A steroidal saponin compound extracted from Paris polyphylla, method and application thereof

Abstract

The present invention belongs to the technical field of anti-tumor drugs, and specifically relates to a steroidal saponin compound extracted from Paris polyphylla, a method and an application thereof. The present invention discloses a steroidal saponin compound named diosgenin-3-O-β-D-fructofuranosyl-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-β-D-pyranoglucoside extracted from Paris polyphylla, a method for extracting the steroidal saponin compound from Paris polyphylla and an application thereof in the preparation of an anti-tumor drug. Steroidal saponin compounds are natural products that can significantly inhibit the proliferation of a tumor cell, especially glioma cells, U251 human glioma cells, capan-2 human pancreatic cancer cells, Hela human cervical cancer cells and HepG2 human liver cancer cells, providing a new direction for drug research and development for treating and curing tumors.

Description

Technical Field

[0001] The invention belongs to the technical field of anti-tumor drugs, and particularly relates to a steroidal saponin compound extracted from Paris polyphyllae, a method and an application thereof. Background Art

[0002] Tumors are caused by the loss of normal regulation of local tissues at the genetic level under the catalytic action of various carcinogenic factors, leading to clonal abnormal proliferation and lesions, which are one of the main causes of human death. The research and development of anti-tumor drugs has always been the focus and difficulty of research in the medical field. Anti-tumor drugs have a single mechanism, large toxic side effects, easy to develop drug resistance and poor prognosis, which has led more and more researchers to turn their attention to natural medicines. Among them, the anti-tumor effect of saponins has attracted much attention. They are widely present in plants and have complex types and characteristics. Among the steroidal saponins, the main ones that have received attention are Paris saponins, such as Paris saponin I., Paris saponin II., Paris saponin V., Paris saponin VI., Paris saponin VII., etc., as well as triterpenoid saponins such as ginsenosides, bupleurum saponins, and glycyrrhizin.

[0003] The Chinese medicine Paris polyphylla is the dried rhizome of Paris polyphylla Smithvar. yunnanensis or P. polyphylla var. chinensis of the genus Paris of the family Liliaceae. It has the effects of clearing away heat and detoxifying, reducing swelling and relieving pain, and cooling the liver. It is often used for furuncle, carbuncle, throat injury, snake bite, trauma, convulsion and other symptoms. It is the main ingredient of the famous Chinese patent medicines Yunnan Baiyao Capsules, Tongxuekang Capsules, Reduqing Tablets, etc. Pharmacological studies have found that the main active substance of Paris polyphylla is steroidal saponins, also known as Paris polyphylla saponins, which are often used in the treatment of inflammation in modern clinical practice.

[0004] Paris polyphyllavar. latifolia belongs to the genus Paris and is a variety of the Paris polyphylla group. It is mainly distributed in Shanxi, Gansu, Anhui and other places in my country, and is also distributed in Huayin City, Yaozhou District, Huanglong, Zhenba County in Shaanxi Province and the north and south slopes of the Qinling Mountains. However, the existing technology still lacks a relatively complete study on its chemical composition and exploration of its pharmacological activity. Summary of the invention

[0005] The invention provides steroidal saponin compounds extracted from Paris polyphylla, a preparation method and uses thereof, and aims to provide a method for extracting steroidal saponin compounds from Paris polyphylla and a prescription for extracting the same.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A steroidal saponin compound extracted from Paris polyphylla, the steroidal saponin compound being: diosgenin-3-O-β-D-fructofuranosyl-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside; the structural formula thereof is as follows:

[0008]

[0009] A method for extracting steroidal saponin compounds from Paris polyphyllae, comprising the following steps:

[0010] Step 1: Take the raw material of Paris polyphylla and crush its rhizome;

[0011] Step 2: Take the rhizomes crushed in step 1, add a proportion of 70% ethanol at a ratio of 1kg:(8-9)L, and perform reflux extraction at preset intervals;

[0012] Step 3: after combining the reflux extracts in step 2, recovering ethanol under reduced pressure to obtain an extract;

[0013] Step 4: Disperse the extract obtained in step 3 in water of the same number of liters as the weight of the crushed rhizomes, and then extract with petroleum ether, performing at least 3 extractions, each extraction of 3L;

[0014] Step 5: The extracted aqueous phase is extracted with water-saturated n-butanol for at least 3 times, 3 L each time, the water-saturated n-butanol extracts are combined, and the n-butanol is recovered under reduced pressure to obtain a total saponin extract;

