A marine fungus-derived steroidal compound, a preparation method thereof and application thereof in preparing an antitumor drug

Abstract

The application discloses a marine fungus-derived steroidal compound, a preparation method thereof and application of the steroidal compound in preparation of an antitumor drug. A structural formula of the steroidal compound is shown as formula (I). The compound has a strong antiproliferation effect on MDA-MB-435S, HepG2, SKOV3, SKOV3R, A2780, A549 and various tumor cells, and can be used for preparing an antitumor drug, especially an anti-melanoma, liver cancer, ovarian cancer and lung cancer drug, thereby providing a new drug for treatment of various tumors. In addition, the compound is derived from a natural microorganism and can be obtained through microbial fermentation, and the preparation method is simple. Therefore, the steroidal compound has good clinical application potential in antitumor treatment and has important application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical compounds. More particularly, it relates to a class of steroidal compounds derived from marine fungi, a preparation method thereof and an application thereof in preparing anti-tumor drugs. BACKGROUND

[0002] According to the WHO report, the number of global deaths due to cancer reached tens of millions in 2020, indicating that cancer is still the main cause of global disease deaths. However, most of the current clinical anti-tumor drugs have great side effects, and there is also the possibility of tumor drug resistance and recurrence. Therefore, in order to solve the tolerance and adverse reactions of anti-tumor drugs, it has become a hot spot in the field of anti-tumor drug research to find new anti-tumor drugs and related novel skeleton type drugs.

[0003] Natural products are an important source of drug lead compounds. Marine fungal-derived natural products have the characteristics of sustainability, environmental friendliness, and rich and diverse metabolites, and have always been an important source for drug screening. At present, a number of new skeleton compounds have been discovered from marine fungi. These natural products with novel skeletons not only have unique chemical structures and significant pharmacological activities, but also can bind to new proteins or targets in the body, thereby helping researchers to discover different mechanisms of action from existing drugs, and thus contributing to new drug development.

[0004] Therefore, it is of great significance to explore new skeleton compounds derived from marine fungi for anti-tumor drug research. SUMMARY

[0005] The present application aims to develop new anti-tumor drug options from natural products. A steroidal compound derived from marine fungi has been obtained, which has a significant effect of inhibiting the proliferation of a variety of tumor cells.

[0006] The first object of the present application is to provide a steroidal compound derived from marine fungi.

[0007] The second object of the present application is to provide a Talaromyces sp. JNQQJ-4 strain and its application.

[0008] The third object of the present application is to provide a preparation method of the above-mentioned steroidal compound.

[0009] The fourth object of the present application is to provide the application of the above-mentioned steroidal compound and the above-mentioned strain.

[0010] The fifth object of the present application is to provide an anti-tumor drug.

[0011] The above-mentioned objects of the present application are achieved by the following technical solutions:

[0012] The application isolates a Talaromyces sp. JNQQJ-4 strain from fresh leaves of Kandelia candel in a mangrove in Jinniu Island, Zhanjiang, Guangdong, and further isolates a new steroidal compound from the fermentation product of the strain after a large amount of research. The compound has a significant effect of inhibiting the proliferation of various tumor cells.

[0013] Therefore, the application provides a Talaromyces sp. JNQQJ-4 strain, which is preserved in the Guangdong Provincial Microbial Culture Collection Center on May 24, 2024, and the preservation number is GDMCC No: 64682.

[0014] Further, the application provides a steroidal compound, and the structural formula of the steroidal compound is shown as formula (I):

[0015]

[0016] The application also provides an application of the Talaromyces sp. JNQQJ-4 strain in the preparation of the above-mentioned steroidal compound.

[0017] The application also provides a preparation method of the above-mentioned steroidal compound, and the method is to isolate the steroidal compound from the fermentation product of the above-mentioned Talaromyces sp. JNQQJ-4 strain.

