Compound with novel enantiokaurane diterpene dimer skeleton structure as well as preparation method and application of compound

Abstract

The invention discloses a compound with a novel enantiokaurane diterpene dimer skeleton structure and a preparation method of the compound. According to the invention, chemical components of a Western African plant I.trichana tuber are deeply studied, and one enantiokaurane diterpene dimer new skeleton compound Bisicacinol A is obtained through separation by virtue of various chromatographic combination methods, wherein the chemical components of the Western African plant I.trichana tuber can be used as an enantiokaurane diterpene dimer new skeleton compound Bisicacinol A; pharmacological experiments show that the monomeric compound Bisicacinol A (1) obtained through separation has remarkable anti-tumor activity, also has better antibacterial activity, and has a potential prospect of being developed into a new drug. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to a preparation method of a novel skeleton compound of an enantiokaurane diterpene dimer and its applications in anti-tumor and anti-bacterial aspects. Background Art

[0002] I. trichantha is a plant endemic to Central and West Africa in the genus Iodes, and is a medicinal plant used by indigenous tribes in Nigeria and surrounding countries. This plant has played an important role in traditional medicine in Nigeria. Local people use its tubers as medicine and widely apply them to the treatment of various diseases, such as poisoning, constipation, inducing vomiting, and treating malaria. The tubers of I. trichantha are rich in starch. During famine years, local people wash the tubers with water repeatedly and then eat them. Modern research shows that I. trichantha has various physiological activities such as hypoglycemic, anti-convulsant, sedative, analgesic, and antibacterial effects. Currently, there is relatively little research on the chemical constituents of I. trichantha. Summary of the Invention

[0003] The object of the present invention is to conduct in-depth research on the active ingredients of the tubers of I. trichantha, extract and separate the potentially active ingredients by various chromatographic combination methods, and provide a scientific basis for clinical application.

[0004] Technical Solution: The present invention has isolated a novel skeleton compound of an enantiokaurane diterpene dimer from the tubers of the plant I. trichantha, and the compound is named Bisicacinol A (1).

[0005] The structural formula of Compound 1 of the present invention is:

[0006]

[0007] The preparation method of the novel skeleton compound of an enantiokaurane diterpene dimer according to the present invention is characterized by including the following steps:

[0008] (1) Weigh the dried tubers of I. trichantha, crush them, add ethanol and water for reflux extraction of the medicinal materials, filter and collect the filtrate, and concentrate it under reduced pressure until there is no alcohol smell to obtain a concentrated solution;

[0009] (2) Extract the concentrated solution obtained in step (1) with petroleum ether, dichloromethane, ethyl acetate, and n-butanol respectively, and concentrate under reduced pressure to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction, an n-butanol fraction, and a residual water fraction respectively;

[0010] (3) Take the dichloromethane fraction obtained in step (2), perform normal pressure column chromatography on AB-8 macroporous resin, and elute with ethanol-water to obtain 3 sub-fractions DCM-1 - DCM-3;

[0011] (4) The eluate fraction DCM-3 obtained in step (3) was subjected to silica gel normal pressure column chromatography and eluted with dichloromethane-methanol to obtain 7 sub-fractions DCM-3-1 - DCM-3-7;

[0012] (5) The fraction DCM-3-5 obtained in step (4) was subjected to medium pressure preparative-MCI column chromatography with gradient elution using methanol-water as the mobile phase to obtain 11 fractions DCM-3-5-1 - DCM-3-5-11;

[0013] (6) Compound 1 was prepared from the fraction DCM-3-5-7 by semi-preparative high performance liquid chromatography.

[0014] As a more limited scheme, the preparation method of the present invention includes the following steps:

[0015] (1) Weigh dry I. trichantha tubers, crush them, add ethanol with a volume concentration of 95% to reflux and extract the medicinal materials with a material-liquid ratio of 1:10, extract 3 times, 2.5 hours each time, filter and collect the filtrate, combine the filtrates, and concentrate under reduced pressure until there is no alcohol smell to obtain an extract;

[0016] (2) The extract obtained in step (1) was made into a suspension with an appropriate amount of water, and then extracted with equal volumes of petroleum ether, dichloromethane, ethyl acetate, and n-butanol, 5 extractions each, to obtain the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction, and residual water fraction respectively.

