An anti-tumor natural medicine composition based on norway maple and a preparation method thereof

Abstract

This invention discloses an antitumor natural drug composition based on Norway maple and its preparation method, relating to the field of biomedical technology. The compound is selected from at least one of the following structures: 3-O-[β-D-glucopyranosyl-(1→4)-[β-D-galactopyranosyl-(1→2)]]-β-D-glucopyranoside-21-O-acetyl-22-O-angeloyl yrutile alcohol, with the molecular formula C2. 55 H 86 O 23 This invention isolates a class of triterpenoid saponins with a rutinol skeleton from the bark of Norwegian maple, enriching the chemical composition library of maple species. The compounds contain unique oligosaccharide chains and diacyl substitution patterns at C-21 and C-22. One compound has a rare branched acyl group (3-hydroxy-4-methylhexanoyl) at C-21, while the other compound contains an arabinofuranose unit in its C-3 trisaccharide chain. These structural features are rare in natural products and demonstrate excellent structural novelty.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an anti-tumor natural drug composition based on Norway maple and its preparation method. Background Technology

[0002] Maple (Acer) plants, commonly known as maple trees, are widely distributed in temperate regions of the Northern Hemisphere. They not only have significant ornamental and economic value but also have a long history of medicinal use in traditional medicine, traditionally used to treat fever, liver diseases, eye diseases, and inflammation. Modern pharmacological studies have shown that extracts from maple plants and their isolated compounds possess various biological activities, including antitumor, antioxidant, anti-inflammatory, antibacterial, α-glucosidase inhibitory, and anti-obesity activities.

[0003] Currently, over 300 compounds have been identified from Acer species, mainly phenols, including tannins, flavonoids, lignans, phenylpropanoids, and phenolic acids. Although there are reports on the chemical components of species such as red maple (A. rubrum), sugar maple (A. saccharum), silver maple (A. saccharinum), and European maple (A. pseudoplatanus), systematic phytochemical studies on Norway maple (A. platanoides), especially on its saponins and their bioactivity, are limited.

[0004] Existing natural antitumor drugs (such as paclitaxel and doxorubicin) suffer from problems in clinical applications, including poor water solubility, significant toxic side effects, and a tendency to induce drug resistance. Therefore, the search for novel, highly effective, and low-toxicity antitumor lead compounds from plants is currently a hot topic in new drug development.

[0005] In response to this, this application proposes an anti-tumor natural drug composition based on Norway maple and its preparation method to solve the above-mentioned problems. Summary of the Invention

[0006] The purpose of this invention is to provide an anti-tumor natural drug composition based on Norway maple and its preparation method, in order to solve the problems of poor water solubility, large toxic side effects, and easy development of drug resistance in the clinical application of existing natural anti-tumor drugs (such as paclitaxel, doxorubicin, etc.).

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, this application provides a triterpenoid saponin compound of the rutinol type, said compound being selected from at least one of the following structures:

[0009] 3-O-[β-D-glucopyranosyl-(1→4)-[β-D-galactopyranosyl-(1→2)]]-β-D-glucopyranoside-21-O-acetyl-22-O-angeloyl rutinol, molecular formula C 55 H 86 O 23 ;

[0010] 3-O-[β-D-glucopyranosyl-(1→4)-[β-D-galactopyranosyl-(1→2)]]-β-D-glucuronide methyl ester-21-O-acetyl-22-O-angeloyl yruvicol, molecular formula C 56 H 88 O 23 ;

[0011] 3-O-[α-L-arabinofuranyl-(1→3)-[β-D-galactopyranoyl-(1→2)]]-β-D-glucuronyl-21-O-(3-hydroxy-4-methylhexanoyl)-22-O-angeloyl yrutile alcohol, molecular formula C 56 H 88 O 23 ;

[0012] 3-O-[α-L-arabinofuranyl-(1→3)-[β-D-galactopyranoyl-(1→2)]]-β-D-glucuronyl-21-O-acetyl-22-O-angeloyl rutinol, molecular formula C 59 H 94 O 23 .

[0013] Furthermore, the compound is a structural analogue in which the acyl group at C-21 and / or C-22 is replaced by a saturated or unsaturated fatty acid acyl group at C2-C8, or a structural analogue in which the type or sequence of monosaccharides in the trisaccharide chain is replaced, or a derivative in which the carboxyl group of glucuronic acid is modified into an amide, hydroxylamine, or a salt-forming form.

