TWI430800B - A triterpenoid compound, its preparation method and uses - Google Patents

Description

Triterpenoid compound, preparation method and use thereof

The present disclosure is broadly related to a novel compound isolated from Ganoderma lucidum, a process for preparing the novel compound, and uses thereof. In particular, this disclosure relates to an oxa-A, B-dihomo-steroidal, isolated from Ganoderma colossum (Fr.) Baker. a novel triterpenoid compound of the skeleton; a method for isolating the novel triterpenoid compound; and the use of the novel triterpenoid compound for the preparation of an anticancer or anti-inflammatory drug.

Food and medicinal fungi are widely used as health foods, among which Ganoderma lucidum is a large one. In Southeast Asia, Ganoderma lucidum fruiting bodies are more commonly used. Since Ganoderma lucidum can initiate many positive reactions in the living body, there are many studies on related active ingredients, and the active ingredients are roughly divided into five categories: triterpenoids, polysaccharides, nucleotides, plants. Phytosterols and polypeptides.

G. colossum is a genus of Ganoderma lucidum. Because of the scarcity of yellow zhizhi , there are not many studies on related active ingredients. The inventor of the present application accidentally discovered a novel Ganoderma lucidum triterpenoid compound during the research of Huangzhi, and confirmed in an in vitro test that the newly discovered Ganoderma lucidum triterpenoid compound can inhibit the growth and metastasis of cancer cells. The efficacy of inflammatory gene expression can be used as a lead compound for future development of anti-tumor growth drugs or anti-inflammatory drugs.

As broadly described and exemplified in the present disclosure, the present disclosure aims to provide a novel compound isolated from yellow cherries and the use of such novel compounds.

Accordingly, a first object of the present disclosure is to provide a novel triterpenoid compound having an oxa-A, B-dihomo-steroidal skeleton separated from yellow lucidum, having the following formula (I) Structure:

According to one embodiment of the present invention, the novel compound of the formula (I) has an effect of inhibiting the growth, metastasis or inhibition of the expression of an inflammatory gene, and thus can be developed into an anticancer or anti-inflammatory drug in the future.

Accordingly, a second object of the present disclosure is to provide a method of isolating the above novel compound of formula (I) comprising the steps of:

(a) extracting a G. colossum fruiting body with a first solution to obtain a first extract, wherein the yellowing fruit body and the first solution are weighted: a volume of about 1:10 to 1 a ratio of 50:

(b) extracting the first extract with a second solution consisting of water and an organic solvent to obtain an aqueous layer and an organic layer extract, wherein the water and the organic solvent are in a volume ratio of about 1:1 to a ratio of 2:3;

(c) extracting the organic layer extract with a third solution in a cartridge column and collecting a first extract;

(d) in a reverse-phase high performance liquid chromatography, the first rush is flushed with a fourth solution having a concentration between 20% and 95% by volume. Lifting liquid to obtain a second extract;

(e) The second extract is concentrated and dried to obtain the compound of the formula (I).

According to an embodiment of the present disclosure, in the step (a), the first solution is 70%-95% (% by volume) of ethanol; in the step (b), the organic solvent is ethyl acetate, and the first The second solution is prepared by mixing an equal volume of water and ethyl acetate; in the step (c), the third solution is composed of hexane and ethyl acetate in a volume ratio of 1:0 to 0:1; In the step (d), the fourth solution is methanol having a concentration of between 60% and 80% by volume.

A third object of the present disclosure is to provide a pharmaceutical composition which inhibits the proliferation, metastasis or inhibition of the expression of an inflammatory gene, comprising an effective amount of a compound of formula (I); and a pharmaceutically acceptable excipient thereof.

According to an embodiment of the present invention, the cancer cells suitable for inhibiting growth or migration by the pharmaceutical composition are liver cancer or lung cancer cells.

Accordingly, a fourth object of the present disclosure is to provide a method of delaying the growth, metastasis or inhibition of inflammatory conditions in a subject, comprising administering to the individual a pharmaceutical composition as described above.