[0017] (3) The dichloromethane fraction obtained in step (2) was mixed with the sample and macroporous resin at a ratio of 1:1.5, and subjected to macroporous resin normal pressure column chromatography, and successively eluted with ethanol-water with a volume ratio of 30:70, 60:40, 90:10, 100:0 as the mobile phase with gradient elution, 5 column volumes for each gradient elution; after analysis by thin layer and ultra-high performance liquid chromatography, it was concentrated and combined using a rotary evaporator to obtain 3 sub-fractions DCM-1 - DCM-3;

[0018] (4) The eluate fraction DCM-3 obtained in step (3) was mixed with the sample and silica gel at a ratio of 1:1.5, and subjected to silica gel normal pressure column chromatography, and successively eluted with dichloromethane-methanol with a volume ratio of 100:0, 50:1, 30:1, 20:1, 10:1, 7:1, 5:1, 3:1, 2:1, 1:1, 1:3, 1:7, 0:100 as the mobile phase with gradient elution, 4 column volumes for each gradient elution; after analysis by thin layer and ultra-high performance liquid chromatography, it was concentrated and combined using a rotary evaporator to obtain 7 sub-fractions DCM-3-1 - DCM-3-7;

[0019] (5) The fraction DCM-3-5 obtained in step (4) was mixed with the sample and MCI at a ratio of 1:1.2, and medium-pressure preparative MCI column chromatography was used for gradient elution with methanol-water as the mobile phase, where A was water and B was methanol; the elution gradient program was as follows: from 0.01 to 20.00 min, 30%-30% B; from 20.00 to 50.00 min, 40%-40% B; from 50.00 to 80.00 min, 45%-45% B; from 80.00 to 110.00 min, 50%-50% B; from 110.00 to 140.00 min, 55%-55% B; from 140.00 to 170.00 min, 60%-60% B; from 170.00 to 200.00 min, 70%-70% B; from 200.00 to 230.00 min, 85%-85% B; from 230.00 to 270.00 min, 100%-100% B. The elution flow rate was 20 mL / min, and the detection wavelengths were 256 nm and 310 nm; after ultra-high performance liquid chromatography analysis, it was concentrated and combined to obtain 11 fractions DCM-3-5-1 - DCM-3-5-11;

[0020] (6) The fraction DCM-3-5-7 of the eluate from 150 - 170 min obtained in step (5) was subjected to semi-preparative high performance liquid chromatography and isocratic elution with pure water (A)-methanol (B) as the mobile phase, and the stock solution of monomer compound 1 was collected in the time period of 47 - 52 min.

[0021] As a preferred embodiment, the stock solution of compound 1 described above was further separated by semi-preparative high performance liquid chromatography. The chromatographic conditions of the semi-preparative high performance liquid chromatography were as follows: the chromatographic column model was Hedera ODS preparative chromatographic column (10 nm, 5 μm, 10×250 mm), the high performance liquid chromatography column pressure was 9.8 MPa, the column temperature was 22 - 26 °C, the injection volume was 100 μL, and the mobile phase was MeOH-H 2 O (v / v, 66:34), the flow rate was 3 mL / min, and the detection wavelength was 256 nm. Monomer compound Bisicacinol A (1) was obtained.

[0022] Beneficial effects:

[0023] In the present invention, the chemical constituents of the tubers of West African plant I. trichantha were deeply studied, and a new skeleton compound Bisicacinol A, an ent-kaurane diterpene dimer, was isolated. Through pharmacological experiments, it was found that the monomer compound Bisicacinol A (1) isolated in the present invention has significant anti-tumor activities (including pancreatic cancer, colon cancer, lung cancer or liver cancer), and also has good antibacterial activities (including Helicobacter pylori and Candida albicans), showing the potential for development as a new drug. Brief Description of the Drawings

[0024] Figure 1 (+)-HR-ESI-MS spectrum of Compound 1;

