Application of solanum nigrum fruit steroid saponin extract in preparation of medicine for preventing or treating tumors and preparation method of solanum nigrum fruit steroid saponin extract

Abstract

The invention discloses an application of a steroid saponin extract of solanum nigrum fruit in preparation of a medicine for preventing and / or treating tumors. The compound is Desmettianoside B and / or Solaniroside I, and / or a hydrolysis product of the Desmettianoside B and the Solaniroside I. The invention further discloses a preparation method of the compound. The medicine at least has any one of the following effects: (1) inhibiting human non-small cell lung cancer; (2) inhibiting precancerous lesions of human oral mucosa; (3) inhibiting the growth of colorectal cancer cells; and (4) inhibiting human liver cancer cell growth activity. Experiments prove that the solanum nigrum fruit steroid saponin extract has good inhibitory activity in inhibiting non-small cell lung cancer cells, human oral mucosa precancerous lesion cells, human colorectal cancer cells, mouse colon cancer cells, human liver cancer cells and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to application of Solanum nigrum L. fruit steroidal saponin extract in preparation of a drug for preventing or treating tumors and a preparation method thereof. BACKGROUND

[0002] Solanum nigrum L., also known as "bitter herbs", "bitter grass", "water eggplant", etc., is an annual herbaceous plant of Solanaceae, and its mature fruit is black purple berry, which is distributed all over the country. Solanum nigrum L. is cold in nature and bitter in taste, and has the effects of clearing heat and resolving toxins, promoting blood circulation and removing blood stasis, and can be used for treating boils and contusions. Studies have shown that Solanum nigrum L. extract also has many effects such as anti-inflammatory and hepatoprotective, antioxidant and the like. In view of this, it is particularly important to explore and separate the active ingredients for the comprehensive development and utilization of Solanum nigrum L.

[0003] Patent CN118453722A discloses Solanum nigrum L. fruit total saponin extract, extraction method and application. The enriched Solanum nigrum L. fruit total saponin is extracted by using ethanol infiltration and petroleum ether extraction combined with macroporous adsorption resin gradient elution method in Solanum nigrum L. fruit. The patent improves the extraction precision of steroidal saponins and steroidal alkaloids without destroying the chemical structure of the effective active ingredients in Solanum nigrum L. fruit. At present, there is still a lack of in-depth separation and research on the specific chemical components of steroidal saponins in Solanum nigrum L. fruit, and relevant monomer compounds have not been screened out and their biological activities have not been studied, which is still the key to current research. SUMMARY

[0004] The purpose of the present application is to provide a Solanum nigrum L. fruit steroidal saponin extract in preparation of a drug for preventing or treating tumors and a preparation method thereof.

[0005] The purpose of the present application is achieved by the following technical solutions.

[0006] In one aspect, the present application provides a Solanum nigrum L. fruit steroidal saponin extract in preparation of a drug for preventing and / or treating tumors.

[0007] Further, the compound is Desmettianoside B and / or Solanigroside I, and / or a hydrolysis product of Desmettianoside B and Solanigroside I.

[0008] Further, the drug at least has any one of the following effects:

[0009] (1) Inhibiting human non-small cell lung cancer;

[0010] (2) Inhibiting oral mucosa precancerous lesions;

[0011] (3) inhibiting colorectal cancer cell growth;

[0012] (4) inhibiting human hepatoma cell growth activity.

[0013] Further, the drug has at least one of the following effects:

[0014] (1) inhibiting human non-small cell lung cancer cell A549 and PC-9 growth;

[0015] (2) inhibiting the proliferation of human oral mucosa precancerous lesion cells DOK;

[0016] (3) inhibiting the proliferation of human colorectal cancer cells DLD1 and SW480;

[0017] (4) inhibiting the proliferation of mouse colon cancer cells CT-26 and MC 38;

[0018] (5) inhibiting the growth activity of human hepatoma cells Hep3B and HepG2.

[0019] Further, the amount of Desmettianoside B and / or Solanigroside I used is a therapeutically effective amount.

[0020] Further, the Desmettianoside B and / or Solanigroside I is prepared by extraction and separation from Solanum nigrum L. fruit powder.

[0021] Further, the Solanum nigrum L. fruit steroidal saponin extract is prepared by extracting effective components from Solanum nigrum L. fruit powder.

[0022] A pharmaceutical composition comprising a therapeutically effective amount of the Desmettianoside B and / or

[0023] Solanigroside I, and / or hydrolysis products of Desmettianoside B and Solanigroside I, and a pharmaceutically acceptable carrier.

[0024] Further, the drug has at least one of the following effects:

[0025] (1) inhibiting human non-small cell lung cancer;

[0026] (2) inhibiting human oral mucosa precancerous lesions;

[0027] (3) inhibiting colorectal cancer cell growth;

[0028] (4) inhibiting human hepatoma cell growth activity.

[0029] Further, the pharmaceutical composition has at least one of the following effects:

[0030] (1) inhibiting the growth of human non-small cell lung cancer cells A549 and PC-9;

[0031] (2) inhibiting the proliferation of human oral mucosa precancerous lesion cells DOK;

[0032] (3) inhibiting the proliferation of human colorectal cancer cells DLD1 and SW480;

[0033] (4) inhibiting the proliferation of mouse colon cancer cells CT-26 and MC 38;

[0034] (5) inhibiting the growth activity of human hepatoma cells Hep3B and HepG2.

[0035] The present application also provides an extraction process of Desmettianoside B and / or Solanigroside I, comprising the following steps:

[0036] (1) taking dry Solanum nigrum fruit powder as the solvent to extract the ethanol extract of Solanum nigrum fruit by ultrasonic extraction method;

[0037] (2) removing impurities and purifying the ethanol extract of Solanum nigrum fruit by AB-8 resin;

[0038] (3) enriching and refining the product obtained in step (2) by MCI column to obtain a crude steroidal saponin;

[0039] (4) separating the crude steroidal saponin by silica gel column chromatography to obtain a steroidal saponin mixture containing Desmettianoside B and Solanigroside I;

[0040] (5) further separating the steroidal saponin mixture by silica gel column chromatography, and recrystallizing the product, i.e. obtaining high-purity Desmettianoside B monomer and Solanigroside I monomer.