According to an embodiment of the invention, the cancer suitable for treatment by the method (i.e., delaying the growth or migration of cancer cells) may be liver cancer, lung cancer or breast cancer; in particular, lung cancer or breast cancer having metastatic ability. An individual suitable for treatment by the methods is a mammal, for example, a human.

The detailed description of the invention, which is set forth below, and the accompanying drawings are merely illustrative of the embodiments of the invention The following text will explain the functions of the embodiments and the steps and sequences of how to construct and operate the embodiments. However, the scope of the present invention is not limited to the disclosed embodiments.

G. colossum is a rare species in Ganoderma lucidum fungi. Because of its rareness, Huangzhi is not a research on its active ingredients. In view of this, the inventors of the present invention used solvent extraction and chromatography techniques, including tantalum column chromatography and reverse-phase high performance liquid chromatography, to isolate a novel from the dried yellow lucidum fruit body. The compound, which was structurally identified, was confirmed to be a Ganoderma lucidum triterpenoid containing an oxa-A, B-dihomo-steroidal skeleton. This novel compound has been shown to inhibit cancer cell growth and inflammatory gene expression in in vitro studies, and may be developed into an anti-cancer or anti-inflammatory drug in the future.

Accordingly, in one aspect of the invention, there is provided a novel Ganoderma lucidum triterpenoid compound isolated from a fruiting body of the yellow lucidum, namely (22 S , 23 R )-A, B-L 2-19--15-β-acetyloxy- 5,23-dihydroxy-4-oxa-3-oxo lanosterol-1,8,19,24-tetraene-26,22-lactone [(22 S ,23 R )-A,B- Dihomo-19-nor-15-β-acetoxy-5,23-dihydroxy-4-oxa-3-oxolanosta-1,8,19,24-tetraen-26,22-olide)] or lanosterol H (Colossolactone H) having the structure of the following formula (I):

Another aspect of the present invention is to provide a method for isolating a compound of the above formula (I) from a fruiting body of a yellow gum, comprising the steps of:

(a) extracting a yellow fruiting body with a first solution to obtain a first extract, wherein the yellow fruiting body and the first solution are mixed in a weight: a volume of about 1:10 to 1:50;

(b) extracting the first extract with a second solution consisting of water and an organic solvent to obtain an aqueous layer and an organic layer extract, wherein the water and the organic solvent are in a volume ratio of about 1:1 to a ratio of 2:3;

(c) extracting the organic layer extract with a third solution in a cartridge column and collecting a first extract;

(d) in a reverse phase high performance liquid chromatography column, the first extract is flushed with a fourth solution having a concentration between 20% and 95% by volume to obtain a second flush Lifting liquid;

(e) The second extract is concentrated and dried to obtain the compound of the formula (I).

According to an embodiment of the present disclosure, in the above step (a), the yellow berry fruit body is preferably subjected to a drying treatment to reduce the water content to prevent the fresh yellow lucidum fruit body from being spoiled and the active compound therein to be changed or decreased. Next, the dried fruit body is pulverized into chips or powder of uniform size before being mixed with the first solution and subjected to extraction to obtain a first extract. Suitable first solutions are alcohols including, but not limited to, ethanol, propanol, isopropanol, n-butanol, and isobutanol. In a preferred embodiment, the first solution is 70% to 95% by volume of ethanol; preferably 95% by volume of ethanol. The dried yellow lucidum fruit body powder and the first solution (eg, ethanol) are preferably mixed in a dry powder weight: first solution volume in a ratio of 1:10 to 1:50, more preferably 1:20 to 1:40. Proportional mixing; optimally mixed in a ratio of from about 1:25 to about 1:35. For example, at least 10 liters of 95% (% by volume) ethanol is added to 1 kg of dried powder of the yellow body fruit. The mixture can be extracted by heating, turbulent dip or room temperature soaking. In a preferred embodiment, the dried powder of the yellow body fruit body and the first solution are mixed at a ratio of 1:24, and then the mixture is immersed at 37 ° C for about 24 hours to obtain a crude extract. Next, the crude extract is filtered, concentrated and dried (the filtration, concentration and drying steps can be carried out in any conventional manner) to form a crude extract.

