Three saponin compounds, methods and applications

Abstract

The application discloses three new saponin compounds, methods and applications, the compounds are named as anthenoside K1, anthenoside K2 and anthenoside K3, the method comprises the following steps: S1, extracting, extracting the saponin compounds of starfish; S2, separation and purification are carried out to obtain anthenoside K1, K2 and K3 pure products; the application verifies that the compounds have obvious inhibiting effects on the Notch signal pathway through the in-vitro inhibiting Notch signal pathway activity screening test of the compounds anthenoside K1-K3, provides scientific basis for screening new compounds with antitumor activity, and has important significance for the development and utilization of Chinese marine medicinal biological resources.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to three new saponin compounds, methods, and applications. Background Technology

[0002] The rimmed starfish (Craspidaster hesperus) is a type of starfish belonging to the family Craspidasteridae in the coastal areas of Guangdong, Fujian, Zhejiang, and the Spratly Islands of my country. Its body is pentagonal, with five narrow, tapering arms reaching over 5 cm in length and approximately 3.5 cm in diameter. The aboral surface is densely covered with small columnar structures, each topped with 1-20 hemispherical granules and surrounded by 7-20 radially arranged small spines connected by membranes. The upper marginal plate typically has around 30 plates, roughly rectangular in shape, large and thick, arranged neatly like a rim. The lower marginal plate is opposite the upper marginal plate, and equal in number.

[0003] The rimmed starfish has extremely high medicinal value [Liao Yulin, ed., Fauna Sinica, Phylum Echinodermata, Class Asteridae [M]. Beijing: Science Press, 1997, 116-117]. Further research and discovery are needed regarding the medicinal value of the rimmed starfish. Summary of the Invention

[0004] To address the aforementioned issues, this invention provides saponin compounds anthenosideK1, K2, and K3 extracted and isolated from rimmed starfish, and verifies that these three compounds inhibit the Notch signaling pathway, providing a new approach for the research of anti-tumor drugs.

[0005] To achieve this objective, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides three novel saponin compounds, named anthenoside K1, anthenoside K2, and anthenoside K3, the chemical structural formulas of which are as follows:

[0007]

[0008] Preferably, the anthenoside K1 is a colorless crystalline powder with the molecular formula C. 41 H 68 O 14 Both the Liebermann-Burchard reaction and the Molish reaction were positive.

[0009] Preferably, the anthenoside K2 is a colorless crystalline powder with the molecular formula C2. 41 H 70 O 14Both the Liebermann-Burchard reaction and the Molish reaction were positive.

[0010] Preferably, the anthenoside K3 is a colorless crystalline powder with the molecular formula C3. 42 H 79 O 14 Both the Liebermann-Burchard reaction and the Molish reaction were positive.

[0011] A second aspect of the present invention provides a method for extracting the novel saponin compounds described herein, comprising the following steps:

[0012] S1. Extraction: Extracting saponin compounds from starfish.

[0013] S2. Separation and purification yielded pure anthenoside K1, K2, and K3.

[0014] Preferably, step S1 includes: pulverizing dried starfish, extracting with ethanol under hot reflux, combining the extracts, concentrating under reduced pressure to obtain a fluid extract, uniformly dispersing the fluid extract in water, first extracting repeatedly with petroleum ether, then extracting the aqueous phase with n-butanol, combining the n-butanol extracts, recovering the n-butanol under reduced pressure, and obtaining a total saponin extract of starfish.

[0015] Preferably, step S2 includes: subjecting the total saponin extract extracted in step S1 to normal-phase silica gel column chromatography, using a gradient elution with a mixed solvent of chloroform:methanol:water = 10:1:0~6:4:0.5, detecting by thin-layer chromatography, collecting the fraction containing saponins, and then passing the fraction containing saponins through a C... 18 Reversed-phase column chromatography was performed with gradient elution using methanol:water at ratios of 30:70 to 90:10. Different filtrates were collected, analyzed by thin-layer chromatography, and concentrated to dryness. The solutions were then subjected to LH-20 dextran gel column chromatography with methanol and water for the first elution. The filtrate was collected, analyzed by thin-layer chromatography, and concentrated to dryness. Finally, semi-preparative high-performance liquid chromatography (HPLC) was performed with methanol and water for the second elution. The fractions were collected and concentrated to dryness to obtain pure anthenoside K1, K2, and K3.

