Euplathylrin A, a phylloxanthanone-type diterpenoid compound, and its preparation method and application

Abstract

The present invention provides a phylloxane-type diterpenoid compound, Euplathylrin A, whose structural formula is shown in formula (1). The present invention also provides a method for preparing the phylloxane-type diterpenoid compound, Euplathylrin A. The preparation method of the present invention is simple, easy to operate, has low equipment cost, uses an environmentally friendly solvent, and is convenient for industrial scale-up production. The Euplathylrin A compound and a pharmaceutical composition thereof can effectively promote apoptosis of human scar fibroblasts and are suitable for the development of anti-scar hyperplasia drugs.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a preparation method and application of a physophane-type diterpenoid compound, Euplathylrin A. Background Art

[0002] Diterpenoids from Leptochloa crus-galli are a class of natural products with potential pharmacological activity. They are widely found in plants, particularly in the Euphorbiaceae family. These compounds have been extensively studied and exhibit numerous biological activities, including anti-inflammatory, anti-tumor, antibacterial, and anti-parasitic properties.

[0003] Numerous methods for preparing diterpenoids from the seed of the Chinese genus Leptochloa have been reported in the prior art. These methods typically involve extracting raw materials from plants, such as the roots, stems, leaves, or rhizomes of plants in the Leptochloa family, and performing extraction, isolation, and purification steps. Subsequently, the extracted compounds are structurally modified through chemical synthesis or semi-synthesis to obtain the target compounds. Although some progress has been made, the existing technology still has some defects in the preparation and application of diterpenoids of Euphorbia pulex, such as: 1. Obtaining natural ingredients, complexity of extraction and separation methods: Diterpenoids of Euphorbia pulex in Euphorbiaceae plants usually exist in low concentrations, so extracting and separating these compounds from plants is a challenging task. Existing extraction methods may require complex operating steps and high-cost reagents, which makes the preparation process complicated and expensive; 2. Difficulty of structural modification: In the existing technology, structural modification of diterpenoids of Euphorbia pulex usually requires multi-step chemical synthesis or semi-synthesis processes, which may have low yield, low selectivity and complex synthesis steps, limiting the output and quality of the target compounds; 3. Limitation of biological activity: Although diterpenoids of Euphorbia pulex show multiple biological activities, no pharmacological activity for treating scar hyperplasia has been reported in the existing technology. Summary of the Invention

[0004] In view of the above technical problems, the object of the present invention is to provide a phylloxanthanone-type diterpenoid compound Euplathylrin A and its preparation method and application.

[0005] To achieve the above-mentioned and other related purposes, the present invention provides a euplathylrin-type diterpenoid compound, Euplathylrin A, whose structural formula is shown in formula (1):

[0006]

[0007] The present invention also provides a method for preparing the above-mentioned euplathylrin-type diterpenoid compound Euplathylrin A, comprising the following steps:

[0008] 1) using the Herba Lycopodii fructus as a raw material, extracting with an organic solvent, and removing the solvent to obtain a crude extract;

[0009] 2) dispersing the crude extract in water, and extracting with petroleum ether and ethyl acetate, respectively, to obtain a petroleum ether extract and an ethyl acetate extract;

[0010] 3) The petroleum ether extract was separated by MCI gel column, and gradient elution was performed with methanol / water gradient eluent in a volume ratio of 5:5 to 10:0, and fraction Fr.VI was collected under the methanol / water eluent in a volume ratio of 8:2 to 9:1;

[0011] 4) Fraction Fr.VI was separated on a silica gel column using a gradient elution of petroleum ether / ethyl acetate in a volume ratio of 5:1 to 1:5, and fraction Fr.VIb was collected using a gradient elution of petroleum ether / ethyl acetate in a volume ratio of 5:4 to 3:5;

[0012] 5) Fraction Fr.VIb was separated on an RP-C18 column using a methanol / water gradient eluent of 6.5:3.5 to 8:2 by volume, and fraction Fr.VIb2 was collected using a methanol / water gradient eluent of 7.5:2.5 to 8:2 by volume;

[0013] 6) Fraction Fr.VIb2 was purified by semi-preparative HPLC chromatography to obtain compound Euplathylrin A.

