Application of compound in tumor treatment

Abstract

The invention provides an application of (1R, 2R, 4S)-beta-elemene in tumor treatment and a method for preparing the (1R, 2R, 4S)-beta-elemene, the (1R, 2R, 4S)-beta-elemene is obtained by heating and converting (-)-gemmarene A, and the (-)-gemmarene A is obtained by fermenting engineering bacteria containing nucleotide of enzyme capable of synthesizing the (-)-gemmarene A. The invention further discloses a preparation method of the (1R, 2R, 4S)-beta-elemene and an application of the (1R, 2R, 4S)-beta-elemene.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to and the benefits of Chinese Patent Application No. 202310770007.0, filed on June 27, 2023, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to the field of medicine, and in particular to the use of a compound (+)-β-elemene in treating tumors and a preparation method thereof. Background Art

[0004] Cancer is currently a major disease that threatens human health. In my country, approximately 1.8 million patients are diagnosed with the disease each year, and the incidence rate is increasing year by year. Currently, cancer treatment mainly relies on surgery, chemotherapy, and radiotherapy. However, a large number of chemotherapy drugs currently used in clinical practice still have problems such as large systemic toxicity and side effects, drug resistance, and low selectivity.

[0005] In recent years, researchers have tried to find natural products from natural plants such as camptothecin, matrine, berberine, paclitaxel, elemene, etc. to overcome the problems of chemotherapy drugs in the treatment of cancer. In the 1970s, people found that zedoary oil extracted from Curcuma zedoaria had antiviral and anti-tumor effects. In the 1990s, elemene was isolated from zedoary oil, and it was found that it included α-elemene, β-elemene, γ-elemene, and δ-elemene. It has been reported that β-elemene has the following three structures (W.Chen.etc., Royal alsociety of chemistry, 2022):

[0006]

[0007] Among them, (-)-β-elemene (i.e. (1S,2S,4R)-β-elemene) has been widely used to treat cancer. It has also been reported that (1S,2S,4S)-β-elemene is used to treat cancer and has better activity than (1S,2S,4R)-β-elemene. However, the synthesis of (1S,2S,4S)-β-elemene is very difficult (W.Chen.etc.,Royalsociety ofchemistry,2022). Not only does it have at least 8 reaction steps, but the yield is also extremely low, less than 10%, making it very difficult to industrialize.

[0008] At present, there are few reports on whether (1R,2R,4S)-β-elemene ((+)-β-elemene) has pharmacological activity, and the extraction or synthesis of (1R,2R,4S)-β-elemene is difficult to meet the needs of industrial applications. Summary of the invention

[0009] In one aspect, the present disclosure provides use of (1R,2R,4S)-β-elemene (ie, (+)-β-elemene) or a pharmaceutical composition comprising (1R,2R,4S)-β-elemene in the preparation of a medicament for treating cancer.

[0010] In another aspect, the present disclosure provides a method for treating cancer in an individual in need thereof, comprising administering to the individual (1R,2R,4S)-β-elemene or a pharmaceutical composition comprising (1R,2R,4S)-β-elemene.

[0011] In yet another aspect, the present disclosure provides (1R, 2R, 4S)-β-elemene or a pharmaceutical composition comprising (1R, 2R, 4S)-β-elemene for treating cancer.

[0012] In yet another aspect, the present disclosure provides use of (1R, 2R, 4S)-β-elemene or a pharmaceutical composition comprising (1R, 2R, 4S)-β-elemene in treating cancer.

[0013] In some embodiments, the cancer is selected from one or more of pancreatic cancer, leukemia, breast cancer, ovarian cancer, colorectal cancer, thyroid cancer, lung cancer, lymphoma, endometrial cancer, renal cell carcinoma, esophageal cancer, prostate cancer, gastric cancer, bladder cancer, brain tumor, liver cancer, cervical cancer, head and neck cancer, bone cancer, malignant melanoma, cancerous pleural effusion, malignant pleural mesothelioma and brain glioma.

[0014] In some embodiments, the cancer is selected from one or more of gastric cancer, brain glioma, pancreatic cancer, ovarian cancer, leukemia, lung cancer, breast cancer, esophageal cancer, prostate cancer, colorectal cancer, and liver cancer.

[0015] In some embodiments, the cancer is selected from one or more of pancreatic cancer, ovarian cancer, leukemia, lung cancer, breast cancer, esophageal cancer, prostate cancer, colorectal cancer, and liver cancer.

[0016] In some embodiments, the cancer is selected from lung cancer, glioma, liver cancer, and gastric cancer. In some embodiments, the cancer is selected from gastric cancer and glioma. In some embodiments, the cancer is selected from gastric cancer. In some embodiments, the cancer is selected from liver cancer.

[0017] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. Examples of the excipient include, but are not limited to, injection oil, carrier, filler, disintegrant, binder, lyophilization protectant, pH regulator, preservative, antioxidant, wetting agent, etc.

[0018] In some embodiments, the pharmaceutical composition can be prepared into various dosage forms, for example, solid dosage forms, semisolid dosage forms, liquid dosage forms and gaseous dosage forms, and the dosage forms can be, for example, liposomes, tablets, capsules, pills, mixtures, gels, ointments, granules, powders, creams, granules, pills, lozenges, solutions, suspensions, emulsions, pellets or injections, or one or more of these, or any other suitable dosage forms.