Next, in the step (b), the first extract obtained in the step (a) is extracted with a second solution composed of water and an organic solvent. Since the second solution is composed of two solvents which are mutually incompatible or different in polarity, in a ratio of about 1:1 to about 2:3, the extraction is divided into two layers, an aqueous layer and an organic layer, respectively. The organic solvent suitable for use in this step is a low carbon number ester, including, but not limited to, ethyl formate, methyl acetate, ethyl acetate, methyl propionate, ethyl propionate and propyl propionate; It is preferably ethyl acetate. In a preferred embodiment, the second solution is formed by mixing an equal volume of water with ethyl acetate, but it is understood that water and ethyl acetate may also be mixed in other suitable ratios, as those skilled in the art may According to the actual operating conditions, through simple tests, find the mixing ratio that is most suitable for the current situation. After extracting the first extract of the above step (a) with the second solution, the extract of the organic layer is retained for successive separation steps (c) to (e).

The above step (b) may be repeated several times, for example, 1, 2 or 3 times, and the organic layer extracts obtained each time are combined for successive separation steps (c) to (e).

In the step (c), the ethyl acetate layer extract of the above step (b) is loaded into a ruthenium tube column, followed by hexane and ethyl acetate in a volume ratio of 1:0 to 0:1. The third solution composed is washed and the resulting first extract is collected. A third solution suitable for use in the stripping of the gum column is composed of hexane and ethyl acetate in a volume ratio of 1:0 to 0:1.

In the step (d), the first extract obtained in the step (c) is further loaded into a reverse phase high performance liquid chromatography column at a flow rate of about 0.5 to 20 ml/min (compared Preferably, it is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ml/min, more preferably about 2, 3, 4, 5, 6, 7, 8 or 9 ml/min), at a concentration between 20% and 95% (% by volume), for example about 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% 70%, 75%, 80%, 85%, 90% or 95%; more preferably between about 50% and 90% (% by volume), for example about 50%, 55%, 60%, 65%, 70% , 75%, 80%, 85% or 90%; optimally between 60% and 80% (% by volume), for example about 60%, 65%, 70%, 75% or 80% of the fourth solution is stripped And collect the resulting extract. A fourth solution suitable for use in flushing the column includes, but is not limited to, water or alcohols including water, methanol, ethanol, propanol, isopropanol, n-butanol, iso-butanol, and the like. In a specific example, the fourth solution is a methanol solution having a concentration between 60 and 80% by volume. In a specific example, the fourth solution is a methanol solution having a concentration between 60 and 80% by volume.

In this example, the rinsed extract can be collected together, concentrated under reduced pressure, and lyophilized to obtain the desired yellow gum compound. In a specific example, the proposed novel compound was identified by NMR, UV, and high resolution electrospray ionization mass spectroscopy (HREIMS) and identified as (22 S , 23 R )-A, B-Like II-19-Lower-15-β-Ethyloxy-5,23-dihydroxy-4-oxa-3-oxo lanosterol-1,8,19,24-four Alkene-26,22-lactone, also known as lanosterol triterpene H. The yield of the novel compound isolated by the process of the invention is about 0.04% (this percentage is the weight of the compound of formula (I) / dry weight of the fruit body). Furthermore, the product can be concentrated and dried in any conventional manner. In one example, the extract is concentrated under a reduced pressure environment, for example, at a pressure of about 0.1 torr, and the concentration treatment is stopped when the solids content in the rinse is 20%. Next, the concentrate is dried, for example, freeze-dried at -30 ° C to -50 ° C.

Accordingly, another object of the present invention is to provide a pharmaceutical composition which inhibits proliferation of cancer cells or inhibits inflammation, comprising an effective amount of a compound of formula (I); and a pharmaceutically acceptable carrier therefor.

