A daphne-type diterpenoid compound Wikstdaphnein A, and a preparation method and application thereof

Abstract

The application discloses a daphne-type diterpenoid compound Wikstdaphnein A, a preparation method and application thereof. The monomer compound is obtained by repeatedly separating and purifying the ethyl acetate part extract of Daphne fedtschenkoi by using various separation fillers and separation technologies. The relative configuration and absolute configuration of the monomer compound are identified by using a spectrum method and a calculation chemistry method, and the molecular formula and structural formula are deduced. Further in-vitro cell activity researches show that the new compound Wikstdaphnein A separated and identified from the ethyl acetate extract of Daphne fedtschenkoi has good inhibitory activity on melanoma cells. The application is helpful for the development and utilization of natural compounds of Daphne fedtschenkoi.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of separation and purification of natural products and application, and particularly relates to a daphnane type diterpenoid compound Wikstdaphnein A, a preparation method and application thereof. BACKGROUND

[0002] Melanoma, usually refers to malignant melanoma. It is a malignant tumor originating from neural crest melanocytes, which is commonly seen in the skin and can also be found in internal organs and mucosa, accounting for about 3% of all tumors. Globally, about 2-3 million cases of skin cancer are diagnosed each year. In recent years, the incidence and mortality of melanoma have increased significantly worldwide. Cutaneous malignant melanoma accounts for about 6.8%-20% of cutaneous malignant tumors. Globally, there are 232,000 new melanoma patients each year, of which 55,000 die each year. And it shows a trend of younger age, and is one of the fastest growing malignant tumors. Compared with other solid tumors, it has a lower age of death. According to the latest Cancer Statistics, 2021 tumor model evaluation, more than 100,000 people in the United States will be newly diagnosed with melanoma in 2021. The Chinese Melanoma Diagnosis and Treatment Guidelines (2015 Edition) points out that the incidence of melanoma in China is increasing, with more than 20,000 new cases each year. Therefore, melanoma has seriously affected human health, so it is of great significance to strengthen the research on melanoma, explore new ways of treating melanoma, and develop and research drugs for treating melanoma.

[0003] At present, the treatment methods for melanoma in clinical practice are traditional surgical treatment and radiotherapy and chemotherapy, as well as emerging targeted therapy and immunotherapy. For example, vemurafenib, a targeted inhibitor of the high-mutation gene BRAF in melanoma, and trametinib (GSK1120212), a targeted MEK1 / 2. Although these targeted drugs have certain efficacy, they generally have problems such as large side effects and drug resistance; emerging immunotherapy, such as ipilimumab, an anti-CTLA-4 antibody, and nivolumab, an anti-PD-1 antibody, only a small number of patients have satisfactory efficacy after immunotherapy. Although new targeted therapy and immunotherapy have reduced some of the suffering of melanoma patients, the long-term survival rate of patients is still very low, so it is of great significance to understand the molecular mechanisms of melanoma development and seek appropriate new drugs for treatment.

[0004] The most studied mechanism of melanoma is the MAPK signaling pathway, and the commonly used targeted drugs are BRAF inhibitors and MEK inhibitors. In recent years, according to the results of tumor biopsy and in vitro culture of melanoma cells, about 70% of melanoma patients have abnormal PI3K / Akt signaling pathway, and the downstream mTOR gene also shows differences. It shows that the phosphatidylinositol-3-kinase / protein kinase B (PI3K / Akt) signaling pathway also plays an important role in melanoma, which provides a new direction for targeted therapy research. At the same time, it is also a classic pathway that inhibits tumor cell proliferation and promotes cell apoptosis, and plays an important role in the occurrence and development of tumors. Therefore, the study of the changes of PI3K / Akt / mTOR signaling pathway has become a new trend in the treatment of advanced malignant melanoma. In May 2019, the FDA in the United States approved the first PI3K inhibitor alpelisib for the treatment of breast cancer, so the research of new drugs targeting the PI3K / Akt signaling pathway may bring light to the treatment of melanoma.