[0019] In some embodiments, the (1R, 2R, 4S)-β-elemene is obtained by converting (-)-germane A by heating.

[0020] In some embodiments, the heating temperature is 100°C-380°C. In some embodiments, the heating temperature is 100°C-200°C. In some embodiments, the heating temperature is 150°C-200°C. In some embodiments, the heating temperature is 150°C.

[0021] In some embodiments, the (-)-germarene A is obtained by a fermentation process.

[0022] In some embodiments, the fermentation method comprises the following steps: culturing the engineered bacteria in a seed culture medium to obtain a seed liquid; then inoculating the seed liquid into a fermentation culture medium for fermentation culture, and recording the product of the fermentation culture as a fermentation system; during the fermentation culture process, adding the fermentation culture medium to the fermentation system.

[0023] In a further embodiment, the fermentation culture comprises yeast fermentation or E. coli fermentation. The yeast fermentation or E. coli fermentation can be performed using methods known in the art.

[0024] In some embodiments, the fermentation culture comprises a two-phase fermentation or a single-phase fermentation.

[0025] In some embodiments, the fermentation culture is carried out at a temperature of 20-40°C. In some embodiments, the fermentation culture is carried out at a temperature of 20-37°C. In some embodiments, the fermentation culture is carried out at a temperature of 37°C. In some embodiments, the fermentation culture is carried out at a temperature of 20-30°C. In some embodiments, the fermentation culture is carried out at a temperature of 25-30°C. In some embodiments, the fermentation culture is carried out at a temperature of 25°C.

[0026] In some embodiments, the fermentation culture is carried out at a rotation speed of 100-500 rpm. In some embodiments, the fermentation culture is carried out at a rotation speed of 200-300 rpm.

[0027] In some embodiments, the fermentation culture time is 1-240 hours. In some embodiments, the fermentation culture time is 40-120 hours.

[0028] In some embodiments, the seed culture medium and fermentation culture medium contain 25 g / L glucose, 15 g / L ammonium sulfate, 6.15 g / L magnesium sulfate heptahydrate, 0.72 g / L zinc sulfate heptahydrate, 8 g / L potassium dihydrogen phosphate, 2 ml / L calcium chloride mother liquor, 10 ml / L trace metal salt mother liquor, 3 ml / L vitamin mother liquor, 100 mL / L succinic acid mother liquor, and the balance is water.

[0029] In some embodiments, the engineered bacteria comprises one or more enzymes capable of synthesizing (-)-germarene A. In further embodiments, the one or more enzymes capable of synthesizing (-)-germarene A are selected from enzymes capable of synthesizing (-)-germarene A having an amino acid sequence at least 80% homologous to the amino acid sequence of SEQ ID NO.2.

[0030] In a further embodiment, the engineered bacteria contains an expression cassette of a nucleotide sequence encoding an enzyme capable of synthesizing (-)-germarene A.

[0031] In some embodiments, the nucleotides encoding the enzyme capable of synthesizing (-)-germarene A are independently selected from nucleotide sequences that are at least 90% homologous to the nucleotide sequence of SEQ ID NO.1.

[0032] In some embodiments, the enzyme capable of synthesizing (-)-germarene A refers to an enzyme capable of converting farnesyl pyrophosphate into germarene.

[0033] In some embodiments, the (-)-germane A is separated and purified by extraction or chromatography. In some embodiments, the organic solvent used for extraction includes but is not limited to n-dodecane, isopropyl myristate, n-hexane, n-heptane, etc. In some embodiments, the organic solvent used for extraction is n-dodecane.

[0034] The inventors of the present application surprisingly found that (+)-β-elemene unexpectedly showed better tumor inhibition in in vitro and / or in vivo models compared to (-)-β-elemene and / or (1S, 2S, 4S)-β-elemene, providing a new option for the treatment of tumors. The present disclosure also provides a method for the biosynthesis of (+)-β-elemene, wherein (-)-gemaene A is obtained by fermentation of an engineered bacterium containing nucleotides encoding an enzyme capable of synthesizing (-)-gemaene A, and then (-)-gemaene A is thermally converted into (+)-β-elemene, thereby solving the problem that (+)-β-elemene is difficult to obtain, and providing an important source for the clinical application and industrial production of (+)-β-elemene. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The graph shows the time-dependent changes in the bioluminescent signal of orthotopic liver cancer tumors after (+)-β-elemene and (-)-β-elemene were administered to mice.

[0036] Figure 2 The inhibition rates of (+)-β-elemene and (-)-β-elemene on liver cancer in situ tumors were shown.

[0037] Figure 3 The graph shows the time-dependent changes in the bioluminescent signal of gastric cancer orthotopic tumors after administration of (+)-β-elemene to mice.

[0038] Specific implementation examples

[0039] The embodiments of the present disclosure are further described in detail below in conjunction with specific examples, but the implementation of the present disclosure is not limited thereto. Equivalent substitutions, combinations, improvements or modifications made to the embodiments of the present disclosure by those skilled in the art based on the description of the present disclosure should all be included in the protection scope of the present disclosure.