By "an effective amount" is meant a dose of a therapeutically effective compound or composition administered to a subject. The measurement of efficacy can be objective (measured by some test or marker) or subjective (the subject gives a narrative of its effects and feelings). The effective dosage of the pharmaceutical compositions of the present invention can range from about 1 mg/kg to about 1000 mg/kg, or from 10 mg/kg to about 100 mg/kg. The effective dose will also vary with the route of administration and the other agents that may be used in combination. Those skilled in the art will recognize that it is possible to use more or less than the above. The particular dose and method of treatment used for a particular patient will be affected by a number of variables, including the activity of the drug used, age, weight, overall health, sex, diet, time of administration, route of administration, frequency of administration, rate of excretion, Combined medication, the course and severity of the disease, the location of the patient's disease, the state of the symptoms, and the treatment and judgment of the doctor.

Accordingly, it is an aspect of the present invention to provide a method of delaying or inhibiting the growth or metastasis of a majority of cancer cells in a subject, comprising at least administering to the individual an effective amount of the pharmaceutical composition such that about 90% of the individual is The growth of these cancer cells is stagnant or does not metastasize, and thus can achieve the purpose of delaying the growth or spread of tumors or cancers. A cancer suitable for treatment with the method or pharmaceutical composition of the invention (i.e., delaying the growth or metastasis of cancer cells) is liver cancer, lung cancer or breast cancer; in particular, lung cancer or breast cancer with metastatic ability.

Another aspect of the present invention is to provide a method for inhibiting or slowing the inflammatory condition of a subject comprising administering to the individual an effective amount of the pharmaceutical composition such that the inflammatory gene expression of the individual is inhibited, thereby Can achieve the effect of slowing down inflammation.

The term "subject" as used herein refers to any animal (eg, human) that can accept a pharmaceutical composition of the invention, including, but not limited to, human, non-human primates, such as mammals, dogs, Cats, horses, sheep, pigs, cattle, etc. In general, the terms "patient" and "subject" are used interchangeably herein.

Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described may be employed in the methods of the invention. The preferred embodiments and materials described herein are for illustrative purposes only. All references cited in this application are hereby incorporated by reference in their entirety to the extent of the disclosure of the disclosure of the disclosure. Moreover, the documents discussed herein merely disclose prior art techniques of the present application. And there is no literature showing that the present invention has been disclosed in the prior art. The actual data obtained in the context of the present invention may differ from the data disclosed in the present disclosure by the present invention.

Unless otherwise indicated, the singular "a", "an", "the" or "the"

Hereinafter, preferred embodiments of the present invention will be described by way of examples.

Example Example 1 Isolation of a compound of formula (I) from a fruiting body of yellow lucidum

Wool sterol H (Colossolactone H)

(22 S ,23 R )-A,B-Like II-19-Lower-15-β-Ethyloxy-5,23-dihydroxy-4-oxa-3-oxo lanosterol-1,8 ,19,24-tetraene-26,22-lactone

(22 S ,23 R )-A,B-dihomo-19-nor-15-β-acetoxy-5,23-dihydroxy-4-oxa-3-oxolanosta-1,8,19,24-tetraen-26, 22-olide

1.1 Preparation of alcoholic crude extract of the fruit body of the yellow lucidum

The 50 g dry weight of the yellow lucidum fruit body was broken into powder by a powder machine and placed in an oscillating extraction device. Next, the dilute liquid was concentrated by adding 1,200 ml of an alcohol solvent to the dry weight of the powder of the yellow berry fruit: solvent volume = 1:24, and digesting at 37 ° C for one day. This extraction and concentration steps were repeated several times, and the resulting extracts were combined and filtered and concentrated under reduced pressure to give 13 g (yield: 26%). Next, the residual solvent in the alcoholic crude extract of the resulting yellow body fruit body was removed by suction filtration overnight in a 40 ° C constant temperature vacuum drying system.