[0018] As an alternative embodiment, the preparation method of the steroidal compound comprises the following steps:

[0019] S1. The Talaromyces sp. JNQQJ-4 strain is inoculated into a seed culture medium for culture to obtain a seed culture solution;

[0020] S2. The seed culture solution obtained in S1 is inoculated into a fermentation culture medium for culture to obtain a fermentation product;

[0021] S3. The fermentation product is filtered to obtain a mycelium, the mycelium is extracted with methanol to obtain an extract solution, the extract solution is concentrated to obtain an extract, the extract is extracted with ethyl acetate to obtain an ethyl acetate crude extract, and the ethyl acetate crude extract is separated by silica gel normal phase chromatography; petroleum ether / ethyl acetate is used for elution, the 40% to 60% ethyl acetate / petroleum ether part is collected, and then gel, silica gel and ND normal phase column chromatography separation technologies are used for separation, so that the steroidal compound shown as formula I is obtained.

[0022] As an alternative embodiment, the composition of the seed medium is: glucose 0.2-0.4%, yeast extract 0.05-0.15%, peptone 0.1%-0.5%, sodium chloride 1.5%-4%, water 94.95-98.15% by weight. Preferably, the composition of the seed medium is: glucose 0.3%, yeast extract 0.1%, peptone 0.5%, sodium chloride 3%, water 96.1% by weight.

[0023] As an alternative embodiment, the culture in step S1 is carried out at 170-200 rpm, 28-35°C for 4-7 days. Preferably, the culture in step S1 is carried out at 180 rpm, 30°C for 6 days.

[0024] As a preferred embodiment, the fermentation medium is a solid rice fermentation medium, which is prepared by mixing rice and seawater at a mass ratio of (6-8):(2-4), more preferably at a mass ratio of 7:3.

[0025] As an alternative embodiment, the culture in step S2 is carried out at 25-35°C for 25-30 days. Preferably, the culture in step S2 is carried out at 30°C for 30 days.

[0026] Preferably, the elution in step S3 is gradient elution using petroleum ether / ethyl acetate.

[0027] More preferably, during the gradient elution in step S3, the volume fraction of petroleum ether changes as follows: 90%→80%→70%→60%→50%→40%→30%; and the volume fraction of ethyl acetate changes as follows: 10%→20%→30%→40%→50%→60%→70%.

[0028] Preferably, the specific method of sequentially separating by gel, silica gel, and ND normal phase column chromatography in step S3 is as follows: the collected components are dissolved with methanol, and then separated by Sephadex LH-20 gel chromatography column with dichloromethane / methanol as eluent (volume ratio of dichloromethane to methanol is 1:1), and the obtained components are sequentially labeled as components 1 and 2 (determined by thin layer chromatography TLC) from early to late; component 2 is dissolved (dissolved with dichloromethane / methanol) and then column-packed after sample mixing with silica gel, and eluted with dichloromethane / methanol (volume ratio of dichloromethane to methanol is 100:1), and the obtained components are sequentially labeled as components Fr.2.1, Fr.2.2, Fr.2.3 (determined by thin layer chromatography TLC) from large to small in polarity, and component Fr.2.2 is the crude sample of compound I. The crude sample of compound I is dissolved with n-hexane / isopropyl alcohol, and then purified by semi-preparative high performance liquid chromatography (ND chiral column) with n-hexane / isopropyl alcohol (v / v 70:30, flow = 1.0 mL / min) as eluent, to obtain the steroidal compound shown in formula I.

[0029] The research of the present application shows that the steroidal compound isolated from the fermentation product of Talaromyces sp. JNQQJ-4 strain has a significant inhibitory effect on a plurality of tumor cells, and therefore the present application further provides the following application schemes:

[0030] The application of the above-mentioned steroidal compound or the above-mentioned strain or the fermentation product of the above-mentioned strain in the preparation of an anti-tumor drug.

[0031] The application of the above-mentioned steroidal compound or the above-mentioned strain or the fermentation product of the above-mentioned strain in the preparation of an anti-melanoma drug.

[0032] The application of the above-mentioned steroidal compound or the above-mentioned strain or the fermentation product of the above-mentioned strain in the preparation of an anti-liver cancer drug.

[0033] The application of the above-mentioned steroidal compound or the above-mentioned strain or the fermentation product of the above-mentioned strain in the preparation of an anti-ovarian cancer drug.

[0034] The application of the above-mentioned steroidal compound or the above-mentioned strain or the fermentation product of the above-mentioned strain in the preparation of an anti-lung cancer drug.

