A terpenoid compound with anti-tumor activity isolated from the tunic of Halocynthia roretzi

Abstract

A novel anti-tumor terpenoid compound was isolated and purified from the tunic of Halocynthia roretzi. This compound can inhibit the proliferation of various tumor cells, especially the hepatocellular carcinoma cell line, and at the same time has less toxicity to normal cells. In the culture medium supplemented with this terpenoid compound, the proliferation of the hepatocellular carcinoma cell line and other types of tumor cell lines is inhibited. And for the hepatocellular carcinoma cell line, with the prolongation of time, the effect of inhibiting its proliferation continues. It shows that this compound is not only a terpenoid compound with a novel structure, but also has good anti-tumor effects in vitro and has the potential to become a leading compound for anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of preparation and application of natural active substances, and particularly relates to a terpene compound with anti-tumor activity isolated from Halocynthia roretzi. Background Art

[0002] With the continuous development of modern biotechnology, the continuous improvement and in-depth understanding of marine resources by people, the utilization of marine resources is more extensive, and a large number of natural-structured active substances in the ocean have been gradually discovered. These natural compounds often have important effects such as anti-tumor, anti-microbial, anti-viral, and immunomodulatory effects. A large number of marine biological products are expected to become effective and specific good medicines for treating human diseases, and many natural products can be used as lead compounds for clinical drugs for research. Malignant tumors seriously threaten human life and health.

[0003] The effectiveness of chemotherapy, one of the tumor treatment methods, has been initially demonstrated. However, for some cancers with relatively high mortality rates, some chemical drugs still have problems such as large toxic side effects, low treatment efficiency, and easy drug resistance of tumor cells. Therefore, it is of great significance to seek anti-tumor chemical drugs with high efficiency, low toxicity, and strong specificity, which has a profound impact on solving cancer diseases.

[0004] Halocynthia roretzi belongs to the phylum Chordata, subphylum Urochordata, and class Ascidiacea. The structure of Halocynthia roretzi can be divided into three parts. It is surrounded by a hard shell on the outside, showing an orange-red color. There is a reproductive root at the top of the shell. Inside the shell is its body tissue, showing an orange color. Previous studies have shown that Halocynthia roretzi not only contains rich mineral elements and various rare amino acids, but also contains a large amount of polyunsaturated fatty acids. In recent years, there have also been reports on the ability of the inner sac of Halocynthia roretzi to prevent diabetes. Due to the special living environment of Halocynthia roretzi, it contains a large number of symbiotic microorganisms and can produce rich secondary metabolites. However, there are few reports on the preparation of natural products with anti-tumor activity using Halocynthia roretzi as a raw material. Summary of the Invention

[0005] The present invention provides a terpene compound with anti-tumor activity isolated from Halocynthia roretzi. The terpene compound provided in the present invention can inhibit the proliferation of multiple tumor cell lines at a lower dose, and at the same time has less toxicity to normal cells, and has the application potential as a lead compound for anti-tumor drugs.

[0006] The present invention first provides an anti-tumor terpene compound isolated from Halocynthia roretzi, and its chemical structural formula is as follows:

[0007]

[0008] wherein R represents a methyl group (CH3);

[0009] The terpenoid compound is prepared by extracting the tunic of Halocynthia roretzi using an ethanol solution or ethyl acetate. The extract is first separated by silica gel column chromatography, and then the fraction eluted with methanol is further separated by a silica gel preparative plate and semi-preparative liquid chromatography.

[0010] The extraction using an ethanol solution or ethyl acetate is carried out by soaking the crushed tunic of Halocynthia roretzi in 95% ethanol or ethyl acetate to obtain the extract of the tunic of Halocynthia roretzi.

[0011] The silica gel column chromatography uses a normal-phase silica gel column filled with normal-phase silica gel powder of 300 - 400 mesh.

[0012] The mobile phase condition for the separation on the silica gel preparative plate is a mixed solution of dichloromethane and methanol, where dichloromethane:methanol = 30:1.