[0015] Step 6: Take the total saponin extract sample obtained in step 5, use hydroxypropyl dextran gel column chromatography, elute with methanol, receive 500 mL as a fraction, detect by thin layer chromatography, combine the 4th to 8th fractions containing steroidal saponin compounds, and evaporate the solvent under reduced pressure to obtain 1 g of sample;

[0016] Step 7: 1 g of the sample was subjected to medium pressure preparative liquid chromatography, eluted with methanol: water at a volume ratio of 1:4 to 1:0, and 500 mL was collected as a fraction, and detected by thin layer chromatography. The 8th to 10th fractions containing steroidal saponin compounds were combined, and the solvent was evaporated under reduced pressure to obtain 120.4 mg of the sample;

[0017] Step 8: obtaining pure steroidal saponin compounds through separation and purification.

[0018] The preset reflux extraction time in step 2 is 2 hours, and the extraction is performed 5 times in total; and the semi-preparative high performance liquid chromatography method is used in step 8 to separate and purify the pure steroidal saponin compounds.

[0019] The steroidal saponin compounds extracted from Paris polyphylla are used in the preparation of anti-tumor drugs.

[0020] The anti-tumor drug is a drug for glioma, cervical cancer, liver cancer or pancreatic cancer.

[0021] The glioma is LN229 human glioma cell or U251 human glioma cell.

[0022] The cervical cancer is Hela human cervical cancer cells.

[0023] The pancreatic cancer is capan-2 human pancreatic cancer cells.

[0024] The liver cancer is HepG2 human liver cancer cell.

[0025] The steroidal saponin compound is used alone or mixed with other drugs to prepare injections, powders, pills, tablets, microcapsules, soft capsules, films, ointments, tinctures, granules or aerosols for clinical use.

[0026] Beneficial effects:

[0027] (1) The present invention provides a method for extracting steroidal saponin compounds from Paris polyphylla and a method for extracting steroidal saponin compounds from Paris polyphylla. Steroidal saponin compounds are natural products that can effectively inhibit the proliferation of tumor cells.

[0028] (2) The steroidal saponin compounds of the present invention are used as new anti-tumor drugs or auxiliary ingredients in anti-tumor drugs, and have obvious tumor inhibition effects.

[0029] (3) The steroidal saponin compounds of the present invention are isolated from Paris polyphylla and have good biological activity. The β-D-fructofuranosyl group in the sugar group of the steroidal saponin compounds is very rare. The steroidal saponin compounds have a significant inhibitory effect on glioma cells, cervical cancer cells, pancreatic cancer cells and liver cancer cells, providing a new direction for drug research and development for the treatment and cure of tumors.

[0030] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 Schematic diagram of the structure of compound PPL1 in the present invention.

[0033] Figure 2 The NMR of the compound PPL1 in the present invention is 1 H-NMR spectrum.

[0034] Figure 3 The NMR of the compound PPL1 in the present invention is 13 C-NMR spectrum.

[0035] Figure 4 The NMR of the compound PPL1 in the present invention is 1 H- 1 H COSY spectrum.

[0036] Figure 5 It is the nuclear magnetic resonance HMBC spectrum of compound PPL1 in the present invention.

[0037] Figure 6 It is the nuclear magnetic resonance NOESY spectrum of the compound PPL1 in the present invention.

[0038] Figure 7 It is the NMR HSQC spectrum of the compound PPL1 in the present invention.

[0039] The compound PPL1 is the abbreviation of diosgenin-3-O-β-D-fructofuranose-(1→6)-[α-L-rhamnose-(1→2)]-β-D-pyranoglucoside. DETAILED DESCRIPTION

[0040] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] The test methods and English abbreviations used in the following embodiments are common knowledge in the industry and are not described here one by one.

[0042] Embodiment 1:

[0043] like Figure 1 As shown, a steroidal saponin compound extracted from Paris polyphylla, the steroidal saponin compound is: diosgenin-3-O-β-D-fructofuranosyl-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-β-D-pyranoglucoside; its structural formula is as follows:

[0044]

[0045] The steroidal saponin compound PPL1 is extracted from Paris polyphylla in nature. This steroidal saponin compound has a significant inhibitory effect on glioma cells, cervical cancer cells, pancreatic cancer cells and liver cancer cells, and can be used in the development of new anti-tumor drugs.