[0014] In some embodiments of the present invention, in step 6), the semi-preparative HPLC chromatography purification is performed using an Eclipse XDB-C18 column. Furthermore, the purification uses a mobile phase of methanol / water in a ratio of 7.5:2.5 to 8:2. The flow rate of the mobile phase is 3 mL / min.

[0015] The present invention also provides use of Leptochloa chinensis in preparing the Leptochloa alkane-type diterpenoid compound Euplathylrin A.

[0016] The present invention also provides a pharmaceutical composition comprising the above-mentioned euplathylrin-type diterpenoid compound Euplathylrin A or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient.

[0017] The present invention also provides a use of the aforementioned phyllane-type diterpenoid compound Euplathylrin A or the aforementioned pharmaceutical composition in the preparation of an anti-scar hyperplasia drug.

[0018] As described above, the preparation method and application of the euphyllane-type diterpenoid compound Euplathylrin A of the present invention have the following beneficial effects:

[0019] The invention uses Leptochloa chinensis as a raw material and obtains the Leptochloa alkane-type diterpenoid compound Euplathylrin A by extraction for the first time.

[0020] The preparation method of the phylloxane-type diterpenoid compound Euplathylrin A of the present invention is simple and easy to operate, the equipment is low-cost, the solvent used is environmentally friendly, and it is convenient for industrial scale-up production.

[0021] The phyllane-type diterpenoid compound Euplathylrin A of the present invention can effectively promote apoptosis of human scar fibroblasts and is expected to be a potential drug for treating scar hyperplasia. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Shown is the extraction and separation flow chart of compound Euplathylrin A.

[0023] Figure 2 Shown is the single crystal structure of the compound Euplathylrin A.

[0024] Figure 3 Shown is the H NMR spectrum of compound Euplathylrin A.

[0025] Figure 4 Shown is the NMR carbon spectrum of compound Euplathylrin A.

[0026] Figure 5 Shown is the two-dimensional HH COSY spectrum of compound Euplathylrin A.

[0027] Figure 6 Shown is the NOESY spectrum of the compound Euplathylrin A.

[0028] Figure 7 Shown is the HSQC spectrum of compound Euplathylrin A.

[0029] Figure 8 Shown is the HMBC spectrum of the compound Euplathylrin A.

[0030] Figure 9 Shown is the mass spectrum of the compound Euplathylrin A.

[0031] Figure 10 Shown is the IR spectrum of compound Euplathylrin A.

[0032] Figure 11Shown are AO / PI double-stained fluorescence micrographs under the action of Euplathylrin A; green fluorescence represents living cells, red fluorescence represents dead cells, and orange represents premature apoptosis.

[0033] Figure 12 Shown is a diagram of the cell cycle phases under the action of Euplathylrin A. DETAILED DESCRIPTION

[0034] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0035] Please refer to the accompanying drawings. It should be noted that the illustrations provided in this embodiment are merely schematic illustrations of the basic concept of the present invention. Therefore, the drawings only show components relevant to the present invention and are not drawn according to the number, shape, and size of components in actual implementation. In actual implementation, the type, quantity, and proportion of each component may be varied arbitrarily, and the component layout may also be more complex.

[0036] The first aspect of the present invention provides a phytholane-type diterpenoid compound Euplathylrin A, wherein the structural formula of the phytholane-type diterpenoid compound Euplathylrin A is shown in formula (1):

[0037]

[0038] A second aspect of the present invention provides a method for preparing the above-mentioned phylloxanthanone-type diterpenoid compound Euplathylrin A, comprising the following steps:

[0039] 1) using the Herba Lycopodii fructus as a raw material, extracting with an organic solvent, and removing the solvent to obtain a crude extract;

[0040] 2) dispersing the crude extract in water, and extracting with petroleum ether and ethyl acetate, respectively, to obtain a petroleum ether extract and an ethyl acetate extract;

[0041] 3) The petroleum ether extract was separated by MCI gel column, and gradient elution was performed with methanol / water gradient eluent in a volume ratio of 5:5 to 10:0, and fraction Fr.VI was collected under the methanol / water eluent in a volume ratio of 8:2 to 9:1;

[0042] 4) Fraction Fr.VI was separated on a silica gel column using a gradient elution of petroleum ether / ethyl acetate in a volume ratio of 5:1 to 1:5, and fraction Fr.VIb was collected using a gradient elution of petroleum ether / ethyl acetate in a volume ratio of 5:4 to 3:5;

[0043] 5) Fraction Fr.VIb was separated on an RP-C18 column using a methanol / water gradient eluent of 6.5:3.5 to 8:2 by volume, and fraction Fr.VIb2 was collected using a methanol / water gradient eluent of 7.5:2.5 to 8:2 by volume;

[0044] 6) Fraction Fr.VIb2 was purified by semi-preparative HPLC chromatography to obtain compound Euplathylrin A.