[0016] Preferably, step S2 includes: a first elution of methanol:water = 2:1, and a second elution of methanol:water = 85:15.

[0017] Preferably, the chemical structural formula of the compound is:

[0018]

[0019] Preferably, the anthenoside K1 is a colorless crystalline powder with the molecular formula C. 41 H68 O 14 Both the Liebermann-Burchard reaction and the Molish reaction were positive.

[0020] Preferably, the anthenoside K2 is a colorless crystalline powder with the molecular formula C2. 41 H 70 O 14 Both the Liebermann-Burchard reaction and the Molish reaction were positive.

[0021] Preferably, the anthenoside K3 is a colorless crystalline powder with the molecular formula C3. 42 H 79 O 14 Both the Liebermann-Burchard reaction and the Molish reaction were positive.

[0022] A third aspect of the present invention provides novel saponin compounds as described in the present invention, and the application of saponin compounds prepared by the method of the present invention in the preparation of Notch signaling pathway inhibitors.

[0023] Preferably, the Notch signaling pathway inhibitor includes an antitumor drug.

[0024] Compared with the prior art, the beneficial effects and significant progress of applying the technical solution of the present invention are as follows:

[0025] 1. This invention extracts three new compounds from the rimmed starfish and names them for the first time anthenoside K1 to K3;

[0026] 2. This invention verifies that compounds anthenoside K1-K3 have significant inhibitory effects on the Notch signaling pathway through in vitro screening experiments. This provides a scientific basis for screening new compounds with anti-tumor activity and is of great significance for the development and utilization of China's marine medicinal biological resources. Attached Figure Description

[0027] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the embodiments of the present invention will be briefly described below.

[0028] Figure 1 This is a statistical chart of the bioactivity of the Notch signaling pathway targeted in Example 3 of the present invention (the concentration of the compound to be detected in each group is 10 μg / mL);

[0029] Figure 2 This is a dose-response diagram showing the inhibitory effect of the compound of Example 3 of this invention on the biological activity of the Notch signaling pathway. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this invention, those skilled in the art can make various alterations and modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0031] To better understand this invention, the technical terms used in this invention will be explained below.

[0032] *Echinodermata*, a species of echinoderm, is commonly found in Guangdong, Fujian, and Zhejiang provinces of my country. It has a robust body with five narrow, elongated arms that taper towards the tip, reaching over 50 mm in length; the ratio of radius to diameter (R:r) is approximately 3.5. The aboral surface is covered with numerous small columnar structures, with the central and peripheral columnar structures being small and densely packed. Each columnar structure has 1-20 hemispherical granules at its apex and 7-20 radially arranged small spines around its periphery, connected by membranes. The upper marginal plate typically has around 30 large, thick, and slightly rectangular structures, arranged neatly and attractively, resembling an inlaid border. The lower marginal plate is opposite the upper marginal plate, with an equal number of structures, except that the radial sections are slightly wider than the upper marginal plate. Both upper and lower marginal plates have glassy, ​​easily detachable fine granules on their surfaces; each plate has small spines along its edges, connected by membranes. The lateral ambulacral plates are small and rhomboid, with a row of 5-6 larger spines along the groove edge; the other three sides have smaller spines, with one larger, thumb-shaped spine in the center. The mouth plates are small and elongated. Each radial portion of the mouth has several ventral plates of varying sizes and irregularly arranged, appearing almost inlaid. The sieve plates also bear granules and small spines, similar in shape to those on the adjacent columnar segments. The opposing marginal plates are purplish-brown, the columnar segments on the aboral side are yellowish-brown, and the mouth surface is pale yellowish-white.

[0033] Saponins are a class of complex glycosides that, when mixed with water and shaken, produce a persistent, soap-like foam. They are widely distributed in the plant kingdom, and many traditional Chinese medicines, such as ginseng, Panax notoginseng, Anemarrhena asphodeloides, Polygala tenuifolia, licorice, Platycodon grandiflorus, and Bupleurum chinense, contain saponins. In the past, soap pods were used in China for washing clothes because they contain saponins. Saponins are composed of a saponin aglycone and sugars, uronic acids, or other organic acids. Common sugars that make up saponins include D-glucose, L-rhamnose, D-galactose, L-arabinose, and L-xylose. Common uronic acids include glucuronic acid and galacturonic acid. These sugars or uronic acids often first combine to form oligosaccharide chains, which then condense with the C3-OH group of the saponin aglycone molecule, or two sugar chains condense with OH groups at two different positions in the saponin aglycone molecule. The -COOH group in the saponin aglycone molecule may also connect with sugars to form ester bonds.