[0040] Unless otherwise specified, the experimental methods used below are routine methods well known to those skilled in the art. For example, they can be implemented using the standard procedures described in the following works: Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd Edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001; Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA, 1995; and Juan S. Bonifacino et al., Current Protocols in Cell Biology, John Wiley and Sons, Inc.

[0041] Identification of (-)-germarene A: Based on the disclosure in Alfred J. Weinheimer et al., Tetrahedron Letters No. 7, pp. 497-500, 1970.

[0042] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0043] The equipment and reagents used in the present disclosure are as follows: microplate reader (Spark JC437), balance (SECURA225D-1CNJC395), centrifuge (MODEL0406-1 GY067), inverted microscope (CKX53SF JC406), cell counter (CountstarRigel S2 JC422), CO2 incubator (371YL110), clean bench (ESCO, SW-CJ-2FD, FZ084), anhydrous ethanol (Sigma WXBC939V), CCK8 (Beyotime061223231013), PBS (ProcellWHB823U071), double antibody (Gibco 2441832), DMEM culture medium (Gibco 2537564), 1640 culture medium (Gibco 2732541), FBS (Sciencell 38890), pancreatic enzyme (Gibco 2764663).

[0044] The cell lines used in the present invention are: human in situ pancreatic adenocarcinoma cell line (BXPC-3), human blast crisis chronic myeloid leukemia cell line (LAMA-84), human lung squamous cell carcinoma cell line (NCI-H226), human colorectal cancer cell line (HCT116), human pancreatic cancer cell line (PANC-1), human ovarian cancer cell line (A2780), human chronic myeloid leukemia cell line (K562), human non-small cell lung cancer cell line (A549), human breast cancer cell line (MDA-MB-231), human esophageal cancer cell line (Eca-109), human prostate adenocarcinoma cell line (PC-3), human liver cancer cell line (HepG-2), human gastric cancer cell line (MKN-28), and human glioma cell line (U251).

[0045] The (+)-β-elemene used in Examples 1 to 7 can be obtained by the preparation method described in Example 8.

[0046] Example 1

[0047] (1) Prepare cell suspension (BXPC-3, density: 8×10 4 / mL), 100 μL of cell suspension was added to each well of a 96-well cell culture plate;

[0048] (2) The 96-well cell culture plate was placed in an incubator at 37°C, 5% CO2 and saturated humidity for 24 h; the drug was diluted with 1640 medium to the required working solution concentration, and 100 μL of drug-containing medium was added to each well (final drug concentration 150 μM). A vehicle control group (1640 medium without drug was added to the vehicle control group) and a blank control group (1640 medium was added only to the blank control group without cells and drugs) were set up at the same time;

[0049] (3) The 96-well cell culture plate was placed in an incubator at 37°C, 5% CO2 and saturated humidity for 24 h;

[0050] (4) Discard the supernatant and add 100 μL of 10% CCK8 detection reagent solution (prepared by adding 0.8 mL of CCK8 to 7.2 mL of serum-free 1640 culture medium and mixing well) to each well. Continue culturing in the incubator for 2.5 h. Observe that the bottom of the 96-well plate containing cells turns yellow.

[0051] At a detection wavelength of 450 nM, the OD value of each well was read using a microplate reader, wherein the tumor cell growth inhibition rate IR (%) = (1-(OD compound-OD blank control) / (OD solvent control-OD blank control))*100%, and the calculated increase in the inhibition rate of (+)-β-elemene relative to (-)-β-elemene was 6.77 times, calculated using the formula: increase factor = ((+)-β-elemene inhibition rate-(-)-β-elemene inhibition rate) / (-)-β-elemene inhibition rate.

[0052] Example 2

[0053] (1) Prepare cell suspension (LAMA-84, density: 3×10 5 / mL), 100 μL of cell suspension was added to each well of a 96-well cell culture plate;

[0054] (2) The 96-well cell culture plate was placed in an incubator at 37°C, 5% CO2 and saturated humidity for 24 h; the drug was diluted with 1640 medium to twice the required working solution concentration, and 100 μL of drug-containing medium was added to each well (final drug concentration 150 μM). A vehicle control group (medium without drug was added to the vehicle control group) and a blank control group (only medium was added without cells and drugs) were set up at the same time;

[0055] (3) The 96-well cell culture plate was placed in an incubator at 37°C, 5% CO2 and saturated humidity for 24 h. 20 μL of 10% CCK8 detection reagent solution (7.2 mL of serum-free 1640 medium plus 0.8 mL of CCK8 mixed) was added to each well, and the cells were cultured in the incubator for 2.5 h. The bottom of the 96-well plate containing cells turned yellow.

[0056] At a detection wavelength of 450 nM, the OD value of each well was read using a microplate reader, wherein the tumor cell growth inhibition rate IR (%) = (1-(OD compound-OD blank control) / (OD vehicle control-OD blank control))*100%, and the calculated increase in the inhibition rate of (+)-β-elemene relative to (-)-β-elemene was 0.54 times, and the calculation method of the increase factor was as described in Example 1.