[0005] Wikstroemia Chamaedaphne Meisn. is a medicinal plant of Thymelaeaceae Wikstroemia, and its dry flower buds are originally from Shanxi, Shaanxi, Hebei and other places. It was first recorded in 'Tu Jing Ben Cao' and included in 'Shanxi Province Chinese Herbal Medicine Standard' (1987 edition) and 'Chinese Pharmacopoeia' (1977 edition) volume one. Wikstroemia Chamaedaphne Meisn. is also known as North Yuhua, which is pungent and warm in nature, with a small amount of toxicity. It belongs to the lung and kidney channels. It is used for edema, water retention, phlegm accumulation, cough and asthma, constipation, and is used externally for scabies, tinea, and carbuncle. In China, the research on Wikstroemia Chamaedaphne Meisn. is mostly on the traditional effect of anti-fertility, and the research on chemical composition is less. Recent studies have shown that the main chemical components of Wikstroemia Chamaedaphne Meisn. are diterpenoids, flavonoids, phenolic acids, glycosides and other components. Modern pharmacological and clinical studies have shown that diterpenoids in Wikstroemia Chamaedaphne Meisn. have anti-fertility, anti-hepatitis B virus, anti-tumor and other activities. For example, pimelotide A, pimelotide B and pimelotide C have strong anti-proliferative activity on human promyelocytic leukemia HL-60, human hepatocarcinoma SMMC-7721, lung cancer A549, breast cancer MCF-7 and colon cancer HT-29; Wikstroelide E has anti-hepatitis B virus activity; Wikstroemia Chamaedaphne Meisn. is used for mid-term labor induction and has good anti-fertility effect; Yuhua ester A inhibits the growth of bladder cancer and colon cancer cells by up-regulating the expression of p21. These seco-abietane diterpenoids as the pharmacodynamic material basis have attracted widespread attention from natural product chemists and pharmacologists. The extraction and separation of new compounds from Wikstroemia Chamaedaphne Meisn. and the screening of anti-melanoma activity lay the foundation for the development of new drugs against melanoma.

[0006] Journal literature (Zhang Zhiqiang. Diterpenoid components of Daphne giraldii and its anti-HBV activity research[D]; Shanxi University, 2017: This paper adopts the traditional separation method to study the chemical composition of Daphne giraldii, 18 compounds are separated, including 8 diterpenoids of daphnane type, 2 diterpenoids of tigliane type, 6 diterpenoids of ingenane type and 2 flavonoid glycosides. Compounds 4, 6, 8-10 were tested for in vitro anti-HBV activity, and the results showed that the test compounds showed certain inhibition of HBsAg production and HBV-DNA replication; Among them, compound 9 showed strong activity in inhibiting the production of HBsAg.)(Wang Chengrui, Huang Huizhu, Han Mei, et al. Separation and identification of antifertility active ingredient shuoxie flower[J]. Chinese herbal medicine, 1981, 12(08): 1-3. It is found that shuoxie flower has good antifertility effect.)(Wang Chengrui, Huang Huizhu, Han Mei, et al. Separation and identification of shuoxie flower[J]. Chinese pharmaceutical journal, 1981, (06): 51-2. Research method: take the plant seed 14 kg, crush, cold soak with ethanol, dilute the concentrated solution with water, extract with chloroform, and evaporate to get 725 g of crude extract. The sample is chromatographed on a silica gel column, and the effective part obtained is further chromatographed on a low pressure column, and silica gel G is used as the filler, and the pure product 430 mg is obtained by eluting with benzene solution containing different proportions of acetic acid ethyl ester, with a yield of 0.0031%.

[0007] At present, there is no Wikstdaphnein A, a daphnane type diterpenoid compound, its preparation method and application like the present application. SUMMARY

[0008] The purpose of the present application is to provide a Wikstdaphnein A, a daphnane type diterpenoid compound, its preparation method and application, aiming at the deficiencies in the prior art.

[0009] In the first aspect, the present application provides an application of a daphnane type diterpenoid compound or its pharmaceutically acceptable salt in the preparation of a drug for treating melanoma, and the structure of the compound Wikstdaphnein A is shown as (I):

[0010]

[0011] Preferably, Wikstdaphnein A can significantly inhibit the proliferation and migration of melanoma cell B16 and promote apoptosis by inducing PI3K / Akt / mTOR pathway.

