A triterpenoid saponin compound of honeysuckle and its preparation method and use

Abstract

The invention relates to the field of natural drug chemistry, and specifically relates to a new triterpenoid saponin compound obtained by extracting and separating the dried flowers of Lonicera japonica as raw materials through a natural product chemical method. The compound has a strong inhibitory effect on Hela, HTC-116, B16 and MDA-MB-468 tumor cells, and can provide a new lead compound for anti-tumor drug screening.

Description

Technical Field

[0001] The invention relates to the field of natural products, in particular to a triterpenoid saponin compound of Lonicera japonica and a preparation method and application thereof. Background Art

[0002] Lonicera macranthoides Hand.-Mazz. is a vine plant of the genus Lonicera in the family Caprifoliaceae. It is a species unique to my country. Its dried flower buds or flowers with initial opening are the main sources of Chinese medicine honeysuckle. It has the effects of clearing away heat and detoxicating, evacuating wind-heat, and can be used to treat carbuncle, furuncle, throat paralysis, erysipelas, pyretic bloody dysentery, wind-heat cold, and fever caused by febrile diseases. Triterpenoid saponin compounds are characteristic components of Lonicera macranthoides, and are mainly found in Lonicera macranthoides flower buds (Li Jinshen et al., Northern Pharmacy, 2014, 11(02):71-73.). Several saponin compounds found in Lonicera japonica have anti-tumor activity, such as Lonicera japonica saponin B, which exhibits significant anti-tumor activity in vitro and in vivo (Wang, J et al. Food Chem. Toxicol. 47, 1716-1721; Shan, Y., et al. Nutr. Cancer. 68, 280-289). Therefore, it is necessary to further explore the anti-tumor active components of Lonicera japonica and develop a new class of anti-cancer drugs for preventing and treating tumor growth. Summary of the invention

[0003] The invention aims to provide a triterpenoid saponin compound of Lonicera japonica, as well as a preparation method and use thereof in antitumor drugs.

[0004] To achieve the purpose of the present invention, the technical solution is as follows:

[0005] A triterpenoid saponin compound of Lonicera japonica, with a chemical name of 3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-23-hydroxy-ol ean-18-ene-28-oic acid, has the following chemical structure:

[0006]

[0007] The above compound is prepared by the following method, comprising the following steps:

[0008] S1 uses the dried flowers of Lonicera japonica as raw materials to prepare the total saponins of Lonicera japonica, and then uses reverse phase silica gel column chromatography with an elution system of ethanol-water;

[0009] S2: The product obtained in S1 is subjected to liquid-mass chromatography, gel column chromatography and preparative liquid chromatography, and the elution system is methanol-water to obtain the compound.

[0010] Furthermore, in S1, the elution fraction with an elution system ethanol: water ratio of 60:40 is taken to perform step S2.

[0011] Furthermore, in S2, the elution fraction of the gel column with a methanol:water ratio of 35:65 is taken and entered into liquid chromatography, and the elution fraction of the eluent system with a methanol:water ratio of 40:60 is taken to obtain the compound.

[0012] In a specific embodiment, the reverse phase silica gel column chromatography is selected from C18 reverse phase column, and the gel column chromatography is selected from Sephadex LH-20.

[0013] Furthermore, the preparation method of total saponins of Lonicera macranthoides Hand.-Mazz. comprises the following steps: using dried flowers of Lonicera macranthoides Hand.-Mazz. as raw materials, using water, alcohol, or a mixture of alcohol and water through a reflux method, a water extraction method or a cold soaking method to obtain an extract; passing the extract through a macroporous adsorption resin column chromatography, and the elution system is ethanol-water.

[0014] Furthermore, macroporous adsorption resin column chromatography was used to obtain the total saponins of Lonicera japonica L. by taking the elution system ethanol: water ratio of 70:30 to 75:15.

[0015] Furthermore, when alcohol or a mixture of alcohol and water is used for extraction, the extracting solution is concentrated before entering the macroporous adsorption resin column for chromatographic separation.