1.2 Preparation of ethyl acetate extract (GCAE) of yellow fruit body

A mixed solution of water and ethyl acetate (H ₂ O: ethyl acetate = 1:1) mixed in an equal volume ratio was added to the crude extract of the yellow lucidum fruit body obtained by the above Example 1.1 in a dry weight of 13.0 g. For extraction, an aqueous layer solution and an ethyl acetate extraction layer solution are obtained, and the ethyl acetate extraction layer solution is concentrated and dried, and then the residual solvent is removed by suction filtration at 40 ° C under a vacuum drying system overnight to obtain a dry weight of 9.5. The acetic acid extract (GCAE) of the yellow lucidum fruiting body has a GCAE yield of about 19% relative to the total alcoholic crude extract of the yellow gum fruit body.

1.3 Separation of the compound of formula (I) by gel column chromatography

The dried ethyl acetate extract (GCAE) obtained from the above Example 1.2 was subjected to chromatography on an open gel column (silica gel 60, 25×3.5 cm) using hexane:ethyl acetate=1:1. After the solvent system is subjected to gradient elution, it is divided into 12 distinguishing layers, which are referred to as layer 1 to layer 12, respectively. Further, the 11th discrimination layer (247.7 mg) was separated and purified by a reversed-pressure high pressure liquid chromatography system (HPLC/Diode array) in a 60%-80% methanol solution system to obtain 18 mg of the compound of the formula (I). The white powdery solid has a yield of the compound of the formula (I) of 0.04% relative to the total alcoholic crude extract of the yellow gum fruit body.

Further, the compound of the formula (I) was identified by IR, UV, ¹ H NMR, ¹³ C NMR, HREIMS and CD, and the relevant data are as follows: mp 149-156 ° C; +48.5 ( c 0.40, MeOH); IR (KBr) ν _max 3418, 2930, 1705, 1576, 1446, 1372, 1248, 1142, 1021 cm ^-1 ; UV (MeOH) λ _max 250 (sh), 340 nm; CD Δε ₂₅₁ -5.446, Δε ₃₅₃ , +0.936 ( c 1.44 × 10 ^-4 mM, MeOH); ¹ H NMR (CD ₃ OD, 500 MHz) δ 7.08 (1H, d, J =10.0 Hz, H-1 ), 6.62 (1H, m, H-24), 6.38 (1H, s, H-19), 5.80 (1H, d, J = 10.0 Hz, H-2), 4.91 (1H, d, brs, J = 7.0 Hz, H-15), 4.41 (1H, dd, J = 10.5, 2.0 Hz, H-23), 2.67 (1H, m, H-16a), 2.34 (1H, m, H-6a), 2.33 ( 2H, m, H-11), 2.24 (1H, m, H-6b), 2.12 (1H, m, H-17), 2.08 (1H, m, H-20), 2.05 (3H, s, H-) 2 ' ), 1.88 (3H, s, H-27), 1.83 (2H, m, H-12), 1.51 (1H, m, H-16b), 1.24 (3H, s, H-29), 1.17 ( 3H, s, H-18), 1.14 (3H, s, H-28), 1.06 (3H, d, J = 5.5 Hz, H-21), 1.03 (3H, s, H-30); ¹³ C NMR (CD ₃ OD, 125 MHz) δ 172.0 (s, C-1 ' ), 167.5 (s, C-3), 167.4 (s, C-26), 150.6 (d, C-1), 150.2 (s , C-8), 147.5 (d, C-24), 141.2 (d, C-19), 134.8 (s, C-10), 129.7 (s, C-25), 127.7 (s, C-9) , 117.0 (d, C-2), 94.9 (s, C-5), 85.8 (d, C-22), 79.9 (s, C-15), 77.9 (s, C-4), 64.1 (d, C-23), 56.6 (s, C-14), 47.2 (d, C-17), 45.9 (t, C-6), 45.7 (s, C-13), 39.2 (t, C-16), 36.9 (d, C-20), 32.6 (t, C-12), 28.9 (t, C-11), 27.6 (t, C-7), 25.1 (q, C-28), 24.7 (q, C) -29), 24.7 (q, C -30), 21.6 (q, C-2 ' ), 17.7 (q, C-18), 16.9 (q, C-27), 13.3 (q, C-21); HRESIMS m/z 577.2767 [M+Na ] ⁺ (Calc. for C ₃₂ H ₄₂ O ₈ Na, 577.2772).

