Grass fruit lignan compound as well as preparation method and application thereof

Abstract

The invention discloses a plant lignan compound, the structural formula of the compound is shown as a formula (I), and the invention also protects a preparation method and application of the compound. According to the invention, an active component for resisting triple-negative breast cancer from amomum tsao-ko is separated for the first time; the biological activity of the compound is equivalent to that of a clinical common drug adriamycin, and the toxicity is much lower. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of natural medicinal chemistry, and in particular to tsaoko lignan compounds and preparation methods and applications thereof. Background Art

[0002] Triple-Negative Breast Cancer (TNBC) is a subtype of breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), accounting for approximately 15-20% of breast cancer cases. TNBC is characterized by strong invasiveness, high metastasis rate, easy recurrence, and significant drug resistance. Currently, the clinical treatment of TNBC mainly relies on traditional chemotherapy drugs such as paclitaxel and anthracyclines, but these drugs have large toxic side effects and are prone to drug resistance. In addition, due to the lack of specific molecular targets, the development of targeted therapeutic drugs for TNBC has been slow, resulting in a significantly lower survival rate for patients than other breast cancer subtypes. Therefore, the development of new anti-TNBC drugs has become an urgent need in the field of tumor treatment. Active ingredients derived from Chinese herbal medicines are one of the research hotspots for new anti-tumor drugs.

[0003] Amomum tsaoko (Ziberaceae) is the dried, mature fruit of the genus Cardamom, belonging to the Zingiberaceae family. It is warm in nature and pungent in flavor, entering the spleen and stomach meridians. It has the effects of drying dampness, warming the middle-Jia, and removing malaria and expectoration. It is commonly used clinically to treat internal cold-dampness obstruction, abdominal distension and pain, and malaria-induced chills and fever. Modern research has shown that tsaoko is rich in volatile oils, flavonoids, lignans, and polysaccharides, exhibiting pharmacological activities such as anti-inflammatory, antioxidant, gut microbiome-regulating, and anti-tumor activities. Lignans, a key active ingredient in tsaoko, have been reported to inhibit tumor cell proliferation and induce apoptosis by regulating signaling pathways such as PI3K / AKT and NF-κB. Summary of the Invention

[0004] The first technical problem to be solved by the present invention is to provide a lignan compound derived from tsaoko.

[0005] The second technical problem to be solved by the present invention is to provide a method for preparing the above compound.

[0006] The third technical problem to be solved by the present invention is to provide the use of the above compound in the preparation of drugs for treating breast cancer.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] A tsaoko lignan compound, the structural formula of which is shown in formula (I), is hereinafter named Amomumlignan B:

[0009]

[0010] The preparation method of the above-mentioned tsaoko lignan compound comprises the following steps:

[0011] a. Extract the tsaoko using ethanol and aqueous solution and concentrate to obtain an extract;

[0012] b. The extract is extracted with n-butanol to obtain an extract;

[0013] c. The extract is concentrated to obtain an n-butanol extract;

[0014] d. The n-butanol extract was mixed with water, solubilized by ultrasound into a suspension, eluted on a macroporous resin column, and the eluate was collected and concentrated to obtain the eluted portion;

[0015] e. The eluted portion was dissolved in methanol, applied to a silica gel column, and gradient eluted with a mixed solution of dichloromethane and methanol, and the eluted fractions were collected;

[0016] f. The eluted fraction was separated by medium pressure column chromatography and separated by preparative liquid phase to obtain the tsaoko lignan compound (I).

[0017] In step a, the tsaoko is crushed before extraction; the concentration of the ethanol aqueous solution is 90-99% v / v (preferably 95% v / v); the extraction is performed by heating and reflux extraction, the number of extractions is 2 to 3 times (preferably 3 times), and each extraction takes 2 to 3 hours (preferably 2 hours); and the concentration is performed by reduced pressure concentration until there is no solvent taste.

[0018] In step b, the volume ratio of n-butanol to extract is 1:1 to 3, preferably 1:2.

[0019] In step c, the concentration is performed under reduced pressure until there is no solvent smell.

[0020] In step d, the volume ratio of the extract to water is 1:1-2, preferably 1:1.3; the macroporous resin is macroporous resin D101; the elution is sequentially performed with water, 30% v / v ethanol aqueous solution, 60% v / v ethanol aqueous solution, and 95% v / v ethanol aqueous solution, and the 30% v / v ethanol aqueous solution eluate is collected; and the concentration is performed under reduced pressure until there is no solvent smell.

[0021] In step e, the volume ratio of the eluted portion to methanol is 1:1-2; the volume ratio of the sample amount to the silica gel column is 1:2; the gradient elution is performed in the order of dichloromethane: methanol = 50:1; 25:1; 10:1; 5:1; 2:1; 1:1; 0:100; and the fraction of dichloromethane: methanol = 5:1 is collected.

