A terpenoid compound and its extraction method and application

Abstract

The present invention provides a terpenoid compound. The terpenoid compound provided by the present invention can weaken the anti-apoptotic protein Bcl-2 and reduce the Bcl-2 / Bax ratio. The terpenoid compound provided by the present invention can promote cell apoptosis by down-regulating β-catenin and up-regulating Caspase-3 and TNF-α. The terpenoid compound provided by the present invention is an Nrf2 inhibitor. The present invention also provides an extraction method and application of the terpenoid compound.

Description

Technical Field

[0001] The invention relates to the field of agricultural waste conversion, and in particular to a terpene compound extracted from agricultural waste, and an extraction method and application thereof.

[0002] Background Camellia is an important economic and phylogenetic member of the Theaceae family. More than 300 species of camellia are widely distributed in China, Japan and tropical and subtropical regions of Asia. Several varieties of camellia have been cultivated in China, and many varieties have been cultivated to meet the growing demand, which are used as raw materials for producing popular commercial products, including tea ingredients that promote health, horticulturally gorgeous flowers, and high-quality edible oils. Due to its high nutritional value and economic value, Camellia oleifera is considered to have the highest production value among Camellia oleifera varieties. This small perennial tree has been cultivated and utilized in China for more than 2,000 years and is also one of the four major woody oil plants in the world. In the food and cosmetics industry, rapeseed is mainly used to produce pure natural edible oil. The Food and Agriculture Organization of the United Nations (FAO) recommends it as a high-quality and healthy vegetable oil because it is rich in unsaponifiable components, such as sterols, fatty alcohols and tocopherols. The medicinal value of Camellia oleifera oil is also very important. For a long time, this oil has been used in folk medicine to treat gastrointestinal pain and skin burns. Modern chemical and pharmacological studies have shown that the oil and its main components, including saponins, polyphenols, unsaturated fatty acids, and squalene, have a wide range of biological functions, such as antitumor, antioxidant, anti-inflammatory, antibacterial, antithrombotic, and liver protective activities.

[0003] Camellia oil seed cake (abbreviated as camellia oil cake) is a residual byproduct of the extraction of tea oil from camellia seeds. Although the oil cake accounts for 80% of the total mass of the seeds, after oil extraction, the oil cake is either burned or discarded, which is a huge waste of organic resources. Fully developing the potential value of camellia oil seed cake can not only generate economic value but also prevent air, water or soil pollution. Saponin is the most important component in camellia oil seed cake, accounting for about 15%-20% by weight. More than 30 saponins have been identified from camellia oil seed cake, and new saponins are constantly being isolated and identified, many of which have good cytotoxicity in human cancer cell lines.

[0004] In view of this, in order to make full use of camellia seed cake resources, it is necessary to further identify and separate substances with better anti-cancer effects and promote the recycling of this waste. Summary of the invention

[0005] The first object of the present invention is to provide a terpene compound.

[0006] The second object of the present invention is to provide a method for extracting terpenoids.

[0007] The second object of the present invention is to provide an application of terpenoid compounds.

[0008] To achieve the above purpose, the present invention adopts the following technical means:

[0009] A terpenoid compound, wherein the terpenoid compound has the following structure:

[0010] .

[0011] The method for extracting terpenoid compounds comprises the following steps:

[0012] Grinding camellia seed cake into powder, and refluxing with a first organic solvent I;

[0013] Then, a second organic solvent is used to extract the crude extract;

[0014] The crude extract was eluted with a macroporous resin column and then concentrated under reduced pressure to obtain total saponins;

[0015] After the total saponins are refluxed II and neutralized, they are extracted with ester compounds, and the ester compound layer is taken to obtain aglycone extract;

[0016] The aglycone extract is eluted III and then purified by semi-preparative chromatography to obtain the product.

[0017] The first organic matter includes petroleum ether;

[0018] The reflux time I is 2 hours;

[0019] The extraction was performed three times;

[0020] The second organic matter includes ethanol;

[0021] The macroporous resin includes AB-8;

[0022] The elution I comprises the step of eluting with a NaOH solution;

[0023] The elution I comprises the step of eluting with a methanol aqueous solution.