Example 2 Analysis of in vitro anticancer activity of a compound of formula (I)

In this experiment, the human liver cancer cell line HepG2 and the human lung cancer cell line A549 were used for in vitro anticancer analysis; after treating the cells with different concentrations of the compound of formula (I), the intracellular acid phosphatase (ACP) was measured. Activity as an indicator of cellular activity.

2.1 Cell culture

DMEM (Dulbecco's Modified Eagle Medium) culture medium was used as the base medium of HepG2 or A549 cell line, and penicillin G (100 units/ml), streptomycin sulfate (100 ng/ml), L-brate was added thereto. Indoleamine, non-essential amino acid, fetal bovine serum (10%) and sodium pyruvate (1%). Cells are usually cultured in a 10 cm culture dish and subcultured as the cells grow to eight minutes. The subculture method was to first aspirate the old culture medium, and then wash the culture cells once with 3 ml of phosphate buffer (PBS), followed by 1 ml of trypsin/EDTA (0.05%/0.025%) at 37 °C. The cells were treated with the solution for 5 minutes, and the cells were detached from the culture plate to be in a suspended state; then, 2 ml of fresh culture solution was added to the suspension cells to neutralize the activity of trypsin. Then, the cells were dispersed to be evenly distributed in the cell suspension, and after a proper ratio of the number of cells was left, the new culture solution was added to an appropriate volume and mixed, and then cultured in a 37 ° C incubator.

2.2 Anticancer activity analysis

Approximately 3,000 HepG2 cells were inoculated into a flat-bottom 96-well plate, and the culture solution in each well was supplemented with a culture medium to a volume of 180 μL, and then the plate was incubated overnight in a 37 ° C incubator. The next day, the above basic medium containing no fetal bovine serum (FBS) was added to dilute to different concentrations of the compound of formula (I), and the final volume in each culture well was 200 μL. The concentration of the compound of formula (I) was 0, 5, 10, 20 and 40 μg/ml, each concentration was triple-coated, placed in a 37 ° C incubator, and assayed for cell activity at 72 hours.

Cell viability assay: A large number of acid phosphatase (ACP) in living cells can convert p -nitrophenyl phosphate ( p- NPP) into p-nitrophenol ( p- nitrophenol, p- NP), p- NP has the maximum absorbance at 405 nm, so the absorbance can be used to quantify the ACP activity in the cell, and then the cell activity can be deduced.

To measure cell viability, first aspirate the culture solution, wash once with 200 μL PBS, and add 100 μl of assay buffer (containing 10 mM p- NPP, 0.1 M sodium acetate, 0.1% Triton X-100, and pH approx. 5.5), after reacting at 37 ° C for 30 minutes, 10 μl of sodium hydroxide (0.1 N) was added to terminate the reaction, and the absorbance was measured by MRX II ELISA Reader (DYN-EX Tech., USA) at a wavelength of 410 nm, and then converted. Cell viability under different conditions. The results are shown in Figures 1 and 2.

As shown in Fig. 1, when the cells are treated with the compound of formula (I), the activity decreases, and as the concentration of the compound of formula (I) increases and the treatment time increases, the decrease in cell activity becomes more pronounced, and the compound of formula (I) is for HepG2. the IC ₅₀ (50% inhibitory concentration of cell viability) is approximately 25 μM. Similar trends were also observed in the A549 cell line, see Fig. 2, 72 hours after treatment, the compound of formula (I) for the A549 cells IC ₅₀ of about 38 μM.

Example 3 In vitro anti-inflammatory activity analysis of a compound of formula (I)

When the cells are stimulated by external stimuli, they activate cyclooxygenase (COX), which converts arachidonic acid into prostaglandin G2 (PG) and then transforms into different metabolites such as prostaglandins. E2 (PGE2), prostaglandin D2 (PGD2), etc., and then cause vasodilation and red, swollen, heat, pain and other inflammatory reactions. There are two kinds of COX enzymes in the cells, COX-1 and COX-2. COX-1 is usually present in cells, and it expresses and synthesizes prostaglandins in various tissues to regulate normal physiological functions, such as regulating renal blood vessels. Resistance and maintenance of microvascular integrity of the gastrointestinal tract. The amount of COX-2 in normal cells is very low, and it is only induced when it is stimulated by inflammatory stimuli or cytokine, and a large amount of prostaglandin is synthesized. COX-2 can be selectively inhibited because it is different from the activation position of COX-1. If a test substance can inhibit the expression of COX-2, it means that the analyte has an activity of inhibiting the inflammatory reaction.