[0034] The Liebermann-Burchard reaction is a color reaction of triterpenoids: dissolving triterpenoids in acetic anhydride and adding a few drops of concentrated sulfuric acid-acetic anhydride (1:20) produces color changes from yellow to red to purple to blue, eventually fading. This alters the color of a chemical substance through a chemical change. Nitric acid reacts with proteins, turning them yellow. This is called the protein color reaction and is often used to identify certain proteins; it is one of the characteristic reactions of proteins.

[0035] The Molish reaction is used to identify glycosides. Sugars dehydrate under the action of concentrated sulfuric acid or concentrated hydrochloric acid to form furfural and its derivatives, which react with α-naphthol to form a purple-red complex. A purple ring is formed between the sugar solution and the surface of concentrated H2SO4, hence it is also called the purple ring reaction.

[0036] Example 1: Preparation of compounds anthenoside K1-K3

[0037] The preparation methods of compounds anthenoside K1 to K3 include the following steps:

[0038] 1.1 Extraction: 1500 g of dried starfish (Aster spp.) from the South China Sea off Hainan Island, China, was pulverized and extracted six times sequentially by hot reflux with twice its weight of 65% ethanol, each extraction lasting one hour. The ethanol extracts were combined, and the ethanol was recovered under reduced pressure to obtain a fluid extract. The fluid extract was dissolved in three times its weight of water, and first extracted repeatedly with petroleum ether. Then, the aqueous phase was extracted again with n-butanol. The n-butanol extracts were combined, and the solvent was recovered under reduced pressure until dry to obtain 19.0 g of total saponins.

[0039] 1.2 Separation and Purification: The total saponins were subjected to normal-phase silica gel column chromatography, eluted with approximately 1500 ml of a mixed solvent (chloroform:methanol:water = 10:1:0–6:4:0.5, lower layer). The fraction containing saponins was collected and then subjected to C24 chromatography. 18 Reversed-phase column chromatography was performed with gradient elution of methanol:water = 30:70 to 90:10, followed by thin-layer chromatography detection. The solution was concentrated to dryness and then subjected to semi-preparative high-performance liquid chromatography (Zorbax 300SB-C18, 5 μm; 250 × 9.4 mm id) with elution of methanol:water = 85:15. Fractions containing anthenoside K1, K2, and K3 were collected, the solvent was recovered to near dryness, and the solutions were evaporated to dryness in a 70°C water bath to obtain anthenoside K1, K2, and K3 of 0.0262 g, 0.036 g, and 0.019 g, respectively.

[0040] Example 2

[0041] The chemical structures and stereoconfigurations of the compounds anthenoside K1 to K3 prepared in Example 1 were determined by various modern spectroscopic analyses, especially by the comprehensive application of multiple advanced two-dimensional nuclear magnetic resonance spectra.

[0042] Anthenoside K1 is a colorless crystalline powder with the molecular formula C1. 41 H 68 O 14 ; -51.04 (c=1, MeOH), both the Liebermann-Burchard reaction and the Molish reaction were positive. Electrospray cation exchange mass spectrometry (ESI-MS) + m / z: 807 [M+Na] + High-resolution mass spectrometry (HRESI-MS) + The precise molecular weight m / z provided by the mid-molecular ion peak is: 807.4534 [M+Na]. + (Calc. Mass 807.4501). IR(KBr)cm -1 : 3386 (hydroxyl group), 1639 (double bond). 13 C NMR and 1 The ¹H NMR data are shown in Table 1. The monosaccharides constituting the saponin sugar chain were obtained by hydrolyzing the saponins with trifluoroacetic acid. These monosaccharides were then prepared as glyconitrile acetate derivatives and analyzed by gas chromatography-mass spectrometry. Comparison with glyconitrile acetate derivatives of standard sugars confirmed that the sugar chain of anthenoside K1 consists of 6-methoxy-D-galactofuranose and 3-methoxy-D-galactopyranose in a 1:1 ratio.

[0043] Table 1. Anthenoside K1 aglycone and glycan moiety 13 C NMR and 1 H NMR data

[0044]

[0045]

[0046] Among them, MeGal f : Methylfuran galactose; MeGal p : Methylpyranogalactose; J: Coupling constant.