[0022] In step f, the medium-pressure column chromatography method is a uniform linear elution of 10 to 90% v / v methanol aqueous solution for 10 hours at a flow rate of 20 mL / min. Starting from 0 o'clock, one fraction is collected every hour, and the third fraction is collected. The preparative liquid phase separation is carried out under the condition of using 27% v / v acetonitrile aqueous solution.

[0023] The use of the above-mentioned tsaoko lignan compounds in the preparation of drugs for treating breast cancer is also within the scope of protection of the present invention.

[0024] Wherein, the breast cancer is triple-negative breast cancer.

[0025] A pharmaceutical preparation comprising the above-mentioned compound (I) and pharmaceutically acceptable excipients; the preferred dosage form of the preparation is granules, tablets, capsules, oral liquids, pills, emulsions, suspensions, injections, infusions or sprays.

[0026] Preferably, the dosage form is a tablet, and pharmaceutically acceptable excipients include diluents, binders, wetting agents, disintegrants, lubricants, and glidants. The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets or multi-layer tablets.

[0027] Preferably, the dosage form is a capsule, and pharmaceutically acceptable excipients include diluents, glidants, binders, and disintegrants. The active ingredient and excipients can be mixed and the mixture can be directly placed in a hard capsule or soft capsule, or the mixture can be made into granules or pellets and then placed in a hard capsule or soft capsule. Among them, the diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate; the wetting agent can be water, ethanol, isopropyl alcohol; the binder can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol; the disintegrant can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate; the lubricant and glidant can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol.

[0028] Preferably, the dosage form is an injection, and water, ethanol, isopropyl alcohol, propylene glycol, or a mixture thereof can be used as a solvent, and appropriate amounts of solubilizers, cosolvents, pH adjusters, and osmotic pressure regulators commonly used in the art can be added. The solubilizer or cosolvent can be poloxamer, lecithin, or hydroxypropyl-β-cyclodextrin; the pH adjuster can be phosphate, acetate, hydrochloric acid, or sodium hydroxide; and the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, or acetate.

[0029] Preferably, colorants, preservatives, spices, flavoring agents or other additives are added to the pharmaceutical preparation as excipients.

[0030] In previous research, the inventors discovered that tsaoko extract inhibits the proliferation of triple-negative breast cancer cells. They further isolated a lignan compound from this extract and, through in vitro and in vivo experiments, confirmed its anti-tsaoko activity. This research not only provides a scientific basis for the further development of tsaoko, but also opens up new avenues for the research and development of innovative drugs or adjuvant therapies for triple-negative breast cancer.

[0031] Compared with the prior art, the present invention has the following significant advantages:

[0032] 1. The present invention is the first to isolate the active ingredient from Amomum villosum that is effective against triple-negative breast cancer;

[0033] 2. The biological activity of this compound is comparable to that of the commonly used clinical drug doxorubicin, and its toxicity is much lower. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0035] Figure 1 It is the separation process of the lignan compound Amomumlignan B;

[0036] Figure 2 This is the carbon spectrum of the lignan compound Amomumlignan B;

[0037] Figure 3 This is the mass spectrum of the lignan compound Amomumlignan B;

[0038] Figure 4 Amomumlignan B, a lignan compound, inhibits the proliferation of breast cancer cells;

[0039] Figure 5 Amomumlignan B, a lignan compound, is less cardiotoxic than doxorubicin;

[0040] Figure 6 The in vivo antitumor effect of the lignan compound Amomumlignan B. DETAILED DESCRIPTION

[0041] Example 1: Isolation and physicochemical characterization of lignan compound Amomumlignan B

[0042] like Figure 1 As shown, in this embodiment, the preparation steps of the lignan compound Amomumlignan B are as follows:

[0043] a. The dried Amomum villosum (12kg) was crushed and soaked in 95% v / v ethanol aqueous solution (80L) for 2h, then heated under reflux and extracted three times for 2 hours each time. The extract was concentrated under reduced pressure until there was no solvent taste to obtain 500g of extract;

[0044] b. The extract was suspended in water and extracted with petroleum ether, ethyl acetate, and n-butanol (the volume ratio of the extract to the organic solvent was 1:2);

[0045] c. Each extract was concentrated under reduced pressure until there was no solvent taste, to give 50.0 g of petroleum ether, 80.0 g of ethyl acetate, and 321.1 g of n-butanol;

[0046] d. The n-butanol extract was dissolved in water at a volume ratio of 1:1.3. Ultrasonication was used to dissolve the resulting suspension, which was then adsorbed onto a macroporous resin D101 column. Elution was performed sequentially with water, 30% v / v ethanol, 60% v / v ethanol, and 95% v / v ethanol. The solvent was recovered under reduced pressure until the solvent was completely absorbed. The resulting fraction was 120 g from the water elution, 75 g from the 30% v / v ethanol elution, 50 g from the 60% v / v ethanol elution, and 40 g from the 95% v / v ethanol elution.