[0013] For the semi-preparative liquid chromatography, the mobile phase system is acetonitrile and pure water, and the gradient elution conditions are as follows: 0 - 10 min, 20% - 50% acetonitrile; 10 - 20 min, 50% - 70% acetonitrile; 20 - 30 min, 70% - 100% acetonitrile; 30 - 45 min, 100% acetonitrile; 45 - 50 min, 100% - 20% acetonitrile.

[0014] Another aspect of the present invention also provides the application of the terpenoid compound in the preparation of products for anti-tumor cells:

[0015] Another aspect of the present invention also provides the application of the terpenoid compound in inhibiting the proliferation of tumor cells;

[0016] Another aspect of the present invention provides an anti-tumor product, which contains the terpenoid compound;

[0017] The tumor cells, as a specific record of an embodiment, are liver cancer cells.

[0018] The present invention isolates and purifies a novel anti-tumor terpenoid compound from the tunic of Halocynthia roretzi. This compound can inhibit the proliferation of various tumor cells, especially hepatocellular carcinoma cell lines, and has relatively low toxicity to normal cells. In the culture medium supplemented with this terpenoid compound, the proliferation of hepatocellular carcinoma cell lines is inhibited, and with the prolongation of time, the inhibitory effect on proliferation persists. It shows that this compound is not only a terpenoid compound with a novel structure but also has good in vitro anti-tumor effects and has the potential to become a leading compound for anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Photograph of the dissection of Halocynthia roretzi, where bar is 1 cm;

[0020] Figure 2 : Separation diagram of the terpenoid compound of the present invention on a preparative silica gel plate;

[0021] Figure 3 : Semi-preparative liquid phase separation diagram of the terpenoid compound of the present invention;

[0022] Figure 4 : Proton nuclear magnetic resonance spectrum diagram of the terpenoid compound of the present invention;

[0023] Figure 5 : Carbon nuclear magnetic resonance spectrum diagram of the terpenoid compound of the present invention;

[0024] Figure 6 : The terpenoid compound of the present invention 1 H- 1 H COSY spectrum diagram;

[0025] Figure 7 : HSQC spectrum diagram of the terpenoid compound of the present invention;

[0026] Figure 8 : HMBC spectrum diagram of the terpenoid compound of the present invention;

[0027] Figure 9 : NOESY spectrum diagram of the terpenoid compound of the present invention;

[0028] Figure 10 : High-resolution mass spectrum diagram of the terpenoid compound of the present invention.

[0029] Figure 11 : Proliferation inhibition effect diagram of the above terpenoid compound on different tumor cell lines;

[0030] Figure 12 : Relationship between the proliferation inhibition of the above terpenoid compound on hepatocellular carcinoma cell line and time. Detailed implementation manners

[0031] The applicant's research found that the extract of the tunic of Halocynthia roretzi has significant activity in inhibiting the proliferation of tumor cells. After silica gel column chromatography separation, preparative silica gel plate separation and semi-preparative liquid phase separation, a novel anti-tumor terpenoid compound was obtained. This compound can inhibit the proliferation of a variety of tumor cells, especially the hepatocellular carcinoma cell line, and the effect of inhibiting the proliferation of the hepatocellular carcinoma cell line continues with the extension of time.

[0032] The present invention will be described in detail below in conjunction with the embodiments and the drawings.

[0033] Example 1: Preparation of anti-tumor terpenoid compound

[0034] The provided anti-tumor terpenoid compound, its preparation method includes the following steps:

[0035] 1. Preparation of materials to be extracted from Halocynthia roretzi:

[0036] Take the tunic of Halocynthia roretzi ( Figure 1 , Tunic) and break it into pieces about 150 mesh size.

[0037] 2. Preparation of the extraction solution of Halocynthia roretzi tunic by alcohol extraction method or ethyl acetate extraction method:

[0038] Soak the broken tunic of Halocynthia roretzi in 95% ethanol or ethyl acetate, shake it from time to time during the soaking process, and the soaking time is one week.