[0046] Embodiment 2:

[0047] A method for extracting steroidal saponin compounds from Paris polyphyllae, comprising the following steps:

[0048] Step 1: Take the raw material of Paris polyphylla and crush its rhizome;

[0049] Step 2: Take the rhizomes crushed in step 1, add a proportion of 70% ethanol at a ratio of 1kg:(8-9)L, and perform reflux extraction at preset intervals;

[0050] Step 3: after combining the reflux extracts in step 2, recovering ethanol under reduced pressure to obtain an extract;

[0051] Step 4: Disperse the extract obtained in step 3 in water of the same number of liters as the weight of the crushed rhizomes, and then extract with petroleum ether, performing at least 3 extractions, each extraction of 3L;

[0052] Step 5: The extracted aqueous phase is extracted with water-saturated n-butanol for at least 3 times, 3 L each time, the water-saturated n-butanol extracts are combined, and the n-butanol is recovered under reduced pressure to obtain a total saponin extract;

[0053] Step 6: Take the total saponin extract sample obtained in step 5, use hydroxypropyl dextran gel column chromatography, elute with methanol, receive 500 mL as a fraction, detect by thin layer chromatography, combine the 4th to 8th fractions containing steroidal saponin compounds, and evaporate the solvent under reduced pressure to obtain 1 g of sample;

[0054] Step 7: 1 g of the sample was subjected to medium pressure preparative liquid chromatography, eluted with methanol: water at a volume ratio of 1:4 to 1:0, and 500 mL was collected as a fraction, and detected by thin layer chromatography. The 8th to 10th fractions containing steroidal saponin compounds were combined, and the solvent was evaporated under reduced pressure to obtain 120.4 mg of the sample;

[0055] Step 8: obtaining pure steroidal saponin compounds through separation and purification.

[0056] The preset reflux extraction time in step 2 is 2 hours, and the extraction is performed 5 times in total.

[0057] Furthermore, in step eight, semi-preparative high performance liquid chromatography is used for separation and purification to obtain pure steroidal saponin compounds.

[0058] The above technical scheme is used to extract the steroidal saponin compounds present in Paris polyphylla. The extracted steroidal saponin compounds have good biological activity and can effectively inhibit the proliferation of tumor cells. The β-D-fructofuranosyl group in the sugar group of the steroidal saponin compounds is very rare.

[0059] The steroidal saponin compounds extracted by the present invention can be used as new anti-tumor drugs or auxiliary components in anti-tumor drugs, have obvious tumor-inhibiting effects, and provide a new drug research and development direction for treating and curing tumors.

[0060] Embodiment three:

[0061] A steroidal saponin compound extracted from Paris polyphyllae is used in the preparation of anti-tumor drugs.

[0062] Furthermore, steroidal saponin compounds extracted from Paris polyphylla are used in the preparation of anti-tumor drugs.

[0063] Furthermore, the anti-tumor drug is a drug for glioma, cervical cancer, liver cancer or pancreatic cancer.

[0064] Furthermore, the glioma is LN229 human glioma cell or U251 human glioma cell; the cervical cancer is Hela human cervical cancer cell.

[0065] Furthermore, the pancreatic cancer is capan-2 human pancreatic cancer cells; the liver cancer is HepG2 human liver cancer cells.

[0066] Furthermore, the steroidal saponin compounds are used alone or mixed with other drugs to prepare injections, powders, pills, tablets, microcapsules, soft capsules, films, ointments, tinctures, granules or aerosols for clinical use.

[0067] Embodiment 4:

[0068] A method for extracting steroidal saponin compounds from Paris polyphyllae.

[0069] The chemical name of the steroidal saponin compound is: diosgenin-3-O-β-D-fructofuranosyl-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-β-D-pyranoglucoside, hereinafter referred to as PPL1.

[0070] Taking Paris polyphylla as a raw material, after crushing its rhizome, taking 2.8 kg, adding 24 L of 70% ethanol for reflux extraction, extracting for 5 times, each time for 2 hours, combining the 5 extracts, and then recovering the ethanol under reduced pressure to obtain an extract; dispersing the extract in 3 L of water, extracting with petroleum ether for 3 times, each time with 3 L; extracting the aqueous phase after extraction with water-saturated n-butanol for 3 times, each time with 3 L, combining the water-saturated n-butanol extracts, and recovering the n-butanol under reduced pressure to obtain a total saponin extract.