[0016] Furthermore, the extraction temperature of the extract is room temperature 25°C to 100°C, the number of extractions is 2 to 3 times, and the time of each extraction is 2h to 7d.

[0017] The alcohol is a small molecule alcohol, preferably methanol or ethanol. In one embodiment, when a mixture of alcohol and water is used, a mixture of ethanol and water is preferably used, with a volume ratio of 70% ethanol to 95% ethanol. In one embodiment, when a reflux method or a water extraction method is used, the extraction temperature is 80 to 100°C for each extraction, and the extraction time is 2 to 3 hours for each extraction. In another embodiment, when a cold soaking extraction is used, the extraction temperature is room temperature for each extraction, and the extraction time is 7 days for each extraction.

[0018] Preferably, the macroporous adsorption resin is selected from any one or more of D101, AB-8, and HP-20. More preferably, the macroporous adsorption resin is selected from D101.

[0019] The present invention also provides the use of the above compound in preparing an anti-tumor drug. Further, the tumor is cervical cancer, colorectal cancer, melanoma or breast cancer. The anti-tumor drug comprises an active ingredient and a pharmaceutically acceptable excipient, and the active ingredient comprises the above compound.

[0020] The beneficial effects of the present invention are as follows: the triterpenoid saponin compounds of Lonicera japonica discovered for the first time in the present invention can inhibit the growth of tumor cells in vitro, and have good results in cell inhibition activity assays on Hela, HTC-116, B16 and MDA-MB-468 tumor cells, and have the potential to develop anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the key HMBC signal diagram of the triterpenoid saponin compounds of Honeysuckle provided by the present invention. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with embodiments, but the embodiments of the present invention are not limited thereto. Without departing from the above-mentioned technical concept of the present invention, various substitutions and changes are made according to common technical knowledge and customary means in the field, which should all be included in the scope of the present invention.

[0023] Example 1 Preparation and identification of target compound 1

[0024] Take 3 kg of dried flowers of Honeysuckle, extract them with 70% ethanol aqueous solution under reflux at 80°C for 3 times, each time for 2 hours, combine the extracts, and then filter with a Buchner funnel to obtain a filtrate;

[0025] The filtrate was then rotary evaporated to obtain 96.4 g of a brown colloidal crude extract without alcohol taste;

[0026] The crude extract was separated by D101 macroporous resin column, and the elution system ethanol: water ratio was 0:100, 30:70, 70:30, 95:5, and four main fractions (Fr.I-Fr.IV) were obtained, among which Fr.III was the total saponins of Lonicera japonica;

[0027] The elution fraction Fr.III (34.2 g) with an elution system ethanol: water ratio of 70:30 was separated using a C18 reverse phase column, and the elution system ethanol: water ratios were 30:70, 40:60, 60:40, and 80:20, respectively, to obtain four subfractions Fr.III-A to D;

[0028] Then, guided by liquid-mass chromatography, the elution fraction of Fr.III-A with an ethanol: water ratio of 60:40 (16.4 g) was taken and separated using polysaccharide gel LH-20 (eluent system with methanol: water ratio of 35:65) and preparative liquid phase (eluent system with methanol: water ratio of 40:60, respectively), and finally the target compound 1 (27 mg) provided by the present invention was obtained.

[0029] Various spectral techniques (MS, NMR, UV, IR) were used to identify the structure of the target compound 1.

[0030] The target compound 1 is a white powder, easily soluble in water, methanol-water, and insoluble in low polar solvents such as ethyl acetate. The optical rotation is [α]20D-28.91 (c 0.11, methanol). The TCL plate is developed and sprayed with vanillin-concentrated sulfuric acid and heated to show blue-purple, indicating that the compound 1 may be a saponin compound. HR-ESI-MS shows m / z of 1097.5529 [M+Na] + (Calculated value: 1097.5503), its molecular formula can be determined to be C 53 H 86 O 22 , and its unsaturation is calculated to be 11.

