Camellia oleifera seed meal extract, extraction method thereof and application thereof in preparation of anti-colon cancer drugs

Abstract

The application provides a camellia oleifera meal extract, an extraction method thereof and application of the camellia oleifera meal extract in preparation of an anti-colon cancer drug, and relates to the technical field of medicines.The extraction method can effectively regulate active ingredients in the camellia oleifera meal extract by performing heat treatment on the camellia oleifera meal before extraction, so that the sensitivity and inhibitory effect on colon cancer cells are improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to an extract of camellia seed meal, its extraction method, and its application in the preparation of anti-colon cancer drugs. Background Technology

[0002] Colorectal cancer is the third most common malignant tumor worldwide. Poor diet, obesity, excessive alcohol consumption, smoking, and insufficient physical activity all increase the risk of developing colorectal cancer. It is projected that 1.92 million new cases of colorectal cancer will be diagnosed globally by 2040, and this burden is expected to increase further in the coming years. Treatment methods for colorectal cancer have expanded with the accumulation of clinical practice, including chemotherapy, surgery, and radiotherapy. However, current chemotherapy strategies for colorectal cancer have many drawbacks, especially the side effects caused by repeated administration. Increasing research indicates that drugs derived from natural products can be used to treat colorectal cancer in various ways, both in vitro and in vivo, using medicinal plant compounds to treat or prevent colorectal cancer. Therefore, the development of drugs with anti-colorectal cancer effects is urgent and important for the prevention and treatment of colorectal cancer. Summary of the Invention

[0003] The purpose of this invention is to provide a camellia seed oil extract, its extraction method, and its application in the preparation of anti-colon cancer drugs. By heat-treating the camellia seed oil before extraction, the active ingredients in the camellia seed oil extract can be effectively regulated, thereby improving the sensitivity and inhibitory effect on colon cancer cells.

[0004] In a first aspect, the present invention provides an extraction method for camellia seed oil extract used in the preparation of anti-colon cancer drugs, wherein the camellia seed oil is heat-treated at 150-170°C for 5-7 hours before extraction.

[0005] The present invention utilizes heat treatment before extracting camellia seed oil meal to effectively regulate the active ingredients within the meal, thereby obtaining more phenolic substances and organic acids, which can enhance the sensitivity and toxicity to colon cancer cells and effectively improve the inhibitory effect on colon cancer cells.

[0006] Optionally, the method includes the following steps: heat-treating camellia seed meal powder at 150-170℃ for 5-7 hours, extracting it in an extraction reagent and separating the extract, then concentrating it under reduced pressure to obtain an extract; dissolving the extract in chloroform and extracting it multiple times with a saturated sodium bicarbonate aqueous solution, then combining the extracts; acidifying the pH of the extract to 2-3 at 0±2℃, and extracting it multiple times with chloroform, then combining the extracts; concentrating the extract under reduced pressure and drying it to obtain camellia seed meal extract.

[0007] Optionally, when using a saturated sodium bicarbonate solution for multiple extractions, the number of extractions may be 2-5.

[0008] Alternatively, when using chloroform for multiple extractions, the number of extractions may be 2-5.

[0009] Optionally, the extraction reagent comprises at least one of an 80-95 vol.% aqueous ethanol solution and methanol.

[0010] Optionally, when the camellia seed meal powder is heat-treated at 150-170℃, the average particle size of the camellia seed meal powder is 0.40-0.50 mm.

[0011] Optionally, when the camellia seed cake powder is heat-treated at 150-170°C, the passing rate of the camellia seed cake powder through a 40-mesh sieve is 100%.

[0012] Optionally, the process of extracting and separating the extract in the extraction reagent includes: mixing the heat-treated camellia seed meal powder with the extraction reagent at a volume ratio of 1:(15-25), extracting at 20-30℃ for 36-60 hours, and then filtering to separate the extract.