[0047] e. The eluted portion with 30% v / v ethanol was dissolved in methanol (the extract to methanol ratio was 1:1.5), then mixed with silica gel (1:2). The solvent was evaporated, and the sample was loaded onto a silica gel column chromatography with a volume ratio of sample to silica gel of 1:2. The fractions Fr1-Fr7 were separated using a gradient elution method using dichloromethane:methanol (50:1, 25:1, 10:1, 5:1, 2:1, 1:1, and 0:100, in this order).

[0048] f. Fr4 (8 g) was subjected to medium pressure column chromatography using a 10-90% v / v methanol-water solution with a uniform linear elution rate for 10 hours at a flow rate of 20 mL / min. Starting at 0 o'clock, one fraction was collected every hour and divided into 10 fractions (Fr4-1 to Fr4-10). A new lignan compound (5 mg) was obtained from Fr4-3 by preparative liquid phase with 27% v / v acetonitrile-water solution.

[0049] The physicochemical properties and spectral data of the lignan compound Amomumlignan B are as follows:

[0050] Physical and chemical properties: The compound is a white powder that turns purple when treated with 10% sulfuric acid ethanol solution.

[0051] Spectral data ( Figures 2-3 ): UV(CH3OH)λ max :254nm. ESI-MSm / z 361.1299[M+H] + , determine the molecular formula C 19 H 20 O7, NMR data see Table 1.

[0052] Table 1 Amomumlignan B NMR data (In CD3OD)

[0053]

[0054] Note: a=600MHz, b=150MHz

[0055] Example 2: Experiment on inhibiting tumor cell proliferation.

[0056] Cell viability was determined using the Cell Counting Kit-8 from Beyotime. The steps are as follows:

[0057] (1) Take MDA-MB-231 and MDA-MB-468 cells in the logarithmic growth phase and use a density of 1x10 4 Each well was inoculated into a 96-well plate and cultured overnight (16 h);

[0058] (2) Two breast cancer cell lines were divided into three groups: a positive control group (doxorubicin, 5 μM), a negative control group, and an experimental group (Amomumlignan B, 5 μM). The volume of the positive control group and the experimental group was 0.1% DMSO, and the negative control group was added with an equal volume of 0.1% DMSO; three replicate wells were set for each sample, and the cell activity was measured at 24 h, 48 h, 72 h, 96 h, and 120 h, respectively. 100 μL of culture medium was used as a blank control, and the cells were placed in an incubator for routine culture; 10 μL of CCK-8 solution was added to each well and the cells were placed at 37°C, 5% CO2 for 2 hours; the absorbance value (OD value) at 450 nm was measured on a microplate reader, and the measurement was continued for 5 days. The cell proliferation curve was drawn, and the experiment was repeated three times independently.

[0059] The results are as follows Figure 4 As shown, Amomumlignan B has an inhibitory effect on the proliferation of human breast cancer cell lines in vitro.

[0060] Example 3: Cardiotoxicity of the lignan compound Amomumlignan B

[0061] (1) H9c2 cells in the logarithmic growth phase were taken at a density of 1x10 4 Each well was inoculated into a 96-well plate and cultured for 24 h;

[0062] (2) The experiment was set up in 6 groups: negative control group, positive control group (doxorubicin), and four dose groups (5, 10, 20, and 50 μM Amomumlignan B). The doxorubicin group was added with the corresponding drug dissolved in 0.1% DMSO, and the experimental concentration was 5 μM. The negative control group was added with an equal volume of 0.1% DMSO. The cells were placed in a conventional incubator for culture.

[0063] (3) After 24 h, add 20 μL of MTS solution to each well and incubate at 37°C for 4 h;

[0064] (4) The absorbance was measured at 450 nm using an enzyme reader, and the cell survival rate was calculated and normalized using the following formula:

[0065] Relative cell survival rate = (A i -A b ) / (A b )×100%

[0066] Among them, A i and A b are the absorbance of the test group and the normal group, respectively.

[0067] The results are as follows Figure 5As shown, compared with the negative control group, Amomumlignan B had no significant effect on the survival rate of H9c2 cells in the concentration range of 5 to 50 μM.