[0045] Wherein, in step 1), the organic solvent is an ethanol solution. In a preferred embodiment of the present invention, the organic solvent is a 95% ethanol solution.

[0046] In a preferred embodiment of the present invention, the extraction in step 1) is performed by percolation extraction.

[0047] In step 1), the Leptochloa chinensis seeds are specifically the dried mature seeds of Euphorbia lathylris L.

[0048] In step 3), the gradient elution is performed by sequentially eluting with methanol / water in volume ratios of 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0.

[0049] In step 3), the petroleum ether extract is separated by an MCI gel column, and gradient elution is performed using MeOH-H2O with a volume ratio of 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0 as the eluent, and fractions Fr.I, Fr.II, Fr.III, Fr.IV, Fr.V, and Fr.VI can be collected; among which fraction Fr.VI is obtained by collecting under a methanol / water eluent volume ratio of 8:2 and 9:1.

[0050] In step 4), the gradient elution is performed by sequentially eluting with petroleum ether / ethyl acetate in a volume ratio of 5:1, 5:2, 5:3, 5:4, 5:5, 3:5, and 1:5.

[0051] In step 4), the fraction Fr.VIb is the eluent under petroleum ether / ethyl acetate eluent with a volume ratio of 5:4, 5:5, and 3:5.

[0052] In step 4), fraction Fr.VI is separated by silica gel column and eluted with petroleum ether / ethyl acetate gradient elution at a volume ratio of 5:1, 5:2, 5:3, 5:4, 5:5, 3:5, and 1:5, respectively, to collect fractions Fr.VIa, Fr.VIb, and Fr.Vic; wherein fraction Fr.VIb is obtained by collecting the eluent at a volume ratio of petroleum ether / ethyl acetate of 5:4, 5:5, and 3:5.

[0053] In step 5), the gradient elution is performed by sequentially eluting with methanol / water in volume ratios of 6.5:3.5, 7:3, 7.5:2.5, and 8:2.

[0054] In step 5), the fraction Fr.VIb2 is an eluent in a methanol / water eluent with a volume ratio of 7.5:2.5 to 8:2.

[0055] In step 5), fraction Fr.VIb is separated on an RP-C18 column using a methanol / water gradient elution with a volume ratio of 6.5:3.5, 7:3, 7.5:2.5, and 8:2, respectively, to collect fractions Fr.VIb1 and Fr.VIb2; wherein fraction Fr.VIb2 is collected using a methanol / water eluent with a volume ratio of 7.5:2.5 to 8:2.

[0056] In step 6), the semi-preparative HPLC chromatography purification uses an Eclipse XDB-C18 column.

[0057] In step 6), the purification uses a mobile phase of MeOH / H2O in a ratio of 7.5:2.5 to 8:2, for example, 7.5:2.5, 7.6:2.4, 7.7:2.3, 7.8:2.2, 7.9:2.1, or 8:2, with a flow rate of 3 mL / min. In a preferred embodiment of the present invention, the mobile phase MeOH / H2O ratio is 8:2.

[0058] A third aspect of the present invention provides use of Leptochloa chinensis in preparing the Leptochloa alkane-type diterpenoid compound Euplathylrin A as described above.

[0059] The fourth aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned phylloxanthanone-type diterpenoid compound Euplathylrin A or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient.

[0060] In the present invention, the term "pharmaceutically acceptable" means that the molecular entities and compositions will not produce adverse, allergic or other untoward reactions when appropriately administered to animals or humans.

[0061] "Pharmaceutically acceptable carriers or excipients" should be compatible with the euphyllane-type diterpenoid compound Euplathylrin A, that is, they can be blended with it without significantly reducing the efficacy of the pharmaceutical composition under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers or excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavorings; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer. These materials are used as needed to aid in the stability of the formulation or to help increase the activity or its bioavailability or to produce an acceptable taste or flavor in the case of oral administration.