[0014] Secondly, this application provides a method for extracting and separating compounds as described in the first aspect, characterized in that it includes:

[0015] The dried bark of Norway maple is crushed, extracted with an aqueous solution of methanol or ethanol, and concentrated to obtain an extract.

[0016] The extract was suspended in water and subjected to liquid-liquid partition extraction with ethyl acetate and n-butanol in sequence, and the n-butanol extract was collected.

[0017] The n-butanol extract was subjected to macroporous adsorption resin column chromatography, eluted with a water-methanol or water-ethanol gradient, and the active fraction was collected.

[0018] The active fraction was subjected to gel column chromatography and eluted with methanol to enrich saponin components.

[0019] The saponin-enriched fractions were subjected to reverse-phase ODS column chromatography, eluted with a methanol-water or acetonitrile-water gradient, and purified by preparative high-performance liquid chromatography to obtain the compounds.

[0020] Thirdly, this application provides an antitumor natural pharmaceutical composition based on Norway maple, comprising a therapeutically effective amount of the compound as described in the first aspect or a pharmaceutically acceptable salt, ester, or prodrug thereof, as well as a pharmaceutically acceptable carrier, diluent, or excipient.

[0021] Furthermore, the dosage form of the pharmaceutical composition is tablets, capsules, granules, oral liquid, injection, lyophilized powder for injection, or liposome injection.

[0022] Furthermore, the compound is the active ingredient in the composition, and its content is determined by high performance liquid chromatography-evaporative light scattering detection or ultra-high performance liquid chromatography-mass spectrometry, wherein the total content of the compound is not less than 20% of the extract.

[0023] Fourthly, this application provides the use of the pharmaceutical composition as described in the third aspect in the preparation of an antitumor drug, characterized in that the tumor is renal cell carcinoma, melanoma, breast cancer, or liver cancer.

[0024] Furthermore, tumor cell apoptosis and cell cycle arrest were induced by regulating the PI3K / Akt / mTOR signaling pathway.

[0025] Fifthly, this application provides the use of the pharmaceutical composition as described in the third aspect in the preparation of a treatment for inflammatory diseases, autoimmune diseases, or as an immunomodulator.

[0026] Compared with existing technologies, this invention provides an antitumor natural drug composition and preparation method based on Norway maple. A class of triterpenoid saponin compounds with a rutin skeleton are isolated from the bark of Norway maple (Acer platanoides), enriching the chemical composition library of maple plants. The compounds simultaneously contain unique oligosaccharide chains and diacyl substitution patterns at C-21 and C-22. Compound 3 has a rare branched acyl group (3-hydroxy-4-methylhexanoyl) at C-21, and compound 4 contains an arabinofuranose unit in its C-3 trisaccharide chain. These structural features are relatively rare in natural products, demonstrating good structural novelty. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0028] Figure 1 This is a schematic diagram of the compound composition structure provided in an embodiment of the present invention. Detailed Implementation

[0029] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0030] As attached Figure 1 As shown:

[0031] Example 1:

[0032] Extraction and separation of compounds:

[0033] 1. Plant materials:

[0034] The bark of the Norway maple (Acer platanoides) was collected in the fall of 2012 at the University of Rhode Island, Kingston. The voucher specimen is deposited at the Medicinal Botanical Garden of the College of Pharmacy, University of Rhode Island.

[0035] 2. Extraction and separation:

[0036] 0.7 kg of dried Norway maple bark was pulverized and extracted three times (1 L each time) with methanol at room temperature. The extracts were combined and concentrated under reduced pressure to obtain 30.0 g of methanol extract. The extract was dissolved in 200 mL of water and extracted successively with ethyl acetate (5 × 200 mL) and n-butanol (5 × 200 mL) to obtain 4.3 g of n-butanol extract.

