Solanum nigrum picoside A as well as preparation method and application thereof

Abstract

The invention belongs to the field of natural medicinal chemistry, and particularly relates to nightshade alkaloid A as well as a preparation method and application thereof. According to the invention, the steroidal glycoside alkaloid, namely Soladineside A, C39H59NO11, which has a novel structure and contains a pyridine structural unit, is separated and identified from solanum nigrum seeds for the first time. The nightshade alkaloid A can effectively inhibit proliferation of cancer cells and has the potential of being developed into anti-cancer drugs.

Description

Technical Field

[0001] The invention belongs to the field of natural drug chemistry, and specifically relates to solanine glycoside A and a preparation method and application thereof. Background Art

[0002] In recent years, research on the anti-tumor effects of Chinese herbal medicines has made great progress. Compared with designed and synthesized small molecule drugs, natural drugs have obvious advantages in structural novelty, biocompatibility, and functional diversity. Therefore, it is of great practical significance to find safe and effective cancer prevention and treatment drugs from Chinese herbal medicines.

[0003] Solanum nigrum L. is a plant of the genus Solanum in the family Solanaceae, and is used as both medicine and food. Solanum nigrum is bitter and cold in nature, and has the effects of clearing away heat and detoxifying, promoting blood circulation and removing blood stasis, promoting diuresis and reducing swelling, and relieving cough and expectoration. The main chemical components in Solanum nigrum seeds include steroidal alkaloids, steroidal saponins, phenols, etc. Among them, alkaloid components are considered to be the main active ingredients of Solanum nigrum seeds.

[0004] Therefore, if novel steroidal glycoalkaloid compounds can be extracted from Solanum nigrum seeds, it will have important scientific research and clinical value. Summary of the invention

[0005] The purpose of the present invention is to provide solanine glycoside A and a preparation method and application thereof.

[0006] In order to achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is: solanine glycoside A, the structural formula of solanine glycoside A is as follows:

[0007]

[0008] Accordingly, the method for preparing solanine glycoside A comprises the following steps:

[0009] (1) defatting Solanum nigrum seeds, and then soaking and extracting them with an ethanol aqueous solution to obtain an extract; adding water to disperse the extract and extracting;

[0010] (2) The aqueous layer obtained after extraction is separated and purified by macroporous resin, normal phase silica gel column chromatography, reverse phase silica gel column chromatography and HPLC to obtain solanine glycoside A.

[0011] Preferably, in step (1), petroleum ether is used for soaking and degreasing.

[0012] Preferably, in step (1), petroleum ether is used for extraction.

[0013] Preferably, in step (2), the macroporous resin separation step comprises: separation by macroporous resin column chromatography, gradient elution with increasing ethanol concentration using ethanol aqueous solutions of different concentrations, and discarding the water elution portion.

[0014] Preferably, in step (2), the normal phase silica gel column chromatography separation step comprises: eluting with a solvent system of ethyl acetate-methanol-water-diethylamine system or dichloromethane-methanol-water-diethylamine system.

[0015] Preferably, in step (2), the reverse phase silica gel column chromatography separation step comprises: gradient elution with increasing methanol concentration using methanol-water solution.

[0016] Preferably, in step (2), during HPLC separation and purification, the mobile phase is methanol-water solution.

[0017] Correspondingly, the solanine glycoside A is a pharmaceutically acceptable salt.

[0018] Correspondingly, medicines, health products, and foods prepared using the solanine glycoside A or the solanine glycoside A prepared by the method or the salt thereof; or; pharmaceutical compositions, health products, and foods comprising the solanine glycoside A or the solanine glycoside A prepared by the method or the salt thereof.