[0035] The present application provides an anti-tumor drug containing the above-mentioned steroidal compound or the fermentation product of the above-mentioned Talaromyces sp. JNQQJ-4 strain.

[0036] The present application has the following beneficial effects:

[0037] The present application provides a marine fungus-derived steroidal compound, which has the effect of significantly inhibiting the proliferation of MDA-MB-435S, HepG2, SKOV3, SKOV3R, A2780 and A549 and other tumor cells, and the IC 50 Can reach 11.11 μM, 27.22 μM, 28.47 μM, 32.66 μM, 21.31 μM and 19.19 μM respectively, and can be used for preparing anti-melanoma, liver cancer, ovarian cancer and lung cancer drugs, and provides a new drug for the treatment of various tumors.

[0038] In addition, the compound is derived from natural microorganisms and can be obtained by microbial fermentation, and the preparation method is simple.

[0039] Therefore, the steroidal compound provided by the present application has good clinical application potential for anti-tumor treatment and has important application value. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 It is a morphological diagram of JNQQJ-4 strain under culture medium.

[0041] Figure 2 It is the nuclear magnetic resonance hydrogen spectrum of compound I.

[0042] Figure 3 It is the nuclear magnetic resonance carbon spectrum of compound I.

[0043] Figure 4 It is the nuclear magnetic resonance HSQC spectrum of compound I.

[0044] Figure 5 It is the nuclear magnetic resonance 1 H- 1 HCOSY spectrum.

[0045] Figure 6 It is the nuclear magnetic resonance HMBC spectrum of compound I.

[0046] Figure 7 It is the nuclear magnetic resonance NOESY spectrum of compound I. DETAILED DESCRIPTION

[0047] The present application is further illustrated below in combination with the drawings and specific examples of the specification, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0048] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0049] Thiazolyl blue (MTT, 3-[4, 5-dimethylthiazol-2-yl]-2, 5diphenyl tetrazolium bromide), molecular formula is C 18 H 16 BrN5S, CAS number: 298-93-1, structural formula is:

[0050]

[0051] Cisplatin (foreign name: Cisplatin, DDP), CAS number: 15663-27-1, structural formula is

[0052] Melanoma cell line MDA-MB-435S, human hepatoma cell line HepG2, human ovarian cancer cell line SKOV3, human ovarian cancer cell line SKOV3R, human ovarian cancer cell line A2780 and human lung cancer cell line A549 were purchased from Shanghai Cell Biology Research Institute of Chinese Academy of Sciences.

[0053] DMEM complete medium: DMEM medium (purchased from Thermo Fisher, Waltham, MA, USA) containing 5% fetal bovine serum (purchased from Thermo Fisher, Waltham, MA, USA), 1% glutamine (purchased from Gibco, Grand Island, NY, USA), 1% streptomycin and penicillin mixture (purchased from Gibco, Grand Island, NY, USA), 1% non-essential amino acids (purchased from Gibco, Grand Island, NY, USA).

[0054] Example 1 Obtaining of marine blue fungus Talaromyces sp. JNQQJ-4

[0055] 1. Isolation and screening of strains

[0056] A single colony was isolated from fresh leaves of mangrove plants Excoecaria agallocha L. in Zhanjiang Jinniu Island, Guangdong, and named JNQQJ-4.

[0057] 2. Identification of strains

[0058] Morphological identification: moderate growth, typical velvet colony and a large number of dark green conidia were produced, and the broom-shaped branches were double-wheel and irregular, arranged closely, and the bottom of the culture medium was red, as shown in Figure 1 .