[0071] The sample was chromatographed on a hydroxypropyl dextran gel column and eluted with methanol. 500 mL of the sample was collected as a fraction and detected by thin layer chromatography. The 4th to 8th fractions containing PFP1 were combined and the solvent was evaporated under reduced pressure to obtain 1 g of the sample.

[0072] 1 g of the sample was subjected to medium-pressure preparative liquid chromatography, eluted with methanol:water in a volume ratio of 1:4 to 1:0, and 500 mL was received as a fraction. Thin layer chromatography was performed for detection, and the 8th to 10th fractions containing PPL1 were combined. The solvent was evaporated under reduced pressure to obtain 120.4 mg of the sample.

[0073] Finally, the pure product of PPL1 was obtained by semi-preparative high performance liquid chromatography (HPLC) separation and purification.

[0074] Embodiment five:

[0075] A method for extracting steroidal saponin compounds from Paris polyphyllae.

[0076] The raw material of Paris polyphylla for preparing PPL1 was collected from Mei County, Baoji City, Shaanxi Province. 2.8 kg of its rhizome was crushed into coarse powder, and then 24 L of 70% ethanol was added for reflux extraction. The extraction was performed 5 times, each time for 2 hours. After the 5 extracts were combined, the ethanol was recovered under reduced pressure to obtain 611 g of extract; the extract was dispersed in 3 L of water, and extracted 3 times with petroleum ether, each time with 3 L; the aqueous phase after extraction was extracted 3 times with water-saturated n-butanol, each time with 3 L, and the water-saturated n-butanol extracts were combined. After the n-butanol was recovered under reduced pressure, 135 g of total saponin extract was obtained.

[0077] The sample was chromatographed on a hydroxypropyl dextran gel (GE-Healthcare) column, eluted with methanol, and received as a fraction of 200 mL, detected by thin layer chromatography, and the 4th to 8th fractions containing PPL1 were combined, and the solvent was evaporated under reduced pressure to obtain 1 g of the sample. The 1 g sample was eluted with methanol: water at a volume ratio of 1:4 to 1:0, and received as a fraction of 500 mL, detected by thin layer chromatography, and the 8th to 10th fractions containing PPL1 were combined, and the solvent was evaporated under reduced pressure to obtain 120.4 mg of the sample.

[0078] Finally, it was separated and purified by high performance liquid chromatography (Gilson) (HPLC conditions: Hedra ODS-2 column 20×250 mm, 40% acetonitrile as mobile phase, flow rate 12 mL / min, 25° C., 206 nm UV detection) to obtain 15.6 mg of pure PPL1.

[0079] Embodiment six:

[0080] like Figure 2-Figure 7 As shown, the structure of the compound PPL1 extracted in Example 5 was identified. The specific identification process is as follows:

[0081] Compound PPL1 is a white powder, soluble in methanol, and positive in Liebermann-Burchard reaction (acetic anhydride-concentrated sulfuric acid reaction) and Molish reaction (purple ring reaction). The color reaction of 20% sulfuric acid ethanol solution is purple-red, indicating that the compound may be a steroidal saponin compound. ESI-MS gives its quasi-molecular ion peak m / z 885.4854 [M+H] + , (calc.forC 45 H 73 O 17 ,885.48), combined 13 C-NMR (200 MHz, deuterated methanol) spectrum (such as Figure 3 ) in the data, as shown in Table 1, its molecular formula is determined to be C 45 H 72 O 17 .

[0082] exist 1 H-NMR spectrum (such as Figure 2 ) shows that there are five characteristic methyl hydrogen signals in the high field region, namely: δ H 0.96(d,J=7.02Hz), 0.80(s), 0.79(d,J=6.49Hz), 1.23(d,J=6.21Hz) and 1.05(s), respectively. C 14.88, 16.77, 17.49, 17.98 and 19.85 signals correspond. 1 olefinic hydrogen proton signal δ H 5.20 and its corresponding olefin carbon signal δ C 122.65(C-6), δ C 141.89 (C-5), 1 hemiacetal quaternary carbon signal δ C 110.60(C-22). 13 There are four quaternary carbon signals in the C-NMR spectrum. C 141.89(C-5),δ C 38.03(C-10), δC 41.43(C-13), δ C 110.60 (C-22), combined with the above data, it can be determined that the aglycone of PPL1 is diosgenin. H 5.38) and H-7(δ H 1.98) 1 H- 1 H COSY (such as Figure 4 The spectrum is shown in Figure 2, indicating that the trisubstituted double bond is Δ 5(6) , in NOESY (such as Figure 6 In the spectrum shown in Figure 2, H3-19β / H-1a(δ H 1.88) and H-1b(δ H 1.10) / H-3(δ H 3.58) shows that 3-OH is in β configuration; the chemical shift values ​​of C-16 and C-17 are δ C 82.22, 63.74, indicating that there is no hydroxyl group at C-17; the chemical shift difference of H2-26 δ Ha -δ Hb =3.44-3.32=0.12<0.48, it can be determined that C 25 It is R configuration.