[0025] Figure 2 UV spectrum of Compound 1;

[0026] Figure 3 For Compound 1 1 1H NMR spectrum (500 MHz, Methanol-d 4 );

[0027] Figure 4 For Compound 1 13 13C NMR spectrum (125 MHz, Methanol-d 4 );

[0028] Figure 5 DEPT 135 spectrum of Compound 1 (125 MHz, Methanol-d 4 );

[0029] Figure 6 For Compound 1 1 1H- 1 1H COSY spectrum (500 MHz, Methanol-d 4 );

[0030] Figure 7 HSQC spectrum of Compound 1 ( 1 1H: 500 MHz, 13 13C: 125 MHz, Methanol-d 4 );

[0031] Figure 8 HMBC spectrum of Compound 1 ( 1 1H: 500 MHz, 13 13C: 125 MHz, Methanol-d 4 );

[0032] Figure 9 NOESY spectrum of Compound 1 (500 MHz, Methanol-d 4 );

[0033] Figure 10 For Compound 1 1 1H- 1 1H COSY, key HMBC and NOESY correlation diagrams. Detailed Description of the Invention

[0034] Example 1

[0035] 1. Instruments and Materials

[0036] 1.1 Instruments

[0037]

[0038]

[0039] 1.2 Experimental Materials

[0040] Hedera ODS preparative chromatography column (10 nm, 5 μm, 10×250 mm); Waters ACQUITY UPLC BEH C 18 (2.1 mm×100 mm, 1.7 μm) chromatography column; MCI GEL (CHP20, 75 - 150 μm), column chromatography silica gel (200 - 300 mesh); Chromatographic grade acetonitrile, methanol and formic acid were purchased from Merck, USA; Analytical grade petroleum ether, ethyl acetate, etc. were purchased from Nanjing Wanqing Chemical Reagent Co., Ltd.

[0041] 2. The preparation method of the compound comprises the following steps:

[0042] (1) Weigh the dried I. trichantha tubers, crush them, add ethanol with a volume concentration of 95% to reflux and extract the medicinal materials, with a material - liquid ratio of 1:10, extract 3 times, 2.5 h each time, filter and collect the filtrate, combine the filtrates, and concentrate under reduced pressure until the alcohol smell disappears to obtain an extract.

[0043] (2) Make the extract obtained in step (1) into a suspension with an appropriate amount of water, then extract with equal volumes of petroleum ether, dichloromethane, ethyl acetate and n - butanol, extract 5 times each to obtain the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n - butanol fraction and residual water fraction respectively.

[0044] (3) For the dichloromethane fraction obtained in step (2), mix the sample with macroporous resin at a ratio of sample:macroporous resin = 1:1.5, use normal - pressure column chromatography with macroporous resin, and elute successively with ethanol - water with volume ratios of 30:70, 60:40, 90:10, 100:0 as the mobile phase gradient, elute 5 column volumes for each gradient; After analysis by thin - layer and ultra - high - performance liquid chromatography, concentrate and combine using a rotary evaporator to obtain 3 sub - fractions DCM - 1 - DCM - 3.

[0045] (4) The eluate fraction DCM-3 obtained in step (3) was mixed with the sample and silica gel at a ratio of 1:1.5, and silica gel normal pressure column chromatography was used. Dichloromethane-methanol with volume ratios of 100:0, 50:1, 30:1, 20:1, 10:1, 7:1, 5:1, 3:1, 2:1, 1:1, 1:3, 1:7, 0:100 was used as the mobile phase for gradient elution, and each gradient was eluted for 4 column volumes; after analysis by thin layer and ultra-high performance liquid chromatography, it was concentrated and combined using a rotary evaporator to obtain 7 sub-fractions DCM-3-1 - DCM-3-7;