[0057] Example 3

[0058] (1) Prepare cell suspension (NCI-H226, density: 8×10 4 / mL), 100 μL of cell suspension was added to each well of a 96-well cell culture plate;

[0059] (2) The 96-well cell culture plate was placed in an incubator at 37°C, 5% CO2 and saturated humidity for 24 h; the drug was diluted with 1640 medium to the required working solution concentration, and 100 μL of drug-containing medium was added to each well (final drug concentration 150 μM). A vehicle control group (a vehicle control group was added with medium without drug) and a blank control group (no cells or drugs were added, only medium was added);

[0060] (3) The 96-well cell culture plate was placed in an incubator at 37°C, 5% CO2 and saturated humidity for 24 h;

[0061] (4) Discard the supernatant and add 100 μL of 10% CCK8 detection reagent solution (7.2 mL of serum-free 1640 culture medium plus 0.8 mL of CCK8 and mix well) to each well. Continue culturing in the incubator for 2 h. Observe that the bottom of the 96-well plate containing cells turns yellow.

[0062] When the drug concentration was 150 μM and the detection wavelength was 450 nM, the OD value of each well was read by a microplate reader, and the inhibition rate was calculated. The tumor cell growth inhibition rate IR (%) = (1-(OD compound-OD blank control) / (OD vehicle control-OD blank control)) * 100%. It was calculated that the inhibition rate of (+)-β-elemene relative to (-)-β-elemene was increased by 3.85 times, and the calculation method of the increase factor was as described in Example 1.

[0063] Example 4

[0064] (1) Preparation of cell suspension: HCT116 density: 4×10 4 / mL, A549 density: 4.5×10 4 / mL, MDA-MB-231 density: 5×10 4 / mL, Eca-109 density: 4.5×10 4 / mL, PC-3 density: 6×10 4 / mL, HepG-2 density: 3.5×10 4 / mL, PANC-1 density: 6×10 4 / mL, A2780 density: 8×10 4 / mL, K562 density: 1×10 5100 μL of cell suspension was added to each well of a 96-well cell culture plate.

[0065] (2) The 96-well cell culture plate was placed in an incubator at 37°C, 5% CO2 and saturated humidity for 24 h; the drug was diluted with 1640 medium to the required working solution concentration, and 100 μL of drug-containing medium was added to each well, so that the final drug concentrations in each group of wells were 3.91 μM, 7.81 μM, 15.63 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, and 1000 μM, respectively. Each group had 3 replicate wells with the same final drug concentration. At the same time, a vehicle control group (a vehicle control group was added with a medium without drug) and a blank control group (no cells or drugs were added, only containing medium) were set up.

[0066] (3) The 96-well cell culture plate was placed in an incubator at 37°C, 5% CO2 and saturated humidity for 72 h.

[0067] (4) Discard the supernatant, add 100 μL of 10% CCK8 detection reagent solution to each well (add 0.8 mL CCK8 to 7.2 mL serum-free 1640 culture medium and mix well), continue to culture in the incubator for 2.5 hours, and observe that the bottom of the 96-well plate containing cells turns yellow. Under the condition of detection wavelength of 450 nM, the OD value of each well is read by microplate reader, and the inhibition rate is calculated, wherein, the tumor cell growth inhibition rate IR (%) = (1-(OD compound-OD blank control) / (OD solvent control-OD blank control)) * 100%. According to the inhibition rate of the drug at each concentration, the IC50 value of the drug is calculated using GraphPadPrism 10 software, that is, the drug concentration value corresponding to the inhibition rate of 50%. The experimental results are shown in Table 1.

[0068] Table 1 Inhibitory effects of different drugs in different cell lines

[0069]

[0070] Example 5

[0071] (1) Prepare cell suspension (MKN-28 density: 8×10 4 / mL, U251 density: 3.5×10 4 / mL), and 100 μL of cell suspension was added to each well of a 96-well cell culture plate.

[0072] (2) The 96-well MKN-28 cell culture plate was placed in an incubator at 37°C, 5% CO2 and saturated humidity for 24 h; the drug was diluted with 1640 medium to the required working solution concentration, and 100 μL of drug-containing medium was added to each well, so that the final drug concentrations in each group of wells were 3.91 μM, 7.81 μM, 15.63 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, and 1000 μM, respectively. Each group had 3 replicate wells with the same final drug concentration. At the same time, a vehicle control group (a medium without drug was added to the vehicle control group) and a blank control group (a blank control group without cells and drugs, containing only 1640 medium) were set up. The 96-well U251 cell culture plate was placed in an incubator at 37°C, 5% CO2 and saturated humidity for 24 hours; the drug was diluted with 1640 medium to the required working solution concentration, and 100 μL of drug-containing medium was added to each well, so that the final drug concentrations in each group of wells were 1.96 μM, 3.91 μM, 7.81 μM, 15.63 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, and 500 μM, respectively, and there were 3 replicate wells with the same final drug concentration in each group. At the same time, a solvent control group (medium without drug was added to the solvent control group) and a blank control group (no cells and drugs were added to the blank control group, and only 1640 medium was contained).

[0073] (3) The 96-well cell culture plate was placed in an incubator at 37°C, 5% CO2 and saturated humidity for 72 h.