[0039] 3. Separation of the extraction solution of Halocynthia roretzi tunic by silica gel column chromatography:

[0040] Take the extraction solution of Halocynthia roretzi tunic. If the concentration of the extraction solution is low, use a rotary evaporator (Tokyo Rika) to concentrate it. Mix the extraction solution of the tunic with silica gel of 200 - 300 mesh. After mixing the extraction solution dry in the 200 - 300 mesh silica gel, perform dry column packing. Pack silica gel of 300 - 400 mesh at the bottom layer of a glass silica gel chromatography column (Xinweier brand), with a height of about 40 cm. Above the 300 - 400 mesh silica gel is the mixed extraction solution sample, and above the sample is silica gel of 60 - 80 mesh for buffering, with a height of about 15 cm. After the silica gel chromatography column is packed, first moisten the chromatography column with the elution solvent with the least polarity (ethyl acetate: petroleum ether = 1:4).

[0041] After the wetting solution wets the whole column, perform gradient elution. During elution, use an ethyl acetate - petroleum ether system to elute in the order of increasing polarity, ethyl acetate: petroleum ether = 1:4 (800 mL), 1:3 (400 mL), 1:2 (200 mL), 100% methanol (500 mL). After the elution is completed, evaporate the eluate to dryness using a rotary evaporator and weigh it. After weighing, dissolve it in methanol again, and perform tumor cell cytotoxicity detection on the samples eluted with each polarity. Among them, the fraction eluted with methanol has tumor cell cytotoxicity. Prepare the sample and store it at 4°C for later use.

[0042] 4. Separation of the eluate obtained by silica gel column chromatography using a preparative silica gel plate

[0043] Dip the cytotoxic sample obtained by silica gel column chromatography into a capillary and then spot it on a preparative silica gel chromatography plate, and dry it with a hot air gun (Delixi Electric Appliance) while spotting. After spotting is completed, perform thin - layer chromatography. The mobile phase condition is dichloromethane - methanol, and the mobile phase ratio is dichloromethane: methanol = 30:1. During chromatography, when the solution front reaches about 2 cm from the top of the silica gel chromatography plate, take out the chromatography plate and dry it with a hot air gun. According to the color of the bands shown on the plate, cut each part of the sample and dissolve it in methanol, and then perform cytotoxicity detection. Samples with tumor cell cytotoxicity such as Figure 2As shown in the marked part (), it was prepared and stored at -20 °C for later use.

[0044] 5. Semi-preparative liquid separation of the sample with tumor cell toxicity in the above process

[0045] Preparation of silica gel plate After obtaining the target sample, the target sample was filtered through a membrane and then subjected to semi-preparative liquid separation conditions. Using chromatographic grade acetonitrile (Sinopharm) and pure water as the mobile phase, the proportion of acetonitrile was linearly increased from 20% to 100% thereafter. The injection volume for each preparation was 200 μL, and samples were manually collected according to the elution time of each sample. Subsequently, the tumor cell toxicity of each group of compound peaks was detected, and the compound peaks with tumor cell toxicity were the above-mentioned anti-tumor terpenoid compounds. Under the above eluent conditions, this compound was eluted at 27.4 minutes ( Figure 3 circled part). The chromatographic column used for preparative liquid was a Kromasil C18 reverse chromatographic column, and the liquid phase model was Hitachi L-2000 liquid phase.