[0037] like Figure 1 As shown, the n-butanol extract was subjected to MCI gel CHP-20P column chromatography (610 cm × 4.0 cm inner diameter) with a water-methanol gradient (100%, 80%, 60%, 40%, 20%, 0% methanol), and the 80% methanol eluent fraction D (470 mg) was collected. Fraction D was subjected to Sephadex LH-20 column chromatography (130 cm × 3.0 cm inner diameter) with methanol elution to obtain saponin-enriched subfraction D1 (126 mg). Subfraction D1 was subjected to ODS column chromatography (500 mm × 30 mm inner diameter) with a methanol-water gradient (80:20 to 100:0), and further purified by preparative HPLC to obtain:

[0038] Compound 1 (9.0 mg): 3-O-[β-D-glucopyranosyl-(1→4)-[β-D-galactopyranosyl-(1→2)]]-β-D-glucopyranoside-21-O-acetyl-22-O-angeloyl yruvicol, molecular formula C 55 H 86 O 23 ;

[0039] Compound 2 (2.5 mg): 3-O-[β-D-glucopyranosyl-(1→4)-[β-D-galactopyranosyl-(1→2)]]-β-D-glucuronide methyl ester-21-O-acetyl-22-O-angeloyl yruvicol, molecular formula C 56 H 88 O 23 ;

[0040] Compound 3 (4.0 mg): 3-O-[α-L-arabinofuranyl-(1→3)-[β-D-galactopyranoyl-(1→2)]]-β-D-glucuronyl-21-O-(3-hydroxy-4-methylhexanoyl)-22-O-angeloyl yrutile alcohol, molecular formula C 56 H 88 O 23 ;

[0041] Compound 4 (4.0 mg): 3-O-[α-L-arabinofuranyl-(1→3)-[β-D-galactopyranoyl-(1→2)]]-β-D-glucuronyl-21-O-acetyl-22-O-angeloyl yruvicol, molecular formula C 59 H 94 O 23 .

[0042] Example 2:

[0043] Evaluation of in vitro antitumor activity (MTT method):

[0044] 1. Experimental materials:

[0045] Human renal cell carcinoma cells (786-O), melanoma cells (A375), breast cancer cells (MCF-7), and liver cancer cells (HepG2) were purchased from ATCC. DMEM culture medium, fetal bovine serum, trypsin, MTT, and doxorubicin (purity >98%) were all purchased from commercial suppliers.

[0046] 2. Experimental apparatus:

[0047] CO2 incubator (Thermo), clean bench, microplate reader (BioTek Instruments, 490 nm wavelength).

[0048] 3. Experimental methods:

[0049] Cells in the logarithmic growth phase were seeded at a density of 8000-10000 cells per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. After cell attachment, the original culture medium was discarded, and 200 μL of DMEM medium containing different concentrations (2.5, 5, 10, 20, 40, 50 μM) of compounds 1-4 were added to each well. A blank control and a positive control (doxorubicin) were also included. Each concentration was used in triplicate. After culturing for another 24 hours, 10 μL of MTT solution (5 mg / mL) was added to each well, and the plates were incubated for 4 hours. The supernatant was carefully aspirated, and 150 μL of DMSO was added to each well. The formazan crystals were dissolved by shaking, and the optical density (OD) of each well was measured at 490 nm using a microplate reader. Cell viability and inhibition rate were calculated, and the half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism 5.01 software. 50 ).

[0050] Cell inhibition rate (%) = (1 - OD value of experimental group / OD value of control group) × 100%

[0051] 4. The experimental results are shown in Table 1 below;

[0052] Table 1. IC50 of compounds 1-4 against four tumor cell lines 50 Values ​​(μM, mean ± SD, n=3).

[0053] compound 786-O A375 MCF-7 HepG2 1 21.9 ± 0.8 24.7 ± 3.0 36.9 ± 5.4 35.0 ± 5.0 2 40.0 ± 5.4 >50 >50 >50 3 9.8 ± 0.5 9.4 ± 0.6 39.5 ± 3.0 11.6 ± 1.0 4 12.1 ± 2.2 11.5 ± 2.0 22.4 ± 2.7 15.5 ± 0.8 Dorothy Star 5.4 ± 0.4 3.7 ± 0.3 34.2 ± 3.3 23.0 ± 1.2

[0054] Conclusion: Compounds 1, 3, and 4 all exhibited varying degrees of cytotoxicity against four types of tumor cell lines. Compound 3 showed the highest IC50 value against A375 cells (melanoma). 50 The concentration was 9.4 μM, indicating strong selective inhibition. Compound 2 showed weaker activity, suggesting that the free state of the carboxyl group at the C-6 position of the glucuronic acid in the C-3 oligosaccharide chain is crucial for maintaining activity.