[0062] In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited and can be prepared into injections, oral liquids, tablets, capsules, dripping pills, sprays, etc., and can be prepared by conventional methods. The choice of pharmaceutical dosage form should match the mode of administration.

[0063] A fifth aspect of the present invention provides use of the aforementioned phylloxanthanone-type diterpenoid compound Euplathylrin A or the aforementioned pharmaceutical composition in the preparation of an anti-scar hyperplasia drug.

[0064] In the following examples, unless otherwise specified, 95% ethanol was used for extraction as an industrial reagent; petroleum ether, ethyl acetate, dichloromethane, methanol, etc. were all analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.; methanol used in HPLC was chromatographic grade and purchased from Fisher Scientific; water used in HPLC was Wahaha purified water; HPLC instrument (Agilent Technologies 1260 series); semi-preparative HPLC instrument (Agilent Technologies 1260 series); rotary evaporator (IKA); GF 254 Thin layer chromatography silica gel (Qingdao Ocean Chemical Company); column chromatography silica gel (Yantai Jiangyou Silica Gel Development Co., Ltd.); automatic receiver (Shanghai Huxi Company).

[0065] Example 1

[0066] Preparation of Euplathylrin A, a phyllane-type diterpenoid

[0067] 1) Using 4 kg of Leptochloa chinensis as the raw material, extract with 95% ethanol (3 × 4 L), and remove the solvent by vacuum distillation to obtain a crude extract;

[0068] 2) The crude extract was dispersed in water and extracted with petroleum ether (3 × 1 L) and ethyl acetate (3 × 1 L) to obtain a petroleum ether extract and an ethyl acetate extract, respectively;

[0069] 3) 60 g of the petroleum ether extract was separated on an MCI gel column using a gradient elution with MeOH-H2O in a volume ratio of 5:5 to 10:0 (gradient elution used MeOH-H2O in a volume ratio of 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0, respectively) to obtain fractions Fr.I, Fr.II, Fr.III, Fr.IV, Fr.V, and Fr.VI; Fraction Fr.VI was collected and used as the eluent with methanol / water in a volume ratio of 8:2 and 9:1;

[0070] 4) Fraction Fr.VI was separated on a silica gel column using a gradient elution with petroleum ether / ethyl acetate in a volume ratio of 5:1 to 1:5 (gradient elution using petroleum ether / ethyl acetate in a volume ratio of 5:1, 5:2, 5:3, 5:4, 5:5, 3:5, and 1:5, respectively) to obtain fractions Fr.VIa, Fr.VIb, and Fr.VIc; fraction Fr.VIb was collected, and fraction Fr.VIb was the eluate using the petroleum ether / ethyl acetate in a volume ratio of 5:5;

[0071] 5) Fraction Fr.VIb was separated on an RP-C18 column using a gradient elution with MeOH-H2O in a volume ratio of 6.5:3.5 to 8:2 (gradient elution used methanol / water in volume ratios of 6.5:3.5, 7:3, 7.5:2.5, and 8:2, respectively) to obtain fractions Fr.VIb1 and Fr.VIb2; fraction Fr.VIb2 was collected, and Fr.VIb2 was the eluent obtained with methanol / water in volume ratios of 7.5:2.5 and 8:2.

[0072] 6) Fraction Fr.VIb2 was purified by semi-preparative HPLC (Eclipse XDB-C18 column) with a mobile phase of 8:2 MeOH / H2O at a flow rate of 3 mL / min to obtain compound Euplathylrin A as a white amorphous powder. Compound represented by formula (1):

[0073]

[0074] The structural identification data of the compound of formula (1) are as follows:

[0075] Eupathylrin A,colorless crystals;mp 96-100℃;[α] 25 D +70.5(c 0.3,MeOH); UV(MeCN)λ max (logε)194(3.12)nm,222(3.42); CD(c 1.0×10 -4 M,MeOH)λ max (Δε)234(Δε-2.6),306(Δε+1.8)nm; IR(KBr)ν max 2956,2923,2854,1692,1456,1021,801cm -1 ; 1 H and 13 CNMR data, see Table 1; HRESIMS ion at m / z 539.2659[M+H] + (calcd for C 31 H 39 O8,539.2645)

