CN115850049A - Separation method and application of terpenoid with anti-tumor activity - Google Patents
Separation method and application of terpenoid with anti-tumor activity Download PDFInfo
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Abstract
The invention provides a preparation method and application of an anti-tumor terpenoid, and the anti-tumor terpenoid is separated and purified from tunicates of Halocynthia Roretzi and has small toxicity to normal cells. The compound can inhibit proliferation of various tumor cells, especially hepatocellular carcinoma cell lines. In the culture medium added with the terpenoid, the expression level of hepatocellular carcinoma cell line development promoting genes c-Myc and c-Met is obviously reduced, and the expression level of genes TP53 and KEAP1 inhibiting the development of hepatocellular carcinoma cell lines is obviously increased. The comparison of cytotoxicity with the clinical antitumor drug doxorubicin shows that the terpenoid compound has similar cytotoxicity to hepatocellular carcinoma cell lines with the same additive dose as doxorubicin. The compound is shown to be a terpenoid compound with a novel structure, has a good in-vitro anti-tumor effect and has the possibility of becoming a lead compound of an anti-tumor medicament.
Description
Technical Field
The invention belongs to the field of preparation and application of natural active substances, and particularly relates to a separation method and application of a terpenoid with anti-tumor activity.
Background
With the continuous development of modern biotechnology, people continuously improve and deepen the understanding of marine resources, the utilization of the marine resources is wider, a large number of active substances with natural structures in the sea are gradually discovered, and the natural compounds often have important effects of resisting tumors, microorganisms, viruses, immunoregulation and the like. A large number of marine biological products are expected to become effective and specific good medicines for treating human diseases, and a plurality of natural products can be used as lead compounds of clinical medicines for research. Malignant tumor seriously threatens the life health of human beings.
The effect of the chemical drug therapy which is one of the tumor treatment methods is shown primarily, but for some cancers with higher mortality rate, some chemical drugs still have the problems of great toxic and side effects, low treatment efficiency, easy drug resistance of tumor cells and the like. Therefore, the search for high-efficiency, low-toxicity and high-specificity anti-tumor chemical drugs has great significance and has profound influence on solving the problem of cancer diseases.
Halocynthia roretzi belongs to the phylum chordata (Chordates), the subdivision Urochordata (Urochordata), the class Ascidiacea (Ascidiacea). The constitution of Halocynthia Roretzi can be divided into three parts. The exterior is surrounded by a hard shell, orange-red. The top of the shell is provided with a reproductive root. The interior of the shell is its body tissue, which is orange in color. Previous studies have shown that Halocynthia Roretzi contains not only abundant mineral elements and various rare amino acids, but also a large amount of polyunsaturated fatty acids, and recently there have been reports that Halocynthia Roretzi inner capsule can prevent diabetes. The eusea squirt contains a large amount of symbiotic microorganisms due to the particularity of living environment, and can generate abundant secondary metabolites, but at present, reports on the preparation of natural products with anti-tumor activity by using the eusea squirt as a raw material are few.
Disclosure of Invention
The invention provides a separation method and application of a terpenoid with anti-tumor activity, and the terpenoid has the effect of inhibiting tumor cell proliferation. The terpenoid provided by the invention is similar to the cytotoxicity of broad-spectrum antitumor drug doxorubicin on hepatocellular carcinoma under the same dosage, and may be used as a lead compound of an antitumor drug in the future.
The invention firstly provides a novel anti-tumor terpenoid, which has the following chemical structural formula:
wherein R represents a methyl group (CH) 3 )；
The terpenoid is prepared by extracting tunicate of Halocynthia Roretzi with ethanol solution, and sequentially separating the extractive solution with silica gel column chromatography, silica gel plate and semi-preparative liquid phase;
wherein the ethanol solution is used for extracting, which is to break the tunica vaginalis and then soak the broken tunica vaginalis in 95 percent ethanol to obtain the ethanol extract of the tunica vaginalis;
wherein the silica gel column chromatography uses a normal phase silica gel column filled with 300-400 meshes of normal phase silica gel powder,
wherein, the semi-preparative liquid phase separation is carried out, wherein, the mobile phase system is methanol and pure water, and the conditions of gradient elution are as follows: 0-10min, 20-50% methanol; 10-20min, 50% -70% of methanol; 20-30min, 70% -100% of methanol; 30-45min,100% methanol; 45-50min, and 100-20% of methanol.