[0068] Example 4: In vivo tumor inhibition effect of the compound

[0069] This experiment used a mouse transplanted tumor animal model to test the inhibitory effect of the compound of the present invention on animal transplanted tumor 4T1:

[0070] (1) First, remove 4T1 cells from liquid nitrogen, quickly revive them in a 37°C water bath, transfer them to RPMI-1640 medium containing 10% fetal bovine serum, and culture them in a 37°C, 5% CO2 incubator. Change the medium every 2-3 days until the cells reach the logarithmic growth phase.

[0071] (2) The cultured 4T1 cells were digested with trypsin to prepare a single cell suspension and the cell concentration was adjusted to 5×10 6 Each nude mouse was subcutaneously inoculated with 0.1 mL of cell suspension in the right axilla.

[0072] (3) After inoculation, the nude mice’s mental state, diet, and tumor growth were observed every day.

[0073] mm 3 When the size is large, medication can be given;

[0074] (4) Nude mice were randomly divided into negative control group, adriamycin group, and Amomumlignan B group, with 5 mice in each group. Before the experiment, the drugs were prepared into a concentration of 3 mg / kg with sterile PBS and administered on the 8th, 10th, and 15th days.

[0075] On day 12, 3 mg / kg doxorubicin and 3 mg / kg Amomumlignan B were injected through the tail vein;

[0076] (5) Before administration, weigh the nude mice and determine the administration volume based on the weight of each nude mouse.

[0077]

[0078] The tumor volume and tumor weight of all mice were measured. Figure 6 As shown in the results, at the same administration concentration, the tumor inhibition effect of the compound is comparable to that of doxorubicin.

[0079] The present invention provides a method and concept for preparing and applying tsaoko lignan compounds, and methods and concepts for their preparation and application. There are many methods and approaches for implementing this technical solution. The above is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A tsaoko lignan compound or a pharmaceutically acceptable salt or glycoside thereof, characterized in that: The structural formula of the compound is shown in formula (I):

2. The method for preparing the tsaoko lignan compound according to claim 1, characterized in that: The preparation method comprises the following steps: a. Extract the tsaoko using ethanol and aqueous solution and concentrate to obtain an extract; b. The extract is extracted with n-butanol to obtain an extract; c. The extract is concentrated to obtain an n-butanol extract; d. The n-butanol extract was mixed with water, solubilized by ultrasound into a suspension, eluted on a macroporous resin column, and the eluate was collected and concentrated to obtain the eluted portion; e. The eluted portion was dissolved in methanol, applied to a silica gel column, and gradient eluted with a mixed solution of dichloromethane and methanol, and the eluted fractions were collected; f. The eluted fraction was separated by medium pressure column chromatography, and then separated by preparative liquid phase to obtain the tsaoko lignan compound (I).

3. The preparation method according to claim 2, characterized in that In step a, the tsaoko is crushed before extraction; the concentration of the ethanol aqueous solution is 90-99% v / v; the extraction is performed by heating and reflux extraction, the number of extractions is 2 to 3 times, and each extraction takes 2 to 3 hours; the concentration is performed by reduced pressure concentration until there is no solvent taste.

4. The preparation method according to claim 2, characterized in that In step b, the volume ratio of n-butanol to extract is 1:1-3.

5. The preparation method according to claim 2, characterized in that In step c, the concentration is performed under reduced pressure until there is no solvent smell.

6. The preparation method according to claim 2, characterized in that In step d, the volume ratio of the extract to water is 1:1-2; the macroporous resin is macroporous resin D101; the elution is sequentially performed using water, 30% v / v ethanol aqueous solution, 60% v / v ethanol aqueous solution, and 95% v / v ethanol aqueous solution, and the 30% v / v ethanol aqueous solution eluate is collected; and the concentration is performed under reduced pressure until there is no solvent smell.

7. The preparation method according to claim 2, characterized in that In step e, the volume ratio of the eluted portion to methanol is 1:1-2; the volume ratio of the sample amount to the silica gel column is 1:2; the gradient elution is performed in the order of dichloromethane: methanol = 50:1; 25:1; 10:1; 5:1; 2:1; 1:1; 0:100; and the fraction of dichloromethane: methanol = 5:1 is collected.

8. The preparation method according to claim 2, characterized in that In step f, the medium-pressure column chromatography is performed by uniform linear elution with 10-90% v / v methanol aqueous solution for 10 hours at a flow rate of 20 mL / min. Starting from 0 o'clock, 1 fraction is collected every hour, and the third fraction is collected.

9. Use of the tsaoko lignan compound according to claim 1 in the preparation of a drug for treating breast cancer; the breast cancer is preferably triple-negative breast cancer.

10. A pharmaceutical preparation, characterized in that Comprising the compound according to claim 1 and pharmaceutically acceptable excipients; preferred dosage forms are granules, tablets, capsules, oral solutions, pills, emulsions, suspensions, injections, infusions or sprays.

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