[0012] In the second aspect, the present application provides a preparation method of a new daphnane type diterpenoid compound, comprising the following steps: using ethanol to extract the flower bud powder of Daphne giraldii to obtain an extract, and then separating and purifying to obtain the diterpenoid compound.

[0013] As a preferred embodiment, the preparation method comprises the following steps:

[0014] ①Take the flower bud powder of yellow flower (10 kg) and extract it with 95% EtOH (50 L, one month each time) for three times at room temperature. After removing the solvent, the crude extract (2.5 kg) is obtained. The extract is suspended in water (3 L) and extracted with petroleum ether (3 x 3 L), ethyl acetate (3 x 3 L) and n-butanol (3 x 3 L) in sequence to obtain three corresponding parts;

[0015] ②Take the ethyl acetate part extract of 800 g obtained in step ① and apply XDA-7 macroporous resin column for preliminary segmentation. Adopt 30%, 60%, 90%, 95% and 100% methanol-water gradient elution to combine similar components to obtain Fr.A-D;

[0016] ③Take Fr.C obtained in step ② and further segment it with MCI column. Elute with 50%-100% gradient methanol-water to obtain two components Fr.CI-CII. Fr.CII (87 g) is treated with silica gel (CHCl3 / MeOH, 1:0 to 0:1, v / v) to obtain Fr.CIIa-Fr.CIIg. Fr.CIIe is subjected to chromatographic analysis on Sephadex LH-20 with CH2Cl2-MeOH (1:1) to obtain three sub-components Fr.CIIe1-Fr.CIIe3. Fr.CIIe1 is treated with silica gel (CHCl3 / MeOH, 100:1 to 5:1, v / v) to obtain Fr.CIIe1a-Fr.CIIe1c. CIIe1b is purified by semi-preparative HPLC (Eclipse XDB-C18 column, MeOH / H2O, 7.5:2.5, 3 mL / min) to obtain compound 1 (34.2 mg, t R 8.5 min).

[0017] In a third aspect, the present application provides an extract containing the diterpenoid compound as described above.

[0018] In a fourth aspect, the present application provides a pharmaceutical composition containing the diterpenoid compound or a pharmaceutically acceptable salt thereof as described above.

[0019] As a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0020] The pharmaceutically acceptable carrier is selected from diluents, preservatives, fillers, flow control agents, penetration enhancers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, antibacterial agents, antifungal agents, lubricants and dispersants.

[0021] According to the present invention, the compounds and extracts of the present invention can be used alone or in the form of pharmaceutical compositions. The administration method can be determined according to the specific circumstances, and can be prepared into dosage forms suitable for oral, rectal, intramuscular injection and other administration methods, such as tablets, capsules, ointments, patches, injections, etc., according to conventional methods in the field of pharmaceuticals.

[0022] The dosage of the compound of the present invention or its pharmaceutically acceptable salt or extract depends on factors such as the age, weight, and type and severity of the disease of the user.

[0023] The advantages of this invention are:

[0024] This invention employs various separation packing materials and techniques to repeatedly separate and purify the ethyl acetate extract of *Daphne genkwa* to obtain monomeric compounds. The relative and absolute configurations of the monomeric compounds were identified using various 1D and 2D NMR spectroscopy and high-resolution mass spectrometry (HR-ESI-MS), leading to the derivation of their molecular and structural formulas. Further in vitro cell activity studies showed that a novel compound, Wikstdaphnein A, isolated from the ethyl acetate extract of *Daphne genkwa*, exhibits good inhibitory activity against melanoma cells. This invention contributes to the development and utilization of natural compounds from *Daphne genkwa*. Attached Figure Description

[0025] Figure 1 The flowchart shows the extraction and separation process of compound 1.