[0083] Ion chromatography and derivatization analysis of compound PPL1 after acid hydrolysis showed that its sugar moieties were D-glucose, L-rhamnose and D-fructose, with a composition ratio of 1:1:1. 1 H-NMR spectrum (such as Figure 2 The terminal hydrogen signals of D-glucose and L-rhamnose are shown in Figure 5 H 4.49 (d, J = 7.84 Hz, Glc H-1′), 5.20 (br s, Rha H-1″), in HSQC (e.g. Figure 7 The corresponding carbon signal (δ C 100.85,102.15), and the carbon signal of D-fructose (δ C 105.15). The Glc terminal hydrogen coupling constant can be used to determine that the glycosidic bond is in β configuration. The chemical shifts of rhamnose C-3 and C-5 can be used to infer that the glycosidic bond configuration is α configuration. Figure 5As shown in the figure, glucose H-1 and aglycone C-3 have long-range correlation signals, indicating that glucose is connected to the C-3 position of the aglycone; rhamnose H-1 and glucose C-2 have long-range correlations, indicating that rhamnose is connected to the C-2 position of glucose; fructose H-1 and glucose Glc C-6 have long-range correlations, indicating that fructose H-1 is connected to the C-6 position of glucose. In summary, the structure of PPL1 was identified as diosgenin-3-O-β-D-fructofuranosyl-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-β-D-pyranoglucoside.

[0084] Table 1:

[0085] Compound 1 H and 13 C NMR data (test solvent: deuterated methanol)

[0086]

[0087] Embodiment seven:

[0088] The invention discloses an application of steroidal saponin compounds extracted from Paris polyphyllae. The steroidal saponin compounds extracted from Paris polyphyllae are applied to the preparation of anti-tumor drugs.

[0089] Furthermore, the anti-tumor drug is a drug for glioma, cervical cancer, pancreatic cancer, liver cancer or gastric cancer.

[0090] Furthermore, the glioma is LN229 human glioma cell or U251 human glioma cell.

[0091] Furthermore, the cervical cancer is Hela human cervical cancer cells.

[0092] Furthermore, the pancreatic cancer is capan-2 human pancreatic cancer cells.

[0093] Furthermore, the liver cancer is HepG2 human liver cancer cells.

[0094] The compound PPL1 separated and purified in Example 5 was subjected to an in vitro anti-tumor test.

[0095] The cells used in the experiment were human glioma cells (LN229, U251), human cervical cancer cells (Hela), human pancreatic cancer cells (capan-2), and human liver cancer cells (HepG2); the conventional CCK-8 method was used for testing.

[0096] The implementation method is as follows: Tumor cells U251, LN229, Hela, HepG2 and capan-2 cells with good growth in the logarithmic growth phase were taken and the cell density was adjusted to 6×104 / mL. The cell suspension was inoculated on a 96-well plate, 100 μL / well, and placed in a constant temperature CO2 incubator for 24 hours before administration. The cells were divided into a blank control group, a positive control group and an experimental group. The concentrations of PPL1, PolyphyllinⅠ, PolyphyllinⅡ and Dioscin were set to 0.5, 1, 2, 4, 8, 16, 32 and 64 μM, and 3 replicates were set for each group. After 24 hours of cell culture, 10 μL of CCK-8 reagent was added to each well according to the instructions for use of the CCK-8 reagent, and the plates were incubated in a constant temperature incubator for 2 hours. The absorbance value (OD value) of each well was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay. The inhibition rate of the drug on tumor cells was calculated (calculation formula: cell inhibition rate = (OD value of the experimental group - OD value of the blank control group) / OD value of the blank control group × 100%). SPSS16.0 was used to calculate the half inhibition concentration (IC50), and IC50>20μM indicated no cytotoxic effect.

[0097] Among them, the Paris saponin I, Paris saponin II and Dioscin are products obtained from the total saponin mixture in the process of preparing the steroidal saponin compound PPL1 according to thin layer chromatography detection and in vitro anti-tumor activity screening.