[0046] (5) The fraction DCM-3-5 obtained in step (4) was mixed with the sample and MCI at a ratio of 1:1.2, and medium pressure preparative-MCI column chromatography gradient elution was used, with methanol-water as the mobile phase, where A is water and B is methanol; the elution gradient program was: 0.01 - 20.00 min, 30% - 30% B; 20.00 - 50.00 min, 40% - 40% B; 50.00 - 80.00 min, 45% - 45% B; 80.00 - 110.00 min, 50% - 50% B; 110.00 - 140.00 min, 55% - 55% B; 140.00 - 170.00 min, 60% - 60% B; 170.00 - 200.00 min, 70% - 70% B; 200.00 - 230.00 min, 85% - 85% B; 230.00 - 270.00 min, 100% - 100% B, the elution flow rate was 20 mL / min, and the detection wavelengths were 256 nm and 310 nm; after analysis by ultra-high performance liquid chromatography, it was concentrated and combined to obtain 11 fractions DCM-3-5-1 - DCM-3-5-11;

[0047] (6) The eluate fraction DCM-3-5-7 from 150 - 170 min obtained in step (5) was separated by semi-preparative high performance liquid chromatography. The chromatographic column model was Hedera ODS preparative chromatographic column (10 nm, 5 μm, 10×250 mm), the high performance liquid chromatography column pressure was 9.8 MPa, the column temperature was 22 - 26 °C, the injection volume was 100 μL, and the mobile phase was MeOH-H 2 O (v / v, 66:34), the flow rate was 3 mL / min, and the detection wavelength was 256 nm. The monomeric compound Bisicacinol A (1) was obtained.

[0048] 3. Structural analysis of the compound

[0049] 3.1 Structural identification of Bisicacinol A (1)

[0050]

[0051] Compound 1 is a white amorphous powder, (c 0.01, MeOH). Combining 13 The \(^{13}\)C NMR data and the quasimolecular ion peak m / z 687.3504 ([M + Na]\(^{+}\), \(C_{36}H_{52}O_{11}Na\) calculated as 687.3509) given by high-resolution electrospray ionization mass spectrometry (HRESIMS) led to the deduction of the molecular formula of the compound as \(C_{36}H_{52}O_{11}\), with an index of hydrogen deficiency of 14. In the \(^1\)H NMR spectrum of compound 1 (Table 1), signals of four methyl protons were observed at δ 39 H 52 O 9 Na calculated as 687.3509), the molecular formula of the compound was deduced to be \(C_{36}H_{52}O_{11}\), with an index of hydrogen deficiency of 14. In the \(^1\)H NMR spectrum of compound 1 (Table 1), signals of four methyl protons were observed at δ 39 H 52 O 9 , and the index of hydrogen deficiency was 14. In the \(^1\)H NMR spectrum of compound 1 (Table 1), signals of four methyl protons were observed at δ H (1.09, s, \(CH_3\)-18; 1.04, s, \(CH_3\)-19; 1.03, s, \(CH_3\)-18′; 0.96, s, \(CH_3\)-19′), combining 3 -18; 1.04, s, \(CH_3\)-19; 1.03, s, \(CH_3\)-18′; 0.96, s, \(CH_3\)-19′), combining 3 -19; 1.03, s, \(CH_3\)-18′; 0.96, s, \(CH_3\)-19′), combining 3 -18′; 0.96, s, \(CH_3\)-19′), combining 3 -19′), combining 13 The \(^{13}\)C NMR spectrum and DEPT spectrum showed that the compound had 39 carbon signals, including four methyl carbons, 13 methylene carbons (including two oxygenated methylenes), 11 methine carbons (including six oxygenated methines), nine quaternary carbons (including one oxygenated carbon), and two carbonyl carbons. Combining the carbon spectrum (Table 1), DEPT spectrum, and HSQC spectrum of the compound indicated that 1 had 39 carbon signals, which could be assigned as follows: four methyl carbons at δ C 21.4 (\(CH_3\)-18), 33.1 (\(CH_3\)-19), 32.5 (\(CH_3\)-18′), 29.2 (\(CH_3\)-19′); 13 methylene carbons (including two oxygenated methylenes) at δ 3 -18), 33.1 (\(CH_3\)-19), 32.5 (\(CH_3\)-18′), 29.2 (\(CH_3\)-19′); 13 methylene carbons (including two oxygenated methylenes) at δ 3 -19), 32.5 (\(CH_3\)-18′), 29.2 (\(CH_3\)-19′); 13 methylene carbons (including two oxygenated methylenes) at δ 3 -18′), 29.2 (\(CH_3\)-19′); 13 methylene carbons (including two oxygenated methylenes) at δ 3 -19′); 13 methylene carbons (including two oxygenated methylenes) δ C 30.1 (C-2), 39.7 (C-3), 21.7 (C-11), 21.2 (C-12), 17.9 (C-17), 64.8 (C-20), 23.8 (C-2′), 32.2 (C-3′), 71.9 (C-7′), 26.8 (C-10′), 22.9 (C-11′), 45.0 (C-15′), and 26.3 (C-17′); 11 methine carbons (including six oxygenated methines) δ C 73.4 (C-1), 62.8 (C-5), 74.7 (C-6), 56.4 (C-9), 41.5 (C-13), 83.4 (C-14), 81.5 (C-1′), 56.3 (C-5′), 103.3 (C-6′), 32.5 (C-9′), and 78.7 (C-16′); nine quaternary carbons (including one oxygenated carbon) δC 34.5 (C-4), 97.7 (C-7), 63.8 (C-8), 41.9 (C-10), 62.4 (C-16), 31.1 (C-1′), 52.6 (C-8′), 147.6 (C-12′) and 136.0 (C-13′); two carbonyl carbons δ C 221.5 (C-15) and 206.9 (C-14′).