In this experimental example, the mouse macrophage RAW264.7 was used as the cell model, and lipopolysaccharide (LPS) treatment was used to increase the expression of COX-2 in the cells. Then, whether the compound of formula (I) can inhibit COX The performance of -2 to assess whether the compound of formula (I) can be used as an anti-inflammatory drug.

3.1 anti-inflammatory response analysis

The mouse macrophage cell line RAW264.7 was cultured in DMEM medium containing 10% fetal calf serum (FBS) and cultured in the same manner as in Example 2.1.

Determination of intracellular mRNA expression In a 6 cm diameter cell culture dish, 3 × 10 ⁵ cells were placed, and after attaching to the bottom of the culture plate, 1, 5, 10, 20 μg/ml were added, respectively. The compound (corresponding to 1.8, 9, 18, 36 μM of the compound of the formula (I)) was cultured at 37 ° C for 1 hour, and then treated with lipopolysaccharide (1 μg / ml) for 7 hours, followed by extraction of RNA in the cells.

2 μg of RNA was subjected to reverse transcription reaction (this reverse transcription reaction solution contained 0.5 mM dNTPs, 10 mM DTT, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl ₂ and 200 unit SuperScript ^TM III RNase H- Reverse transcriptase), reacted at 50 ° C for 2 hours to completely reverse transcribe mRNA into cDNA. Next, an instant polymerase chain reaction was performed to obtain 200 ng of cDNA with a pair of primers with a final concentration of 400 nM, namely 5'-CCT CTG CGA TGC TCT TCC GA-3' (SEQ ID NO: 1) and 5'-CAC TTA TAC TGG TCA AAT CC-3' (sequence number: 2), mix well, add 10 μl of reaction buffer (containing dNTP mixture, buffer, Mg ²⁺ , Tag polymerase and SYBR Green I and reference pigment) Add water to a final volume of 20 μL. The GADPH gene expression was used as the calibration standard, and the untreated drug cell group was used as the control group, and the fluorescence change of the PCR product was determined by using the Applied Biosystems 7500 Fast Real Time PCR (Applied Biosystems 7500 Fast Real Time PCR). And based on this, the performance of each gene is released. The amount of change in each gene was calculated based on the software provided by the manufacturer. The results are shown in Figure 3.

As shown in Fig. 3, as the concentration of the compound of formula (I) increases, the ability to inhibit the expression of the COX-2 gene increases as compared to the control group (i.e., cells treated with LPS only). According to the results of the present experiment, the compound of the formula (I) has an activity of inhibiting an inflammatory reaction, and thus it can be developed into an anti-inflammatory drug in the future.

Example 4 In vitro inhibition of cancer metastasis activity of a compound of formula (I)

Malignant tumor metastasis is one of the reasons why cancer is difficult to heal, so the ability of compounds to inhibit cancer metastasis is of great medical value. We used three cancer cell lines with high metastatic potential, including lung cancer cell lines PC9 and CL1-5, and breast cancer cell line MDA-MB231, to analyze the ability of cancer cell metastasis in vitro by wound-healing assay. .

4.1 Wound healing test

Cell culture spacers (Culture-Insert, purchased from ibidi, Gmbh, Germany) were placed in 24-well culture dishes, and 70 μl of cell suspension (containing 3×10 ⁴ cells) was added to each well with the cell culture spacer inserted. Cells), shake the cells to make the cells evenly distributed, and place the culture dish in a carbon dioxide incubator at 37 ° C. After the cells are attached, remove the cell culture spacers, and you can see the separation of the cells by the culture spacers (called " Wounds), the medium was replaced with DMEM medium containing 1% FBS, and the compound of formula (I) was added, and the "wound" was observed by the microscope at 10 hours, 12 hours, 24 hours, and 48 hours, respectively. The situation and taking pictures, and then quantitative analysis of the "wound" healing with Image-J software. The results are shown in Figures 4 and 5.