[0024] The solution used for the neutralization includes NaOH solution.

[0025] The ester compound includes ethyl acetate.

[0026] The elution III comprises the step of eluting with petroleum ether and ethyl acetate in a weight ratio of 30:1-1:1;

[0027] The purification comprises the step of eluting with MeCN aqueous solution;

[0028] The volume ratio of MeCN to water in the MeCN aqueous solution is 70:30.

[0029] The terpenoid compound is used for preparing medicine for treating cancer.

[0030] The drug for treating cancer includes an Nrf2 inhibitor.

[0031] The drug for treating cancer includes a drug that can reduce the expression level of p-STAT3 / STAT3; or

[0032] The cancer treatment drug includes a drug that can weaken the anti-apoptotic protein Bcl-2 and reduce the Bcl-2 / Bax ratio; or

[0033] The cancer treatment drugs include drugs that can downregulate β-catenin and upregulate Caspase-3 and TNF-α.

[0034] Compared with the prior art, the present invention brings the following technical effects:

[0035] The terpenoid compound provided by the present invention can weaken the anti-apoptotic protein Bcl-2 and reduce the Bcl-2 / Bax ratio.

[0036] The terpenoid compound provided by the invention can promote cell apoptosis by down-regulating β-catenin and up-regulating Caspase-3 and TNF-α.

[0037] The terpenoid compound provided by the invention is an Nrf2 inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0039] Figure 1(a) shows the positive ion HR-ESI–MS spectrum of C1.

[0040] Figure 1(b) shows the positive ion HR-ESI–MS spectrum of C1.

[0041] Figure 2 The 1H NMR spectrum of C1 is shown.

[0042] Figure 3 The 13C NMR spectrum of C1 is shown.

[0043] Figure 4 The 1H 1H COSY spectrum of C1 (solvent: CDCl4) is shown.

[0044] Figure 5 The HSQC spectrum of C1 (solvent: CDCl4) is shown.

[0045] Figure 6 The HMBC spectrum of C1 (solvent: CDCl4) is shown.

[0046] Figure 7 The NOESY spectrum of C1 is shown.

[0047] Figure 8 The 1H NMR spectrum of C2 is shown.

[0048] Fig. 9 The 13C NMR spectrum of C2 is shown.

[0049] Fig.10 The 1H 1H COSY spectrum of C2 is shown.

[0050] Fig.11 The HSQC spectrum of C2 is shown.

[0051] Fig.12 The HMBC spectrum of C2 is shown.

[0052] Fig.13 The NOESY spectrum of C2 is shown.

[0053] Fig.14 The 1H NMR spectrum of C3 is shown.

[0054] Fig.15 The 13C NMR spectrum of C3 is shown.

[0055] Fig.16 The 1H 1H COSY spectrum of C3 is shown.

[0056] Fig.17 The HSQC spectrum of C3 is shown.

[0057] Fig.18 The HMBC spectrum of C3 is shown.

[0058] Fig.19 The NOESY spectrum of C3 is shown.

[0059] FIG20( a ) shows the HRESIM spectrum of C4.

[0060] FIG20( b ) shows the HRESIM spectrum of C4.

[0061] Fig.21 The 1H NMR spectrum of C4 is shown.

[0062] Fig. 22 The 13C NMR spectrum of C4 is shown.

[0063] Fig.23 The 1H 1H COSY spectrum of C4 is shown.

[0064] Fig.24 The HSQC spectrum of C4 is shown.

[0065] Fig.25 The HMBC spectrum of C4 is shown.

[0066] Fig.26 The NOESY spectrum of C4 is shown.