[0052] The relative configuration of compound 1 was determined by analyzing the NOESY spectrum ( Figure 9 ). So far, it has been reported that H-5 of ent-kaurane diterpenoids is β-oriented, and H-1 / H-5 / CH 3 -19, H-5 / H-9 correlations can be observed in the NOESY spectrum, indicating that H-1, H-9, and CH3-19 are all β-oriented; further, the correlations of H-6 / CH 3 -18 / H-20a, H-20b / H-14 / H-13 were used to deduce that H-6, H-13, H-14, and CH 3 -18 are all α-oriented; the cross-peaks of H-13 / H-17α and H-17β / H-14′ indicate that H-14′ is β-oriented; the correlations of H-1′ / H-9′ / CH 3 -19 / H-5′ indicate that the protons of the four are coplanar; the correlation between H-6 and CH 3 -18′ indicates that they have the same orientation. However, due to the lack of NOESY correlations between units A and B, the stereochemistry of H-1′, H-5′, H-6′, and H-9′ remains unknown. To analyze the absolute configuration of H-1′, H-5′, H-6′, and H-9′ in unit B of compound 1, through the measurement and calculation of ECD, the absolute configuration of compound 1 was finally determined to be 1S, 5R, 6S, 7S, 8R, 9S, 10S, 13S, 14R, 16R, 1′S, 5′R, 6′R, 8′S, 9′R, 16′S. Therefore, the complete structure of the new skeleton compound 1 was determined and named Bisicacinol A. Compound Bisicacinol A is the first ent-kaurane diterpenoid dimer with a new skeleton compound of trans / cis / cis / cis 6 / 6 / 5 / 6 / 7 connected to a cage-like 3,3-dimethyl-9,10-dioxatricyclo[4.3.1.0 2,7 system.

[0053] Table 1 1 H and 13 C NMR data of compound 1 ( 1 H, 500 MHz; 13 C, 125 MHz)

[0054]

[0055]

[0056]

[0057] Example 2

[0058] The anti-tumor cell activity test research of the present invention was carried out according to the following steps:

[0059] 1. Tumor cell culture

[0060] The pancreatic cancer cell line MIA PaCa-2, colon cancer cell line HT-29, lung cancer cell line A549 and liver cancer cell line HepG2 (Cell Bank of the Chinese Academy of Sciences) were cultured in DMEM culture medium (Gibco) containing 10% fetal bovine serum at 37 °C and 5% CO 2 under the condition. The culture media of HT-29, A549 and HepG2 cells were supplemented with 10% FBS and 1% PSN, and the culture medium of MIA PaCa-2 cells was supplemented with 10% FBS, 2.5% HS and 1% PSN.