[0026] Figure 2 The correlation is between (A) HMBC(→) and COSY(-) of compound 1; and (B) NOESY of compound 1. Related;

[0027] Figure 3 CD and UV of compound 1 (the arrows indicate the electric transition dipoles of the chromophore);

[0028] Figure 4 The IC50 of compound 1 inhibits the proliferation of B16 cells 50 ;

[0029] Figure 5 The effect of compound 1 on apoptosis in B16 cells;

[0030] Figure 6 shows the results of Annexin V-FITC / PI assay for the promotion of apoptosis in B16 cells by compound 1; **p<0.01, ***p<0.001 indicates a significant difference compared with the control group;

[0031] Figure 7 shows the effect of compound 1 on the cell cycle; **p<0.01, ***p<0.001 indicates a significant difference compared with the control group;

[0032] Figure 8 Effect of compound 1 on migration ability; ***p<0.001 indicates significant difference compared with the control group;

[0033] Figure 9 is the effect of compound 1 on the expression level of apoptosis proteins;

[0034] Figure 10 is the change in the expression level of PI3K / Akt / mTOR pathway related proteins under the action of compound 1. DETAILED DESCRIPTION

[0035] The separation and purification method described in the present application will be further described below in combination with specific examples, but the scope of the present application is not limited thereto.

[0036] Preparation of compound (1) Wikstdaphnein A

[0037] 1. Experiment

[0038] 1.1 Instruments and reagents

[0039] 1.1.1 Main instruments

[0040] High performance liquid chromatograph (Agilent Technologies 1260 series); semi-preparative high performance liquid chromatograph (Agilent Technologies 1260 series); XDA-7 macroporous resin adsorption resin (Shaanxi Leibo Biochemical Technology Co., Ltd.); ultrasonic cleaner (Shanghai Yiqing Ultrasonic Instrument Co., Ltd.); Sephadex LH-20 (GE healthcare Bio-science AB Co.); rotary evaporator (IKA Co.); GF254 thin layer chromatography silica gel (Qingdao Marine Chemical Co.); column chromatography silica gel (Yantai Jiangyou Silica Gel Development Co., Ltd.); automatic receiver (Shanghai Huxi Co.); and the like.

[0041] 1.1.2 Main reagents

[0042] Reagents: 95% ethanol for extraction is an industrial reagent; petroleum ether, ethyl acetate, dichloromethane, chloroform, methanol, acetone, and the like are all analytical pure, and are purchased from the National Pharmaceutical Group Chemical Reagent Co., Ltd.; methanol and acetonitrile used for high performance liquid chromatography are chromatographically pure, and are purchased from Fisher Scientific Co.; water used for high performance liquid chromatography is Wahaha purified water; TLC developer: 10% H2SO4-ethanol solution.

[0043] 1.1.3 Medicinal materials

[0044] The yellow daphne flower (approximately 10 kg) was purchased from the Bozhou City Medicinal Materials Market in Anhui Province. The place of origin is Taiyuan City, Shanxi Province. It was identified by Zhu Jianyong, Deputy Chief Pharmacist of the Seventh People's Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, as the flower bud of the plant *Wikstroemia chamaedaphne* Meissn., belonging to the genus *Wikstroemia* of the family Thymelaeaceae.

[0045] 1.1.4 Extraction and Separation

[0046] 10 kg of powdered flower buds of *W. chamaedaphne* was extracted three times at room temperature using 95% EtOH (50 L, once a month). After removing the solvent, 2.5 kg of crude extract was obtained. The extract was suspended in water (3 L) and extracted sequentially with petroleum ether (3 × 3 L), ethyl acetate (3 × 3 L), and n-butanol (3 × 3 L) to obtain three corresponding fractions. 800 g of the ethyl acetate fraction extract of *W. chamaedaphne* was initially fractionated using an XDA-7 macroporous resin column, eluted with a methanol-water gradient of 30%, 60%, 90%, 95%, and 100%, respectively. Similar fractions were combined to obtain Fr. AD. Fr. C was further fractionated using an MCI column, eluted with a methanol-water gradient of 50%–100% to obtain Fr. CI and CⅡ fractions. Fr.CII (87g) was treated with silica gel (CHCl3 / MeOH, 1:0 to 0:1, v / v) to obtain Fr.CIIa-Fr.CIIg. Fr.CIIe was analyzed by chromatographic analysis on Sephadex LH-20 with CH2Cl2-MeOH (1:1) to obtain three subfractions: Fr.CIIe1-Fr.CIIe3. Fr.CIIe1 was treated with silica gel (CHCl3 / MeOH, 100:1 to 5:1, v / v) to obtain Fr.CIIe1a-Fr.CIIe1c. CIIe1b was purified by semi-preparative HPLC (Eclipse XDB-C18 column, MeOH / H2O, 7.5:2.5, 3mL / min) to obtain 1 (34.2mg, t R (8.5 min), see Figure 1 .