[0031] The target compound 1 1 In the H-NMR spectrum, δ H 1.09(3H,s,H-24),δ H 0.86(3H,s,H-25),δ H 1.01(3H,s,H-26),δ H 0.88(3H,s,H-27),δ H 1.11(3H,s,H3-29) and δ H 1.03 (3H, s, H3-30) are 6 methyl hydrogen signals. 13 C-NMR spectrum shows δ C 13.9(C-24),δ C 17.4(C-25),δ C 16.3(C-26),δ C 15.2(C-27),δ C 30.8 (C-29) and δ C 29.3 (C-30) is the 6 corresponding methyl carbon signals. In addition, according to δ C 81.2(C-3) and δ C The chemical shift of 179.3 (C-28) can confirm that the sugar chain is connected to the C-3 of the aglycone. H 5.24 (H, s, H-19) olefin protons and δC 41.6(C-13),δ C 48.5(C-17),δ C 32.4(C-20),δ C 34.2(C-21),δ C 30.8 (C-29) is related to the five carbon atoms, and δ C 139.1(C-18) and δ H 2.68 (H, d, H-13) correlation, indicating that the double bonds are located at C-18 and C-19. Further analysis of 2D-NMR (COSY, ROESY, HSQC and HMBC) revealed that the aglycone part of the target compound 1 is 23-hydroxyolean-18-ene-28-oic acid.

[0032] The C-3 monosaccharide residue of target compound 1 is H 5.24(H,d,H-1′),δ H 6.27(H,s,H-1′),δ H 5.43 (H, d, H-1′) and δ H 5.18(H,d,H-1′) and δ C 104.8(C-1′),δ C 101.4(C-1′),δ C 106.7(C-1′),δ C 104.9 (C-1””), indicating that it contains 4 monosaccharides. The trimethylsilyl derivative of the target compound 1 after acid hydrolysis was subjected to gas chromatography-mass spectrometry analysis with raw sugar. The results showed that the sugar part was composed of L-Ara, L-Rha and D-Glc (ratio of 1:1:2). The β-terminal configuration of the glucose unit is composed of J 1,2 The coupling constant (7.8-8.0 Hz) was determined, and the α-terminal configuration of the arabinose unit was determined by J 1,2 The coupling constant (6.27 Hz) was determined, and the α-terminal configuration of the rhamnose unit was determined by the chemical shift δ C 69.6 (C-5″). The order of the tetrasaccharide chain at C-3 of the glycogen was deduced by the following HMBC correlation: δ H 5.24(H,d,H-1') and aglycone δ C 81.2(C-3) related, Rha δ H 6.27 (H, s, H-1") and Ara's δ C 75.3 (C-2') related, δ of Glc I H 5.43 (H, d, H-1”') and Rha δ C83.5 (C-3") related, δ of Glc II H 5.18 (H, d, H-1””) and δ of Glc I C 81.1 (C-4”'). The above NMR data show that the sugar moiety of the target compound 1 is the same as the tetrasaccharide moiety of macranthoside B. Through HSQC and HMBC spectrum analysis, all 1 H and 13 The C NMR signal was finally identified as 3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-23-hydroxy-olean-18-ene-28-oic acid (1). The specific hydrogen and carbon spectrum data of the target compound 1 are shown in Table 1.

[0033] Table 1. Hydrogen and carbon spectrum data of target compound 1 provided by the present invention

[0034] Table 1 1 H and 13 C NMR spectral data of 1

[0035]

[0036]

[0037] Data were measured at 600MHz for 1 H and 150MHz for 13 C in Pyridine-d 5 ,δin ppm,J inHz.