[0059] Molecular biology identification: strain JNQQJ-4 was subjected to molecular biology ITS sequencing, and the ITS sequencing result was:

[0060] GCGCTTTTTTGATATGCTTAAGTTCAGCGGGtaACTCCTACCTGATcCGAGGTCAACCGTGGTAAAAAATACAGTTGTGACAGACGTGCGCAGGTCCTTCCCGAGCGAGTGACAAAGCCCCATACGCTCGAGGACCGGACGCGACGTCGCCGCTGCCTTTCGGGCAGGTCCCCCCGCCAGGGAGGGGACCACACCCAA CACACAAGCCGTGCTTGAGGGCAGAAATGACGCTCGGACAGGCATGCCCCCCGGAATGCCAGGGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACGGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAGATCCATTGTTGAAAGTTTTGACAATTTTCATTTATAACTCAGACAGCCCATATTCATCAGGGTTCATGAGGCGCATCGGCGGGCGCGGACCCGAGGGCACAGAGGCCCCCCGGCGACCGGACCGAAGCCCAGTGGGCCCGCCGAAGCAACAGGGTATGTTTAAAACACGGGTGGGAGGTTGGGCTCACGAAGGAGCCCTCACTCGGTAATGTCTtCCGCAGgTttCACcTACGGAAACCTTGTTACGACTTTTACATCCA(SEQ ID NO.1).

[0061] The ITS sequencing result is identified as belonging to Talaromyces sp.

[0062] In summary, strain JNQQJ-4 is identified as belonging to Talaromyces sp., and is preserved in Guangdong Microbial Culture Collection Center (GDMCC) on May 24, 2024, with the preservation number of GDMCC No: 64682, and the classification name of Talaromyces sp. The address of the preservation unit is No. 59, Building 5, Guangzhou, Guangdong Province.

[0063] Example 2 Isolation of Steroid Compounds

[0064] A steroid compound isolated from the fermentation product of marine fungus Talaromyces sp. JNQQJ-4.

[0065] The specific separation and preparation method of the steroid compound is as follows:

[0066] S1. Seed liquid culture of marine fungus Talaromyces sp. JNQQJ-4: marine fungus Talaromyces sp. JNQQJ-4 is inoculated into a seed culture medium, and cultured at 30℃ for 6 days at a shaking speed of 180 rpm to obtain a seed culture liquid; the seed culture medium is composed of 0.3% of glucose, 0.1% of yeast extract, 0.5% of peptone, 3% of sodium chloride, and 96.1% of water by weight;

[0067] S2. Fermentation culture of marine fungus Talaromyces sp. JNQQJ-4: the seed culture liquid is inoculated into a solid rice fermentation medium (rice and seawater are mixed at a mass ratio of 7:3), and cultured at room temperature for 30 days at 30℃ to obtain a fermentation product;

[0068] S3. The fermentation product is filtered to obtain mycelium, which is extracted with methanol for 3 times, and the 3 times of extraction liquids are combined and concentrated to obtain an extract, which is extracted with ethyl acetate to obtain an ethyl acetate crude extract; the ethyl acetate crude extract is separated by silica gel normal phase chromatography; gradient elution is performed with petroleum ether / ethyl acetate (the volume fraction of petroleum ether changes as follows during the elution process: 90%→80%→70%→60%→50%→40%→30%; the volume fraction of ethyl acetate changes as follows during the elution process: 10%→20%→30%→40%→50%→60%→70%), and the part with elution polarity of 40%-60% ethyl acetate / petroleum ether is collected. The collected component is dissolved with methanol, and separated by Sephadex LH-20 gel chromatography column with dichloromethane / methanol as the eluent (the volume ratio of dichloromethane to methanol is 1:1), and the obtained components are sequentially labeled as components 1 and 2 (determined by thin layer chromatography TLC) in the order from first to last; component 2 is dissolved with dichloromethane / methanol, and separated by column chromatography with silica gel, and eluted with dichloromethane / methanol (the volume ratio of dichloromethane to methanol is 100:1), and the obtained components are sequentially labeled as components Fr.2.1, Fr.2.2, Fr.2.3 (determined by thin layer chromatography TLC) in the order from large to small polarity, and component Fr.2.2 is the crude sample of compound I. The crude sample of compound I is dissolved with n-hexane / isopropyl alcohol, and purified by semi-preparative high performance liquid chromatography (ND chiral column) with n-hexane / isopropyl alcohol (v / v 70:30, flow=1.0 mL / min) as the eluent, to obtain pure compound I.