[0074] (4) Discard the supernatant, add 100 μL of 10% CCK8 detection reagent solution to each well (add 0.8 mL CCK8 to 7.2 mL serum-free 1640 culture medium and mix well), continue to culture in the incubator for 2.5 hours, and observe that the bottom of the 96-well plate containing cells turns yellow. Under the condition of detection wavelength of 450 nM, the OD value of each well is read by microplate reader, and the inhibition rate is calculated, wherein, the tumor cell growth inhibition rate IR (%) = (1-(OD compound-OD blank control) / (OD solvent control-OD blank control)) * 100%. According to the inhibition rate of the drug at each concentration, the IC50 value of the drug is calculated using GraphPadPrism 10 software, that is, the drug concentration value corresponding to the inhibition rate of 50%. The experimental results are shown in Table 2.

[0075] Table 2 Inhibitory effects of different drugs in different cell lines

[0076]

[0077] Example 6

[0078] 1. Experimental Materials

[0079] 1.1 Animals: BALB / c female nude mice, 6-8 weeks old, weighing 17-23 grams, 18 mice, supplied by Shanghai Bikeway Biotechnology Co., Ltd. 1.2 Test drugs:

[0080] 1) Test substance: (+)-β-elemene (2% ( W / V )) or (-)-β-elemene (2% ( W / V )) and soybean oil (4.8% ( W / V )) was mixed as the oil phase, albumin solution (6% ( W / V )) is used as the aqueous phase and subjected to shear high-pressure homogenization treatment to obtain a stable aqueous dispersion system with an average particle size of the dispersed phase ≤1μm.

[0081] 2) Reference substance: 0.9% sodium chloride injection (Shijiazhuang Siyao Co., Ltd., national standard: H13023200)

[0082] 2. Experimental process

[0083] 1) Cell culture: Human hepatoma Hep3B-Luc (Hep3B, ATCC-HB-8064) cells were cultured in vitro in a monolayer in DMEM medium + 10% FBS and 1% Antibiotic-Antimycolic acid at 37°C in a 5% CO2 incubator. The cells were routinely digested and passaged using trypsin-EDTA. When the cell saturation reached 80%-90% and the number reached the required level, the cells were harvested, counted, and inoculated.

[0084] 2) Tumor cell inoculation: The anesthetized mouse was placed in supine position, and the abdominal skin was disinfected with 75% ethanol to keep the surgical area sterile. A transverse incision was made on the abdominal wall to fully expose the left lobe of the liver. 20 μl of 3×10 6 A cell suspension of Hep3B-Luc cells (15% Matrigel) was injected into the left liver lobe, and the injection site was checked for leakage. The left liver lobe was returned to the abdominal cavity and sutured, and the mice were continuously observed until they fully woke up.

[0085] 3) Experimental method: After inoculation, mice were intraperitoneally injected with 15 mg / mL of fluorescein solution at 10 μL / g body weight (the fluorescein injection dose for each animal was 150 mg / kg). Ten minutes later, the mice were pre-anesthetized with a mixture of oxygen and isoflurane. After the mice were deeply anesthetized, they were moved to the imaging room of IVIS (Lumia II) to start bioluminescence detection, and the bioluminescence signals in the animals and the generated image information were detected and recorded. Six days after tumor inoculation, group dosing began when the average fluorescence value reached 2.29E+07, with each group containing 6 mice. The day of grouping was defined as Day 0, and dosing began on Day 0 (i.e., dosing treatment began on the day of grouping). Dosing began on Day 0 and ended on Day 28. The dosage, route of administration, and frequency of administration for the three groups are as described in Table 3. Tumor growth inhibition rate TGI% calculation = [(1-(average bioluminescence signal of a treatment group at the end of drug administration-average bioluminescence signal of the treatment group at the beginning of drug administration)) / (average bioluminescence signal of the vehicle control group at the end of treatment-average bioluminescence signal of the vehicle control group at the beginning of treatment)] x 100%.

[0086] Table 3 Administration route and frequency

[0087] Group Number of animals Drug group Dosage (mg / kg) Route of administration Dosing frequency 1 6 0.9% sodium chloride injection (control group) / iv QD×28 2 6 (+)-β-elemene 150 iv QD×28 3 6 (-)-β-elemene 150 iv QD×28

[0088] Note: iv: intravenous injection; QD: once a day;

[0089] 3. Experimental results

[0090] Figure 1 The figure shows the changes of bioluminescent signals over time after administration of drugs to each group of mice. The tumor growth inhibition rate (TGI%) of each group was calculated by bioluminescent signals. The results are shown in Figure 2 shown.

[0091] Example 7

[0092] 1. Experimental Materials

[0093] 1.1 Animals: BALB / c female nude mice, 6-8 weeks old, weighing 19-23 g, 12 mice supplied by Beijing Ankai Yibo Biotechnology Co., Ltd.

[0094] 1.2 Drugs under investigation:

[0095] 1) Test substance: (+)-β-elemene (2% ( W / V )) and soybean lecithin (2% ( W / V )) added to soybean oil (5% ( W / V )) and heated to form an oil phase; the oil phase was added to a mixture containing sodium oleate (0.1% ( W / V)) and an appropriate amount of glycerol in an aqueous solution, and after shearing and high-pressure homogenization treatment, a stable aqueous dispersion system with an average particle size of the dispersed phase ≤1μm is obtained.