[0047] 2. Structural identification of compounds

[0048] 2.1 Identification of the structure of new compounds

[0049] 2.1.1 Structural Identification of Wikstdaphnein A

[0050]

[0051] Compound 1 is white amorphous powder, and its molecular formula is C + (calcd for C 34 H 40 O 10 Na, 631.2514) by HR-ESI-MS m / z 631.2487 [M+Na] 34 H 40 O 10 , and its unsaturation is 15. According to the infrared spectrum, it is indicated that the compound contains hydroxyl group (3394 cm -1 ), carbonyl group (1720 cm -1 ) and olefin (1645 cm -1 ) and other groups, 1 H NMR spectrum shows that there are three methyl groups [δ H 1.88 (3H, s), 0.94 (3H, d, J = 6.5 Hz), 0.90 (3H, d, J = 7.1 Hz)], one terminal double bond [δ H 5.15 (3H, d, J = 7.1 Hz)], four aromatic hydrogen signals [δ H 8.11 (1H, m), 7.62 (1H, m), 7.58 (1H, m), 7.51 (1H, m)]. 13 C NMR spectrum combined with DEPT spectrum analysis structure, it is known that the compound has 34 carbons, including three methyl carbon signals, four methylene carbon signals (including one oxygen carbon and one olefin), 14 methine carbon signals (including four oxygen carbons and six olefins) and nine quaternary carbon signals (including two ester carbonyl groups, four oxygen carbons and one olefin). Combined with the unsaturation, it is judged that the compound should contain two ester carbonyl groups, two benzene rings and one double bond, and it is speculated that there is an oxygen-containing ring.

[0052] Through further data comparison, it is found that compound 1 is the same as known compound genkwanin Ⅷ [1]The structures of 1 and genkwanin VIII are very similar and have the same molecular formula, so it is considered that the difference is in the substituents. HMBC and HMQC data show that the substituents at C-3 and C-14 are different. The carbon signals of genkwanin VIII are 74.2 (C-3) and 72.2 (C-14), while those of 1 are 76.2 (C-3) and 73.5 (C-14). The coupling constant of H-2 / H-3 is 10.2 Hz, indicating that there is a cis relationship between H-2 and H-3. In the NOESY spectrum, the NOE correlations of H-1α / H-3, H-3 / H-5, H-5 / H-10, H-10 / CH3-18, H-2 / H-10 and H-3 / H-10 indicate that H-2, H-3, H-5, H-10 and CH3-18 are in the α configuration, and the NOE correlations of CH3-19 / H-3' / 7', H-7 / H2-20, H-7 / H-14, H-7 / H-8 and H-8 / H-1 indicate that CH3-19, H-7, H-8, H-14 and H2-20 are in the β configuration. The absolute configuration of 1 was determined by CD curve. The obtained CD spectrum shows a negative effect at 235 nm and a positive effect at 220 nm, indicating a negative chirality between the two benzoate groups, and the transition dipole moments of the two chromophores are oriented in a counterclockwise direction Figure 3 ). ECD calculation determines the absolute configuration as 2S,3S,4S,5R,6R,7R,8R,9R,10S,11R,13R,14R Figure 2 ), therefore, compound 1 is named Wikstdaphnein A.