[0069] Structure identification of compound I in Example 3

[0070] Compound I was analyzed by nuclear magnetic resonance hydrogen spectrum, carbon spectrum, 1 H- 1 H COSY, HSQC, HMBC, NOESY spectrum, and then determined the structure of Compound I by high resolution mass spectrometry, infrared, etc., and determined the absolute configuration of Compound I by X-ray single crystal diffraction combined with ECD calculation, and the nuclear magnetic resonance hydrogen spectrum, carbon spectrum, HSQC spectrum, 1 H- 1 HCOSY spectrum, HMBC spectrum, NOESY spectrum of Compound I were respectively as shown in Figures 2 to 7

[0071] Compound I was subjected to structural test analysis, and the following experimental data were obtained:

[0072] Compound I: C 44 H 56 O7, HRESI-MS: 719.3923 [M+H] + (calculated value 719.3923).

[0073] The NMR data of Compound I are shown in Table 1.

[0074] Table 1 NMR data of Compound I (CDCl3, 600MHz / 150MHz, ppm)

[0075]

[0076]

[0077]

[0078] According to the above data results, it is confirmed that Compound I is a steroid compound, and the structural formula is as shown in formula (I):

[0079]

[0080] Example 4 Anti-tumor effect study of steroid compound I

[0081] I. Experimental method

[0082] 1. Resuscitation and culture of cells

[0083] ​a. Revival: The cells were quickly taken out from liquid nitrogen, carefully put into sterile water, and quickly transferred into a water bath pot of 37°C sterile water for quick thawing. After 3-4 min, when no obvious ice block was observed, 800 rpm room temperature centrifugation was performed for 4 min. In the super-clean bench, the cryopreservation tube was opened, the cryopreservation liquid was discarded, 1 mL of fresh DMEM complete medium was used to resuspend the cells, which were transferred into a 15 mL sterile centrifuge tube, 5 mL of fresh DMEM complete medium was added, and 800 rpm room temperature centrifugation was performed again for 4 min. After centrifugation, 1 mL of fresh DMEM complete medium was used to resuspend the cells and blow them evenly, which were transferred into a 100 mm culture dish containing 6-8 mL of fresh DMEM complete medium, shaken and placed in a 37°C, 5% CO2 incubator for culture. After 12 h of culture, the old culture medium was discarded, 1xPBS was washed once, 1xPBS was discarded, 6-8 mL of fresh DMEM complete medium was replaced, and the cells were placed back into the incubator for culture.

[0084] b. Culture: All tumor cells were cultured using DMEM complete medium in a 37°C, 5% CO2 incubator. The morphology, density, etc. of the cells were observed under a microscope, and the medium was replaced or the cells were passaged as needed. During the culture process, mycoplasma was detected regularly, and if mycoplasma-positive cells appeared, anti-mycoplasma drugs were used to treat until negative or the positive cells were discarded to ensure the state of the experimental cells and the stability of the experiment.

[0085] The revival and culture methods of the cells of the present embodiment were used to revive and culture melanoma cell strain MDA-MB-435S, human liver cancer cell strain HepG2, human ovarian cancer cell strain SKOV3, human ovarian cancer cell strain SKOV3R, human ovarian cancer cell strain A2780, and human lung cancer cell strain A549.

[0086] 2. Cell counting

[0087] After the cells were digested into a single cell suspension, the cells were repeatedly blown and mixed evenly, 10 μL of the cell suspension was taken and added to a cell counting plate for counting, and the number of cells in the large squares in 4 directions was counted under a microscope. The cell concentration calculation formula is as follows:

[0088]

[0089] (C represents the number of cells per milliliter, and N0 represents the total number of cells in the large squares in 4 directions).

[0090] 3. Preparation of DMEM complete medium containing the test compound

[0091] Prepare 5 1.5 mL tubes, 4 tubes add 600 μL DMEM complete medium, and the last tube prepare 900 μL DMEM complete medium containing 50 μM steroid compound I, dilute the compound I by 3 times gradient, ultrasonic emulsification, filter sterilization, and store at 4°C.

[0092] Prepare 5 1.5 mL tubes, 4 tubes add 600 μL DMEM complete medium, and the last tube prepare 900 μL DMEM complete medium containing 50 μM cisplatin, dilute the cisplatin by 3 times gradient, ultrasonic emulsification, filter sterilization, and store at 4°C.