In another aspect, the invention also provides an application of the terpenoid in preparing an anti-tumor cell product, wherein the application comprises the following steps:
in still another aspect, the invention also provides the use of the terpenoid in inhibiting tumor cell proliferation;
in yet another aspect, the present invention provides an anti-tumor preparation, which comprises the terpenoid;
the tumor cell is specifically described as a liver cancer cell as an example.
The invention separates and purifies a novel anti-tumor terpenoid compound from tunicates of Halocynthia Roretzi, and the compound has small toxicity to normal cells. The compounds are capable of inhibiting the proliferation of a variety of tumor cells, particularly hepatocellular carcinoma cell lines. In the culture medium added with the terpenoid, the expression level of hepatocellular carcinoma cell line development promoting genes c-Myc and c-Met is obviously reduced, and the expression level of genes TP53 and KEAP1 inhibiting the development of hepatocellular carcinoma cell lines is obviously increased. The comparison of cytotoxicity with the clinical antitumor drug doxorubicin shows that the terpenoid compound has similar cytotoxicity to hepatocellular carcinoma cell lines with the same additive dose as doxorubicin. The compound is shown to be a terpenoid compound with a novel structure, has a good in-vitro anti-tumor effect and has the possibility of becoming a lead compound of an anti-tumor medicament.
Drawings
FIG. 1: photograph of dissecting Halocynthia Roretzi with bar of 1cm;
FIG. 2: nuclear magnetic hydrogen spectra of terpenoids of the invention;
FIG. 3: nuclear magnetic carbon spectrum of the terpenoid of the invention;
FIG. 4: process for preparing terpenoids of the invention 1 H- 1 H COSY spectrogram;
FIG. 5: an HSQC spectrum of the terpenoid of the invention;
FIG. 6: HMBC spectra of the terpenoid of the invention;
FIG. 7: NOESY spectra of terpenoids of the invention;
FIG. 8: high resolution mass spectra of the terpenoids of the invention;
FIG. 9: the terpenoids have proliferation inhibiting effect on different tumor cell lines;
FIG. 10: the terpenoid cytotoxicity is compared with doxorubicin and cytomorphogram, and tumor cell line is HepG-2 cell, p <0.01, p <0.001;
FIG. 11: the expression amount of the genes related to the development of the hepatocellular carcinoma after the action of the terpenoids is shown in a graph, wherein c-Myc and c-Met genes are hepatocellular carcinoma development promoting genes, TP53 and KEAP1 genes are hepatocellular carcinoma development inhibiting genes, and the tumor cell lines are HepG-2 cells, p <0.01, p <0.001 and p <0.05.
Detailed Description
The applicant researches and discovers that the echinacea purpurea bursa ethanol extract has the activity of obviously inhibiting the proliferation of tumor cells, and a novel anti-tumor terpenoid compound is obtained by silica gel column chromatography separation, preparation grade silica gel plate separation and semi-preparation liquid phase separation. The compound can inhibit the proliferation of various tumor cells, obviously change the expression of genes related to the development of hepatocellular carcinoma, and has cytotoxicity on a hepatocellular carcinoma cell line similar to that of a clinical antitumor drug doxorubicin at the same dosage.
The present invention will be described in detail below with reference to examples and the accompanying drawings.
Example 1: preparation of antitumor terpenoid
The preparation method of the provided anti-tumor terpenoid comprises the following steps:
1. preparing a material to be extracted from the Halocynthia Roretzi:
the Halocynthia Roretzi is broken into pieces of 150 mesh by the tunic (figure 1).
2. Preparing ethanol extract of tunicaria auriculata by an alcohol extraction method:
the Halocynthia Roretzi is soaked in 95% ethanol after being broken, and the Halocynthia Roretzi is shaken from time to time during the soaking process, and the soaking time is one week.