[0041] As a preferred technical solution of the present application, the Solanum nigrum fruit powder in step (1) is selected from the dry fruits of Solanum nigrum L. in Solanaceae, which is crushed into a particle size of 24-65 mesh;

[0042] The Solanum nigrum fruit powder is extracted by ethanol for multiple times (such as 2-6 times), and each ultrasonic extraction time is 20-60 min, preferably 20-40 min, and in a preferred embodiment, the ultrasonic extraction time is 30 min;

[0043] The ratio of the fruit of Solanum nigrum to ethanol is 1g:3-10mL, preferably 1g:4-6mL, and in a preferred embodiment, the ratio of the fruit of Solanum nigrum to ethanol is 1g:5mL;

[0044] The ethanol has a concentration of 50%-70%.

[0045] As a preferred technical solution of the present application, step (2) uses AB-8 macroporous resin column to remove impurities and purify, and the ratio of the wet weight of the resin to the dry weight of the medicinal material is 1:1 (w / w);

[0046] Before sampling, the ethanol extract is diluted to an ethanol volume percentage of 20%-30%, and when the resin column is purified, 25% ethanol is first eluted for 2-4 times the column volume, and then 50%-70% ethanol is eluted for 7-9 times the column volume, and the 50%-70% ethanol eluate is collected.

[0047] In a preferred embodiment, 25% ethanol is first eluted for 3 times the column volume, and then 50%-70% ethanol is eluted for 8 times the column volume, and the 50%-70% ethanol eluate is collected.

[0048] As a preferred technical solution of the present application, step (3) uses SBC MCI GEL F resin (Chengdu Kepu Biological) for column chromatography, and the volume ratio of the dry weight of the medicinal material to the resin is 2g:1-3mL, preferably 2g:1mL;

[0049] Before loading, the sample is diluted to an ethanol volume percentage of 20%-30%, and when the MCI column is operated, 25% ethanol is first eluted for 2-4 times the column volume, and then anhydrous ethanol is eluted, and the anhydrous ethanol eluate is collected, concentrated to dryness under reduced pressure, to obtain a crude steroidal saponin.

[0050] As a preferred technical solution of the present application, when step (4) is performed by silica gel column chromatography, the crude steroidal saponin is dissolved in methanol and mixed with silica gel, and gradient elution is performed with dichloromethane:isopropanol=3:1, 2:1, 1:1 and pure isopropanol in terms of volume ratio;

[0051] The elution part of dichloromethane:isopropanol=2:1→1:1 is collected to obtain a mixture containing Desmettianoside B and Solanigroside I.

[0052] As a preferred technical solution of the present application, when step (5) is performed by silica gel column chromatography, elution is performed with dichloromethane:isopropanol=2:1→1:1 in terms of volume ratio to obtain a crude product with a purity of >80%, and then recrystallization with methanol is performed to obtain pure Desmettianoside B and Solanigroside I with a purity of >95%.

[0053] As a preferred technical scheme of the present application, the mass ratio of the mixture sample to silica gel in step (5) is 1:80-100.

[0054] A solanum fruit steroidal saponin extract is prepared by the above-mentioned extraction process, and contains Desmettianoside B monomer and Solanigroside I monomer with purity ≥95%.

[0055] The structural formula of Desmettianoside B is as follows:

[0056]

[0057] The structural formula of the compound produced by hydrolysis of Desmettianoside B (demethylation) is as follows:

[0058]

[0059] The structural formula of Solanigroside I is as follows:

[0060]

[0061] The structural formula of the compound produced by hydrolysis of Solanigroside I (demethylation) is as follows:

[0062]

[0063] Experiments have verified that the solanum fruit steroidal saponins Desmettianoside B and Solanigroside I of the present application can significantly inhibit the growth of non-small cell lung cancer cells A549 and PC-9. When the concentration of Desmettianoside B reaches 64 μg / mL, the inhibition rate of A549 cells is 92.85%, and the inhibition rate of PC-9 cells is 80.46%, and the corresponding IC50 values are 6.749 μg / mL and 0.8672 μg / mL, respectively; when the concentration of Solanigroside I reaches 16 μg / mL, the inhibition rate of A549 cells is 95.2%, and when the concentration of Solanigroside I reaches 4 μg / mL, the inhibition rate of PC-9 cells is 85.49%, and the corresponding IC50 values are 1.962 μg / mL and 1.097 μg / mL, respectively.

[0064] The Desmettianoside B and Solanigroside I of the application can significantly inhibit the proliferation of human oral mucosa precancerous lesion cells DOK, and the inhibition shows obvious concentration dependence. When the concentration of Desmettianoside B reaches 16 μg / mL, the inhibition rate of Desmettianoside B on DOK cells is 68.52%, and when the concentration of Solanigroside I is 8 μg / mL, the inhibition rate of Solanigroside I on DOK cells is 70.9%, and the corresponding IC50 values are 3.647 μg / mL and 1.439 μg / mL, respectively.

[0065] The Desmettianoside B and Solanigroside I of the application can significantly inhibit the proliferation of human colorectal cancer cells DLD1 and SW480, and the inhibition shows obvious concentration dependence. When the concentration of Desmettianoside B reaches 16 μg / mL, the inhibition rate of Desmettianoside B on DLD1 cells is 70.33%, and the inhibition rate of Desmettianoside B on SW480 cells is 78.06%, and the corresponding IC50 values are 2.29 μg / mL and 2.864 μg / mL, respectively; when the concentration of Solanigroside I reaches 8 μg / mL, the inhibition rate of Solanigroside I on DLD1 cells is 70.65%, and the inhibition rate of Solanigroside I on SW480 cells is 83.88%, and the corresponding IC50 values are 1.128 μg / mL and 0.7404 μg / mL, respectively.

[0066] The Desmettianoside B and Solanigroside I of the application can significantly inhibit the proliferation of mouse colon cancer cells CT-26 and MC 38, and the inhibition shows obvious concentration dependence. When the concentration of Desmettianoside B reaches 16 μg / mL, the inhibition rate of Desmettianoside B on CT-26 cells is 80.97%, and when the concentration is 8 μg / mL, the inhibition rate of Desmettianoside B on MC38 cells is 73.59%, and the corresponding IC50 values are 1.679 μg / mL and 1.684 μg / mL, respectively; when the concentration of Solanigroside I reaches 4 μg / mL, the inhibition rate of Solanigroside I on CT-26 cells is 78.74%, and when the concentration is 8 μg / mL, the inhibition rate of Solanigroside I on MC38 cells is 72.6%, and the corresponding IC50 values are 0.9833 μg / mL and 1.042 μg / mL, respectively.