[0093] Obtain DMEM complete medium containing different concentrations of steroid compound I and containing different concentrations of cisplatin (0 μM, 0.617 μM, 1.851 μM, 5.556 μM, 16.667 μM, 50 μM).

[0094] 4. MTT living cell detection method for determining the antitumor activity of the compound

[0095] MTT can be reduced to blue or blue-purple formazan crystals by succinate dehydrogenase in living cells, and dimethyl sulfoxide (DMSO) can dissolve the formazan crystals and present a purple solution, and the absorbance value is determined at an excitation wavelength of 490 nm by a spectrophotometer. Since the succinate dehydrogenase in the mitochondria of dead cells degrades and disappears, it cannot reduce MTT to formazan crystals, so the survival and death status of cells can be reflected by the size of the absorbance value.

[0096] The specific steps of the MTT method are as follows:

[0097] (1) Plating

[0098] According to the cell culture method of the present embodiment, take the cells in the logarithmic growth phase, trypsinize the cells into single cells, resuspend with 5 mL of DMEM complete medium, count, and inoculate 8000-10000 cells per well (adjust appropriately according to the cell growth rate) into a 96-well plate, and add 100 μL of DMEM complete medium to each well.

[0099] (2) Compound treatment

[0100] The next day, discard the original culture medium, and add 180 μL of DMEM complete medium containing the test compound (compound I, cisplatin) prepared at the preset concentration, set 3 replicate wells for each concentration, and reserve a blank control group (without MTT group) and an experimental control group (DMSO group). After the drug is added, the cells are placed in a 37°C, 5% CO2 incubator for culture.

[0101] (3) Absorbance detection

[0102] After 48h of drug addition, 25μL of MTT (5mg / mL) was added to all wells except the blank control group, and the cells were incubated for another 4-6h at 37℃ in a 5% CO2 incubator. All the culture medium in the wells was carefully aspirated with a vacuum pump, and 160μL of DMSO was added to each well. The plate was shaken on a shaker until the formazan crystals were completely dissolved. The absorbance value at 490nm excitation was read using a multifunctional microplate reader.

[0103] (4) Calculation of cell inhibition rate and IC 50

[0104]

[0105] (B represents the average absorbance value of the blank control group, C represents the average absorbance value of the experimental control group, and T represents the average absorbance value of the experimental group)

[0106] IC 50 The inhibition rate at each concentration was calculated in Microsoft Excel 2019 (Microsoft, WA, USA), and the concentration corresponding to the half inhibition rate was calculated using the Forecast formula.

[0107] II. Experimental results

[0108] IC of steroidal compound I inhibiting various tumor cell lines 50 The results showed that steroidal compound I could effectively inhibit the proliferation of human melanoma cell line MDA-MB-435S, human hepatoma cell line Hep G2, human ovarian cancer cell line SKOV3, SKOV3R and A2780 cell line, and lung cancer cell line A549.

[0109] Table 2 IC of steroidal compound I inhibiting various tumor cell lines 50

[0110]

[0111] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A steroid compound, characterized in that: The structural formula of the steroid compound is shown in formula (I): Formula (I).

2. One plant Talaromyces sp. JNQQJ-4 strain, characterized in that described Talaromyces sp. JNQQJ-4 strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on May 24, 2024, with the deposit number GDMCC No: 64682.

3. The method according to claim 2 Talaromyces Use of the strain JNQQJ-4 in the preparation of the steroid compound according to claim 1.

4. The method for preparing the steroid compound according to claim 1, characterized in that: According to claim 2 Talaromyces The steroid compounds were isolated and obtained from the fermentation product of strain sp. JNQQJ-4.

5. Use of the steroid compound according to claim 1 in the preparation of anti-melanoma drugs.

6. Use of the steroid compound according to claim 1 in the preparation of anti-liver cancer drugs.

7. Use of the steroid compound according to claim 1 in the preparation of anti-ovarian cancer drugs.

8. Use of the steroid compound according to claim 1 in the preparation of anti-lung cancer drugs.

9. An anti-tumor drug, characterized in that: Containing the steroid compound according to claim 1.

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