3. Separating the ethanol extract of the tunicaria auriculata by silica gel column chromatography:
collecting ethanol extractive solution of tunicatum Haliotidis, and concentrating with rotary evaporator (Tokyo physicochemical evaporator) if the concentration of ethanol extractive solution is low. Mixing the ethanol extract of radix corydalis Bungeanae with 200-300 mesh silica gel. The amount of the substance contained in the ethanol extract used for sample mixing was calculated from the concentration of the weighed ethanol extract, and the mass of the 200-300 mesh silica gel used was about 4 times the amount of the substance contained in the ethanol extract. Mixing the two parts of ethanol extract in 200-300 mesh silica gel, drying, and packing. 300-400 mesh silica gel is loaded on the lowest layer of glass silica gel chromatographic column (Xinweier brand), the height is about 40cm, the stirred ethanol extract sample is above 300-400 mesh silica gel, 60-80 mesh silica gel is used for buffering above the sample, and the height is about 15cm. After the silica gel chromatographic column is well assembled, the chromatographic column is firstly soaked by an elution solvent with the minimum polarity,
gradient elution was performed after the whole column was wetted with the impregnating solution. The elution is carried out by an ethyl acetate-petroleum ether system according to the order of polarity from small to large. Ethyl acetate petroleum ether =1 (800 mL), 1 (3 (400 mL), 1.
4. Separating the eluent obtained by silica gel column chromatography separation by using a preparative silica gel plate:
dipping a sample with cytotoxicity obtained by silica gel column chromatography separation into a capillary, then, dropping the sample on a preparative silica gel chromatography plate, and blowing the sample with a heat drying gun (Delixi electric appliance) while dropping. After spotting, thin layer chromatography was performed under mobile phase conditions of dichloromethane-methanol with mobile phase ratio of dichloromethane to methanol = 30. During chromatography, when the front end of the solution reaches about 2cm of the top of the silica gel chromatography plate, the chromatography plate is taken out and dried by a hot drying gun, each part of sample is cut according to a strip displayed on the plate and then dissolved in methanol, cytotoxicity detection is carried out, and the sample with tumor cytotoxicity is stored at-20 ℃ for later use.
5. Semi-preparative liquid phase separation of samples with tumor cytotoxicity in the above procedure:
after preparing the silica gel plate to obtain the target sample, filtering the target sample by a membrane, and performing semi-preparative liquid phase separation. Chromatographic grade methanol (national medicine) and pure water are used as a mobile phase, and then the proportion of methanol is linearly increased from 20% to 100%. The sample amount of each sample is 200 mu L during preparation, and the samples are manually inoculated according to the peak emergence time of each sample. And then carrying out tumor cytotoxicity detection on each group of compound peaks, wherein the compound peak with tumor cytotoxicity is the anti-tumor terpenoid. The chromatographic column used for preparing the liquid phase is a Kromasil C18 reverse chromatographic column, and the liquid phase model is a Hitachi L-2000 type liquid phase.
Example 2: structural identification of terpenoids
As can be seen from the hydrogen spectrum and HSQC spectrum (fig. 1, fig. 5) of the compound having tumor cytotoxicity prepared in example 1, five methyl groups were contained in the compound, and the methyl group at 2.28ppm may be linked to an unsaturated carbon; two groups of methylene signals also exist in the high field area, and the chemical inequivalence characteristics of the signals indicate that two methylene groups are possibly positioned in a ring structure; in addition, there is a methine hydrogen signal near 3.93ppm and three aromatic or olefinic hydrogen signals in the range of 6.00-8.00ppm in the low field region. The nmr spectrum (fig. 2) shows that the compound has a total of 18 carbons, seven quaternary carbons in addition to the 11 carbons attached to hydrogen described above, with a signal at 199.9ppm indicating the presence of an aldehyde or carbonyl group in the compound. In the COSY spectrum (FIG. 4), the correlation between H-2/3/4 suggests that the compound contains a-CH 2-CH (OH) -CH 2-structural fragment, and the cyclohexane moiety fragment contained in the structure is presumed by combining the correlation signal of the HMBC spectrum (FIG. 6) between CH3-15/CH3-16 and C-1/2/6 and the correlation signal of the HMBC spectrum between CH3-17 and C-4/6. In addition, in the COSY spectrum, the correlation between H-10/11/12 indicates that the structure contains two adjacent double bonds, and the correlation between H-12 and C-10/13 and the correlation between H-14 and C-12/13 in the HMBC spectrum indicate that the methyl C-14 is connected with the unsaturated carbonyl group and is connected with the end of C-12. The correlation of H-10 and C-18 in the HMBC spectrum and the HMBC correlation between H-18 and C-8/9 further improve the side chain structure. In addition to the quaternary carbon of C-8, there is a quaternary carbon signal, eventually suggesting that C-8/9 forms an alkynyl group to link the cyclohexane moiety to the side chain moiety. In the NOESY spectrum (FIG. 7), CH3-18 is associated with the presence of NOE in H-11/14 and between H-10 and H-12, so that the configuration of the double bond between C-9/10 is the E configuration. Terpenoids were prepared and the above conclusions were verified by high resolution mass spectrometry (figure 8). In summary, the planar structure of the present invention is determined.