[0067] The Desmettianoside B and Solanigroside I of the present application can significantly inhibit the proliferation of human liver cancer cells Hep3B and HepG2, and the inhibition effect shows obvious concentration dependence. When the concentration of Desmettianoside B reaches 16 μg / mL, the inhibition rate of Hep3B cells is 88.52%, and the inhibition rate of HepG2 cells is 77.02%, and the corresponding IC50 values are 4.774 μg / mL and 6.255 μg / mL respectively; when the concentration of Solanigroside I reaches 8 μg / mL, the inhibition rate of Hep3B cells is 78.37%, and the inhibition rate of HepG2 cells is 77.75%, and the corresponding IC50 values are 1.78 μg / mL and 1.276 μg / mL respectively.

[0068] Compared with the prior art, the present application has the following beneficial effects:

[0069] The present application is verified by in vitro cell experiments that Desmettianoside B and Solanigroside I show strong inhibition effect on multiple tumor cell lines: IC 50 values are generally not more than 5 μg / mL (especially the IC 50 of Solanigroside I on SW480 colorectal cancer cells is as low as 0.74 μg / mL). Desmettianoside B and Solanigroside I are expected to be well applied in the preparation of anti-non-small cell lung cancer drugs, anti-oral mucosa precancerous lesion drugs, anti-colorectal cancer drugs, anti-colon cancer drugs and anti-liver cancer drugs.

[0070] The present application successfully extracts two specific steroidal saponins Desmettianoside B and Solanigroside I from Solanum nigrum by adopting the process combination of ethanol ultrasonic extraction, AB-8 resin impurity removal, MCI column enrichment and gradient silica gel column chromatography separation. Especially, by adopting dichloromethane-isopropanol gradient silica gel column chromatography (3:1→2:1→1:1→pure isopropanol), and directional collection of 2:1→1:1 elution part, Desmettianoside B and Solanigroside I are effectively separated, and the chemical purity is verified to be ≥95% by HPLC-ELSD, which provides a standardized active ingredient for subsequent drug development. BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1 HPLC-ELSD diagram of Desmettianoside B;

[0072] Figure 2HPLC-ELSD chromatogram of Solanigroside I;

[0073] Figure 3 Total ion current chromatogram (TIC) of Desmettianoside B in water;

[0074] Figure 4 Extracted ion chromatogram of m / z 1243.5954 of Desmettianoside B in water;

[0075] Figure 5 Tandem mass spectrometry (MS / MS) chromatogram of m / z 1243.5954 ion of Desmettianoside B in water;

[0076] Figure 6 Total ion current chromatogram (TIC) of Solanigroside I in water;

[0077] Figure 7 Extracted ion chromatogram of m / z 1329.6321 of Solanigroside I in water;

[0078] Figure 8 Tandem mass spectrometry (MS / MS) chromatogram of m / z 1329.6321 ion of Solanigroside I in water;

[0079] Figure 9 Inhibitory effect of Desmettianoside B (A) and Solanigroside I (B) on lung cancer cell growth;

[0080] Figure 10 Inhibitory effect of Desmettianoside B (A) and Solanigroside I (B) on human oral mucosa precancerous lesion cell DOK growth;

[0081] Figure 11 Inhibitory effect of Desmettianoside B (A) and Solanigroside I (B) on colorectal cancer cell growth;

[0082] Figure 12 Inhibitory effect of Desmettianoside B (A) and Solanigroside I (B) on mouse colon cancer cell growth;

[0083] Figure 13 Inhibitory effect of Desmettianoside B (A) and Solanigroside I (B) on human hepatoma cell Hep3B and HepG2 growth. DETAILED DESCRIPTION

[0084] The present invention will be described in further detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereby.

[0085] Unless otherwise specified, all experimental materials and reagents used were commercially purchased.

[0086] Example 1

[0087] The preparation method of Solanum nigrum fruit steroidal saponin extract (Desmettianoside B and Solanigroside I) is as follows:

[0088] The Solanum nigrum fruit was crushed into coarse powder, and the dried Solanum nigrum fruit powder was taken and ultrasonically extracted 4 times with 5 times the amount of 60% ethanol, each ultrasonication for 30 minutes. The extract was filtered, and the filtrates of the 4 extractions were combined. An appropriate amount of pure water was added to the filtrate to dilute it to an alcohol content of 25%, and then applied to an AB-8 macroporous resin column. The wet weight of the resin was roughly equal to the dry weight of the medicinal material (that is, the ratio of the wet weight of the resin to the dry weight of the medicinal material was 1:1 (w / w)). It was first eluted with 25% ethanol for 3 times the column volume, and then eluted with 60% ethanol for 8 times the column volume. The 60% ethanol eluate was collected and diluted with an appropriate amount of pure water to an alcohol content of 25%.

[0089] The diluted solution was filtered through an SBC MCI GEL (F type, 70 μm) column with a resin dosage of 2 g medicinal material: 1 ml (m:V, i.e., 2 g medicinal material uses 1 ml resin). The solution was first eluted with 25% ethanol for 3 times the column volume, then with anhydrous ethanol. The anhydrous ethanol eluate was collected and concentrated to dryness under reduced pressure to obtain a crude steroidal saponin with a yield of 1.8%.

[0090] The crude steroidal saponin was dissolved in methanol and mixed with silica gel. The product was then separated by column chromatography on a silica gel column using gradient elution using dichloromethane:isopropanol = 3:1, 2:1, 1:1, and pure isopropanol. The fraction eluting from dichloromethane:isopropanol = 2:1 → 1:1 was collected to obtain a mixture containing desmettianoside B and solanigroside I.

[0091] The mixture was mixed with silica gel and subjected to silica gel column chromatography again (sample:silica gel = 1:80), eluting with dichloromethane:isopropanol = 2:1 → 1:1 to obtain crude desmettianoside B and solanigroside I with a purity of approximately 80%. After recrystallization from methanol, pure desmettianoside B and solanigroside I were obtained with a purity greater than 95%, with weight yields of approximately 0.08% and 0.05%, respectively.

[0092] The purity was detected by HPLC-ELSD, and the specific conditions were as follows: detector: evaporative light scattering detector (ELSD), drift tube temperature was 50℃, carrier gas pressure was 3.5 bar, and gain was 6. Chromatographic column: Ultimate XB-C18 chromatographic column (4.6 x 250 mm, 5 μm); column temperature was 25℃; 0.1% formic acid-water solution was used as mobile phase A, and acetonitrile was used as mobile phase B for gradient elution (0 min-20 min, 3%-20% B; 20 min-42 min, 20%-30% B; 42 min-55 min, 30%-50% B; 55 min-65 min, 50%-65% B; 65 min-75 min, 65%-90% B; 75 min-80 min, 90%-100% B. The HPLC-ELSD chromatogram of Desmettianoside B is shown in Figure 1 The HPLC-ELSD chromatogram of Solanigroside I is shown in Figure 2 .