[0038] The purity of the target compound 1 was determined by LC-MS. The LC chromatographic conditions were as follows: chromatographic column Agilent Poroshell120SB-AQ C18 (3.0×100 mm, 2.7 um); column temperature 30° C.; mobile phase 0.1% formic acid solution (A)-methanol (B), gradient elution (0-3 min, 10%-45% B; 3-12 min, 45%-60% B; 12-18 min, 60%-95% B; 18-20 min, 95% B), flow rate 0.3 mL min -1The injection volume was 5 μL. The mass spectrometry conditions were as follows: ESI ion source, negative ion mode, capillary voltage of 3.5 kV, drying gas temperature of 350 °C, fragmentation voltage of 0-16 min, 135 V; 16-30 min, 175 V; 30-38 min, 210 V, secondary voltage of 0-30 min, 30 V, 30-38 min, 40 V, nebulizer gas of N 2 , product ion scanning range was m / z 100-1700, nebulizer gas pressure was 50 psi, and drying gas flow rate was 10.0 L·min -1 , Skimmer voltage 65 V. The retention time of 3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-23-hydroxy-olean-18-ene-28-oicacid(1) was 31.24 min, and the purity of the product was 98.74% calculated by the area normalization method.

[0039] Example 2 Preparation of target compound 1 using water extraction method

[0040] Take 1 kg of dried flowers of Honeysuckle, and extract them three times with water heating, each time with 10 L of water, each time with 2 h of extraction time, and each time with 100 °C of extraction temperature;

[0041] The obtained extract was adsorbed by macroporous resin HP-20, and the elution system ethanol: water ratio was 0:100, 30:70, 70:30, 95:5, and the 70% ethanol eluate was decompressed and the solvent was recovered to obtain 47 g of total saponins of Lonicera japonica.

[0042] The obtained total saponins of Lonicera japonica were subjected to C-18 reverse phase silica gel column chromatography, and the ratio of ethanol to water in the mobile phase was 30:70, 40:60, 60:40, 80:20, and 0:100, respectively;

[0043] The third fraction was eluted with an ethanol: water ratio of 60:40. The fraction was separated using Sephadex LH-20 (eluent system was methanol: water ratio of 35:65) and preparative liquid phase (eluent system was methanol: water ratio of 40:60) in a liquid-mass separation manner to obtain 12 mg of 3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-23-hydroxy-olean-18-ene-28-oicacid (1) with a yield of 0.0012%. The purity of the product was 98.2% as determined by LC-MS.

[0044] Example 3: Preparation of target compound 1 using methanol cold extraction method

[0045] Take 1 kg of dried flowers of Honeysuckle, extract them with methanol for three times, the amount of methanol used each time is 20 liters, the extraction time each time is 7 days, the extraction temperature each time is room temperature, and the extract is concentrated to an extract without alcohol taste (dry weight is 210 g);

[0046] The obtained extract was dissolved in 10 times the volume of water, and filtered with filter paper to remove water-insoluble matter;

[0047] The obtained filtrate was adsorbed by macroporous resin AB-8, and the elution system ethanol: water ratio was 0:100, 15:75, 75:15, and the elution solution with ethanol: water ratio of 75:15 was taken and concentrated to obtain 54 g of total saponins of Lonicera japonica.

[0048] The obtained total saponins of Lonicera japonica were subjected to C-18 reverse phase silica gel column chromatography, and the ratio of ethanol to water in the mobile phase was 30:70, 40:60, 60:40, 80:20, and 0:100, respectively;

[0049] The third fraction was eluted with ethanol and water in a ratio of 60:40. The fraction was separated using Sephadex LH-20 (eluent system was methanol and water in a ratio of 35:65) and preparative liquid phase (eluent system was methanol and water in a ratio of 40:60) in a liquid-mass separation manner to obtain 13 mg of 3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-23-hydroxy-olean-18-ene-28-oicacid(1) with a yield of 0.0013%. The purity of the product was 98.5% as determined by LC-MS.