[0055] Example 3:

[0056] Effect of compound 3 on the cell cycle of A375 cells (flow cytometry):

[0057] 1. Experimental Methods: A375 cells in logarithmic growth phase were seeded at 1×10^5 cells / well in 6-well plates and cultured for 24 hours. Compound 3 was added at final concentrations of 5, 10, and 20 μM, respectively, with a blank control group included. After culturing for another 24 hours, cells were collected, washed twice with pre-cooled PBS, and fixed overnight with 70% ethanol. After centrifugation and discarding the ethanol, RNase A (50 μg / mL) and propidium iodide (PI, 50 μg / mL) were added, and the cells were stained in the dark for 30 minutes. Cell cycle distribution was detected by flow cytometry.

[0058] 2. Experimental Results:

[0059] Compared with the control group, the proportion of cells in the G0 / G1 phase and the proportion of cells in the S and G2 / M phases were significantly increased in the compound 3 treatment group in a dose-dependent manner. This indicates that compound 3 can arrest A375 cells in the G0 / G1 phase.

[0060] Example 4:

[0061] Effects of compound 3 on the PI3K / Akt / mTOR signaling pathway (Western blot):

[0062] 1. Experimental Methods:

[0063] After A375 cells were treated with different concentrations of compound 3 (0, 5, 10, 20 μM) for 24 hours, the cells were collected, and total protein was extracted using RIPA lysis buffer. Protein concentration was determined by BCA method. Equal amounts of protein were separated by SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked with 5% skim milk powder for 1 hour, and then incubated overnight at 4°C with primary antibodies against PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, and GAPDH, respectively. After washing with TBST, the membrane was incubated at room temperature for 1 hour with HRP-labeled secondary antibody, developed by ECL, and photographed using a chemiluminescence imaging system.

[0064] 2. Experimental Results:

[0065] Compound 3 dose-dependently inhibited the phosphorylation levels of PI3K, Akt, and mTOR without significantly affecting total protein expression. This suggests that compound 3 may induce tumor cell apoptosis by inhibiting the PI3K / Akt / mTOR signaling pathway.

[0066] Example 5:

[0067] Preparation of pharmaceutical compositions (lyophilized powder for injection):

[0068] Take 10 g of compound 3, add 800 mL of water for injection, add 50 g of mannitol as a lyophilization excipient, stir to dissolve, adjust the pH to 6.5-7.0 with 0.1 mol / L hydrochloric acid or sodium hydroxide, add water for injection to 1000 mL, filter through a 0.22 μm microporous membrane for sterilization, dispense into vials (2 mL per vial), and freeze-dry to obtain the lyophilized powder for injection. This product is used in the preparation of antitumor drugs, with each vial containing 20 mg of compound 3.

[0069] As shown above, this invention has isolated a class of triterpenoid saponins with a rutin skeleton from the bark of *Acer platanoides*, enriching the chemical composition library of *Acer* species. The compounds contain unique oligosaccharide chains and diacyl substitution patterns at C-21 and C-22. Compound 3 has a rare branched acyl group (3-hydroxy-4-methylhexanoyl) at C-21, and compound 4 contains an arabinofuranose unit in its C-3 trisaccharide chain. These structural features are relatively rare in natural products, demonstrating good structural novelty.

[0070] The extraction and separation method provided by this invention is simple to operate, employing a combination of conventional liquid-liquid extraction, macroporous adsorption resin, gel chromatography, and reversed-phase ODS chromatography to efficiently enrich target saponin components. This method exhibits good reproducibility and is suitable for large-scale preparation, providing a material basis for subsequent pharmacodynamic studies and drug development.

[0071] The compounds of this invention exhibit selective cytotoxicity against various tumor cell lines, particularly melanoma, renal cell carcinoma, breast cancer, and hepatocellular carcinoma. Preliminary structure-activity relationship analysis indicates that the presence of the arabinose unit in the oligosaccharide chain at C-3 and the introduction of a specific branched acyl group at C-21 play a crucial role in maintaining and enhancing antitumor activity. These findings provide a lead compound with independent intellectual property rights for the development of antitumor drugs.

[0072] Preliminary mechanistic studies in this invention have confirmed that the compound can induce G0 / G1 phase arrest in tumor cells and promote apoptosis by regulating the PI3K / Akt / mTOR signaling pathway. This signaling pathway is a key pathway in tumor development and progression, suggesting that the compound of this invention has the potential for targeted therapy and may have a synergistic mechanism with existing chemotherapeutic drugs, providing new possibilities for overcoming drug resistance.