[0093] Further, Desmettianoside B will undergo hydrolysis reaction after being dissolved in water, and the methoxy group at C-22 position is changed into hydroxyl group, and the hydrolysis product formed is: (5α, 22α, 25R)-26-O-(β-D-glucopyranosyl)-22-hydroxy-furost-3β, 26-diol-3-O-β-D-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→4)-O-β-D-galactopyranoside.

[0094] The structural formula is:

[0095]

[0096] The molecular weight of the compound is 1244.6037, and the molecular formula is: C 57 H 96 O 29 The structure is highly similar to Desmettianoside B (molecular weight is 1258.6188), and only differs in C-22 position. The mass spectrometry data ESI(-) m / z: 1243.5954 [M-H] - , calculated value: 1243.5959. m / z: 1081.53 [M-H-glc] - , m / z: 757.43 [M-H-glc-glc-glc] - , see Figure 5 .

[0097] The specific steps of sample preparation are as follows: 0.501 mg of Desmettianoside B powder is accurately weighed into a 10 mL volumetric flask, dissolved with ultrapure water and diluted to the mark, mixed thoroughly, and a standard solution with a concentration of 50.1 μg / mL is obtained. After the solution is placed at room temperature for 2 h, centrifugation is performed at 4℃ and 12000 rpm for 15 min, and the supernatant is used for liquid chromatography-mass spectrometry analysis.

[0098] The specific detection conditions of liquid chromatography-mass spectrometry are as follows: an ultra-high performance liquid chromatography-linear ion trap / orbitrap combined high-resolution mass spectrometer (UPLC-LTQ-Orbitrap-MS / MS) is used for analysis. The chromatographic column is ACQUITY UPLC BEH C 18 The chromatographic column is 100 mm x 2.1 mm, 1.7 μm, Waters, the column temperature is 40℃, the injection volume is 10 μL. The mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is 0.1% formic acid acetonitrile solution, the flow rate is 0.3 mL / min, and the gradient elution program is as follows: 0-10 min: 3%-20% B; 10-21 min: 20%-30% B; 21-23 min: 30%-50% B; 23-33 min: 50%-65% B; 33-37 min: 65%-90% B; 37-40 min: 90%-100% B; 40-45 min: 100%-3% B.

[0099] The mass spectrometry analysis uses an electrospray ion source (Electrospray ionization, ESI) in negative ion scanning mode. The capillary spray voltage is 3.2 kV, the ion source temperature is 400℃, the capillary temperature is 350℃, the sheath gas flow rate is 35 arb, the auxiliary gas flow rate is 15 arb, and high-purity nitrogen gas (purity > 99.999%) is used as the atomizing and auxiliary gas. Fourier transform (FT) is used for full scan of the primary mass spectrometry, the resolution is 60,000, and the scan range is m / z 50-1800 Da; the secondary mass spectrometry uses data-dependent acqusition (DDA) mode, the resolution is 30,000, the collision mode is collision induced dissociation (CID), and the collision energy is 35 eV.

[0100] The total ion chromatogram (TIC), m / z 1243.5954 extracted ion chromatogram, and tandem mass spectrometry (MS / MS) chromatogram of m / z 1243.5954 ion are specifically shown in Figures 3-5 .

[0101] Carbon spectral data of the hydrolysis product of Desmettianoside B is: 13 C-NMR data (100 MHz, C5D5N) δ: 37.2, 29.9, 77.5, 34.8, 44.7, 28.9, 32.4, 35.2, 54.4, 35.8, 21.3, 40.2, 41.1, 56.4, 32.4, 81.1, 64, 16.4, 12.3, 40.7, 16.7, 110.6, 37.2, 28.4, 34.3, 75.2, 17.5, 102.4, 73.2, 75.6, 80.2, 75.3, 60.6, 104.9, 75.3, 78.6, 71.6, 78.6, 62.3, 104.5, 76.1, 77.9, 70.9, 78.6, 63, 104.9, 81.5, 88.5, 70.8, 77.4, 62.3, 105.1, 75.2, 78.6, 71.7, 78.5, 62.8.

[0102] Carbon spectral data of Desmettianoside B is: 13 C-NMR data (100 MHz, C5D5N) δ: 37.2, 29.9, 77.5, 34.8, 44.6, 28.9, 32.4, 35.2, 54.4, 35.8, 21.2, 40.0, 41.1, 56.3, 32.1, 81.3, 64.3, 16.3, 12.3, 40.5, 16.5, 112.6, 30.8, 28.2, 34.2, 75.2, 17.1, 102.4, 73.2, 75.6, 80.2, 75.3, 60.6, 104.9, 75.3, 78.6, 71.6, 78.6, 62.3, 104.5, 76.1, 77.9, 70.9, 78.6, 63.0, 105.0, 81.4, 88.4, 70.8, 77.4, 62.3, 105.1, 75.2, 78.6, 71.7, 78.5, 62.9, 47.3.

[0103] The results show that the molecular ion peak of m / z 1243.59 is detected in the negative ion mode of the mass spectrum of the aqueous solution of Desmettianoside B; at the same time, no ion signal of m / z 1257.59 (corresponding to the molecular ion peak of Desmettianoside B) is detected. The above results suggest that Desmettianoside B will undergo demethylation under aqueous solution conditions, converting the methoxyl group at C-22 into a hydroxyl group, thereby generating compound (5α, 22α, 25R)-26-O-(β-D-glucopyranosyl)-22-hydroxy-furost-3β, 26-diol-3-O-β-D-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→4)-O-β-D-galactopyranoside.

[0104] Further, Solanigroside I will undergo hydrolysis after being dissolved in water to form the hydrolyzed compound

[0105] Solanigroside J:

[0106] (22α, 25R)-26-O-β-D-glucopyranosyl-22-hydroxyl-5α-furost-3β, 26-diol-3-O-α-L-arabinopyranosyl-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→4)-O-[α-L-rhamnopyranosyl-(1→2)]-β-D-galactopyranoside, abbreviated as Solanigroside J.

[0107] The structural formula is:

[0108]

[0109] The molecular weight of the compound is 1330.6405, and the molecular formula is: C 61 H 102 O 31 The structure is highly similar to that of Solanigroside I (molecular weight 1344.6562), with only a difference at C-22. Solanigroside I is converted into Solanigroside J after being dissolved in water, and its mass spectrometry data are: ESI(-) m / z: 1329.6321 [M-H] -, calculated: 1329.6327. m / z: 1197.58 [M-H-Ara] - , m / z: 1065.54 [M-H-Ara-Xyl] - , m / z: 903.49 [M-H-Ara-Xyl-Glc] - , see Figure 8 .