[0047] anthenoside K2, a colorless crystalline powder, molecular formula C 41 H 70 O 14 ; -64.69 (c=1, MeOH), positive for both Liebermann-Burchard and Molish reactions. Electrospray cation exchange mass spectrometry (ESI-MS) + m / z: 809 [M+Na] - High-resolution mass spectrometry (HRESI-MS) + The precise molecular weight m / z provided by the mid-molecular ion peak is 809.4638 (Calc. Mass 809.4658). IR (KBr) cm⁻¹ -1 : 3421.10 (hydroxyl group). 13 C NMR and 1 The ¹H NMR data are shown in Table 2. The monosaccharides constituting the saponin sugar chain were obtained by hydrolyzing the saponins with trifluoroacetic acid. These monosaccharides were then prepared as glyconitrile acetate derivatives and analyzed by gas chromatography-mass spectrometry. Comparison with glyconitrile acetate derivatives of standard sugars confirmed that the sugar chain of anthenoside K1 consists of 6-methoxy-D-galactofuranose and 3-methoxy-D-galactopyranose in a 1:1 ratio.

[0048] Table 2. Anthenoside K2 aglycone and glycan moiety 13 C NMR and 1 H NMR data

[0049]

[0050]

[0051] Among them, MeGal f : Methylfuran galactose; MeGal p : Methylpyranogalactose; J: Coupling constant.

[0052] anthenoside K3 is a colorless crystalline powder with the molecular formula C3. 42 H 70 O 14 ; -53.78 (c=1, MeOH), both the Liebermann-Burchard reaction and the Molish reaction were positive. Electrospray cation exchange mass spectrometry (ESI-MS) + m / z: 821 [M+Na] + High-resolution mass spectrometry (HRESI-MS) + The precise molecular weight m / z provided by the mid-molecular ion peak is: 821.4721 [M+Na]. + (Calc. Mass 807.4698). IR(KBr)cm -1 : 3383 (hydroxyl group), 1641 (double bond). 13C NMR and 1 The ¹H NMR data are shown in Table 3. The monosaccharides constituting the saponin sugar chain were obtained by hydrolyzing the saponins with trifluoroacetic acid. These monosaccharides were then prepared as glyconitrile acetate derivatives and analyzed by gas chromatography-mass spectrometry. Comparison with glyconitrile acetate derivatives of standard sugars confirmed that the sugar chain of anthenoside K1 consists of 6-methoxy-D-galactofuranose and 3-methoxy-D-galactopyranose in a 1:1 ratio.

[0053] Table 3. Anthenoside K3 aglycone and glycan moiety 13 C NMR and 1 H NMR data

[0054]

[0055]

[0056] Among them, MeGal f : Methylfuran galactose; MeGal p : Methylpyranogalactose; J: Coupling constant

[0057] Example 3

[0058] Experimental methods

[0059] 3.1 Establishment and identification of HEK293 cell lines stably expressing TRE-luciferase

[0060] Lentiviral cells carrying the TRE-luciferase expression element were constructed and infected with HEK293 cells. After 5-6 passages of selection using a puromycin gradient and monoclonal culture, stable HEK293 cell lines expressing TRE-luciferase were obtained. At the cellular level, positive clones were screened by extracting genomic DNA and detecting puromycin and luciferase genes using PCR. Following these identifications, 14 HEK293 cell lines integrating TRE-luciferase were obtained.

[0061] 3.2 HEK293 (TRE-luciferase) cell viability assay

[0062] Using the previously reported positive Notch signaling pathway inhibitor GSI, TRE-luciferase activity was detected to verify the activation and inhibition of the cell in the Notch signaling pathway. The most sensitive HEK293 (TRE-luciferase) cell line was selected and seeded in 96-well plates (dedicated fluorescent plates - black outer frame, transparent bottom) for culture.

[0063] 3.3 Screening of bioactive compounds targeting the Notch signaling pathway

[0064] Anthenoside K1–K3 were diluted to 10, 1, 0.1, and 0.01 μg / mL, respectively, and then added to HEK293 (TRE-luciferase) cells and cultured for 24 hours. A normal control group and a GSI control group were also set up, with three replicates for each group. The 96-well plates containing the cultured cells were removed from the incubator, and an equal volume of GloLysis buffer was added to each well. The plates were incubated for 5 minutes to allow for complete cell lysis. The relative light unit (RLU) values ​​were recorded using a TECANsafire II multi-mode microplate reader to detect the luciferase.