[0019] The present invention has the following beneficial effects: the present invention separates and identifies for the first time a novel steroidal glycoside alkaloid containing a pyridine structural unit from Solanum nigrum seeds: Solanum nigrum glycoside A. Through further in vitro cell activity studies, the inventors found that Solanum nigrum glycoside A can effectively inhibit the proliferation of cancer cells and has the potential to be developed as an anticancer drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The compounds of the present invention 1 H NMR spectrum;

[0021] Figure 2 The compounds of the present invention 13 C NMR spectrum;

[0022] Figure 3 is the HSQC spectrum of the compound of the present invention;

[0023] Figure 4 is the HMBC spectrum of the compound of the present invention;

[0024] Figure 5 It is the HRESIMS spectrum of the compound of the present invention. DETAILED DESCRIPTION

[0025] The present invention provides a new steroidal glycoside alkaloid compound containing a pyridine structural unit, the molecular formula of which is: 39H 59 NO 11 , the chemical structure is as follows:

[0026]

[0027] The compound is extracted from Solanum nigrum seeds and has the effects of preventing and treating tumors and cancers.

[0028] The present invention also provides a preparation (extraction) process of the compound, comprising the following steps:

[0029] 1. Dry and crush the Solanum nigrum seeds, soak and defatted them in petroleum ether at room temperature for 2 to 3 times, each time for 10 to 16 hours. Add the defatted Solanum nigrum seed powder into 80% to 85% (v / v) ethanol aqueous solution and soak and extract for 3 times, each time for 70 to 74 hours. Combine the extracts, recover the ethanol under reduced pressure until there is no alcohol taste, and obtain the extract. Add 3 to 4 times the weight of the extract to disperse in water, and extract with an equal volume of petroleum ether to obtain the petroleum ether extraction layer and the water layer.

[0030] 2. Separate the aqueous layer by macroporous resin column chromatography (HPD-100), perform gradient elution with ethanol-water (volume ratios: 0:100, 20:80, 90:10), discard the water elution portion, collect and concentrate.

[0031] 3. Separate the concentrated extract using normal phase silica gel column chromatography (100-200 mesh) and perform gradient elution with a solvent system of ethyl acetate-methanol-water-diethylamine system (by volume ratio, 100:0:0:0-1:1:0.1:0.01).

[0032] 4. Separate again by normal phase silica gel column chromatography and elute with a dichloromethane-methanol-water-diethylamine (volume ratio, 100:0:0:0 to 2:1:0.1:0.01) system.

[0033] 5. Continue separation by normal pressure RP-C18 column chromatography, gradient elution with methanol-water (volume ratios: 50:50, 60:40, 70:30, 80:20, 100:0). Purify by preparative HPLC, with the mobile phase being methanol-water (78:22), to obtain the desired compound.

[0034] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The data obtained are all average values ​​obtained after at least 3 repetitions, and all the data obtained in each repetition are valid data.

[0035] Example 1: Extraction and identification of compounds

[0036] 1. Extraction

[0037] Crush the dried Solanum nigrum seeds (50kg), pass through a No. 2 sieve, and soak and defatted them in petroleum ether at room temperature for 3 times, each time for 12 hours. Add the defatted medicinal powder to 85% (v / v) ethanol aqueous solution (200L) and soak and extract for 3 times, each time for 72 hours. Combine the extracts, recover the ethanol under reduced pressure until there is no alcohol taste, and obtain the extract (4.6kg). Disperse the extract in 15L of water, and extract with an equal volume of petroleum ether to obtain the petroleum ether extraction layer and the water layer.

[0038] The aqueous layer (3.9 kg) was separated by macroporous resin column chromatography (HPD-100) with ethanol-water (0:100, 20:80, 90:10) for gradient elution. The water elution was discarded and the 20% and 90% ethanol-water elutions were collected and concentrated to obtain two fractions, FA and Fr.B (90% ethanol-water elution corresponds to the Fr.B fraction, which contains more steroidal alkaloid components).