[0076] Table 1 H-spectrum (400 MHz) and C-spectrum (100 MHz) NMR data of compound 1 (CDCl3, δ C in ppm,J in Hz)

[0077]

[0078] The molecular formula of the compound of formula (1) is C 31 H 38 O8, by the attached Figure 9 The HRESIMS ion is at m / z 539.2659 [M+H] + (Calculated as C 31 H 39 O8, 539.2645) is determined, which is equivalent to 13 degrees of unsaturation. Figure 10 The infrared spectrum shows the carbonyl absorption band (1733cm -1 ). Figure 3 of 1 The H NMR spectrum showed six methyl signals [δ H 2.16 (3H, s); 2.06 (3H, s); 1.91 (3H, d, J = 1.2 Hz); 1.20 (3H, s); 1.09 (3H, s), and 1.01 (3H, d, J = 6.7 Hz)], one olefin proton δ H 6.95 (1H, d, J = 11.1 Hz), aromatic signal [δH 8.09 (2H, dd, J = 7.8, 1.2 Hz); 7.60 (2H, t, J = 7.3 Hz); 7.48 (2H, t, J = 7.8 Hz)] and a series of aliphatic methylene or methyl multiplets. Figure 4 of 13 C NMR spectroscopy, combined with DEPT experiments, revealed 31 carbon atoms that can be assigned to a keto group (δ C 194.1), a benzoyl group (δ C 165.1,130.2,129.6×2,128.5×2,131.1), two carbonyl groups (δ C 170.8 and 169.2), a trisubstituted double bond (δ C 143.8 and 134.9), six methyl groups, six sp3 methyl groups (including two containing oxygen), four sp3 methylene groups (including one containing oxygen), and three sp3 quaternary carbons (including two containing oxygen). Since 9 of the 13 unsaturations are composed of a benzoyl group, two carbonyl groups, a ketone, and a double bond, the remaining unsaturations require that the compound of formula (1) is a tetracyclic ring system. Due to their common biological relationship, the above information is very similar to that of the lathyrane diterpenoid 3-benzoy-15-acety-jolkinol-5',6'-oxide (2), the structural formula of which is shown in formula (2):

[0079]

[0080] The main difference of compound 1 compared with compound 2 is due to the additional signal of the acetyl group [δ C 170.8(C), 20.8(CH3); δ H 1.09(s)], indicating that compound 1 is an acetylated derivative of compound 2. Figures 5 to 8 The planar structure of compound 1 was further determined by detailed interpretation of its 2D NMR data. The relative configuration of compound 1 was established on the basis of NOESY experiments. The key NOESY interaction of H-11 / CH3-20 determined the Δ12(13) double bond to be in E configuration. In the NOESY spectrum, the correlation of H-9 / H-11 indicated that the dimethylcyclopropane molecule was cis. Therefore, the NOE correlations of H-3 / CH3-16, H-3 / H-4 and H-9 / CH2-17 determined H-3, H-4, H3-16 and CH2-17 to be α. The structure including the absolute configuration (AC) of compound 1 was confirmed by single crystal X-ray crystallography using anomalous scattering of Cu Ka radiation, as shown in the attached figure. Figure 2Therefore, the structure of Euplathylrin A was determined to be the compound shown in formula (1).

[0081] Example 2

[0082] Study on the anti-scarring activity of Euplathylrin A prepared in Example 1

[0083] 1. Isolation of primary human scar fibroblasts

[0084] Take fresh scar tissue, remove fat, separate the epidermis and dermis, cut the dermis into long strips, wash it 2-3 times in DMEM containing 5% double antibody, and then cut it into 0.5-1.0mm strips. 3 Transfer tissue blocks to 25 cm 2 After culturing for 4 hours, 1 mL of DMEM (containing 1% double-antibody, 20% FBS) was added to each flask and the cells were placed upright in the incubator for continued culture. Thereafter, the medium was changed every two days, with 5 mL of DMEM (containing 1% double-antibody, 10% FBS) added to each flask. After culturing for 5 to 7 days, fibroblasts were allowed to dissociate from the scar tissue. When they had grown to 80% of the flask, they were isolated and purified using their sensitivity to trypsin. Cells from passages 2 to 5 were used for subsequent experiments.