[0065] 3.4 Statistical Analysis: SPSS 15.0 software was used for statistical analysis. Experimental data are expressed as x±SD. The t-test was used to compare differences between groups, and P<0.05 was considered statistically significant.

[0066] Experimental results

[0067] like Figure 1 and Figure 2 As shown, compared with the blank control group, the relative luciferase intensity was significantly reduced after adding 10 μg / mL of compounds 1 (anthenoside K1), 2 (anthenoside K2), and 3 (anthenoside K3) (P<0.05). This indicates that the addition of starfish saponins anthenoside K1–K3 can effectively mediate the activation of the luciferase gene, significantly reducing its luciferase value and significantly inhibiting the Notch signaling pathway. Among them, compound 2 showed better inhibitory effects on the Notch signaling pathway than compounds 1 and 3.

[0068] Different doses of the substances to be tested were added to cultured HEK293 (TRE-luciferase) cells, and their effects on the activation activity of the Notch signaling pathway were detected using a luciferase assay system. Anthenoside K1–K3 all showed dose-dependent inhibitory effects on the Notch signaling pathway.

[0069] The above experiments demonstrate that the compounds anthenoside K1-K3 of this invention, after in vitro screening experiments to inhibit Notch signaling pathway activity, show significant inhibitory effects on the Notch signaling pathway. The Notch signaling pathway used in the experiments includes Notch receptors, ligands, modifications, and transcription factors. The simplicity of Notch pathway regulation lies in the fact that after the receptor binds to the ligand, the activated Notch receptor detaches from the cell membrane and can directly translocate to the nucleus to bind to transcriptional regulators and activate target genes without a second messenger. Activation can trigger multiple signals, and various regulatory factors can regulate the signal through different mechanisms. Numerous studies have demonstrated that the Notch signaling pathway is closely related to the occurrence and development of human tumors. Abnormal expression or abnormal activation of Notch signaling, as well as dysregulation of receptor and ligand molecule expression, exist in various malignant tumors. RBP-Jk protein is a transcription factor and the most direct downstream regulatory protein of the Notch signaling pathway. The vector used in this experimental model is the miniCMV-TRE (tandem repeats of the RBP-Jk transcriptional response element)-mediated luciferase gene. Therefore, activation of the Notch signal will definitely mediate the initiation of the luciferase gene. Thus, the activity of the Notch signaling pathway can be tested based on the intensity of luciferase activity. The Notch pathway plays an important role in tumorigenesis and development. Investigating its mechanism of action in tumorigenesis and development, establishing a screening model for bioactivity targeting the Notch signaling pathway, and utilizing this activity screening model will provide a scientific basis for screening new compounds with antitumor activity. This is of great significance for the development and utilization of China's marine medicinal biological resources.

[0070] The applicant declares that, in the process of describing the above-mentioned specification:

[0071] The terms "this embodiment," "an embodiment of the present invention," "as shown," "further," and "further improved technical solutions," etc., indicate that the specific features, structures, materials, or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms are not necessarily directed at the same embodiment or example, and the specific features, structures, materials, or characteristics described can be combined or combined in any suitable manner in one or more embodiments or examples. Furthermore, without causing contradiction, those skilled in the art can combine or combine the different embodiments or examples described in this specification and the features of the different embodiments or examples.

[0072] Finally, it should be noted that:

[0073] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them;

[0074] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Non-essential improvements, adjustments or substitutions made by those skilled in the art based on the content of this specification are all within the scope of protection claimed by the present invention.

Claims

1. The application of a saponin compound in the preparation of Notch signaling pathway inhibitors, characterized in that, The compounds were named anthenoside K1, anthenoside K2, and anthenoside K3, and their chemical structural formulas are as follows: 。 2. The application as described in claim 1, characterized in that, The anthenoside K1 is a colorless crystalline powder with the molecular formula C. 41 H 68 O 14 Both the Liebermann-Burchard reaction and the Molish reaction were positive.

3. The application as described in claim 1, characterized in that, The anthenoside K2 is a colorless crystalline powder with the molecular formula C2. 41 H 70 O 14 Both the Liebermann-Burchard reaction and the Molish reaction were positive.

4. The application as described in claim 1, characterized in that, The anthenoside K3 is a colorless crystalline powder with the molecular formula C3. 42 H 79 O 14 Both the Liebermann-Burchard reaction and the Molish reaction were positive.

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