[0067] Fig. 27 The figure shows the effect of C3 and 4 on the activity of three typical tumor cell lines. Fig. 27 (A) shows the spectrum obtained by using the calmodulin AM / PI method to detect the cytotoxicity of C3 and 4 in A549 cells; Fig. 27 (B) shows that C3 promotes apoptosis by upregulating Bax, and C4 promotes apoptosis of A549 cells by downregulating p-STAT3 and Bcl-2. Fig. 27 (C) shows the calmodulin AM / PI method used to detect the cytotoxicity of C3 and 4 in U251 cells. Fig. 27 (D) in the middle shows that C3 promotes apoptosis by downregulating p-mTOR and p-STAT3, and C4 promotes apoptosis by downregulating p-STAT3 and Bcl-2 in U251 cells. Fig. 27 (E) shows the calmodulin AM / PI method used to detect the cytotoxicity of C3 and 4 in PAN02 cells. Fig. 27 (F) shows that C3 and 4 promote apoptosis by downregulating p-mTOR, p-STAT3 and Bcl-2 in PAN02 cells.

[0068] Fig.28 The effect of C4 on apoptosis of HepG2 cells was observed by flow cytometry.

[0069] Fig.29 The effect of C4 on the expression of typical proteins in apoptosis-related signaling pathways in HepG2 cells is shown. P < 0.05

[0070] Fig.30 Shown is the LPS-induced expression of C4 on the Nrf2 and NF-κB pathways in HepG2 cells. Fig.30 (A) shows the protein bands; Fig.30 (B) shows Nrf2; Fig.30 (C) shows HO-1; Fig.30 (D) shows NF-κB; Fig.30 (E) shows iNOS; Fig.30 (F) shows COX-2.LPS+3.125 μM, LPS+6.250 μM compared to LPS, *P<0.05.

[0071] Fig.31 Schematic diagram showing the anti-cancer principle of C4. DETAILED DESCRIPTION

[0072] In order to identify and separate new compounds with anti-tumor activity from camellia seed cakes and further develop saponin resources in camellia seed cakes, the present invention separates a variety of terpenoid compounds from camellia seed cakes.

[0073] The present invention studies the extract of tea seed cake. It is found from the cytotoxicity screening test that the acid-treated component has good cytotoxic activity in tumor cell lines compared with the total saponin component. Furthermore, the present invention separates four tea saponin derivatives from the total saponin by combining pre-acid hydrolysis treatment and activity-guided separation, including two new tea saponin derivatives. The specific structure is as follows:

[0074]

[0075] In the following, C1, C2, C3 and C4 are used to represent the above four compounds respectively.

[0076] The present invention further studies the in vitro cytotoxic activities of the above four compounds on five human tumor cell lines, namely Huh-7, HepG2, Hela, A549 and SGC7901, and explains their mechanism of action.

[0077] Specifically, the characterization method used in the present invention is as follows:

[0078] The UV spectra were recorded by a PERSEE UV spectrophotometer T9 (Beijing, China).

[0079] IR spectra were recorded by a Thermo Nicolet Nexus 470 FT-IR spectrometer (Thermo Fisher Scientific, MA, USA).

[0080] NMR spectra were recorded by a Bruker AVANCE III 400 NMR spectrometer (Bruker, Germany) using CDCl 3 as the solvent, while the chemical changes are referred to the residual peak of the solvent.

[0081] HRESIMS data were acquired using an Agilent 6250TOF LC / MS system.

[0082] Silica gel (China 200-300 mesh, Qingdao Ocean Chemical Co., Ltd., China) and AB-8 macroporous adsorption resin (Solarbio Life Science Co., Ltd., Beijing, China) were used for open column chromatography (CC) characterization.

[0083] High performance liquid chromatography analysis was performed using an XBridge Shield RP18 (3.5um 4.6×150mm) high performance liquid chromatography column and a Waters Acouity high performance liquid chromatography system.

[0084] Semi-preparative RP-HPLC was performed on a Waters 2695-2489 system and a GLP-ID (150 mm x 450 mm) preparative column.

[0085] TLC analysis was performed on pre-coated silica gel GF254 plates.

[0086] All solvents were of analytical grade.

[0087] Specifically, the experimental materials used in the present invention are tea oil seeds collected from Shejiang Town, Meizhou City, Guangdong Province, China in 2020. Tea oil seed cakes are produced by squeezing edible tea oil and then used for extraction.