[0098] The test results are shown in Table 2 below.

[0099] Table 2

[0100] Comparative experimental data of the inhibitory effects of compounds PPL1, Paris saponin I, Paris saponin II and dioscin on five tumor cell lines LN229, U251, Capan-2, Hela and HepG2 (IC50 value, μM)

[0101]

[0102] The results showed that the compound PPL1 had stronger effects on the five tumor cells than Paris saponin II and Dioscorea saponin. The inhibitory effect on HepG2 (human liver cancer cells) was slightly weaker than Paris saponin I, and the inhibitory effect on Hela (human cervical cancer cells) was equivalent to Paris saponin I, while the inhibitory effects on the rest of the tumor cells were stronger than Paris saponin I.

[0103] Summary: The steroidal saponin compound PPL1 of the present invention has a significant inhibitory effect on several human tumor cell lines including LN229 (human glioma cells), U251 (human glioma cells), capan-2 (human pancreatic cancer cells), Hela (human cervical cancer cells) and HepG2 (human liver cancer cells), and the half inhibitory concentrations (IC50 values) are 4.18±0.31, 3.85±0.44, 3.26±0.34, 3.30±0.38 and 4.32±0.51, respectively.

[0104] Embodiment eight:

[0105] The invention discloses an application of steroidal saponin compounds extracted from Paris polyphyllae. The steroidal saponin compounds are used alone or mixed with other drugs to prepare injections, powders, pills, tablets, microcapsules, soft capsules, films, ointments, tinctures, granules or aerosols for clinical use.

[0106] In the absence of conflicts, technicians in this field can combine the relevant technical features in the above examples according to actual conditions to achieve corresponding technical effects. The specific combinations are not described here one by one.

[0107] It should be noted that all directional indications in the embodiments of the present invention (such as up, down, left, right, front, back, etc.) are only used to explain the relative position relationship, movement status, etc. between the components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indication will also change accordingly.

[0108] In addition, the descriptions of "first", "second", etc. in the present invention are only for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" or "second" may explicitly or implicitly include at least one of the features.

[0109] The above are only preferred embodiments of the present invention. The present invention will not be limited to the embodiments shown herein, but should conform to the widest scope consistent with the principles and novel features disclosed herein. Any simple modification, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for extracting steroidal saponin compounds from Paris polyphylla, characterized in that: The following steps are included: Step 1: Take the raw material of Paris polyphylla and crush its rhizome; Step 2: Take the rhizomes crushed in step 1 and add 70% ethanol in a proportion of 1kg: (8-9)L, and perform reflux extraction at preset intervals; Step 3: after combining the reflux extracts in step 2, recovering ethanol under reduced pressure to obtain an extract; Step 4: Disperse the extract obtained in step 3 in water of the same number of liters as the weight of the crushed rhizomes, and then extract with petroleum ether, performing at least 3 extractions, each extraction of 3L; Step 5: The extracted aqueous phase is extracted with water-saturated n-butanol for at least 3 times, 3 L each time, the water-saturated n-butanol extracts are combined, and the n-butanol is recovered under reduced pressure to obtain a total saponin extract; Step 6: Take the total saponin extract sample obtained in step 5, use hydroxypropyl dextran gel column chromatography, elute with methanol, receive 500 mL as a fraction, detect by thin layer chromatography, combine the 4th to 8th fractions containing steroidal saponin compounds, and evaporate the solvent under reduced pressure to obtain 1 g of sample; Step 7: 1 g of the sample was subjected to medium pressure preparative liquid chromatography, eluted with methanol: water at a volume ratio of 1:4 to 1:0, and 500 mL was collected as a fraction, and detected by thin layer chromatography. The 8th to 10th fractions containing steroidal saponin compounds were combined, and the solvent was evaporated under reduced pressure to obtain 120.4 mg of the sample; Step 8: obtaining pure steroidal saponin compounds through separation and purification.

2. The method for extracting steroidal saponin compounds from Paris polyphylla as claimed in claim 1, characterized in that: The preset reflux extraction time in step 2 is 2 hours, and the extraction is performed 5 times in total; and the semi-preparative high performance liquid chromatography method is used in step 8 to separate and purify the pure steroidal saponin compounds.

3. A method for extracting steroidal saponin compounds from Paris polyphylla as claimed in claim 1 or 2, characterized in that: The steroidal saponin compound is: diosgenin-3-O-β-D-fructofuranosyl-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside; its structural formula is as follows: 。

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