[0061] 2. Preparation of experimental drugs

[0062] An appropriate amount of Compound 1 prepared in Example 1 above was weighed and dissolved in DMSO to make the final concentration of the stock solution 40 mM, and stored in a refrigerator at 4 °C. Before the experiment, the stock solution was diluted with DMEM medium to make the drug concentration 20 μM, and it was ensured that the final concentration of DMSO was less than 0.1%. Different volumes of DMEM medium were added to dilute the compound into different concentrations. At the same time, DMEM medium containing 0.1% DMSO was used as a negative control.

[0063] 3. Toxicity of drugs to tumor cell lines

[0064] The tumor cells were suspended with the culture medium. MIA PaCa-2, HT-29 and HepG2 were inoculated into 96-well plates at a cell density of 8000, and A549 was inoculated into 96-well plates at a cell density of 3000 (100 μL / well), and cultured at 37 °C and 5% CO 2 under the condition for 24 h. At the logarithmic growth phase of the tumor cell lines, different concentrations of the compound (0, 0.004, 0.04, 0.4, 4, 40 μM) were added, and cultured at 37 °C and 5% CO 2 under the condition for 72 h.

[0065] 4. Detection of cell viability by CCK-8 method

[0066] After the drug has acted on tumor cells for 72 h, add 10 μL of CCK-8 solution to each well and incubate in a sterile incubator for 1 h. Take out and measure the OD value at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the IC 50 (half maximal inhibitory concentration) of the compound using GraphPad Prism 8 software, with 5-FU and Carboplatin as positive controls.

[0067] 5. The experimental results are shown in Table 2

[0068] Table 2. Cytotoxic activity of Compound 1 (IC50: μM)

[0069]

[0070] Experimental conclusion: Through the evaluation of the cytotoxic activity of Compound 1, it was found that Compound 1 has strong activity against adenocarcinoma cell line MIAPaCa-2, colon cancer cell line HT-29, lung cancer cell line A549, and liver cancer cell line HepG2. In particular, it has a significant anti-proliferative effect on MIA PaCa-2 and A549 cells, and its effect is stronger than that of the positive drugs 5-FU and Carboplatin, showing the potential to be developed into a new anti-cancer drug.

[0071] Example 3

[0072] The antibacterial activity test study of the present invention was carried out according to the following steps:

[0073] 1. Experimental materials and reagents

[0074] Helicobacter pylori (G27 and HP129) and Candida albicans (SC5314 and C5) were both from the laboratory of Professor Bi Hongkai at Nanjing Medical University. Main culture media and main reagents: Columbia medium, LB medium, selective antibiotics (metronidazole and amphotericin B), serum, etc.

[0075] 2. Preparation of culture media

[0076] Columbia liquid medium: Accurately weigh 29.0 g of Columbia liquid medium, heat and dissolve it in 1000 mL of double-distilled water. After the medium is completely dissolved, autoclave it at 121 °C for 15 min and reserve it for use.

[0077] Columbia blood agar solid medium: Accurately weigh 39.0 g of Columbia blood agar solid medium, heat and dissolve it in 1000 mL of double-distilled water. After the medium is completely dissolved, autoclave it at 121 °C for 15 min. When it is naturally cooled to about 50 °C after autoclaving, quickly add 5% sterile defibrinated sheep blood, mix well, and pour it into a sterile petri dish while it is still hot.

[0078] LB medium: glucose 15 g / L, yeast extract 10 g / L, peptone 5 g / L, sodium chloride 10 g / L.

[0079] 3. Recovery and culture of test strains

[0080] Take out the standard strain of Helicobacter pylori from the -80 °C refrigerator, place it at room temperature, accurately pipette 200 μL of the standard strain and transfer it onto the solid medium, and spread the strain evenly with an L-shaped glass rod. Place the culture dish containing the strain into a culture bag, then put a microaerophilic gas-generating bag into the culture bag, quickly seal the culture bag and place it in a 37 °C constant temperature incubator for 72 h. After the culture is completed, first identify Helicobacter pylori, then scrape off the bacterial lawn on the solid medium and transfer the bacterial lawn to 50 mL of liquid medium, which is used as the original bacterial solution.