[0085] 2. MTT assay to detect the inhibitory activity of Euplathylrin A on the proliferation of primary human scar fibroblasts

[0086] Primary human scar fibroblasts were cultured at 5×10 3 100 μL was inoculated into a 96-well culture plate at a density of cells / mL and cultured for 24 hours at 37°C, 5% CO2, and 90% RH. Different concentrations of Euplathylrin A (40, 20, 10, 7.5, 5, 3.75, and 2.5 μM) were added to the experimental group, the blank control group received culture medium without the drug, and the negative control group received 100 μL of DMSO solution. Three replicate wells were set up and cultured at 37°C, 5% CO2, and 90% RH for 24 hours. Then, 20 μL of 5 mg / mL MTT solution was added and incubated for 4 hours. The plate was centrifuged, the supernatant was discarded, and 150 μL of DMSO was added to dissolve the MTT crystals. After shaking the plate for several minutes, the absorbance OD value was measured at a wavelength of 570 nm using an enzyme-linked immunosorbent assay to calculate the inhibition rate. The IC was calculated using GraphPad Prism 8 software with compound concentration as the horizontal axis and inhibition rate as the vertical axis. 50 Finally, the IC of Euplathylrin A was obtained by fitting 50 It is 6.33μM.

[0087] 3. Study on Euplathylrin A-induced apoptosis of human scar fibroblasts

[0088] AO / PI staining (acridine orange / and propidium iodide staining) was used to detect the apoptosis of primary human scar fibroblasts after the addition of Euplathylrin A. Primary human scar fibroblasts were cultured at 3×10 4 The cells were seeded in a 6-well plate at a density of 10 cells per well and cultured in DMEM medium containing fetal bovine serum for 24 hours. The culture medium was discarded and the cells were washed twice with PBS. The experimental group was added with serum-free culture medium containing different concentration gradients of Euplathylrin A (1.58, 3.16, 6.33, 12.66 μM), the control group was added with 350 μM asiaticoside serum-free culture medium, and the blank group was added with serum-free culture medium. The cells were incubated at 37°C for 24 hours. After the incubation, the culture medium was discarded, the cells were washed twice with PBS to fully remove the culture medium, and 100 μL each of 200 μM AO and PI dyes were added for staining. The cells were incubated at room temperature in the dark for 5 minutes, the dyes were aspirated and discarded, the cells were washed twice with PBS, 200 μL of PBS was added, and the cells were observed and photographed under a fluorescence microscope at 200 times. The results are shown in the attached figure. Figure 11 As shown, when the concentration of Euplathylrin A was 6.33 μM and 12.66 μM, most cells emitted orange or red fluorescence, indicating that the apoptosis of human scar fibroblasts at medium and high concentrations was obvious, and the apoptosis was more obvious than that at asiaticoside.

[0089] 4. Study on the cell cycle of Euplathylrin A inhibiting proliferation of human scar fibroblasts

[0090] Human scar fibroblasts were cultured at 3×10 4 Cells were seeded at a density of 10 cells per well in a 6-well plate and cultured in serum-containing DMEM for 24 hours. The culture medium was discarded and the cells were washed twice with PBS. The experimental group was added with serum-free culture medium containing different concentration gradients of Euplathylrin A (1.58, 3.16, 6.33, and 12.66 μM), the control group was added with serum-free culture medium containing 350 μM asiaticoside, and the blank group was added with serum-free culture medium. The cells were incubated at 37°C for 24 hours. After the incubation period, the culture medium was discarded, and the cells were digested with EDTA-free trypsin and collected. The cells were centrifuged at 1000 rpm at room temperature for 5 minutes, and then suspended in pre-cooled PBS and counted. The cell concentration was adjusted to 1×10 6 / mL, take 100μL cell suspension to 1.5mL centrifuge tube and centrifuge, discard the supernatant and add 1mL pre-cooled 70% ethanol solution at 4℃ for 2h, then centrifuge at 1000rpm for 5min, discard the supernatant, add 1mL pre-cooled PBS solution to resuspend, centrifuge again and add 500μL PI staining solution, incubate at room temperature for 15min and then detect on flow cytometer. The results are shown in the attached figure. Figure 12 The results showed that Euplathylrin A arrested human scar fibroblasts in the G2 / M phase of cell division at low and medium concentrations, and arrested human scar fibroblasts in the S phase of cell division at high concentrations.