[0110] The specific steps for sample preparation are as follows: 0.591 mg of Solanigroside I powder is accurately weighed into a 10 mL volumetric flask, and purified water is added to constant volume and mixed thoroughly to obtain a standard solution with a concentration of 59.1 ug / mL. The standard solution after 2 hours of dissolution is centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant is taken for sample analysis. The instrument conditions are as follows. The results show that after Solanigroside I is dissolved in water, a compound with a molecular weight of 1329.63 is detected in the negative mode, which is consistent with Solanigroside J, and no ion peak of Solanigroside I with a molecular weight of 1243.63 is detected. Therefore, after Solanigroside I is dissolved in water, demethylation reaction occurs, converting the methoxy group at position 22 to a hydroxyl group.

[0111] 0.591 mg of Solanigroside I powder is accurately weighed into a 10 mL volumetric flask, dissolved with ultrapure water and constant volume to the mark, mixed thoroughly to obtain a standard solution with a concentration of 50.1 ug / mL. After the solution is placed at room temperature for 2 hours, it is centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant is used for liquid chromatography-mass spectrometry analysis.

[0112] Liquid chromatography-mass spectrometry detection conditions: analysis using ultra-high performance liquid chromatography-linear ion trap / orbitrap combined high-resolution mass spectrometry (UPLC-LTQ-Orbitrap-MS / MS). The chromatographic column uses ACQUITY UPLC BEH C 18 The chromatographic column is 100 mm x 2.1 mm, 1.7 μm, Waters, the column temperature is 40°C, the injection volume is 10 μL. The mobile phase A is 0.1% formic acid in water, the mobile phase B is 0.1% formic acid in acetonitrile, the flow rate is 0.3 mL / min, and the gradient elution program is as follows: 0-10 min: 3%-20% B; 10-21 min: 20%-30% B; 21-23 min: 30%-50% B; 23-33 min: 50%-65% B; 33-37 min: 65%-90% B; 37-40 min: 90%-100% B; 40-45 min: 100%-3% B.

[0113] Mass spectrometry analysis used an electrospray ion source (ESI) in negative ion scan mode. The capillary spray voltage was 3.2 kV, the ion source temperature was 400 °C, the capillary temperature was 350 °C, the sheath gas flow rate was 35 arb, and the auxiliary gas flow rate was 15 arb, with high purity nitrogen (purity > 99.999%) used as the atomizing and auxiliary gas. The first mass spectrometry used Fourier Transform (FT) for full scan with a resolution of 60,000 and a scan range of m / z 50-1800 Da; the second mass spectrometry used a data-dependent acquisition (DDA) mode with a resolution of 30,000 and a collision mode of collision-induced dissociation (CID) with a collision energy of 35 eV.

[0114] The total ion current (TIC), m / z 1243.5954 extracted ion chromatogram, and tandem mass spectrometry (MS / MS) chromatogram of m / z 1243.5954 ions are shown in Figures 6-8 .

[0115] The carbon spectrum data of solanigroside J is: 13 C-NMR data (100 MHz, C5D5N) δ: 37.2, 29.9, 77, 34.4, 44.6, 28.9, 32.4, 35.2, 54.4, 35.9, 21.2, 40.2, 41 1, 5.3, 32.3, 81.1, 63.9, 16.7, 12.4, 40.6, 16.4, 110.6, 37.1, 28.3, 34.2, 75.3, 17.4, 100.1, 77.3, 76.1, 81.4, 75, 60.3, 101.6, 72.4, 72.7, 74, 69.6, 18.4, 105.4, 81.5, 87.8, 70.3, 77.6, 62.8, 105.8, 73.2, 74.7, 69.7, 67.3, 104.9, 75.1, 78.7, 70.6, 67.3, 104.9, 75, 78.5, 71.6, 78.4, 62.8.

[0116] The carbon spectrum data of solanigroside I is: 13C-NMR data (100 MHz, C5D5N) δ: 37.2, 29.9, 77.0, 34.4, 44.6, 28.9, 32.4, 35.2, 54.4, 35.9, 21.2, 39.9, 41.1, 56.3, 32.1, 81.3, 64.3, 16.5, 12.4, 40.5, 16.3, 112.6, 30.8, 28.1, 34.2, 75.2, 17.1, 47.2, 100.0, 77.4, 76.1, 81.5, 75.0, 60.3, 101.6, 72.4, 71.7, 74.0, 69.6, 18.5, 105.4, 81.5, 87.8, 70.3, 77.6, 62.8, 105.8, 73.2, 74.7, 69.7, 67.3, 104.9, 75.1, 78.7, 70.6, 67.3, 104.9, 75.0, 78.6, 71.7, 78.5, 62.9.

[0117] The results show that the aqueous solution of Solanigroside I detects a molecular ion peak of m / z 1329.63 in the negative ion mode of mass spectrometry, and the molecular weight is consistent with that of Solanigroside J; at the same time, no ion signal of m / z 1343.63 (corresponding to the molecular ion peak of Solanigroside I) is detected. The above results suggest that Solanigroside I will undergo demethylation under aqueous solution conditions, convert the methoxy group at C-22 into a hydroxyl group, and thus generate compound Solanigroside J.

[0118] Example 2

[0119] Experimental of the inhibitory activity of compounds Desmettianoside B and Solanigroside I on the growth of human non-small cell lung cancer cells A549 and PC-9.

[0120] Experimental materials: human non-small cell lung cancer cells A549 and PC-9 were purchased from the Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences. RPMI1640, fetal bovine serum, penicillin and streptomycin were purchased from Invitrogen Company of the United States. CCK-8 reagent was purchased from Sigma Company of the United States.

[0121] Experimental method: human non-small cell lung cancer cells A549 and PC-9 were inoculated into RPMI1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, and placed in a 37°C, 5% CO2 and saturated humidity incubator for culture. The cells were passaged by 0.25% membrane protease digestion, and the cells in the logarithmic growth phase were used for experiments.

[0122] The above logarithmic growth phase cells were prepared into a cell suspension (8 x 10 4 cells / mL), and human non-small cell lung cancer cells A549 and PC-9 were inoculated into 96-well plates at a cell concentration of 8 x 10 3 cells / well, and cultured. When the cells were well attached and grew well, the original culture medium was discarded, and a culture medium containing 10% serum was replaced, and different concentrations of desmettianoside B and Solanigroside I were added to the culture medium and incubated with the cells for 48 hours. Then 10 μL CCK8 was added to the culture medium, and the reaction was carried out in the incubator for 4 hours. The culture medium was discarded, 100 μL DMSO was added for complete dissolution, and finally the absorbance was measured at 450 nm wavelength. The absorbance of the measured substance was obtained by subtracting the absorbance of the blank from the measured absorbance.

