which Paris saponin H has the strongest activity.

[0090] Table 3. IC50 of 10 active ingredients in inhibiting esophageal cancer cells 50 value

[0091]

[0092]

[0093] In Table 3, 1: Paris saponin V; 2: Diosgenin; 3: Paris saponin I; 4: Fine diosgenin; 5: Paris saponin II; 6: Paris saponin VI; 7: 17-hydroxy diosgenin; 8: Paris saponin H; 9: 17-hydroxy fine diosgenin; 10: Paris saponin VII.

[0094] Example 7: Evaluation of the anti-esophageal cancer metastasis activity of active components (PPY-3 and PF-3) and active ingredient (Paris saponin H) of Paris polyphylla and Paris polyphylla spheroidosa.

[0095] 1. Experimental Methods

[0096] 1.1 Scratch and Transwell cell migration experiments

[0097] The culture methods for esophageal cancer cell lines EC109 and KYSE520 were the same as in Example 3. The effects of the active components of *Paris polyphylla* and *Gnaphalium affine* on the invasion of esophageal cancer cell lines EC109 and KYSE520 were evaluated using a Scratch cell migration assay (see LI L, YUE GG, LEE JK, et al. The adjuvant value of Andrographis paniculata metastatic esophageal cancer treatment-from preclinical perspectives[J]. SciRep, 2017, 7(1):854). 5 1.5 × 10⁹ EC10⁹ cells and 1.5 × 10⁹ EC10⁹ cells 5 One KYSE520 cell was seeded in RPMI 1640 medium (containing 10% FBS and 1% PS) in a 24-well plate and cultured for 24 h. The RPMI 1640 medium was then replaced with RPMI 1640 medium containing 1% FBS and 1% PS to starve the cells. After 24 h, two vertical cross-scratches were made on the monolayer of cells in each well using a 200 μL pipette tip, and the scratch locations were photographed under a microscope. Then, RPMI 1640 medium (containing 10% FBS and 1% PS) containing the active components of Paris polyphylla PPY-3, PF-3, or Paris polyphylla saponin H were added, respectively. The final concentrations of the active components PPY-3 and PF-3 were 0, 0.75, 1.5, and 3 μg / mL, respectively; the final concentrations of Paris polyphylla saponin H in EC109 cells were 0, 2, 3, and 4 μM, respectively; and the final concentrations in KYSE520 cells were 0, 1, 2, and 3 μM, respectively. Cisplatin was used as a positive control, with a final concentration of 8 μM in EC109 cells and 50 μM in KYSE520 cells. After culturing for 36 h (EC109 cells) or 24 h (KYSE520 cells), images of the scratched areas were taken again. The area change of the scratched region was calculated using Tscratch software.

[0098] The effects of the active components of *Paris polyphylla* and *Gnaphalium affine* on the invasion of esophageal cancer cell lines EC109 and KYSE520 were evaluated using Transwell cell migration assays (see YUE GG, LEE JK, LI L, et al. *Andrographis paniculata* elicits anti-invasion activities by suppressing TM4SF3 gene expression and by anoikis-sensitization in esophageal cancer cells[J]. Am J Cancer Res, 2015, 5(12):3570-87). 5 × 10⁶ cells were used. 4 One EC109 cell and one KYSE520 cell were resuspended in 100 μL of RPMI 1640 medium (containing only 1% PS) and added to the upper chamber of a Tanswell assay. Then, 100 μL of RPMI 1640 medium (containing only 1% PS) containing the active components of Paris polyphylla PPY-3, PF-3, or Paris saponin H were added to the upper chamber, respectively. The final concentrations of the active components PPY-3 and PF-3 were 0, 0.75, 1.5, and 3 μg / mL, respectively; the final concentrations of Paris saponin H in EC109 cells were 0, 2, 3, and 4 μM, respectively; and the final concentrations in KYSE520 cells were 0, 1, 2, and 3 μM, respectively. Cisplatin was a positive control, with a final concentration of 8 μM in EC109 cells and 50 μM in KYSE520 cells. Then, add 500 μL of RPMI 1640 medium (containing 10% FBS and 1% PS) to the lower compartment of the Transwell. Culture for 16 h (EC109 cells) or 6 h (KYSE520 cells). Remove the upper compartment of the Transwell, fix with methanol for 3 min, stain with hematoxylin for 6 min, gently wipe away the cells on the outside of the upper compartment with cotton, and then photograph the cells on the outside of the upper compartment under a microscope to count the number of migrating cells.