[0096] 2) Reference substance: 0.9% sodium chloride injection (Shijiazhuang Siyao Co., Ltd., national standard: H13023201)

[0097] 2. Experimental process

[0098] 1) Cell culture: Human gastric cancer NCI-N87-Luc (NCI-N87, ATCC-CRL-5822) cells were cultured in a monolayer in vitro in RPMI 1640 medium + 10% FBS and 2 μg / mL puromycin at 37°C in a 5% CO2 incubator. The cells were routinely digested and passaged using trypsin-EDTA. When the cell saturation was 80%-90% and the number reached the required level, the cells were harvested, counted, and inoculated.

[0099] 2) Tumor cell inoculation: The anesthetized mouse was placed in supine position, and the abdominal skin was disinfected with 75% ethanol to keep the surgical area sterile. A longitudinal incision was made on the left side of the abdominal midline to expose the mouse stomach. 50 μl of the solution containing approximately 5×10 6 A cell suspension of NCI-N87-Luc cells (15% Matrigel) was injected into the subserosa of the gastric glandular area. A sterile cotton swab soaked in normal saline was used to press the injection site for about 1 minute. The injection site was checked for leakage. The stomach was returned to the abdominal cavity, the abdominal wall and skin were sutured in layers, and the mice were continuously observed until they fully woke up.

[0100] 3) Experimental method: After inoculation, mice were intraperitoneally injected with 15 mg / mL of fluorescein solution at 5 μL / g body weight (the fluorescein injection dose for each animal was 75 mg / kg). Ten minutes later, the mice were pre-anesthetized with a mixture of oxygen and isoflurane. After the mice were deeply anesthetized, they were moved to the imaging room of IVIS (Lumia III) to start bioluminescence detection, and the bioluminescence signals in the animals and the generated image information were detected and recorded. Nine days after tumor inoculation, group dosing began when the average fluorescence value reached 26E+07, with each group containing 6 mice. The day of grouping was defined as Day 0, and dosing began on Day 1 (i.e., dosing treatment began on the first day after grouping). Dosing began on Day 1 and ended on Day 28. The dosage, route of administration, and frequency of administration for one group are as described in Table 4. Tumor growth inhibition rate TGI% calculation = [(1-(average bioluminescence signal of a treatment group at the end of drug administration-average bioluminescence signal of the treatment group at the beginning of drug administration)) / (average bioluminescence signal of the vehicle control group at the end of treatment-average bioluminescence signal of the vehicle control group at the beginning of treatment)] x 100%, and the calculated TGI% value is greater than 40%.

[0101] Table 4 Administration route and frequency

[0102] Group Number of animals Drug group Dosage (mg / kg) Route of administration Dosing frequency 1 6 0.9% sodium chloride injection (control group) / po QD×28 2 6 (+)-β-elemene 200 po BID×28

[0103] Note: po: oral; QD: once a day; BID: twice a day; QW: once a week

[0104] 3. Experimental results

[0105] Figure 3 Shown are the changes in bioluminescent signals over time after drug administration in mice.

[0106] Example 8 Preparation of (+)-β-elemene

[0107] 1. Preparation of (-)-Gemarene

[0108] 1.1 Plasmid preparation

[0109] The nucleotide sequence encoding MmoTPS was synthesized according to the nucleotide sequence in Table 5 in a conventional manner known to those skilled in the art. In order to construct an expression vector for the fusion protein, GGGGS was selected as a connecting peptide (Qi Liu.et al, 2023), and the ERG20 gene fragment (SEQ ID NO: 3), SSA1 promoter fragment (SEQ ID NO.7) and ADH1 terminator fragment (SEQ ID NO.8) were obtained from Saccharomyces cerevisiae by conventional methods. The ERG20 gene fragment and the synthesized MmoTPS gene fragment were used as templates by OE-PCR to obtain the fusion protein MmoTPS-GGGGS-ERG20 gene fragment (amino acid sequence and nucleotide sequence are shown in Table 6). The full-length gold seamless cloning kit was used The MmoTPS-GGGGS-ERG20 gene fragment, SSA1 promoter fragment, and ADH1 terminator fragment were ligated with the yeast expression vector pRS425 (Miaoling Biotechnology, China) using the Seamless Cloning and Assembly Kit (Quanshijin, China), and the ligation products were transformed into Trans 5α competent cells (Quanshijin, China), spread on LB-Amp resistant plates, and cultured at 37°C for 16 h. After that, a single clone was picked and cultured at 37°C overnight, the plasmid was extracted and sequenced for verification.

[0110] Table 5 Enzyme related information

[0111] Enzyme name Enzyme species source Enzyme nucleotide sequence Enzyme amino acid sequence MmoTPS Micromonosporasp.MH33 SEQ ID NO.1 SEQ ID NO.2

[0112] Table 6 Fusion protein sequences

[0113] Fusion Protein Amino acid sequence Nucleotide sequence MmoTPS-GGGGS-ERG20 SEQ ID NO.4 SEQ ID NO.5