[0053] 2.2 Spectroscopic and physicochemical data of new compounds

[0054] Wikstdaphnein A (1): amorphous solid; [α] 25 D -18(c 0.1, MeOH); UV (MeOH) λ max (log ε) 193 (3.28), 228 (3.74) nm; ECD (c 1.0 x 10 -4 M, MeOH) λ max (Δ ε) 199 (-16.02), 219 (+1.57) 234 (-6.96) nm; IR (KBr) ν max 3395, 2921, 1721, 1702, 1646, 1453, 1260, 1099, 1025, 801, 710 cm -1 ; 1 H and 13C NMR (CD3OD) data, see Table 1; HRESIMS m / z 631.2487 [M+Na] + (calcd for C 34 H 40 O 10 Na,631.2514)。

[0055] Table 1 1 H NMR spectrum and 13 C NMR spectrum specific data

[0056]

[0057]

[0058] Example 2 Compound (1) Wikstdaphnein A in vitro anti-melanoma activity study

[0059] 1. Experimental materials

[0060] Test substance: compound 1. Cell strain: A375 (human melanoma cells), accession number: A375-CSP-533; B16 (mouse melanoma cells), accession number: B16-SCSP-5096; HFF-1 (human fibroblast cells), accession number: HFF-1-SCSP-656, all purchased from the Chinese Academy of Sciences Culture Collection Cell Bank.

[0061] 2. Experimental method

[0062] 2.1 Cell viability detection

[0063] MTT method was used to detect cell viability. A375 cells, B16 cells, and HFF-1 cells were inoculated in 96-well plates at a density of 5×10 3 cells / well for 24 hours. The culture solution was aspirated, the experimental group was added with prepared culture solution containing compound 1 at a concentration of 30 μM, the blank control group was added with fresh culture solution, and the negative control group was added with 150 μL DMSO solution. Three replicate wells were set for each group. 20 μL of MTT (5 mg / mL, dissolved in PBS) solution was added to each well, and the incubator was incubated in the dark for 4 hours. 150 μL of DMSO was added to each well, and the absorbance OD value was measured at a wavelength of 560 nm by a microplate reader. The inhibition rate was calculated.

[0064] 3. Experimental results

[0065] 3.1 Compound 1 primary screening results

[0066] Compound 1 had greater than 50% inhibition rate on B16 cells and A375 cells, and less than 30% inhibition rate on HFF-1 cells when the concentration of the compound was 30 μM, as shown in Table 2. Figure 4 .

[0067] Table 2 Wikstdaphnein A (1) in vitro cell activity test results

[0068]

[0069] Example 3 Apoptosis induction effect of compound 1 on melanoma cells B16

[0070] 1. Experimental materials

[0071] Test substance: Compound 1, HPLC analysis purity greater than 98%.

[0072] Main reagents: PVDF membrane (Shanghai Chuangxiang Biological Technology Co., Ltd.); Phosphatase inhibitor cocktail A 50x, PMSF, RIPA, 10% SDS, Glycine, Tris (Shanghai Biyun Tian Biological Technology Co., Ltd.); BCA protein quantification kit (Shanghai absin company); SDS-PAGE gel preparation kit (Shanghai Yaenzyme Biological Company); Protein loading buffer (Solarbio, USA); skimmed milk powder, BSA (Shanghai Kameng Biological Technology Co., Ltd.); Marker (Thermo, USA); hypersensitive chemiluminescence reagent (Affinity, USA).

[0073] Antibodies: Bcl-2; Bax; Phospho-Akt (Ser473); β-actin; Akt; PI3 Kinase (p85); mTOR; all purchased from Cell Signaling Technology, USA.

[0074] Main instruments: electrophoresis instrument (Bio-rad, USA); Fluorchem FC3 gel imager (Protein Simple, USA); THZ-C constant temperature oscillator (Taicang Experimental Equipment Factory, Jiangsu); ice maker (Changshu Xueke Electrical Appliance Co., Ltd.) and the like.

[0075] 2. Experimental methods

[0076] 2.1 AO / PI staining for detecting cell apoptosis

[0077] B16 cells were inoculated in 6-well plates, and after 24 hours of culture, the culture solution was discarded, and culture solution containing 0, 1.85, 3.70, 14.80, 29.60 μM Wikstdaphnein A was added for incubation for 24 hours. The culture solution was discarded, and the cells were carefully washed twice with PBS, 200 μL of AO and PI each was added for staining, and the cells were incubated at room temperature for 5 minutes in the dark, and then washed with PBS, and observed and photographed under a fluorescence microscope.