[0088] Specifically, the extraction, acid hydrolysis and separation process of saponin in the present invention is as follows:

[0089] 5 kg of camellia seed cake was ground into powder and refluxed with petroleum ether for 2 hours.

[0090] The defatted seed cake was dried and further extracted three times with 75% ethanol.

[0091] The crude extract was then loaded onto an AB-8 macroporous resin column and eluted with 1% aqueous sodium hydroxide solution and then with sequential mixtures of methanol-water (0%, 10%, 30%, 80%).

[0092] The fraction eluted with 80% methanol was concentrated under reduced pressure to obtain a total saponin fraction (251 g).

[0093] Dissolve in 3N HCl (methanol–H 2 The total saponin solution in the mixture was refluxed for 5 h.

[0094] The reaction mixture was neutralized with 10% aqueous sodium hydroxide solution, and then extracted three times with EtOAc.

[0095] The EtOAc layer was then dried, and a total of 40 g of aglycone extract was obtained from the camellia seed cake.

[0096] The total aglycone extract was loaded onto a silica gel column and eluted with petroleum ether EtOAc (30:1-1:1, gradient system) to generate two fractions, Fr.a (0.8 g) and Fr.B (1.4 g).

[0097] Using MeCN-H 2 Fr.A was further purified by semi-preparative RP-HPLC with 0 (70:30, v / v) mobile phase to obtain C3 (231 mg) and C4 (70 mg).

[0098] Fr.B was separated by semi-preparative RP-HPLC (85% isovolumetric MeCN in H2O) to afford C1 (52 mg) and C2 (234 mg).

[0099] The test methods of the parameters involved in the present invention are as follows:

[0100] Cytotoxicity bioassay

[0101] The cytotoxicity of C1, C2, C3, and C4 was evaluated in five human cancer cell lines: Huh-7, HepG2, Hela, A549, and SGC7901. The viability of cells treated with the isolates was determined by the MTT assay. Three independent experiments were performed for each treatment group.

[0102] Apoptosis detection

[0103] Cells were cultured in 12-well plates with different treatments for 4 h, then washed twice with cold PBS and resuspended in binding buffer at a concentration of 1×106 cells / mL. Next, 100 μL of the resuspended cells were transferred to a 5 mL tube, 5 μL of Annexin V-FITC and 5 μL of PI were added, and the cells were gently swirled. After incubation at room temperature for 15 min, 400 μL of binding buffer was added to each tube, and the cells were analyzed by flow cytometry within 30 min.

[0104] Cell counterstaining

[0105] The cells were cultured in DMEM medium containing 10% FBS and 1% diabody and then washed with 5 mL PBS. Next, 350 mL trypsin was added and the culture flask was quickly transferred to a 5% CO2 chamber at 37 °C. 2 After the cells are rounded, quickly add 2 mL of culture medium to stop digestion. Transfer to a 15 mL centrifuge tube, centrifuge at 1500 RPM for 5 min, and resuspend the cells with fresh culture medium. Dilute the cell suspension to 1 × 10 6 cells / mL, at 37°C and 5% CO 2After 24 h of incubation, the compound was added. After 48 h of incubation, 100 μl of calcein AM / PI was added and incubated for 30 min, followed by image analysis using a high-content cell imaging analysis system. Six transcription factors were selected. GAPDH or actin was used as a reference gene.

[0106] Western blot analysis

[0107] A549 U251 PAN02 and HepG2 cells (1×10 6 Cells) were pre-cultured in 6-cm culture dishes for 24 hours and then starved for 2.5 hours in serum-free medium to eliminate the effects of FBS. Then, cells were treated with MV for 2 hours or not before being exposed to C3 or C4. The collected cells were lysed and the supernatant was boiled for 5 minutes. Protein concentration was determined using a dye-binding protein detection kit (Beyotime Biotechnology Research Institute) according to the manufacturer's manual. Equal amounts of lysed proteins were subjected to 10% SDS-PAGE and electrophoretically transferred to a PVDF membrane (Amershan Pharmacia Biotech, Little Chalfont, UK). After blocking, the membrane was incubated with a specific primary antibody overnight at 4°C and then incubated with an HRP-conjugated secondary antibody for 1 hour (Cell Signaling Technology, Beverly, USA). The bound antibody was detected using an ECL system with a Lumi Vision PRO machine (TAITEC, Chaidamu, Japan). The relative amount of protein associated with a specific antibody was quantified using LumiVision Imager software.