[0039] The Fr.B part (2.1 kg) was separated by normal phase silica gel column chromatography (200 mesh, 10 kg). The solvent system: ethyl acetate-methanol-water-diethylamine system (by volume ratio: 100:0:0:0, 50:1:0.01:0.01, 40:1:0.01:0.01, 30:1:0.01:0.01, 20:1:0.05:0.01, 10:1:0.05:0.01, 5:1:0.05:0.01, 4:1:0.1:0.01, 3:1:0.1:0.01, 2:1:0.1:0.01, 1:1:0.1:0.01) was used for gradient elution, and 80 L of solvent was used for each gradient concentration, and the solvent was distilled under reduced pressure once every 5 L. The fractions were combined under the guidance of TLC analysis and twelve components were separated, namely Fr.B1 to Fr.B12.

[0040] The content of steroidal alkaloid components in each component was detected by thin layer chromatography (TLC), and the component with the highest content of steroidal alkaloid components (Fr.B7, 157 g) was selected and separated again by normal phase silica gel (200-300 mesh, 3 kg) column chromatography, and then eluted with dichloromethane-methanol-water-diethylamine (volume ratios were: 100:0:0:0, 50:1:0.01:0.01, 40:1:0.01:0.01, 30:1: The mixture was eluted with 15 L of solvent for each gradient concentration and distilled under reduced pressure once for every 1 L of solvent; the mixture was combined under the guidance of TLC analysis and ten fractions were separated, Fr.B7a~Fr.B7j.

[0041] The content of steroidal alkaloid components in each component was detected by thin layer chromatography (TLC), and the component with the highest content of steroidal alkaloid components (Fr.B7h, 63.3 g) was selected and further separated by atmospheric pressure RP-C18 column chromatography, with methanol-water (volume ratios of 50:50, 60:40, 70:30, 80:20, 100:0) for gradient elution to obtain nine fractions, Fr.B7h1-Fr.B7h9.

[0042] The content of steroidal alkaloid components in each component was detected by thin layer chromatography (TLC), and the component with the highest content of steroidal alkaloid components (Fr.B7h8, 3.6 g) was selected and purified by preparative HPLC with methanol-water (78:22, 60 mL / min) as the mobile phase to obtain the desired compound (52 mg, t R =25.3min), white amorphous powder.

[0043] 2. Structural identification

[0044] The compound extracted in step 1 was developed with silica gel thin layer, and the potassium bismuth iodide reaction was positive, indicating that it was an alkaloid compound.

[0045] High resolution mass spectrometry (HRESIMS) gave a quasi-molecular ion peak of m / z 718.4166 [M+H] + (Calculated value C 39 H 60 NO 11 + ,718.4161), combined 13 C NMR data confirmed that its molecular formula is C 39 H 59 NO 11 .

[0046] The compound 1 H NMR is shown in Table 1. In Table 1, a means "overlapping with other signals". 1 HNMR spectrum Figure 1 As shown, 13 C NMR spectrum Figure 2 As shown, the HSQC spectrum is Figure 3 As shown, the HMBC spectrum is Figure 4 As shown, the HRESIMS spectrum is as follows Figure 5 shown.

[0047] Table 1. Compounds 1 H(400MHz) and 13 C(100MHz) NMR data(δppm,J Hz,C 5 D 5 N)

[0048]

[0049] According to Table 1: there are three methyl singlet signals δ H 0.94(3H,s,CH 3 -19),δ H 1.17(3H,s,CH 3 -18) and δ H 2.12(3H,s,CH 3 -27); two methyl doublet signals δ H 1.41(3H,d,J=7.2Hz,CH 3 -21) and 1.72 (3H, d, J = 6.0 Hz, CH 3 -Rha); four olefin proton signals δ H 5.33 (1H, br s, H-6), 7.27 (1H, d, J = 7.6 Hz, H-23), 7.46 (1H, d, J = 7.6 Hz, H-24) and 8.30 (1H, br s, H-26); two sugar terminal proton signals δ H 4.96(1H,d,J=7.6Hz,Glc-H-1'), 5.90(1H,s,Rha-1”). 13 C NMR showed that there were 39 carbon signals in the DEPT and HSQC spectra, 12 of which were attributed to sugar carbon signals, including two sugar terminal carbon signals (δ C 102.5, 102.7), 27 aglycone carbon signals, including 7 olefin carbon signals (δ C121.4, 121.9, 130.8, 138.0, 140.9, 148.5, 165.0), two oxygen-carbon signals (δ C 78.3,71.8). The above NMR data analysis showed that the compound was a steroidal alkaloid disodium glycoside.