[0078] 2.2 Western blotting analysis

[0079] The cells were incubated with 0, 1.85, 3.70, 14.80, 29.60 μM Wikstdaphnein A for 24 hours. The culture solution was discarded, and the cells were carefully washed twice with PBS, 150 μL of RIPA lysis solution containing 1% PMSF and 2% phosphatase inhibitor was added to each well, and the lysis solution was prepared immediately before use. After complete lysis, the supernatant was scraped. The centrifuge was centrifuged at 12000 rpm at 4°C for 20 minutes. The protein concentration was determined by BCA quantification method, and the protein concentration was unified. The protein was denatured by heating at 100°C for 5 minutes.

[0080] According to the different molecular weights of the proteins, 10% or 12% SDS-PAGE separation gel was selected for electrophoresis, and the voltage was adjusted to 80V for 30 minutes and then to 120V. The PVDF membrane with a pore size of 0.22 μM was used for membrane transfer, and the membrane was transferred at a constant current of 220 mA for 110 minutes. After membrane transfer, 5% skimmed milk powder was blocked for 1.5 hours to remove impurities. TBST was washed for 3 times, each for 10 minutes, and the excess milk powder was washed away. According to the different molecular weights, the membrane was cut according to the marker, and the primary antibody was incubated at 4°C overnight. The corresponding secondary antibody was incubated at room temperature for 2 hours, and then washed with TBST for 3 times. The ECL developing solution was used for development.

[0081] 2.3 Statistical method

[0082] Single factor analysis of variance was used for statistics. When the data was normally distributed, and the differences between different groups were the same, ANNOVA parameter analysis was used, and Dunnett's test was used, and p value less than 0.05 was considered statistically significant.

[0083] 3. Experimental results

[0084] 3.1 AO / PI staining

[0085] The results are as follows Figure 5As shown, the cell membrane of the control group cells was complete, and the nucleus was dyed green (as the attached drawings of the specification present gray scale, the same below, which is explained); as the concentration of Wikstdaphnein A increased, the morphology of the cells began to change, the cell membrane began to shrink, and the nuclear chromatin began to show pyknosis. When the concentration of Wikstdaphnein A increased to 14.80 and 29.60 μM, the cell membrane of the B16 cells ruptured, and the nucleus was dyed red, as shown by the yellow arrows in the figure. In summary, Wikstdaphnein A can induce apoptosis of B16 cells, and the dose-effect relationship is obvious.

[0086] 3.2 Annexin V-FITC / PI detection of cell apoptosis

[0087] Wikstdaphnein A on the apoptosis of B16 cells was further determined by Annexin V-FITC / PI staining. As shown in Figure 6, as the concentration of the drug increased, the proportion of apoptotic cells also increased, and when the highest concentration of the drug was 29.60 μM, the proportion of apoptotic cells was 83.68%, which was consistent with the result observed when AO / PI staining showed that most of the cells were in the late stage of apoptosis.

[0088] 3.3 Compound 1 blocks the cell cycle

[0089] To explore whether Wikstdaphnein A can inhibit the proliferation of melanoma cells by blocking the cell cycle of B16 cells, as shown in Figure 7, the drug concentrations were 1.85, 3.70, 14.80, and 29.60 μM, respectively. As the concentration of the drug increased, the number of G0 / G1 phase cells in B16 cells increased significantly compared with the control group, from 49.86% to 64.01%, 71.87%, 76.55%, and 83.60%, while the number of S phase and G2 / M phase cells gradually decreased, indicating that Wikstdaphnein A can inhibit cell proliferation by blocking B16 cells in the G0 / G1 phase.