[0108] Calcein AM / PI was incubated for 30 min, and then imaged using a high-content cell imaging system.

[0109] Statistical analysis

[0110] All data were analyzed using SPSS software. All samples were measured three times, and the data were expressed as the mean ± SD of three different experiments. One-way ANOVA was used to measure the statistical differences between the mean values ​​of each experiment. P < 0.05 was considered a significant statistical difference.

[0111] Extraction, Identification and Characterization of Tea Saponin Derivatives from Camellia oleifera

[0112] Considering the chemical structure of saponin, the present invention pre-acid-treated the total saponin part in the camellia seed cake. The screening test results showed that the acid-treated components showed better cytotoxic activity in most cancer cell lines (Table 1). Then, the camellia seed cake was extracted with 75% methanol, eluted through a silica gel column, and finally detected by semi-preparative RP-HPLC. Finally, four main compounds, C1, C2, C3 and C4, were separated and identified. Among them, two new tea saponin derivatives C1 and C4 were found to exist in oil tea for the first time.

[0113] Table 1

[0114]

[0115] In order to verify the accurate chemical structures of C1, C2, C3 and C4, the present invention conducted 1H and 13CNMR analysis on the above compounds. As shown in Figure 1(a) and Figure 1(b), the positive ion HR-ESI–MS spectrum of C1 shows a [M+H]+ peak at M / z 571.4025 (calculated value 571.3999), corresponding to the molecular formula C35H54O6. The infrared spectrum shows the presence of hydroxyl groups (3525cm -1 ) and α,β-unsaturated ester groups (1710 and 1650 cm -1 ). The 1H NMR spectrum of C1 showed a formyl proton at δH 9.37 (1H, s, H-23), an aromatic singlet proton at δH 5.31 (1H, t, J = 3.9 Hz, H-12), two isolated oxygen-containing methylene proton signals at δH 3.64 (1H, d, J = 11.3 Hz, H-28) and three isolated oxygen-containing methoxyl protons at δH 3.38, three oxygen-containing methylene protons at δH 3.75 (1H, dd, J = 11.4, 4.6 Hz, H-3), two isolated oxygen-containing methylene proton signals at δH 5.80 (1H, dd, J = 12.8, 5.4 Hz, H-22), and δH 3.70 (1H, dd, J = 13.9, 5.8 Hz, H-3). 1.05 (3H, s, H-24), 0.98, H-4') and 1.87 (3H, s, H-5')].

[0116] The 13C NMR spectrum showed resonances of 35 carbons, including two carbonyl carbons (δC 207.3 and 169.5), four olefinic carbons (δC 141.4, 139.2, 123.7, and 129.1), four oxygenated carbons (δC 76.6, 73.2, 72.0, and 71.5), eight methyl groups (33.1, 27.3, 24.9, 16.7, 16.0, 14.7, 12.5, and 9.1), eight methylene groups (δC 46.4, 44.1, 38.4, 32.1, 31.8, 26.2, 23.5, and 20.8), and three methyl groups (δC 76.6, 73.2, 72.0, and 71.5) supported by the HSQC results. The data showed that the C1 molecule contained an oleanolic acid structure, which was consistent with other reports (Zhou, Wang, Ye, & Chen, 2014; Zong et al., 2015).