[0081] 4. Determination of MIC

[0082] (1) Prepare the sample solution of compound 1 obtained in Example 1 with a concentration of 2 mg / mL.

[0083] (2) Preparation of MIC plate: First add 173.6 μL of medium to the first well, then add 6.4 μL of antibacterial drug, and serially dilute to the 7th well; do not add drug to the 8th well, and retain 90 μL of medium as the control for adding bacteria without adding drug.

[0084] (3) Preparation of bacterial solution: Take Helicobacter pylori growing in the logarithmic phase on the solid plate and make a bacterial suspension with BHI medium, adjust the concentration OD 600 to 0.3 (1×10 8 CFU / mL), dilute 10 times to 1×10 7 CFU / mL for standby.

[0085] (4) Inoculation of bacterial solution: Take 10 μL and add it to wells 1-8 (the concentration of bacterial solution in each well is about 1.0×10 6 CFU / mL). Incubate for 72 h to judge the results. The drug concentrations in wells 1 to 7 are 64, 32, 16, 8, 4, 2, and 1 μg / mL respectively.

[0086] (5) Result judgment: The lowest drug concentration that completely inhibits bacterial growth in the small well is the MIC. When bacteria grow significantly in the positive control well 8 (i.e., without antibiotics), the test is meaningful. When a single skip well appears in the microdilution method, the highest drug concentration that inhibits bacterial growth should be recorded. If multiple skip wells appear, the results should not be reported and the test needs to be repeated. Each drug is tested 3 times.

[0087] 5. The experimental results are shown in Table 3 and Table 4.

[0088] Table 3 Inhibitory effects of Compound 1 on different strains (MIC: μg / mL)

[0089] Strain Compound 1 MTZ H.pylori G27 ＞32 2 H.pylori 129 ＞32 8(R)

[0090] MTZ, metronidazole; R, resistant strain

[0091] Table 4 Synergistic inhibitory effects of Compound 1 and amphotericin B (AMB) on different strains (MIC: μg / mL)

[0092]

[0093] The above experimental results show that Compound 1 has certain antibacterial activity against two Helicobacter pylori strains; when used alone, Compound 1 has no significant inhibitory effect on the growth of Candida albicans, but when used in combination with AMB, it can significantly reduce its own MCI and at the same time reduce the MCI of AMB to below 0.25 μg / mL, and it can be used to prepare anti-Candida albicans drugs or as a lead compound for the development of anti-Candida albicans drugs.

Claims

1. A compound having a novel skeleton structure of an enantio-kaurane diterpene dimer, characterized in that: It has the following structural formula:

2. The method for preparing the compound according to claim 1, characterized in that: The following steps are involved: (1) Weigh the dried I. trichantha tubers, crush them, add ethanol water to reflux and extract the medicinal materials, collect the filtrate after filtering, and concentrate it under reduced pressure until there is no alcohol taste to obtain a concentrated solution; (2) extracting the concentrated solution obtained in step (1) with petroleum ether, dichloromethane, ethyl acetate and n-butanol, respectively, and concentrating under reduced pressure to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction, an n-butanol fraction and a raffinate fraction, respectively; (3) taking the dichloromethane fraction obtained in step (2), applying AB-8 macroporous resin to atmospheric pressure column chromatography, and eluting with ethanol-water to obtain three sub-fractions DCM-1 to DCM-3; (4) The eluate fraction DCM-3 obtained in step (3) was subjected to silica gel atmospheric pressure column chromatography and eluted with dichloromethane-methanol to obtain 7 sub-fractions DCM-3-1-DCM-3-7; (5) The fraction DCM-3-5 obtained in step (4) was subjected to medium pressure preparative MCI column chromatography with gradient elution and methanol-water as the mobile phase to obtain 11 fractions DCM-3-5-1-DCM-3-5-11; the fraction DCM-3-5-7 was subjected to semi-preparative HPLC to obtain compound 1.