[0046] Example 2: Structure identification of terpenoid compounds

[0047] Combined with the 1H NMR and HSQC spectra of the compound with tumor cell toxicity prepared in Example 1 ( Figure 4 、 Figure 7 ), it can be seen that there are five methyl groups in this compound, and the methyl group at 2.28 ppm may be connected to an unsaturated carbon; there are also two sets of methylene signals in the high field region, and their chemically non-equivalent characteristics indicate that the two methylene groups may be located in a cyclic structure; in addition, there is a methine hydrogen signal near 3.93 ppm, and there are three aromatic hydrogen or olefinic hydrogen signals in the low field region within the range of 6.00 - 8.00 ppm. The 13C NMR spectrum ( Figure 5 ) shows that this compound has a total of 18 carbons. In addition to the 11 carbons connected to hydrogen mentioned above, there are seven quaternary carbons. The signal at 199.9 ppm indicates the presence of an aldehyde group or a carbonyl group in the compound. In the COSY spectrum ( Figure 6 ), the correlation between H-2 / 3 / 4 indicates that the compound contains a -CH2-CH(OH)-CH2- structural fragment. Combining the HMBC spectrum between CH3-15 / CH3-16 and C-1 / 2 / 6 ( Figure 8Based on the relevant signals of ( ) and the relevant signals of the HMBC spectrum between CH3-17 and C-4 / 6, the cyclohexane partial fragment contained in the structure was deduced. Additionally, in the COSY spectrum, the correlation between H-10 / 11 / 12 indicates the presence of two adjacent double bond structures. Combining the correlations between H-12 and C-10 / 13 and between H-14 and C-12 / 13 in the HMBC spectrum shows that the methyl C-14 is connected to the unsaturated carbonyl group and is connected to the end of C-12. The correlation between H-10 and C-18 in the HMBC spectrum, as well as the HMBC correlations between H-18 and C-8 / 9, further perfect the side chain structure. In addition to the quaternary carbon of C-8, there is also a quaternary carbon signal. Finally, it is speculated that C-8 / 9 form an alkynyl group to connect the cyclohexane part and the side chain part. In the NOESY spectrum ( Figure 9 ), there is a NOE correlation between CH3-18 and H-10 and no correlation with H-11. Therefore, the double bond between C-9 / 10 is in the Z configuration. Based on the NOE correlation between H-10 and H-12 and the large coupling constant (15.6 Hz) between H-11 and H-12, it is speculated that the double bond between C-11 / 12 is in the E configuration. The prepared terpene compound was verified by high-resolution mass spectrometry ( Figure 10 ), and its planar structure was determined.

[0048] Example 3: Anti-tumor cell effect of terpene compounds

[0049] 1. Detection of the cytotoxicity of terpene compounds on different tumor cell lines by CCK-8 method

[0050] The CCK-8 method was used to detect the proliferation inhibition rate ( Figure 11 ) of terpene compounds on tumor cell lines such as HepG-2, BHT-101, Hela, Mcf-7, Sk-Hep-1, Bel-7402, Huh-7 and normal cell line (L929). When subculturing the cells, a part of the cell suspension was used for cell seeding, and then seeded according to the experimental requirements. After culturing at 37 °C for 24 h, when the cell density grew to about 60%, the old culture medium was aspirated, and different test samples were added. After continuing to culture at 37 °C for 48 h, the old culture medium was aspirated, and the prepared CCK-8 staining solution was added. Incubate at 37 °C for 2 h. After incubation, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance (450 nm) and calculate the relative cell viability.

[0051] The results showed that with the increase in the concentration of the terpene compound, its inhibitory effect on tumor cells gradually increased. After treatment with the terpene compound at a concentration of 25 μM for 48 hours, the proliferation inhibition rates of the terpene compound on cells such as HepG2, BHT-101, Hela, Mcf-7, Sk-Hep-1, Bel-7402, and Huh-7 were 74.60% ± 0.8723%, 30.17% ± 1.856%, 49.14% ± 2.523%, 26.41% ± 2.649%, 51.43 ± 2.664%, 56.98 ± 0.5692%, and 37.71 ± 1.638% respectively. After treatment with the terpene compound at a concentration of 25 μM for 48 hours, the proliferation inhibition rate of the terpene compound on the normal cell line (L929) was less than 20%.