[0117] In the HMBC experiments, the correlations between H-24 and C-3 / C-4 / C-5 / C-23, H-25 and C-1 / C-5 / C-9 / C-10, H-26 and C-7 / C-8 / C-9 / C-14, H-27 and C-8 / C-13 / C-14 / C-15, and H-29 / H-30 and C-19 / C-20 / C-21 further confirmed the presence of the oleanolic acid skeleton. The HMBC correlations between H-1 / H-2 / H-24 and C-3 (δC 72.0), H-18 / H-21 / H-28 and C-22 (δC 76.6), and H-16 / H-18 / H-22 and C-28 (δC 73.2) were used to place the three hydroxyl groups on C-3, C-22, and C-28, respectively. The presence of angelyl substituents was further supported by HMBC correlations between H-3' / H-5' and C-1', H-3' / H-4' / H-5' and C-2', H-4' / H-5' and C-3', and NOESY correlations between H-4' and H-5'. Figure 2 ). Based on the HMBC correlation between H-16 and C-1', the position of the angeloyl group at C-16 can be elucidated. The relative configuration of C1 was determined by NOESY experiments.

[0118] The NMR data of compounds 1-4 are shown in Table 2. The solvent was CDCl3 (δ in ppm, J in Hz, 500 MHz for 1H NMR, 125 MHz for 13C NMR).

[0119] Table 2

[0120]

[0121] Based on the 13C NMR and HRESIMS data of C4 (m / z 859.4821 [m+H]+, calculated value of C47H71O14, 859.4844), the molecular formula of C4 was predicted to be C47H70O14, indicating the presence of 13 hydrogen deficiency indicators. The infrared spectrum of C4 has absorption bands at 1708 and 1646 cm-1, representing carboxyl and α,β-unsaturated esters, and broad bands at 3447 and 1044 cm-1 indicate the presence of glycoside structures (Zhou, Wang, Ye, & Chen, 2014; Zong et al., 2015). The data were further supported by the results of nuclear magnetic resonance spectroscopy (Table 1). Detailed analysis of the 1D and 2D NMR data showed that the structure of C4 was similar to that of compound 1. The significant difference lies in the additional signals of the glycoside structure [δH 4.27 (1H, d, J = 7.5 Hz, Ha), 3.33 (1H, m, Hb), 3.50 (1H and m, Hc), 3.66 (1H and m, Hd), 3.87 (1H, m, He), 3.80 (3H, s, Hf-Me); δc 104.1 (ca), 73.3 (cb), 75.5 (cc), 71.4 (cd), 71.7 (ce), 169.9 (cf), 52.9 (cf-Me) 4 oyl substituents. The glycoside structure is connected to C-3 of the oleanolic acid backbone, as evidenced by the HMBC correlations between Ha and C-3 and H-3 and Ca ( Figure 2 ). According to the HMBC correlations between H-21 and C-1', H-22 and C-1, the two angeloyl groups may be located at C-21 and C-22, respectively. Therefore, the structure of C4 can be obtained.

[0122] C2 and C3 are both white amorphous powders, and their MS and NMR data analysis show that they are all oleanolic acid triterpenoids with a compact tea saponin skeleton. Compared with C1, the NMR spectrum data of compounds 2 and 3 show the presence of angelica alcohol groups [δH 6.93 (1H, qd, J = 7.0, 1.1 Hz, H-3'), 1.82 (3H, dd, J = 9.0, 1.1 Hz, H-4'dd, J = 7.2, 1.4 Hz, H-4') and 1.89 (3H, t, J = 1.4, H-5'), δC 169.0 (C-1'), 127.7 (C-2'), 139.2 (C-3'), 16.0 (C-4'), 20.8 (C-5') for 3]. In compound 2, the HMBC correlation between H-16 and C-1' was used to locate the angelyl substituent at C-16. Combined with the HMBC correlation, compound 2 was identified as camellia safflower A16 hornoside. Similarly, the structure of compound 3 was determined to be camellia safflower A22 hornoside due to the HMBC correlation between H-22 and C-1'. Although C2 and C3 are known compounds that have been characterized previously in some places, their 1H and 13C NMR data are fully published for the first time.

[0123] The application effects of C1, C2, C3 and C4 are described below.