[0050] Example 4 Tumor cell inhibitory activity assay

[0051] In this example, the MTT method was used to test the in vitro activity of the target compound 1, and the anticancer drug 5-fluorouracil was used as a positive control. The tumor cells selected were Hela, HTC-116, B16 and MDA-MB-468 tumor cells. The specific method is as follows:

[0052] Hela, HTC-116, B16 and MDA-MB-468 tumor cells in logarithmic growth phase were seeded in 96-well culture plates at a density of 5000 cells per well in 100 μL. They were treated with target compound 1 at concentrations of 10, 5, 2.5, 1.25, 0.625 and 0.3125 μmol / L at 37°C and 5% CO. 2 Incubate in an incubator. After 72 hours, add 10 μL of MTT (5 mg / mL, PBS) to each well, continue incubating in the incubator for 4 hours, centrifuge at 1000 rpm for 5 minutes, and carefully discard the supernatant. Add 100 μL of DMSO to each well, shake for 10 minutes, and use an ELISA reader to measure the absorbance of each well at a wavelength of 570 nm and calculate the cell growth inhibition rate. The cell growth inhibition rate calculation formula is as follows:

[0053] Cell growth inhibition rate (%) = (1-OD value of experimental group / OD value of cell control group) × 100

[0054] The positive control was 5-fluorouracil at concentrations of 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 μmol / L. IC was calculated using DPS software. 50 The measurement results are shown in Table 2.

[0055] Table 2. Inhibitory activity of target compound 1 on Hela, HTC-116, B16 and MDA-MB-468 tumor cells (IC 50 ,μM)

[0056]

Claims

1. A triterpenoid saponin compound of Lonicera japonica, characterized in that: The triterpenoid saponin compound of honeysuckle has the following chemical structural formula:

2. A method for preparing a triterpenoid saponin compound of Lonicera japonica, characterized in that: The compound is the triterpenoid saponin compound of Lonicera japonica as claimed in claim 1, comprising the following steps: S1 uses the dried flowers of Lonicera japonica as raw materials to prepare the total saponins of Lonicera japonica, and then uses reverse phase silica gel column chromatography, and the elution system is ethanol-water; S2 uses liquid-mass-mass-guided gel column chromatography and preparative liquid chromatography, with the elution system being methanol-water, to obtain the compound; In S1, the elution fraction with an elution system ethanol: water ratio of 60:40 is taken to perform step S2; In S2, the elution fraction of the gel column with a methanol:water ratio of 35:65 is taken and entered into liquid chromatography, and the elution fraction of the eluent system with a methanol:water ratio of 40:60 is taken to obtain the compound.

3. The method for preparing a triterpenoid saponin compound of Lonicera japonica L. according to claim 2, characterized in that: The reverse phase silica gel column chromatography is selected from C18 reverse phase column, and the gel column chromatography is selected from Sephadex LH-20.

4. The method for preparing a triterpenoid saponin compound of Lonicera japonica L. according to claim 2, characterized in that: The preparation method of total saponins of Lonicera japonica comprises the following steps: using dried flowers of Lonicera japonica as raw materials, using water, alcohol, or a mixture of alcohol and water through a reflux method, a water extraction method or a cold soaking method to obtain an extract; and subjecting the extract to macroporous adsorption resin column chromatography, wherein the elution system is ethanol-water.

5. The method for preparing a triterpenoid saponin compound of Lonicera japonica L. according to claim 4, characterized in that: The macroporous adsorption resin column chromatography is used to obtain the elution fraction with the elution system ethanol: water ratio of 70:30 to 75:15 to obtain the total saponins of Lonicera japonica.

6. The method for preparing a triterpenoid saponin compound of Lonicera japonica L. according to claim 4, characterized in that: When alcohol or a mixture of alcohol and water is used for extraction, the extracting solution is concentrated before entering the macroporous adsorption resin column for chromatography separation.

7. The method for preparing a triterpenoid saponin compound of Lonicera japonica L. according to claim 4, characterized in that: The extraction temperature of the extract is room temperature 25°C to 100°C, the number of extractions is 2 to 3 times, and the time of each extraction is 2h to 7d.

8. An application of a triterpenoid saponin compound of Lonicera japonica in the preparation of an anti-tumor drug, characterized in that: The compound is the triterpenoid saponin compound of Lonicera japonica as claimed in claim 1, and the tumor is cervical cancer, colorectal cancer, melanoma or breast cancer.

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