[0114] 1.2 Construction of Saccharomyces cerevisiae strain CEN124

[0115] In order to realize the synthesis of germaene A in Saccharomyces cerevisiae, CEN.PK2-1D (Euroscarf, Germany) was selected for transformation, DonorDNA was constructed by conventional means and gene editing was performed as shown in Table 7 (Amanda Reider Apel et al, 2016), by knocking out LPP1 and GAL80 genes and inserting EfmvaE (GEN BANK: WP_002382276) and IDI1 genes (GEN BANK: NP_015208), inserting URA3 (GEN BANK: NP_010893), ERG12 (GEN BANK: NP_013935), ERG8 (GEN BANK: NP_013947) and ERG19 genes (GEN BANK: NP_014441) at the neutral site YPRC15C, and inserting HIS3 (GEN BANK: NP_01622b) at the neutral site 1622b. BANK:WP_020863086), ERG13 (GEN BANK:NP_013580) and EfmvaE genes, backfilled the TRP gene in situ (GEN BANK:NP_010290), replaced the ERG9 promoter with HXT1 (GENBANK:CP092951) and then replaced it with the original ERG9 promoter (GEN BANK:CP026287), and finally obtained the Saccharomyces cerevisiae engineered strain CEN124.

[0116] Table 7

[0117]

[0118] 1.3 Preparation of competent CEN124 yeast

[0119] The CEN124 monoclone was picked up and cultured in 3 mL SD-Trp His Ura (Panjinuo, China) liquid medium at 30°C for 24 h, and then transferred to 50 mL YPD liquid medium at a transfer volume of 5% and cultured until OD 600 =0.6-0.8, the bacteria were collected and CEN124 competent cells were prepared using ZYMO Frozen-EZ Yeast Transformation II Kit (Zymo Research, USA) according to the kit operating procedures.

[0120] 1.4 Construction of engineered bacteria

[0121] According to the operating procedures of the ZYMO Frozen-EZ Yeast Transformation II Kit (Zymo Research, USA), the plasmid (see Table 8) was transformed into the above-mentioned CEN124 competent cells, coated on SD-Leu plates, and cultured at 30°C for 48 hours to obtain the corresponding engineered bacteria (see Table 8).

[0122] Table 8 Engineering bacteria and corresponding plasmids

[0123]

[0124] 1.5 Preparation of (-)-Gemarene A

[0125] 1.5.1 Culture medium preparation

[0126] Calcium chloride mother solution: prepared according to Example 3 of prior art CN108060092B.

[0127] Trace metal salt mother solution: prepared according to Example 3 of the prior art CN108060092B.

[0128] Vitamin mother solution: 0.2g / L biotin, 0.8g / L sodium p-aminobenzoate, 4g / L niacin, 4g / L calcium pantothenate, 4g / L pyridoxine hydrochloride, 4g / L thiamine hydrochloride, 100g / L inositol.

[0129] Succinic acid mother liquor: 60 g / L succinic acid, adjust pH to 5.05 with NaOH.

[0130] F2 fermentation medium: 25g / L glucose, 15g / L ammonium sulfate, 6.15g / L magnesium sulfate heptahydrate, 0.72g / L zinc sulfate heptahydrate, 8g / L potassium dihydrogen phosphate, 2ml / L calcium chloride mother liquor, 10ml / L trace metal salt mother liquor, 3ml / L vitamin mother liquor, 100mL / L succinic acid mother liquor.

[0131] 1.5.2 Fermentation of engineered yeast Saccharomyces cerevisiae

[0132] The engineered bacteria monoclones obtained in the construction of 1.4 engineered bacteria were respectively selected to 3mL SD-Leu (Panjinuo, China) liquid medium, and cultured at 30°C, 250rpm for 24h; the bacterial solution cultured for 24h was transferred to a 100mL triangular flask containing 20mL F2 fermentation medium at a transfer volume of 5%, and cultured at 30°C, 250rpm for 40h. 750μL of the fermentation liquid after 40h was placed in a 2mL centrifuge tube, 300μL of n-dodecane was added, and then glass beads were added to grind for 30min, centrifuged, and the n-dodecane liquid on the upper layer was taken and diluted 10 times with ethanol for yield detection. The yield of (-)-germaene A in yeast was 362.25mg / L.

[0133] 2. Preparation of (+)-β-elemene

[0134] Take the fermentation broth containing (-)-germane A obtained above, add n-dodecane at a volume ratio of 1:2 (V n-dodecane: V fermentation broth), and stir and extract for 16 hours. Take the organic phase and heat it at 150°C for 2 hours to obtain a conversion liquid. The conversion liquid is separated and purified by silver nitrate silica gel and silica gel column chromatography to obtain (+)-β-elemene (yield ≥ 95%). 1 H NMR(CDCl3,400MHz):1.03(3H,s,H-15), 1.48(3H,m,H-2,3a), 1.64(1H,m,H-5a), 1.56(2H ,m,H-3b,5b), 1.74(3H,s,H-11), 1.77(3H,s,H-14), 1.96(1H,m,H-6), 2.04(1H,m,H-4), 4 .61(1H,s,H-10a), 4.73(1H,s,H-10b), 4.74(1H,brs,H-13a), 4.85(1H,brs,H-13b), 4.92 (1H, d, J = 11.2 Hz, H-8a), 4.93 (1H, d, J = 17.6 Hz, H-8b), 5.85 (1H, dd, J = 17.6, 11.2 Hz, H-7).