[0124] Antitumor activities of C1, C2, C3, and C4 in typical cancer cell lines

[0125] The present invention uses the MTT method to evaluate the cytotoxicity of all isolates to five human tumor cell lines (human hepatocellular carcinoma cells Huh-7 and HepG2, human cervical cancer cells Hela, human lung cancer cells A549 and human gastric cancer cells SGC7901). Cisplatin (DDP), a widely used anticancer drug, was used as a positive control. As shown in Table 3, all isolated tea saponin derivatives showed significant cytotoxic activity in human tumor cell lines, especially C4. C4 showed strong cytotoxicity with an ED50 range of 1.5 to 11.3 μM, which is comparable to DDP. In the preliminary structure-activity relationship analysis, the additional substitution of the aldehyde group at the C-23 position in compound 1 can increase the cytotoxic activity compared with compound 2, so further structural modification of the tea saponin scaffold C-23 is likely to greatly improve its cytotoxicity.

[0126] Table 3

[0127]

[0128] DDPa was used as a positive control.

[0129] Effects of C3 and C4 on the activity of three typical cancer cell lines

[0130] Taking into account the cytotoxic effects of the tested tea saponin derivatives and the importance of C3 and C4 as potential anti-tumor compounds, the present invention further studied the effects of C3 and C4 on the expression of intracellular survival, proliferation, migration and invasion-related signaling pathway proteins, such as survival JAK2 / STAT3, proliferation inhibitors, proliferation inhibitory proteins, proliferation inhibitory genes, proliferation inhibitory signaling pathway proteins, proliferation-related signaling pathways and invasion inhibitory proteins β-catenin mTOR, apoptosis Bcl-2 / Bax Caspase3, and redox mediator NF-κB / Nrf2 pathways.

[0131] According to the results of calcein AM / PI staining, it can be found that A549, U251 and PAN02 cells in C3 and C4 have significant apoptotic effects. In the three cell models, a large amount of orange fluorescence representing dead cells was observed in the C3 and C4 groups and compared with the control group. The death rate of A549 and PAN02 cells in the C4 group was higher than that in the C3 group. However, in U251 cells, the cell death rate in the C3 group was higher than that in the C4 group. These results show that C3 and C4 significantly inhibit the proliferation of cancer cells, and C4 is more effective than C3.

[0132] Fig. 27 The expression of C3 and C4 in three apoptosis-related proteins is shown. C3 and C4 promote apoptosis by regulating different proteins in these cells. C3 is expressed by upregulating Bax expression, and C4 is expressed by downregulating p-STAT3 and Bcl-2 in A549 cells. C3 is expressed by downregulating p-mTOR and p-STAT3 in U251 cells, and C4 is expressed by downregulating p-STAT3 and Bcl-2. Compounds 3 and 4 downregulate p-mTOR, p-STAT3, and Bcl-2 in PAN02 cells. The results show that both C3 and C4 have obvious pro-apoptotic effects. At the same time, the effect of C4 is better than that of C3. Compared with control cells, C4 significantly reduces the expression level of p-STAT3 / STAT3 in A549, U251, and PAN02 cells. At the same time, it can also weaken the anti-apoptotic protein Bcl-2 and reduce the Bcl-2 / Bax ratio.

[0133] Effect of C4 on apoptosis of HepG2 cells

[0134] C4 was chosen to confirm its antitumor effect because C4 is more pro-apoptotic than C3. According to the results of flow cytometry, the apoptosis rate of C4 was definitely enhanced in a dose-dependent manner. In order to obtain more accurate molecular evidence of the antitumor activity of C4, typical apoptosis-related proteins were detected in HepG2 cells. Figure 4The results in the Figure 4 showed that the expression of Caspase-3 and TNF-α decreased significantly compared with the blank group to promote cell apoptosis. At the same time, the expression of β-catenin was significantly enhanced compared with the blank group to promote cell apoptosis. However, there was no significant change in JAK2. These results indicate that C4 promotes cell apoptosis by downregulating β-catenin and upregulating Caspase-3 and TNF-α.