[0099] 1.2 Western blot experiment

[0100] The esophageal cancer cell line EC109 was cultured using the same method as in Example 3. 8 × 10⁸ cells were cultured. 5EC109 cells were resuspended in 8 mL of RPMI 1640 medium (containing 10% FBS and 1% PS) and seeded in cell culture dishes for overnight culture. The medium was then replaced with RPMI 1640 medium (containing 10% FBS and 1% PS) containing either the active components of Paris polyphylla PPY-3, PF-3, or Paris polyphylla saponin H. The final concentrations of PPY-3 and PF-3 were 0, 1.5, 3, and 6 μg / mL, respectively; the final concentrations of Paris polyphylla saponin H were 0, 2, 3, and 4 μM, respectively. Cells were cultured for 24 h and 48 h, and total cellular protein was extracted. Protein concentration was determined using the BCA method, and the loading amount was adjusted accordingly. Western blot analysis was performed to determine the expression of proteins related to the TGF-βRI / Smad and TGF-βRI / RhoA / ROCK1 pathways, as well as the expression of EMT-related proteins.

[0101] 1.3 Mouse model of esophageal cancer xenograft

[0102] Animals: 6-8 week old male BALB / c nude mice, provided by the Laboratory Animal Service Centre of the Chinese University of Hong Kong, weighing 22.0-28.0g.

[0103] 5×10 6 EC109 cells were suspended in 200 μL PBS and injected intraperitoneally into mice. The following day, mice were divided into a control group, a positive drug treatment group (5-fluorouracil 85 mg / kg and cisplatin 3 mg / kg), and a Paris polyphylla active component treatment group (PF-3 30, 60 mg / kg; PPY-3 60, 120 mg / kg; or Paris polyphylla saponin H 7, 13.2 mg / kg). Administered the drugs daily by gavage for 21 days. The positive drug treatment group received the drugs intraperitoneally on days 9 and 13. During the treatment, mouse weight was measured twice weekly. After 21 days, mice were anesthetized by intraperitoneal injection of 75 mg / kg ketamine and 10 mg / kg toluenethiazide, followed by cardiac biopsy. Mice were then euthanized by cervical dislocation, and lung and liver tissues were removed, fixed in 10% formalin solution, and used for histological analysis.

[0104] 1.4 Metastasis of tumors in mouse liver and lung tissues

[0105] Mouse liver and lung tissues were fixed in 10% formalin for 3 days, then dehydrated, embedded, and sectioned. Liver tissue was stained with hematoxylin and eosin (HE), and lung tissue was stained with CK8. Microscopic imaging was used to evaluate tumor metastasis in the liver and lung tissues.

[0106] 1.5 Data Statistics

[0107] All in vitro data are expressed as mean ± standard deviation, and all in vivo data are expressed as mean ± standard error. Data were statistically processed using GraphPadprism software. One-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant.

[0108] 2 Experimental Results

[0109] 2.1 Effects of the active components of Paris polyphylla and Paris polyphylla spheroides on the invasion and migration of esophageal cancer cells

[0110] The results showed that both PPY-3 and PF-3 significantly inhibited the invasion and migration of esophageal cancer cells EC109 and KYSE520, and the effect was statistically significant (P<0.01). Figures 2-3 ).

[0111] 2.2 Expression of esophageal cancer metastasis-related proteins by active components of Paris polyphylla and Paris polyphylla spheroides.

[0112] The results showed that PPY-3 and PF-3 dose-dependently inhibited the expression of key TGF-β pathway proteins, including TGF-β receptor I, RhoA, Smad2 / 3, p-smad2 / 3, Smad4, ROCK1, and RhoA. Figure 4 That is, the active components of Paris polyphylla and its spherical septum can inhibit the expression of proteins related to the classical TGF-β / smad pathway and the non-classical TGF-β / RhoA / ROCK1 pathway.

[0113] 2.3 Effects of active components of Paris polyphylla and Paris polyphylla spheroides on the expression of EMT-related proteins

[0114] The results showed that the expression of E-cadherin increased in a dose-dependent manner, while the expression of β-catenin, MMP9, ZEB1, and twist decreased in a dose-dependent manner, indicating that PPY-3 and PF-3 inhibited the EMT process, thereby inhibiting the metastasis of esophageal cancer cells. Figure 5 ).

[0115] 2.4 Effects of the active components of Paris polyphylla and Paris polyphylla spheroidae on tumor growth and tumor metastasis in liver and lung tissues of mice

[0116] The results showed that PPY-3 at 120 mg / kg and PF-3 at 60 mg / kg significantly inhibited tumor growth in mice. Figure 6Liver tissue sections stained with hematoxylin and eosin (HE) and lung tissue sections stained with CK8 were compared with the control group. The results showed that treatment with PPY-3 (120 mg / kg) and PF-3 (60 mg / kg) significantly reduced the area of ​​metastatic tumors in the lung tissue of mice. Treatment with PPY-3 (60 and 120 mg / kg) and PF-3 (30 and 60 mg / kg) also significantly reduced the area of ​​metastatic tumors in the liver. Figure 6 In summary, both *Paris polyphylla* and *Paris stolonifera* active components significantly reduced tumor metastasis in liver and lung tissues in mice, and from a dosage perspective, the active components of *Paris stolonifera* showed greater potential.