[0090] 3.4 Compound 1 affects cell migration

[0091] The effect of Wikstdaphnein A on the migration ability of melanoma cells was reflected by the scratch test. As shown in Figure 8, compound 1 can inhibit the migration of melanoma cells B16, and the dose-effect relationship is obvious. Figure 8

[0092] 3.5 Western blotting analysis of apoptosis pathway protein expression

[0093] ​To verify the effect of Wikstdaphnein A on B16 cell apoptosis, the expression of apoptosis-related proteins was detected by Western blotting. The results of Figure 9 show that with the increase of drug concentration, the expression of anti-apoptotic protein Bcl-2 decreases, and the expression of pro-apoptotic protein Bax increases, indicating that Wikstdaphnein A can promote B16 cell apoptosis.

[0094] 3.6PI3K / Akt / mTOR pathway related protein expression

[0095] As shown in Figure 10, the drug concentration of Wikstdaphnein A was 3.70, 7.40 and 14.8 μM, respectively, and the protein expression of p-AkT, p-PI3K and p-mTOR decreased, while the total protein expression of Akt, PI3K and mTOR did not decrease, indicating that Wikstdaphnein A may inhibit B16 cell proliferation through PI3K / Akt / mTOR pathway.

[0096] References:

[0097] [1]LI S, CHOU G, HSEU Y, et al. Isolation of anticancer constituents from flos genkwa (Daphne genkwa Sieb. et Zucc.) through bioassay-guided procedures [J]. Chemistry Central journal, 2013, 7(1): 159.

[0098] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the method of the present application, a number of improvements and supplements can also be made, which should be considered as the protection scope of the present application.

Claims

1. Use of a daphnane diterpenoid compound or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating melanoma, characterized in that, The Wikstdaphnein A has a structural formula as shown in (I).

2. Use according to claim 1, characterized in that, Wikstdaphnein A can obviously inhibit the proliferation and migration of melanoma B16 cells and promote cell apoptosis by inducing the PI3K / Akt / mTOR pathway.

3. A method for preparing a novel diterpene of the daphnane type, characterized in that, The method comprises the following steps: The diterpenoid compound is obtained by extracting the flower bud powder of Daphne fedtschenkoi using ethanol, and then separating and purifying the extract, which comprises the following steps: ① 10 kg of the flower bud powder of Daphne fedtschenkoi is extracted three times with 50 liters of 95% ethanol at room temperature, and 2.5 kg of the crude extract is obtained after removing the solvent; the extract is suspended in 3 liters of water and extracted three times with 3 liters of petroleum ether, 3 liters of ethyl acetate and 3 liters of n-butanol, respectively; the same solvent is combined and concentrated to dryness to obtain three corresponding parts; ② 800 g of the ethyl acetate extract obtained in step ① is subjected to preliminary segmentation using an XDA-7 macroporous resin column, and gradient elution is performed using 30%, 60%, 90%, 95% and 100% methanol-water; similar components are combined to obtain Fr. A-D; Fr.C obtained in step 2 was further fractionated on a MCI column to give two fractions, Fr.CI and Fr.CII, by elution with 50% to 100% gradient of methanol in water; 87 g of Fr.CII was further fractionated on a silica gel column by elution with CHCl3:MeOH from 1:0 to 0:1 by volume to give Fr.CIIa to Fr.CIIg; among them, Fr.CIIe was further fractionated on a Sephadex LH-20 column by elution with CH2Cl2:MeOH = 1:1 by volume to give three sub-fractions, Fr.CIIe1 to Fr.CIIe3; Fr.CIIe1 was further fractionated on a silica gel column by elution with CHCl3:MeOH from 100:1 to 5:1 by volume to give Fr.CIIe1a to Fr.CIIe1c; among them, Fr.CIIe1b was purified by a semi-preparative HPLC column of Eclipse XDB-C18 by using MeOH:H2O = 7.5:2.5 as the mobile phase at a flow rate of 3 mL / min to give 34.2 mg of compound (I) with a retention time of t R 8.5 min. The compound (I) has a structural formula as shown below 。 4. A pharmaceutical composition, characterized by, The diterpenoid compound or a pharmaceutically acceptable salt thereof as claimed in claim 1.

5. The pharmaceutical composition of claim 4, wherein, It further comprises a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5, wherein, The pharmaceutically acceptable carrier is selected from diluents, preservatives, fillers, flow regulators, penetration enhancers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, antibacterial agents, antifungal agents, lubricants and dispersants.

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