[0123] The calculation formula of cell inhibition rate is as follows:

[0124] As: experimental hole (culture medium containing cells, CCK-8 solution and drug solution)

[0125] Ac: control hole (culture medium containing cells and CCK-8 solution)

[0126] Ab: blank hole (culture medium and CCK-8 solution without cells)

[0127] Inhibition rate = [(Ac-As) / (Ac-Ab)] x 100%

[0128] Experimental results: the results are shown in Figure 9 After treating human non-small cell lung cancer cells A549 and PC-9 with different concentrations of Desmettianoside B and Solanigroside I, the biological activity of human non-small cell lung cancer cells A549 and PC-9 decreased significantly. When the concentration of Desmettianoside B reached 64 μg / mL, the inhibition rate of A549 cells was 92.85%, and the inhibition rate of PC-9 cells was 80.46%, and the IC50 values were 6.749 μg / mL and 0.8672 μg / mL, respectively. When the concentration of Solanigroside I reached 16 μg / mL, the inhibition rate of A549 cells was 95.2%, and when the concentration of Solanigroside I reached 4 μg / mL, the inhibition rate of PC-9 cells was 85.49%, and the IC50 values were 1.962 μg / mL and 1.097 μg / mL, respectively.

[0129] The results show that the Desmettianoside B and Solanigroside I can significantly inhibit the proliferation of human non-small cell lung cancer cells A549 and PC-9, and the inhibition effect shows obvious concentration dependence, that is, the higher the concentration of the two, the stronger the inhibition effect on the growth activity of human non-small cell lung cancer cells A549 and PC-9.

[0130] Example 3

[0131] The experiment of the compound Desmettianoside B and Solanigroside I inhibiting the growth activity of human oral mucosa precancerous lesion cells DOK.

[0132] Experimental materials: human oral mucosa precancerous lesion cells DOK are purchased from Shanghai Institute of Biochemistry and Cell Biology of Chinese Academy of Sciences. RPMI1640, fetal bovine serum, penicillin and streptomycin are purchased from Invitrogen Company of the United States. CCK-8 reagent is purchased from Sigma Company of the United States.

[0133] Experimental method: human oral mucosa precancerous lesion cells DOK are inoculated on RPMI1640 medium containing 10% fetal bovine serum, 100U / mL penicillin and 100ug / mL streptomycin, and are placed in a 37℃, 5%CO2 and saturated humidity incubator for culture, and are digested and subcultured with 0.25% membrane protease, and the cells in logarithmic growth phase are used for experiment.

[0134] The above logarithmic growth phase cells are prepared into cell suspension (8x10 4 6 / mL), and the DOK cells are inoculated into 96-well plates at a cell concentration of 8x10 3 4 / well, and are cultured, and when the cells are well adhered and grow well, the original culture medium is discarded, and the culture medium containing 10% serum is replaced, and different concentrations of compounds desmettianoside B and Solanigroside I are added to the culture medium and incubated with cells for 48 hours; then 10ul CCK8 is added to the culture medium, and is placed in the incubator for reaction for 4 hours, and then the culture medium is discarded, and 100ul DMSO is added for full dissolution, and finally the absorbance is measured at 450nm wavelength, and the measured absorbance is subtracted from the absorbance of the blank to obtain the absorbance of the test substance.

[0135] The calculation formula of cell inhibition rate is as follows:

[0136] As: experimental hole (culture medium containing cells, CCK-8 solution and drug solution)

[0137] Ac: control hole (culture medium containing cells and CCK-8 solution)

[0138] Ab: Blank well (culture medium and CCK-8 solution without cells)

[0139] Inhibition rate = [(Ac-As) / (Ac-Ab)] × 100%

[0140] Experimental results: The results are as follows Figure 10 As shown, the bioactivity of human oral mucosal precancerous lesion DOK cells was significantly decreased after treatment with different concentrations of Desmettianoside B and Solanigroside I. When the concentration of Desmettianoside B reached 16 μg / mL, its inhibition rate on DOK cells was 68.52%, and when the concentration was 8 μg / mL, the inhibition rate of Solanigroside I on DOK cells was 70.9%, with IC50 values ​​of 3.647 μg / mL and 1.439 μg / mL, respectively.

[0141] The results showed that Desmettianoside B and Solanigroside I of the present invention can significantly inhibit the proliferation of human oral mucosal precancerous lesion cells DOK, and the inhibitory effect shows obvious concentration dependence, that is, the higher the concentration of the two, the stronger the inhibitory effect on the growth activity of human oral mucosal precancerous lesion cells DOK.

[0142] Example 4

[0143] Experiment on the inhibition of the growth activity of human colorectal cancer cells DLD1 and SW480 by the compounds Desmettianoside B and Solanigroside I.

[0144] Experimental Materials: Human colorectal cancer cells DLD1 and SW480 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai. RPMI1640, fetal bovine serum, penicillin, and streptomycin were purchased from Invitrogen, USA. CCK-8 reagent was purchased from Sigma, USA.

[0145] Experimental method: Human colorectal cancer cells DLD1 and SW480 were inoculated into RPMI1640 culture medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, respectively, and cultured in an incubator at 37°C, 5% CO2 and saturated humidity. The cells were digested and passaged with 0.25% trypsin, and the cells in the logarithmic growth phase were used for the experiment.

[0146] Take the cells in the logarithmic growth phase and prepare a cell suspension (8×10 4 / mL), 8×10 3Human colorectal cancer cells DLD1 and SW480 were seeded into 96-well plates at a cell concentration of 100 μL / well and cultured. When the cells were well attached and growing, the original culture medium was discarded and replaced with culture medium containing 10% serum. Different concentrations of desmettianoside B and solanigroside I were added to the culture medium and incubated with the cells for 48 hours. 10 μL CCK8 was then added to the culture medium, and the cells were reacted in an incubator for 4 hours before the culture medium was discarded. 100 μL DMSO was added to fully dissolve the cells, and the absorbance was measured at a wavelength of 450 nm. The absorbance of the analyte was obtained by subtracting the absorbance of the blank from the measured absorbance.