[0052] 2. Detection of the inhibitory effect of terpene compounds on the proliferation of hepatocellular carcinoma cell lines over time by CCK-8 method

[0053] The CCK-8 method was used to detect the proliferation inhibition rates of terpene compounds on hepatocellular carcinoma cell lines such as HepG-2, Sk-Hep-1, Bel-7402, and Huh-7 ( Figure 12 ). When subculturing the cells, a part of the cell suspension was used for cell seeding, and then seeded according to the experimental requirements. After culturing at 37 °C for 24 h, when the cell density reached about 60%, the old culture medium was aspirated, and different test samples were added. After continuing to culture at 37 °C for 24 h, 48 h, and 72 h, the old culture medium was aspirated, and the prepared CCK-8 staining solution was added. After incubating at 37 °C for 2 h, after the incubation was completed, the absorbance (450 nm) was measured using a microplate reader, and the relative cell activity was calculated.

[0054] The results showed that with the prolongation of the action time, the inhibitory effect on the proliferation of hepatocellular carcinoma cell lines also continued to increase. After treatment with the above-mentioned terpenoid compounds at a concentration of 20 μM for 24 hours, the proliferation inhibition rates of the above-mentioned terpenoid compounds on cells such as HepG2, Sk-Hep-1, Bel-7402, and Huh-7 were 46.53% ± 1.503%, 9.620% ± 3.606%, 26.89% ± 1.026%, and 19.27% ± 1.415% respectively; after treatment with the above-mentioned terpenoid compounds at a concentration of 20 μM for 48 hours, the proliferation inhibition rates of the above-mentioned terpenoid compounds on cells such as HepG2, Sk-Hep-1, Bel-7402, and Huh-7 were 72.22% ± 0.7576%, 56.10% ± 1.446%, 46.01% ± 2.335%, and 24.85% ± 3.206% respectively; after treatment with the above-mentioned terpenoid compounds at a concentration of 20 μM for 72 hours, the proliferation inhibition rates of the above-mentioned terpenoid compounds on cells such as HepG2, Sk-Hep-1, Bel-7402, and Huh-7 were 79.43% ± 0.5153%, 62.11% ± 2.076%, 63.60% ± 2.330%, and 58.13% ± 0.4278% respectively.

[0055] The above results indicate that the anti-tumor terpenoid compound has low toxicity to normal cells. This compound can inhibit the proliferation of various tumor cells, especially hepatocellular carcinoma cell lines. At the same time, with the prolongation of time, this compound can continuously inhibit the proliferation of hepatocellular carcinoma cell lines. It shows that this compound is not only a terpenoid compound with a novel structure, but also has good in vitro anti-tumor effects and can be used as a lead compound for anti-tumor drugs.

Claims

1. A method for preparing a terpene compound, characterized in that, The chemical structural formula of the terpene compound is as follows: ； wherein R is a methyl group; The terpene compound is prepared by extracting the tunic of Halocynthia roretzi with an ethanol solution or ethyl acetate. The extract is first separated by silica gel column chromatography, and then the eluted fractions are separated by silica gel preparative plates and semi-preparative liquid chromatography.

2. The method for preparing a terpene compound according to claim 1, characterized in that, The ethanol solution is a 95% ethanol solution.

3. The preparation method of the terpene compound according to claim 1, wherein, The silica gel column chromatography is carried out using a normal-phase silica gel column filled with 300-400 mesh normal-phase silica gel powder for separation.

4. The method for preparing the terpene compound according to claim 3, characterized in that, The eluted fraction of the silica gel column chromatography is the fraction eluted with methanol.

5. The method for preparing the terpene compound according to claim 1, wherein, The mobile phase conditions for the silica gel preparative plate separation are a mixed solution of dichloromethane and methanol, wherein dichloromethane:methanol = 30:

1.

6. The preparation method of the terpene compound according to claim 1, wherein, For the semi-preparative liquid chromatography separation, the mobile phase system is acetonitrile and pure water, and the gradient elution conditions are as follows: 0-10 min, acetonitrile 20%-50%; 10-20 min, acetonitrile 50%-70%; 20-30 min, acetonitrile 70%-100%; 30-45 min, 100% acetonitrile; 45-50 min, acetonitrile 100%-20%.

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