[0135] 13 C NMR(CDCl3,100MHz):16.6(C-15), 21.1(C-14), 24.8(C-11), 26.8(C-5), 32.9(C-3), 39.8(C-1), 39.9(C-2) , 45.7(C-6), 52.7(C-4), 108.2(C-13), 109.8(C-8), 112.1(C-10), 147.7(C-9), 150.3(C-12), 150.4(C-7).

Claims

1. Use of (1R, 2R, 4S)-β-elemene or a pharmaceutical composition comprising (1R, 2R, 4S)-β-elemene in the preparation of a medicament for treating cancer, wherein the (1R, 2R, 4S)-β-elemene has the following structure:

2. A method for treating cancer in an individual in need thereof, comprising administering to the individual (1R, 2R, 4S)-β-elemene or a drug comprising A pharmaceutical composition of (1R,2R,4S)-β-elemene.

3. (1R,2R,4S)-β-elemene or a pharmaceutical composition comprising (1R,2R,4S)-β-elemene for treating cancer.

4. (1R,2R,4S)-β-elemene or a pharmaceutical composition comprising (1R,2R,4S)-β-elemene for treating cancer according to claim 1, the method according to claim 2, or according to claim 3, wherein the cancer is selected from pancreatic cancer, leukemia, breast cancer, ovarian cancer, colorectal cancer, thyroid cancer, lung cancer, lymphoma, endometrial cancer, renal cell carcinoma, esophageal cancer, prostate cancer, gastric cancer, bladder cancer, brain tumor, liver cancer, cervical cancer, head and neck cancer, bone cancer, malignant melanoma, carcinoma One or more of pleural effusion, malignant pleural mesothelioma and brain glioma; or, the cancer is selected from one or more of gastric cancer, brain glioma, pancreatic cancer, ovarian cancer, leukemia, lung cancer, breast cancer, esophageal cancer, prostate cancer, colorectal cancer and liver cancer; or, the cancer is selected from one or more of pancreatic cancer, ovarian cancer, leukemia, lung cancer, breast cancer, esophageal cancer, prostate cancer, colorectal cancer and liver cancer; or, the cancer is selected from one or more of lung cancer, liver cancer, gastric cancer and brain glioma; or, the cancer is selected from liver cancer or gastric cancer.

5. (1R,2R,4S)-β-elemene or a pharmaceutical composition comprising (1R,2R,4S)-β-elemene for treating cancer according to claim 1 or 4, or the method according to claim 2 or 4, or according to claim 3 or 4, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, such as an injection oil, a carrier, a filler, a disintegrant, a binder, a lyoprotectant, a pH regulator, a preservative, an antioxidant and a wetting agent.

6. The use according to claim 1, 4 or 5, the method according to claim 2, 4 or 5, or the (1R, 2R, 4S)-β-elemene or the pharmaceutical composition comprising (1R, 2R, 4S)-β-elemene for treating cancer according to claim 3, 4 or 5, wherein the pharmaceutical composition can be formulated into solid dosage forms, semisolid dosage forms, liquid dosage forms and gaseous dosage forms, such as one or more of liposomes, tablets, capsules, pills, mixtures, gels, ointments, granules, powders, creams, granules, pills, lozenges, solutions, suspensions, emulsions, pellets or injections.

7. (1R,2R,4S)-β-elemene or a pharmaceutical composition comprising (1R,2R,4S)-β-elemene for treating cancer according to any one of claims 1 and 4-6, or the method according to any one of claims 2 and 4-6, or according to any one of claims 3 and 4-6, wherein the (1R,2R,4S)-β-elemene is obtained by heating and converting (-)-germane A, and the (-)-germane A is obtained by fermenting an engineered bacterium containing a nucleotide encoding an enzyme capable of synthesizing (-)-germane A, preferably the fermentation comprises the following steps: culturing the engineered bacterium in a seed culture medium to obtain a seed solution; then inoculating the seed solution into a fermentation culture medium for fermentation culture, and recording the product of the fermentation culture as a fermentation system; during the fermentation culture, adding the fermentation culture medium to the fermentation system, preferably the enzyme capable of synthesizing (-)-germane A has SEQ ID NO: 2 or at least the same sequence as SEQ ID NO: NO:2 has an amino acid sequence with 80% homology, preferably, the nucleotide encoding the enzyme capable of synthesizing (-)-germane A has SEQ ID NO:1 or a nucleotide sequence with at least 90% homology to SEQ ID NO:

1.

8. (1R,2R,4S)-β-elemene or a pharmaceutical composition comprising (1R,2R,4S)-β-elemene for treating cancer according to the use, method or method of claim 7, wherein the seed culture medium and the fermentation culture medium contain 25 g / L glucose, 15 g / L ammonium sulfate, 6.15 g / L magnesium sulfate heptahydrate, 0.72 g / L zinc sulfate heptahydrate, 8 g / L potassium dihydrogen phosphate, 2 ml / L calcium chloride mother liquor, 10 ml / L trace metal salt mother liquor, 3 ml / L vitamin mother liquor, 100 mL / L succinic acid mother liquor, and the balance is water.

9. The (1R,2R,4S)-β-elemene or a pharmaceutical composition comprising (1R,2R,4S)-β-elemene for treating cancer according to any one of claims 7 to 8, wherein the (-)-germarene A is heated at 100°C-380°C or 150°C.

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