[0147] The calculation formula of cell inhibition rate is as follows:

[0148] As: Experimental wells (culture medium containing cells, CCK-8 solution, and drug solution)

[0149] Ac: control well (culture medium containing cells and CCK-8 solution)

[0150] Ab: Blank well (culture medium and CCK-8 solution without cells)

[0151] Inhibition rate = [(Ac-As) / (Ac-Ab)] × 100%

[0152] Experimental results: The results are as follows Figure 11 As shown, treatment of human colorectal cancer cells DLD1 and SW480 with different concentrations of desmettianoside B and solanigroside I significantly decreased the bioactivity of these cells. At a concentration of 16 μg / mL, desmettianoside B exhibited an inhibitory rate of 70.33% against DLD1 cells and 78.06% against SW480 cells, with IC50 values ​​of 2.29 μg / mL and 2.864 μg / mL, respectively. At a concentration of 8 μg / mL, solanigroside I exhibited an inhibitory rate of 70.65% against DLD1 cells and 83.88% against SW480 cells, with IC50 values ​​of 1.128 μg / mL and 0.7404 μg / mL, respectively.

[0153] The results showed that the two Solanum nigrum fruit steroidal saponins of the present invention can significantly inhibit the proliferation of human colorectal cancer cells DLD1 and SW480, and the inhibitory effect showed obvious concentration dependence, that is, the higher the concentration of Desmettianoside B and Solanigroside I, the stronger the inhibitory effect on the growth activity of human colorectal cancer cells DLD1 and SW480.

[0154] Example 5

[0155] Experiment on the inhibition of the growth activity of mouse colon cancer cells CT-26 and MC38 by the compounds Desmettianoside B and Solanigroside I.

[0156] Experimental Materials: Mouse colon cancer cells CT-26 and MC 38 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai. RPMI1640, fetal bovine serum, penicillin, and streptomycin were purchased from Invitrogen, USA. CCK-8 reagent was purchased from Sigma, USA.

[0157] Experimental methods: Mouse colon cancer cells CT-26 and MC 38 were inoculated into RPMI1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, respectively, and cultured in an incubator at 37°C, 5% CO2, and saturated humidity. The cells were digested and passaged with 0.25% trypsin, and cells in the logarithmic growth phase were used for experiments.

[0158] Take the cells in the logarithmic growth phase and prepare a cell suspension (8×10 4 / mL), 8×10 3 Mouse colon cancer cells CT-26 and MC 38 were seeded into 96-well plates at a cell concentration of 100 μL / well and cultured. When the cells were well attached and growing, the original culture medium was discarded and replaced with culture medium containing 10% serum. Different concentrations of desmettianoside B and solanigroside I were added to the culture medium and incubated with the cells for 48 hours. 10 μL CCK8 was then added to the culture medium, and the cells were reacted in an incubator for 4 hours before the culture medium was discarded. 100 μL DMSO was added to fully dissolve the cells, and the absorbance was measured at a wavelength of 450 nm. The absorbance of the analyte was obtained by subtracting the absorbance of the blank from the measured absorbance.

[0159] The calculation formula of cell inhibition rate is as follows:

[0160] As: Experimental wells (culture medium containing cells, CCK-8 solution, and drug solution)

[0161] Ac: control well (culture medium containing cells and CCK-8 solution)

[0162] Ab: Blank well (culture medium and CCK-8 solution without cells)

[0163] Inhibition rate = [(Ac-As) / (Ac-Ab)] × 100%

[0164] Experimental results: The results are as follows Figure 12As shown, treatment of mouse colon cancer cells CT-26 and MC 38 with different concentrations of desmettianoside B and solanigroside I significantly decreased the bioactivity of these cells. Desmettianoside B at a concentration of 16 μg / mL exhibited an inhibition rate of 80.97% against CT-26 cells, and at a concentration of 8 μg / mL, an inhibition rate of 73.59% against MC38 cells, with IC50 values ​​of 1.679 μg / mL and 1.684 μg / mL, respectively. Solanigroside I at a concentration of 4 μg / mL exhibited an inhibition rate of 78.74% against CT-26 cells, and at a concentration of 8 μg / mL, an inhibition rate of 72.6% against MC38 cells, with IC50 values ​​of 0.9833 μg / mL and 1.042 μg / mL, respectively.

[0165] The results showed that Desmettianoside B and Solanigroside I of the present invention can significantly inhibit the proliferation of mouse colon cancer cells CT-26 and MC 38, and the inhibitory effect shows obvious concentration dependence.

[0166] Example 6

[0167] Experiment on the inhibition of the growth activity of human liver cancer cells Hep3B and HepG2 by the compounds Desmettianoside B and Solanigroside I.

[0168] Experimental Materials: Human hepatocellular carcinoma cells Hep3B and HepG2 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai. RPMI1640, fetal bovine serum, penicillin, and streptomycin were purchased from Invitrogen, USA. CCK-8 reagent was purchased from Sigma, USA.

[0169] Experimental method: Human liver cancer cells Hep3B and HepG2 were inoculated into RPMI1640 culture medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, respectively, and cultured in an incubator at 37°C, 5% CO2 and saturated humidity. The cells were digested and passaged with 0.25% trypsin, and the cells in the logarithmic growth phase were used for the experiment.

[0170] Take the cells in the logarithmic growth phase and prepare a cell suspension (8×10 4 / mL), 8×10 3The human liver cancer cells Hep3B and HepG2 were inoculated into 96-well plates at a cell concentration of 1 cell / hole, and were cultured. When the cells were well attached and grew in a good state, the original culture solution was discarded, and the culture solution containing 10% serum was replaced, and different concentrations of Desmettianoside B and Solanigroside I were added to the culture solution and incubated with the cells for 48 hours. Then, 10 μL CCK8 was added to the culture solution, and the culture solution was discarded after reaction in a culture box for 4 hours. Then, 100 μL DMSO was added for full dissolution. Finally, the absorbance was measured at a wavelength of 450 nm, and the absorbance of the blank was subtracted from the measured absorbance to obtain the absorbance of the tested substance.

[0171] The calculation formula of the cell inhibition rate is as follows:

[0172] As: experimental hole (culture medium containing cells, CCK-8 solution and drug solution)

[0173] Ac: control hole (culture medium containing cells and CCK-8 solution)

[0174] Ab: blank hole (culture medium and CCK-8 solution without cells)

[0175] Inhibition rate = [(Ac-As) / (Ac-Ab)]x100%

[0176] Experimental results: the results are shown in Table 1. Figure 13 After the human liver cancer cells Hep3B and HepG2 were treated with different concentrations of Desmettianoside B and Solanigroside I, the biological activity of the human liver cancer cells Hep3B and HepG2 was significantly decreased. When the concentration of Desmettianoside B reached 16 μg / mL, the inhibition rate of Hep3B cells was 88.52%, and the inhibition rate of HepG2 cells was 77.02%, and the IC50 values were 4.774 μg / mL and 6.255 μg / mL, respectively. When the concentration of Solanigroside I reached 8 μg / mL, the inhibition rate of Hep3B cells was 78.37%, and the inhibition rate of HepG2 cells was 77.75%, and the IC50 values were 1.78 μg / mL and 1.276 μg / mL, respectively.