3. The method for preparing the compound according to claim 2, characterized in that: The following steps are involved: (1) Weigh the dried I. trichantha tubers, crush them, add ethanol with a volume concentration of 80-95% to reflux and extract the medicinal materials, with a solid-liquid ratio of 1:6-20, extract 1-3 times, each time for 1-3 hours, filter and collect the filtrate, combine the filtrates, and concentrate under reduced pressure until there is no alcohol taste to obtain an extract; (2) The extract obtained in step (1) is made into a suspension with an appropriate amount of water, and then extracted with equal volumes of petroleum ether, dichloromethane, ethyl acetate and n-butanol, each extracted 3 to 6 times, to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction, an n-butanol fraction and a raffinate fraction, respectively. (3) The dichloromethane fraction obtained in step (2) was mixed with a macroporous resin, and subjected to macroporous resin atmospheric pressure column chromatography, with ethanol-water as the mobile phase in a volume ratio of 30:70, 60:40, 90:10, and 100:0, respectively, and each gradient elution was 3 to 6 column volumes; after thin layer chromatography and ultra-high performance liquid chromatography analysis, the fractions were concentrated and combined to obtain three sub-fractions DCM-1 to DCM-3; (4) The eluate fraction DCM-3 obtained in step (3) was mixed with silica gel and subjected to silica gel atmospheric pressure column chromatography, with dichloromethane-methanol as the mobile phase in a volume ratio of 100:0, 50:1, 30:1, 20:1, 10:1, 7:1, 5:1, 3:1, 2:1, 1:1, 1:3, 1:7, 0:100 as gradient elution, each gradient elution being 3 to 6 column volumes; after thin layer chromatography and ultra-performance liquid chromatography analysis, the fractions were concentrated and combined to obtain 7 sub-fractions DCM-3-1 to DCM-3-7; (5) The fraction DCM-3-5 obtained in step (4) was mixed with MCI and subjected to gradient elution by medium pressure preparative MCI column chromatography, with methanol-water as the mobile phase, wherein A is water and B is methanol; the elution gradient program is: 0.01-20.00 min, 30%-30% B; 20.00-50.00 min, 40%-40% B; 50.00-80.00 min, 45%-45% B; 80.00-110.00 min, 50%-50% B; 110.00-140.00 min, 55%-55% B; 140.00-170.00min, 60%-60% B; 170.00-200.00min, 70%-70% B; 200.00-230.00min, 85%-85% B; 230.00-270.00min, 100%-100% B, elution flow rate 20mL / min, detection wavelengths of 256nm and 310nm; after ultra-performance liquid chromatography analysis, concentrated and combined to obtain 11 fractions DCM-3-5-1-DCM-3-5-11; (6) The 150-170 min eluate fraction DCM-3-5-7 obtained in step (5) was subjected to semi-preparative high performance liquid chromatography, with pure water A-methanol B as the mobile phase for isocratic elution, and the fraction was collected in the time period of 47-52 min to obtain monomer compound 1.

4. The method for preparing the compound according to claim 3, characterized in that: The chromatographic conditions of the semi-preparative HPLC in step (6) are as follows: the chromatographic column model is a Hedera ODS preparative chromatographic column with a specification of 10 nm, 5 μm, 10×250 mm, the HPLC column pressure is 9.8 MPa, the column temperature is 22-26° C., the injection volume is 100 μL, the mobile phase is MeOH-H2O with a volume ratio of 66:34, the flow rate is 3 mL / min, and the detection wavelength is 256 nm.

5. Use of the compound according to claim 1 in the preparation of anticancer drugs.

6. Use of the compound according to claim 1 in the preparation of drugs for treating pancreatic cancer, colon cancer, lung cancer or liver cancer.

7. Use of the compound according to claim 1 in the preparation of antibacterial and antifungal drugs.

8. Use of the compound according to claim 1 in the preparation of anti-Helicobacter pylori drugs.

9. Use of the compound according to claim 1 in combination with amphotericin B in the preparation of an anti-Candida albicans drug.

10. A pharmaceutical preparation, characterized in that The compound according to claim 1 and a pharmaceutically acceptable carrier are prepared into a pharmaceutical preparation.

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