[0117] 2.5 Effects of Paris polyphylla saponin H on the invasion and migration of esophageal cancer cells

[0118] The results showed that Paris saponin H significantly inhibited the invasion and migration of esophageal cancer cells EC109 and KYSE520, and the effect was statistically significant (P<0.01). Figure 7 ).

[0119] 2.6 Paris saponin H on the expression of esophageal cancer metastasis and EMT-related proteins

[0120] The results showed that Paris saponin H inhibited the expression of key proteins in the TGF-β pathway, including TGF-β receptor I, RhoA, Smad2 / 3, p-smad2 / 3, Smad4, ROCK1, and RhoA, in a dose-dependent manner. Figure 8 Specifically, the active ingredient, Paris saponin H, can inhibit the expression of proteins related to both the classical TGF-β / Smad pathway and the non-classical TGF-β / RhoA / ROCK1 pathway. E-cadherin expression increased in a dose-dependent manner, while the expression of β-catenin, MMP9, ZEB1, and twist decreased in a dose-dependent manner, indicating that Paris saponin H inhibits the EMT process, thereby suppressing the metastasis of esophageal cancer cells.

[0121] 2.7 Effects of Paris saponin H on tumor growth and tumor metastasis in liver and lung tissues of mice

[0122] The results showed that Paris polyphylla saponin H did not significantly inhibit tumor growth in mice, but it significantly reduced the metastatic area of ​​tumors in liver and lung tissues. Figure 9 That is, Paris saponin H can significantly reduce tumor metastasis in liver and lung tissues in vivo, and has anti-esophageal cancer metastasis activity. Moreover, Paris saponin H is an active ingredient of the active components of Paris polyphylla and Paris polyphylla spherical bud.

[0123] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Active components or active ingredients from Paris L. plants that inhibit esophageal cancer metastasis, characterized in that... The active component comprises PPY-3 or PF-3, an active component from the Paris genus. The active ingredient is obtained from PPY-3 or PF-3 and is one or more of the following: Paris saponin V, diosgenin, Paris saponin I, fibrous diosgenin, Paris saponin II, Paris saponin VI, 17-hydroxydiosgenin, Paris saponin H, 17-hydroxyfibrous diosgenin, and Paris saponin VII. The PPY-3 or PF-3 and the active ingredient are prepared by a method comprising the following steps: 1) Add an aqueous ethanol solution to Paris polyphylla plant material, heat under reflux to extract, obtain an extract, and then concentrate the extract to obtain a paste. 2) The extract was completely dissolved and purified by column chromatography to obtain the active components PPY-3 and PF-3 from Paris polyphylla plants; 3) The active component obtained in step 2) is subjected to column chromatography and semi-preparative high performance liquid chromatography in sequence to obtain the active ingredient.

2. Active ingredient or active ingredients according to claim 1, characterized in that The Paris species material is the rhizome of a Paris species, preferably the Yunnan Paris (P. polyphylla var. yunanensis) and / or the ball-shaped Paris (P. fargesii).

3. Active ingredient or active ingredients according to claim 1, characterized in that The active component PPY-3 contains pinotoid steroidal saponins and diosgenin steroidal saponins, with a total saponin content of 50-60%.

4. The active component or active ingredient according to claim 3, characterized in that, In the active ingredients obtained from the active component PPY-3, the content of Paris saponin V is 0.1-0.4%; the content of diosgenin is 5%-9%; the content of Paris saponin I is 14%-22%; the content of diosgenin fibrous is 0.5%-2%; the content of Paris saponin II is 14%-19%; the content of Paris saponin VI is 0-0.1%; the content of both 17-hydroxydiosgenin and 17-hydroxydiosgenin fibrous is 0.7%-2%; the content of Paris saponin H is 2%-6%; and the content of Paris saponin VII is 3%-11%.

5. The active ingredient or active ingredient according to claim 1, characterized in that, The active component PF-3 contains phenotype steroidal saponins, with a total saponin content of 50-70%.

6. Active ingredient or active ingredients according to claim 5, characterized in that In the active ingredients obtained from the active component PF-3, the content of Paris saponin VI is 8% to 22.1%; the content of 17-hydroxydiosgenin or 17-hydroxydiosgenin is 2% to 5%; the content of Paris saponin H is 22% to 35.1%; and the content of Paris saponin VII is 14% to 18%.

7. A pharmaceutical composition for preventing esophageal cancer metastasis, comprising any one of the active components or active ingredients of the Paris genus as described in any one of claims 1-6.

8. Use of any of the active components or active ingredients of the Paris genus as described in any one of claims 1-6 in the preparation of a medicament for treating esophageal cancer metastasis.

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