[0050] The compound was subjected to acid hydrolysis, and the sugar portion and sugar standard were analyzed by TLC, showing that the sugar portion was composed of glucose and rhamnose, and according to the glucose terminal proton coupling constant value ( 3 J 1,2 >7.0Hz), it was determined to be β-glucose. C 72.9) and C-5 (δ C 70.4), which was determined to be α-rhamnose.

[0051] The absolute configuration of each sugar was determined by HPLC analysis after derivatization of the sugar moiety and was identified as D-glucose and L-rhamnose, respectively.

[0052] Further analysis of the compound 1 H and 13 C NMR spectrum data showed that it has structural similarity with the known compound cissoline. The two are exactly the same in the sugar chain and the AD ring of the aglycone. The difference lies mainly in the E and F rings of the aglycone. Compared with cissoline, the chemical shift values ​​of C-22 to C-26 of this compound move downfield to δ C 121.4 to 165.0, indicating that C-22 to C-26 are all olefin carbons, thus forming an F ring of a pyridine structural unit, and the E ring cleaves from the C16-O-C22 oxygen bridge, and the C-16 position is replaced by a free hydroxyl group.

[0053] The above speculation is based on the δ H 2.12(CH 3 -27) and δ C 138.0(C-24),148.5(C-26);δ H 7.27 (H-23) and δ C 165.0(C-22),138.0(C-24),130.8(C-25); δ H 7.46 (H-24) and δ C 165.0(C-22),121.4(C-23),148.5(C-26); δ H 8.30 (H-26) and δ C 165.0(C-22),138.0(C-24),130.8(C-25); δ H 1.41(CH3 -21) and δ C 165.0(C-22),63.3(C-17),38.9(C-20); δ H 3.65(H-20) and δ C 165.0(C-22),121.4(C-23),63.3(C-17); δ H 1.44 (H-15), 1.51 (H-17) and δ C The long-distance correlation signal between 71.8 (C-17) was further confirmed.

[0054] At the same time, the δ H 2.68(H-4) and δ C 30.3(C-2),37.0(C-10),78.3(C-3),140.9(C-5),121.9.3(C-6); δ H 0.94(H-19) and δ C 37.5(C-1),50.5(C-9),140.9(C-5); δ H 1.17(H-18) and δ C The long-range correlation signals between 40.3 (C-12), 54.9 (C-14), and 63.3 (C-17) also further confirmed the aglycone structure of the compound.

[0055] In the two-dimensional HMBC spectrum, H-1" (δ H 5.90) ​​and C-4'(δ C 78.3), glucose H-1'(δ H 4.98) and C-3 (δ C 78.3), further confirming that the sugar chain substitution position is at the C-3 position of the aglycone and the connection order is rhamnose-(1→4)-glucose.

[0056] NOESY correlation spectrum analysis showed that the configuration of this compound at C-3, C-8, C-9, C-10, C-13, C-14, C-16, C-17 and C-20 was the same as that in cissolane.

[0057] In summary, the chemical structures of the compounds extracted in identification step 1 are as follows:

[0058]

[0059] The compound was named as Soladineside A.

[0060] Example 2: Demonstration of anticancer effect of solanine glycoside A

[0061] 1. Cell culture: Gastric cancer cell lines BGC-827 and HGC-27 were cultured in RPMI-1640 medium containing 10% fetal bovine serum, streptomycin (100 μg / mL) and penicillin (100 U / mL) in a cell culture incubator with a 5% CO 2 , 37°C. When the cell density reaches 70%, subculture is performed, and after the cell state is stable, cells in the logarithmic phase are taken for testing.