Example 3: antitumor effect of terpenoids
1. MTT (thiazole blue) colorimetric method for detecting cytotoxicity of terpenoid on different tumor cell lines
The proliferation inhibition rate of terpenoids on tumor cell lines such as HepG-2, BHT-101, hela, mcf-7, sk-Hep-1, bel-7402, etc. was determined by MTT in vitro assay (FIG. 9). When the cells are passaged, a part of cell suspension is used for cell plating, then the cell is plated according to the experimental requirements, the cell is cultured for 24 hours at 37 ℃, old culture solution is sucked off when the density of the cells is about 60 percent, and different samples to be detected are added. After culturing for 48h at 37 ℃, the old culture solution is aspirated, and the prepared MTT dye solution is added. After incubation at 37 ℃ for 4h, the staining solution was aspirated, 100. Mu.L of DMSO was added to each well, and after incubation at 37 ℃ for 5 minutes, absorbance (490 nm) was measured using a microplate reader, and the relative activity of the phase cells was calculated. The result shows that the inhibition effect of the terpenoid on tumor cells is gradually enhanced along with the increase of the action concentration of the terpenoid, and the proliferation inhibition rates of the terpenoid on cells such as HepG2, BHT-101, hela, mcf-7, sk-Hep-1, bel-7402 and the like after the terpenoid is treated for 48 hours at the concentration of 80 mu M are 71.41% + -2.658%, 50.87% + -1.692%, 50.90% + -1.271%, 30.45% + -2.239%, 49.94 + -3.817% and 35.95 + -2.835%, respectively. Comparison of cytotoxicity of terpenoids and doxorubicin results showed that the cytotoxicity of terpenoids at a concentration of 60 μ M on HepG2 cells was significantly higher than that of doxorubicin at 16 μ g/ml (fig. 10).
2. Fluorescent quantitative PCR (polymerase chain reaction) detection of gene expression condition in tumor cells after terpenoid is added
RNA was extracted from HepG-2 cells treated with 60. Mu.M H-VI for 16 hours, using Novozam R223-01
And carrying out reverse transcription on the extracted RNA by using the II Q RT Supermix reverse transcription kit to obtain cDNA, and carrying out qRT-PCR on the cDNA obtained by reverse transcription to serve as a pre-experiment. According to the Ct value obtained in the preliminary experiment, 2. Mu.l c is takenDNA, using dd water to dilute Ct value of cDNA to 20, as optimal cDNA concentration required by different target gene qRT-PCR experiment. qRT-PCR primers were designed using Primer Premier 5.0 software. The primers used in the experiments are shown in table 1.
qRT-PCR Using Novozam ChamQ SYBR Color qPCR Master Mix kit, the PCR amplification pre-denaturation temperature was 95 ℃ for 5min, and 40 cycles (95 ℃ 30s,59 ℃ 30s,72 ℃ 30 s). The melting curve was then determined (95 ℃ 15s,60 ℃ 60s,95 ℃ 15 s). The beta-actin gene is used as a reference gene of qRT-PCR. The-2 delta. Ct method was used for calculating the relative expression amount of the gene, and the control group was normalized.
Table 1: primer sequence table of gene detected by qRT-PCR experiment
According to the qRT-PCR result (figure 11), liver cancer development inhibiting genes TP53 and KEAP1 in HepG-2 cells treated by 40 mu M of the terpenoid for 16 hours are obviously up-regulated, and liver cancer development promoting genes c-Myc and c-Met are obviously down-regulated.