[0177] The results show that Desmettianoside B and Solanigroside I of the application can significantly inhibit the proliferation of human liver cancer cells Hep3B and HepG2, and the inhibition effect shows obvious concentration dependence.

[0178] The foregoing description of the embodiments has been presented for the purpose of illustration and description. It is not intended to be exhaustive or to limit the application to the precise form disclosed. Modifications and variations are possible in light of the above teachings or can be acquired from practice of the application. As well, the description is presented in the context of the preferred embodiments as a number of alternatives. It is not intended to limit the application to the precise form described.

Claims

1. A use of a steroidal saponin extract from Solanum nigrum fruit in the preparation of a drug for preventing and / or treating tumors, characterized in that: The Solanum nigrum fruit steroidal saponin extract is Desmettianoside B and / or Solanigroside I, and / or the hydrolysis product of Desmettianoside B and Solanigroside I.

2. The use according to claim 1, characterized in that The drug has at least one of the following effects: (1) Inhibit human non-small cell lung cancer; (2) inhibiting precancerous lesions of the human oral mucosa; (3) Inhibit the growth of colorectal cancer cells; (4) Inhibit the growth activity of human liver cancer cells.

3. The use according to claim 2, characterized in that The drug has at least one of the following effects: (1) Inhibit the growth of human non-small cell lung cancer cells A549 and PC-9; (2) Inhibit the proliferation of human oral mucosal precancerous lesion cells DOK; (3) inhibiting the proliferation of human colorectal cancer cells DLD1 and SW480; (4) Inhibit the proliferation of mouse colon cancer cells CT-26 and MC 38; (5) Inhibit the growth activity of human liver cancer cells Hep3B and HepG2.

4. The use according to any one of claims 1 to 3, characterized in that The amount of the Solanum nigrum fruit steroidal saponin extract Desmettianoside B and / or Solanigroside I used is a therapeutically effective amount.

5. The use according to claim 1, characterized in that The desmettianoside B and / or solanigroside I are prepared by extraction and separation using Solanum nigrum fruit powder as raw material.

6. A pharmaceutical composition, characterized in that The invention comprises a therapeutically effective amount of Desmettianoside B and / or Solanigroside I, and / or a hydrolyzate of Desmettianoside B and Solanigroside I according to claim 1, and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition has at least one of the following effects: (1) Inhibit human non-small cell lung cancer; (2) inhibiting precancerous lesions of the human oral mucosa; (3) Inhibit the growth of colorectal cancer cells; (4) Inhibit the growth activity of human liver cancer cells.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition has at least one of the following effects: (1) Inhibit the growth of human non-small cell lung cancer cells A549 and PC-9; (2) Inhibit the proliferation of human oral mucosal precancerous lesion cells DOK; (3) inhibiting the proliferation of human colorectal cancer cells DLD1 and SW480; (4) Inhibit the proliferation of mouse colon cancer cells CT-26 and MC 38; (5) Inhibit the growth activity of human liver cancer cells Hep3B and HepG2.

9. The extraction process of Desmettianoside B and / or Solanigroside I according to claim 6, characterized in that, The following steps are involved: (1) taking dried Solanum nigrum fruit powder and using ethanol as solvent to obtain Solanum nigrum fruit ethanol extract by ultrasonic extraction; (2) removing impurities and purifying the Solanum nigrum fruit ethanol extract by passing it through AB-8 resin; (3) enriching and refining the product obtained in step (2) through an MCI column to obtain a crude steroidal saponin; (4) separating the crude steroidal saponin product by silica gel column chromatography to obtain a steroidal saponin mixture containing desmettianoside B and solanigroside I; (5) subjecting the steroidal saponin mixture to silica gel column chromatography separation again or multiple times, and recrystallizing the product to obtain a steroidal saponin extract rich in desmettianoside B and / or solanigroside I.

10. The extraction process according to claim 10, characterized in that The Solanum nigrum fruit powder in step (1) is selected from the dried fruit of Solanum nigrum of the Solanaceae family, and is crushed into particles with a size of 24 to 65 meshes; The Solanum nigrum fruit powder is subjected to multiple ultrasonic extractions with ethanol, each ultrasonic extraction time being 20-60 minutes, preferably 20-40 minutes; The solid-liquid ratio of the Solanum nigrum fruit to ethanol is 1g:3-10mL, preferably 1g:4-6mL; The ethanol has a concentration of 50% to 70%.

11. The extraction process of the Solanum nigrum fruit steroidal saponin extract according to claim 9, characterized in that: Step (2) using an AB-8 macroporous resin column for impurity removal and purification, with the ratio of resin wet weight to medicinal material dry weight being 1:1 (w / w); Before injection, the ethanol extract was diluted to 20%-30% ethanol by volume. During resin column purification, 2-4 column volumes were first eluted with 25% ethanol, and then 7-9 column volumes were eluted with 50%-70% ethanol. The 50%-70% ethanol eluate was collected.

12. The extraction process according to claim 10, characterized in that Step (3) filtration was performed using an SBC MCI GEL F resin column, with a volume ratio of the medicinal material dry weight to the resin of 2 g: 1-3 mL; Before injection, the sample is diluted to 20%-30% by volume of ethanol. When operating the MCI column, first elute with 25% ethanol for 2-4 times the column volume, then elute with anhydrous ethanol, collect the anhydrous ethanol eluate, and concentrate to dryness under reduced pressure to obtain the crude steroidal saponin.

13. The extraction process according to claim 10, characterized in that In step (4), when performing silica gel column chromatography separation, the crude steroidal saponin is dissolved in methanol and then mixed with silica gel. Gradient elution is performed using dichloromethane:isopropanol = 3:1, 2:1, 1:1 and pure isopropanol, respectively, in a volume ratio; The elution fractions at the ratio of dichloromethane:isopropyl alcohol = 2:1→1:1 were collected to obtain a mixture containing desmettianoside B and solanigroside I.

14. The extraction process according to claim 10, characterized in that In step (5), the mass ratio of the mixture sample to the silica gel is 1:80-100; In step (5), silica gel column chromatography was used for elution with a volume ratio of dichloromethane:isopropanol = 2:1→1:1 to obtain a crude product with a purity >80%, which was then recrystallized from methanol to obtain pure products of desmettianoside B and solanigroside I with a purity greater than 95%.

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