[0135] Effects of C4 on redox-related Nrf2 and NF-κB pathways in LPS-induced HepG2 cells

[0136] In order to clarify the molecular mechanism of C4 anti-tumor effect, typical apoptosis-related proteins were detected in LPS-induced HepG2 cells. Nrf2 and its downstream protein HO-1 were detected. Inflammatory factors NF-κB, iNOS and COX-2 were detected. Figure 5 As shown, the LPS group increased significantly compared with the blank group. This indicates that the inflammation model has been successfully established. Figure 5 As shown in the figure, the expression of Nrf2 and HO-1 in the 3.125 and 6.250 groups was significantly decreased compared with the LPS group. Therefore, C4 is a suitable Nrf2 inhibitor that can enable anti-tumor drugs to exert synergistic effects in cancer cells. The expression of NF-κB, iNOS, and COX-2 was significantly enhanced in the 6.250 group, and the expression in the 3.125 group was enhanced but not significantly. Based on these, C4 may be a suitable inflammatory inducer that can be used to regulate proliferation.

[0137] In summary, the above results indicate that C4 can trigger apoptosis through the Bcl-2 / Caspase 3 and JAK2 / STAT3 pathways and stimulate cell proliferation through the NF-κB / iNOS / COX-2 pathway. More importantly, C4 can enhance the resistance to anticancer drugs by inhibiting the Nrf2 / HO-1 pathway.

[0138] In the present invention, four cytotoxic tea saponin derivatives, including two novel ones (C1 and C4), are provided, which are extracted from camellia seed cake by combining pre-acid hydrolysis treatment and activity-oriented separation. Meanwhile, molecular evidence shows that C4 has excellent anti-tumor ability, can significantly inhibit inflammatory pathways and targets, and regulate intracellular REDOX balance.

[0139] In the present invention, the anti-tumor activity and molecular mechanism of C4 were studied, and it was found that C4 exerted its powerful anti-tumor performance by inducing apoptosis pathway and inhibiting Nrf2 / HO-1 pathway.

[0140] The development of cancer is closely related to inflammation. For example, inflammation-related DNA damage in cancer stem-like cells leads to the development of cancers with aggressive clinical features. Some cancers are preceded by an inflammatory response in the body. However, apoptosis of cancer cells can induce inflammatory pathways, stimulate cell proliferation, and prevent cell death. It can be seen that C4 can significantly stimulate the NF-kB inflammatory pathway and its targets, and inhibit the Nrf2 / HO-1 pathway to regulate the intracellular REDOX balance, indicating that C4 can be developed into a drug for the treatment of cancer. The present invention compared C3 and found that C3 also has anti-tumor activity, but not as effective as C4. This shows that glycoside ligands are an important source of C4's anti-cancer activity.

[0141] At the same time, C4 can reduce the activity of Nrf2, and the effect is significant. This shows that C4 is an Nrf2 inhibitor, which can enhance the sensitivity of cancer cells to anticancer drugs and improve the efficacy.

Claims

1. A method for extracting terpenoid compounds, characterized in that: The steps include: Grind the camellia seed cake into powder and reflux with petroleum ether I; Then, the crude extract was extracted using ethanol; The crude extract was eluted with AB-8 macroporous resin column and then concentrated under reduced pressure to obtain total saponins; After the total saponins are refluxed II and neutralized, they are extracted with ester compounds, and the ester compound layer is taken to obtain aglycone extract; The aglycone extract was eluted III and then purified by semi-preparative chromatography to obtain terpenoids; The elution III comprises the step of eluting with petroleum ether and ethyl acetate in a weight ratio of 30:1-1:1; The purification comprises the step of eluting with MeCN aqueous solution; The structural formula of the terpenoid compound is as follows: 。 2. The method for extracting terpenoids according to claim 1, wherein: The reflux time I was 2 hours.

3. The method for extracting terpenoids according to claim 1, wherein: The extraction was performed three times; The elution I comprises the step of eluting with a NaOH solution; The elution I comprises the step of eluting with a methanol aqueous solution.

4. The method for extracting terpenoids according to claim 1, wherein: The solution used for the neutralization includes a NaOH solution; The ester compound includes ethyl acetate.

5. The method for extracting terpenoids according to claim 1, characterized in that: The volume ratio of MeCN to water in the MeCN aqueous solution is 70:30.

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