[0062] 2. Detection of gastric cancer cell proliferation inhibition activity: Human gastric cancer cell lines BGC-827 and HGC-27 were cultured to the logarithmic growth phase, digested with 0.25% trypsin to form a single cell suspension, and 5×10 3 Cells were seeded at an average density per well in a 96-well plate. Incubate at 37°C and 5% CO 2 The cells were cultured under the same conditions for 24 hours, and then different concentrations of test samples (0, 1, 2.5, 5, 10, 20, 50, 100 μM) were added, with three replicate wells in each group. Three treatments were set for the test samples, including: solanine glycoside A, doxorubicin (positive control 1, drug control), and cytochrome Pyrrolidone (positive control 2, structural control). After the addition of each group of drugs (test samples), the cells were incubated for 48 hours under the same conditions. After the incubation, the drug-containing culture medium was removed, and fresh RPMI-1640 culture medium was added, and then CCK8 reagent was added at a volume of 10 μL / well. The cells were incubated at 37°C in the dark for 2 hours, and the absorbance of each well was measured at 450 nm, and the IC 50 The results were averaged and are shown in Table 2.

[0063] Table 2 Experimental results of the inhibitory effect of the compounds of the present invention on the proliferation of gastric cancer cells

[0064]

[0065] The results showed that solanine glycoside A had a certain inhibitory activity on the proliferation of gastric cancer cells BGC-827 and HGC-27, indicating that solanine glycoside A has good anti-gastric cancer efficacy. At the same time, compared with the known compound cissolani, solanine glycoside A has a significantly stronger inhibitory effect on gastric cancer cell proliferation, indicating that steroidal glycoside alkaloids with pyridine structural units in the F ring have better anti-gastric cancer activity. Therefore, solanine glycoside A has the potential to be used in the development of anticancer drugs.

[0066] The embodiments described above are only descriptions of the preferred modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, modifications, and substitutions made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Solanine glycoside A, characterized in that: The structural formula of solanine glycoside A is as follows:

2. The method for preparing solanine glycoside A according to claim 1, characterized in that: The method comprises the following steps: (1) defatting Solanum nigrum seeds, and then soaking and extracting them with an ethanol aqueous solution to obtain an extract; adding water to disperse the extract and extracting; (2) The aqueous layer obtained after extraction is separated and purified by macroporous resin, normal phase silica gel column chromatography, reverse phase silica gel column chromatography and HPLC to obtain solanine glycoside A.

3. The preparation method according to claim 2, characterized in that: In step (1), petroleum ether is used for soaking and degreasing.

4. The preparation method according to claim 2, characterized in that: In step (1), petroleum ether is used for extraction.

5. The preparation method according to claim 2, characterized in that: In step (2), the macroporous resin separation step comprises: separation by macroporous resin column chromatography, gradient elution with increasing ethanol concentration using ethanol aqueous solutions of different concentrations, and discarding the water elution portion.

6. The preparation method according to claim 2, characterized in that: In step (2), the normal phase silica gel column chromatography separation step includes: eluting with ethyl acetate-methanol-water-diethylamine system and dichloromethane-methanol-water-diethylamine system.

7. The preparation method according to claim 2, characterized in that: In step (2), the reverse phase silica gel column chromatography separation step comprises: performing gradient elution with increasing methanol concentration using a methanol-water solution.

8. The preparation method according to claim 2, characterized in that: In step (2), during HPLC separation and purification, the mobile phase is methanol-water solution.

9. The pharmaceutically acceptable salt of solanine glycoside A according to claim 1.

10. Medicines, health products, and foods prepared using the solanine glycoside A described in claim 1 or the solanine glycoside A prepared by the method described in any one of claims 2 to 8, or the salt described in claim 9; or; pharmaceutical compositions, health products, and foods comprising the solanine glycoside A described in claim 1 or the solanine glycoside A prepared by the method described in any one of claims 2 to 8, or the salt described in claim 9.

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