The results show that the antitumor terpenoid compound has low toxicity to normal cells, and can inhibit the proliferation of various tumor cells, especially hepatocellular carcinoma cell lines. In a culture medium added with the terpenoid, the expression level of hepatocellular carcinoma cell line development promoting genes c-Myc and c-Met is obviously reduced, and the expression level of genes TP53 and KEAP1 for inhibiting the development of hepatocellular carcinoma cell lines is obviously increased. The comparison of cytotoxicity with the clinical antitumor drug doxorubicin shows that the terpenoid compound has similar cytotoxicity to hepatocellular carcinoma cell lines with the same additive dose as doxorubicin. The compound is shown to be a terpenoid compound with a novel structure, has a good in-vitro anti-tumor effect and has the possibility of becoming a lead compound of an anti-tumor medicament.
The above are specific embodiments of the present invention, which are only exemplary cases, and the present invention is not limited to the above embodiments. Any equivalent modifications and substitutions to the practice are also within the scope of the invention. Accordingly, any equivalent alterations, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (10)
1. A terpenoid characterized by the following chemical structural formula:
wherein R represents a methyl group.
2. The terpenoid of claim 1, wherein the terpenoid is prepared by extracting tunicates of Halocynthia roretzi with ethanol solution, and separating the extractive solution by silica gel column chromatography, silica gel preparative plate separation and semipreparative liquid phase separation.
3. The terpenoid of claim 2, wherein the ethanol solution is obtained by crushing the tunicates of Halocynthia Roretzi and soaking in 95% ethanol to obtain the ethanol extract of the tunicates of Halocynthia Roretzi.
4. The terpenoid of claim 2, wherein the normal phase silica gel column chromatography is performed using a normal phase silica gel column packed with a normal phase silica gel powder of 300-400 mesh.
5. The terpenoid of claim 2, wherein the semi-preparative liquid phase separation is performed in a mobile phase system of methanol and pure water under the following conditions: 0-10min, 20-50% methanol; 10-20min, 50% -70% of methanol; 20-30min, 70% -100% of methanol; 30-45min,100% methanol; 45-50min, and 100-20% of methanol.
6. Use of the terpenoids and derivatives thereof according to claim 1 for inhibiting tumor cell proliferation.
7. Use of the terpenoids and derivatives thereof according to claim 1 for the preparation of a preparation for inhibiting tumor cell proliferation.
8. The use of claim 6 or 7, wherein the tumor cell is a hepatoma cell.
9. An anti-tumor product, comprising the terpenoid and the derivative thereof according to claim 1.
10. The anti-tumor article of claim 9, wherein the anti-tumor article is an anti-hepatoma cell article.
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| CN104059040A (en) * | 2014-07-08 | 2014-09-24 | 福建师范大学 | Sesquiterpene compounds with antitumor activity and preparation method of sesquiterpene compounds |
| CN104402898A (en) * | 2014-12-18 | 2015-03-11 | 福州大学 | Citrinin compound penicitrinol M derived from Penicillium citrinum, preparation method and application of citrinin compound penicitrinol M |
| CN110642827A (en) * | 2019-09-09 | 2020-01-03 | 广东省微生物研究所（广东省微生物分析检测中心） | Compounds of photosteroids A and B, preparation method thereof and application thereof in preparing antitumor drugs |
| CN111004251A (en) * | 2019-10-31 | 2020-04-14 | 广东省微生物研究所（广东省微生物分析检测中心） | Marine-derived heteroterpene compounds I and II, preparation method and application thereof in preparation of antitumor drugs |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104059040A (en) * | 2014-07-08 | 2014-09-24 | 福建师范大学 | Sesquiterpene compounds with antitumor activity and preparation method of sesquiterpene compounds |
| CN104402898A (en) * | 2014-12-18 | 2015-03-11 | 福州大学 | Citrinin compound penicitrinol M derived from Penicillium citrinum, preparation method and application of citrinin compound penicitrinol M |
| CN110642827A (en) * | 2019-09-09 | 2020-01-03 | 广东省微生物研究所（广东省微生物分析检测中心） | Compounds of photosteroids A and B, preparation method thereof and application thereof in preparing antitumor drugs |
| CN111004251A (en) * | 2019-10-31 | 2020-04-14 | 广东省微生物研究所（广东省微生物分析检测中心） | Marine-derived heteroterpene compounds I and II, preparation method and application thereof in preparation of antitumor drugs |
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