[0091] In summary, the present invention, using the mature seeds of Euphorbia pulex (Euphratica chinensis) as raw material, extracts the phyllane-type diterpenoid Euplathylrin A for the first time. The extraction method is simple and easy to operate, employs inexpensive equipment, uses environmentally friendly solvents, and is amenable to industrial scale-up. Euplathylrin A, the phyllane-type diterpenoid, can effectively promote apoptosis in human scar fibroblasts and is a potential therapeutic agent for the treatment of scar hyperplasia. Therefore, the present invention effectively overcomes the shortcomings of the prior art and possesses high industrial value.

[0092] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A phylloxanthanane-type diterpenoid compound, Euplathylrin A, characterized in that: The structural formula of the euplathylrin-type diterpenoid compound Euplathylrin A is shown in formula (1):

2. The method for preparing the phylloxetine-type diterpenoid compound Euplathylrin A according to claim 1, wherein: The following steps are involved: 1) using the Herba Lycopodii fructus as a raw material, extracting with an organic solvent, and removing the solvent to obtain a crude extract; 2) dispersing the crude extract in water, and extracting with petroleum ether and ethyl acetate, respectively, to obtain a petroleum ether extract and an ethyl acetate extract; 3) The petroleum ether extract was separated by MCI gel column, and gradient elution was performed with methanol / water gradient eluent in a volume ratio of 5:5 to 10:0, and fraction Fr.VI was collected under the methanol / water eluent in a volume ratio of 8:2 to 9:1; 4) Fraction Fr.VI was separated on a silica gel column using a gradient elution of petroleum ether / ethyl acetate in a volume ratio of 5:1 to 1:5, and fraction Fr.VIb was collected using a gradient elution of petroleum ether / ethyl acetate in a volume ratio of 5:4 to 3:5; 5) Fraction Fr.VIb was separated on an RP-C18 column using a methanol / water gradient eluent of 6.5:3.5 to 8:2 by volume, and fraction Fr.VIb2 was collected using a methanol / water gradient eluent of 7.5:2.5 to 8:2 by volume; 6) Fraction Fr.VIb2 was purified by semi-preparative HPLC chromatography to obtain compound Euplathylrin A.

3. The method for preparing the phylloxetine-type diterpenoid compound Euplathylrin A according to claim 2, wherein: In step 1), the organic solvent is an ethanol solution.

4. The method for preparing the phylloxetine-type diterpenoid compound Euplathylrin A according to claim 2, wherein: The gradient elution in step 3) is eluted with methanol / water in a volume ratio of 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0, respectively; and / or, the gradient elution in step 4) is eluted with petroleum ether / ethyl acetate in a volume ratio of 5:1, 5:2, 5:3, 5:4, 5:5, 3:5, and 1:5, respectively; and / or, the fraction Fr.VIb in step 4) is an eluent under a petroleum ether / ethyl acetate eluent with a volume ratio of 5:4, 5:5, or 3:5; And / or, the gradient elution in step 5) is eluted with methanol / water in a volume ratio of 6.5:3.5, 7:3, 7.5:2.5, and 8:2 in sequence; and / or, the fraction Fr.VIb2 in step 5) is the eluent under a methanol / water eluent in a volume ratio of 7.5:2.5 to 8:

2.

5. The method for preparing the phylloxetine-type diterpenoid compound Euplathylrin A according to claim 2, wherein: In step 6), the semi-preparative HPLC chromatography purification uses an Eclipse XDB-C18 column.

6. The method for preparing the phylloxetine-type diterpenoid compound Euplathylrin A according to claim 2, wherein: In step 6), the purification uses a mobile phase of methanol / water in a ratio of 7.5:2.5 to 8:

2.

7. The method for preparing the phylloxetine-type diterpenoid compound Euplathylrin A according to claim 6, characterized in that: The flow rate of the mobile phase was 3 mL / min.

8. Use of Leptochloa chinensis in the preparation of the Leptochloa alkane-type diterpenoid compound Euplathylrin A as claimed in claim 1.

9. A pharmaceutical composition, characterized in that The invention comprises the euplathylrin A or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable carrier and / or excipient.

10. Use of the phylloxanthanone-type diterpenoid compound Euplathylrin A according to claim 1 or the pharmaceutical composition according to claim 9 in the preparation of an anti-scar hyperplasia drug.

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