As shown in Fig. 4, as the concentration of the compound of formula (I) increases, its ability to inhibit the crawling of PC9 lung cancer cells increases as compared to the control group (i.e., cells that have not been treated with the compound of formula (I)). According to the results of the present experiment, as shown in Fig. 5, the compound of the formula (I) has an activity of inhibiting the crawling of two lung cancer cells, PC9 and CL1-5, and MDA-MB231 breast cancer cells, and thus can be developed into a drug for anti-cancer metastasis in the future.

The present disclosure has been disclosed in the above embodiments, and is not intended to limit the scope of the disclosure, and various modifications and changes may be made without departing from the spirit and scope of the disclosure. For example, the above description, the embodiments and the data are provided to provide a complete description of the structure and the embodiments of the present invention. Any one skilled in the art can refer to the above one or more without departing from the spirit and scope of the present invention. Various modifications and changes may be made to the specific embodiments, and such modifications or variations are considered to be within the scope of the appended claims.

A brief description of the dissolution patterns in this disclosure is provided below, where:

1 is a schematic diagram of inhibiting the growth of HepG2 liver cancer cells with a compound of formula (I) according to an embodiment of the present invention;

2 is a schematic diagram of inhibiting the growth of A549 lung cancer cells with a compound of formula (I) according to an embodiment of the present invention;

Figure 3 is a schematic illustration of inhibition of the inflammatory index COX-2 gene expression by a compound of formula (I) in accordance with one embodiment of the present invention;

Figure 4 is a schematic diagram of inhibiting cancer metastasis of PC9 lung cancer cells in vitro by a compound of formula (I) according to an embodiment of the present invention;

Figure 5 is a graph showing quantification of in vitro cancer metastasis activity of PC9 and CL1-5 lung cancer cells and MDA-MB231 breast cancer cells by a compound of formula (I) according to an embodiment of the present invention.

Claims (8)

A compound having the structure of the following formula (I): A method of preparing a compound according to claim 1, comprising: (a) extracting a G. colossum fruiting body with a first solution to obtain a first extract, wherein the yellow chrysanthemum fruit body and the The first solution is mixed in a weight: a volume of about 1:10 to 1:50, and the first solution is 70%-95% (% by volume) of ethanol; (b) is composed of water and an organic solvent. Extracting the first extract with a second solution to obtain an aqueous layer and an organic layer extract, wherein the water and the organic solvent are mixed in a volume ratio of about 1:1 to 2:3, and the organic solvent Is the ethyl acetate; (c) the organic layer extract is loaded into a column of a rubber tube, and is extracted with a third solution, and a first extract is collected, wherein the third solution is hexane And ethyl acetate or a combination thereof; (d) loading the first extract into a reverse phase high performance liquid chromatography column at a concentration between 20% and 95% by volume And distilling to obtain a second extract, wherein the fourth solution is methanol; and (e) concentrating and drying the second extract to obtain the compound according to claim 1 . The method of claim 2, wherein in the step (a), the yellow body organ and the first solution are mixed in a ratio of a weight: about 1:20 to 1:40. The method of claim 2, wherein the second solution in step (b) is formed by mixing an equal volume of water with ethyl acetate. The method of claim 2, wherein the third solution in step (c) is composed of hexane and ethyl acetate in a volume ratio of 1:1. The method of claim 2, wherein the fourth solution in step (d) is methanol having a concentration between 60% and 80% by volume, and the steps (c) to (e) are continued. The method of claim 2, wherein the yield of the compound of claim 1 in step (e) is about 0.04%. A pharmaceutical composition for inhibiting cell proliferation, cell metastasis or inhibiting inflammation comprising an effective amount of a compound of claim 1; and a pharmaceutically acceptable excipient thereof.