[0073] The pharmaceutical composition provided by this invention can be quality controlled by methods such as thin-layer chromatography, high-performance liquid chromatography-evaporative light scattering detection, or ultra-high-performance liquid chromatography-mass spectrometry to ensure the stability of the content of active saponin components. The compound can be prepared into various dosage forms, including oral and injectable formulations, exhibiting good drug-like properties. Furthermore, the compound can also be used to prepare drugs for treating inflammatory diseases and autoimmune diseases, or as an immunomodulator, demonstrating broad prospects for pharmaceutical applications.

[0074] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A triterpenoid saponin compound of the rutinol type, characterized in that, The compound is selected from at least one of the following structures: 3-O-[β-D-glucopyranosyl-(1→4)-[β-D-galactopyranosyl-(1→2)]]-β-D-glucopyranoside-21-O-acetyl-22-O-angeloyl rutinol, molecular formula C 55 H 86 O 23 ; 3-O-[β-D-glucopyranosyl-(1→4)-[β-D-galactopyranosyl-(1→2)]]-β-D-glucuronide methyl ester-21-O-acetyl-22-O-angeloyl yruvicol, molecular formula C 56 H 88 O 23 ; 3-O-[α-L-arabinofuranyl-(1→3)-[β-D-galactopyranoyl-(1→2)]]-β-D-glucuronyl-21-O-(3-hydroxy-4-methylhexanoyl)-22-O-angeloyl yrutile alcohol, molecular formula C 56 H 88 O 23 ; 3-O-[α-L-arabinofuranyl-(1→3)-[β-D-galactopyranoyl-(1→2)]]-β-D-glucuronyl-21-O-acetyl-22-O-angeloyl rutinol, molecular formula C 59 H 94 O 23 .

2. The triterpenoid saponin compound of the yumyl alcohol type according to claim 1, characterized in that, The compound is a structural analogue in which the acyl group at C-21 and / or C-22 is replaced by a saturated or unsaturated fatty acid acyl group at C2-C8, or a structural analogue in which the type or sequence of monosaccharides in the trisaccharide chain is replaced, or a derivative in which the carboxyl group of glucuronic acid is modified into an amide, hydroxylamine, or a salt-forming form.

3. A method for extracting and separating compounds as described in claim 1 or 2, characterized in that, include: The dried bark of Norway maple is crushed, extracted with an aqueous solution of methanol or ethanol, and concentrated to obtain an extract. The extract was suspended in water and subjected to liquid-liquid partition extraction with ethyl acetate and n-butanol in sequence, and the n-butanol extract was collected. The n-butanol extract was subjected to macroporous adsorption resin column chromatography, eluted with a water-methanol or water-ethanol gradient, and the active fraction was collected. The active fraction was subjected to gel column chromatography and eluted with methanol to enrich saponin components. The saponin-enriched fractions were subjected to reverse-phase ODS column chromatography, eluted with a methanol-water or acetonitrile-water gradient, and purified by preparative high-performance liquid chromatography to obtain the compounds.

4. A natural antitumor drug composition based on Norway maple, characterized in that, It comprises a therapeutically effective amount of the compound as described in claim 1 or 2 or a pharmaceutically acceptable salt, ester, prodrug thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

5. The pharmaceutical composition according to claim 4, characterized in that, The dosage form of the pharmaceutical composition is tablets, capsules, granules, oral liquid, injection, lyophilized powder for injection, or liposome injection.

6. The antitumor natural drug composition based on Norway maple according to claim 4, characterized in that, The compound is the active ingredient in the composition, and its content is determined by high performance liquid chromatography-evaporative light scattering detection or ultra-high performance liquid chromatography-mass spectrometry, and the total content of the compound is not less than 20% of the extract.

7. The use of the pharmaceutical composition as described in claim 4 in the preparation of an antitumor drug, characterized in that, The tumor is kidney cancer, melanoma, breast cancer, or liver cancer.

8. The application according to claim 7, characterized in that, It induces tumor cell apoptosis and cell cycle arrest by regulating the PI3K / Akt / mTOR signaling pathway.

9. The use of the pharmaceutical composition as described in claim 4 in the preparation of a treatment for inflammatory diseases, autoimmune diseases, or as an immunomodulator.

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