[0013] Optionally, when preparing the extract by vacuum concentration, the extraction reagent is recovered.

[0014] Optionally, chloroform can be recovered when the extract is concentrated under reduced pressure.

[0015] Secondly, the present invention also provides an extract of camellia seed meal extracted using any of the above-mentioned optional extraction methods.

[0016] Thirdly, the present invention also provides the application of camellia seed extract obtained by any of the above-mentioned optional extraction methods in the preparation of anti-colon cancer drugs.

[0017] In fact, the camellia seed extract promotes apoptosis by increasing the ROS content in colon cancer cells.

[0018] In fact, the camellia seed extract promotes apoptosis by reducing SOD activity in colon cancer cells.

[0019] In fact, the camellia seed extract promotes apoptosis by increasing the MDA content in colon cancer cells.

[0020] In fact, the camellia seed extract promotes apoptosis by reducing the mitochondrial membrane potential in colon cancer cells.

[0021] In fact, the camellia seed extract promotes apoptosis by increasing mitochondrial ROS in colon cancer cells.

[0022] Optionally, the colon cancer cells include HCT-116 cells. Attached Figure Description

[0023] Figure 1A schematic flowchart of a method for extracting camellia seed meal extract provided by the present invention;

[0024] Figure 2 The total ion current chromatograms are those of TP-ACC extracted in Example 1 and ACC extracted in Comparative Example 1.

[0025] Figure 3 The toxicity curves of TP-ACC extracted in Example 1 and ACC extracted in Comparative Example 1 on HCT-116 cells at different concentrations are shown.

[0026] Figure 4 The ROS content of HCT-116 cells in different experimental groups after TP-ACC extracted in Example 1 of this invention was applied to different experimental groups.

[0027] Figure 5 The above describes the SOD activity of HCT-116 cells in different experimental groups after TP-ACC extracted in Example 1 of this invention was applied to them.

[0028] Figure 6 The MDA content of HCT-116 cells in different experimental groups after TP-ACC extracted in Example 1 of this invention was applied.

[0029] Figure 7 The mitochondrial membrane potential of HCT-116 cells in different experimental groups after TP-ACC extracted in Example 1 of this invention was applied to different experimental groups.

[0030] Figure 8 The mitochondrial ROS content of HCT-116 cells in different experimental groups after TP-ACC extracted in Example 1 of the present invention was applied to different experimental groups.

[0031] Figure 9 The apoptosis rate of HCT-116 cells in different experimental groups after TP-ACC extracted in Example 1 of this invention was applied. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0033] See Figure 1 This invention provides a method for extracting camellia seed cake extract. By pre-heating the camellia seed cake before extracting the active ingredients, the active ingredients in the camellia seed cake can be effectively controlled, so that the extracted camellia seed cake extract has an anti-colon cancer effect, and thus the camellia seed cake extract can be used to prepare anti-colon cancer drugs.

[0034] Specifically, when heat-treating camellia seed cake, keeping it at 150-170℃ for 5-7 hours can increase the content of phenolic substances and organic acids in the camellia seed cake, thereby making the camellia seed cake extract more sensitive to colon cancer cells and improving its inhibitory effect and toxicity on colon cancer cells.

[0035] In practice, to improve the overall heat treatment effect of camellia seed cake during heat treatment, it can be segmented, for example, by cutting large pieces into smaller blocks with dimensions of 3-5 cm. Specifically, the camellia seed cake used can be a byproduct remaining after cold pressing for tea oil, which not only promotes the reuse of agricultural byproducts but also improves the economic benefits of camellia tree cultivation.

[0036] In fact, after heat treatment of camellia seed cake, in order to improve the extraction efficiency of active ingredients in the camellia seed cake, it can be pulverized to obtain camellia seed cake powder. Specifically, the camellia seed cake can be pulverized into particles with an average particle size of 0.40-0.50 mm using pulverizing equipment, or the pulverized material can be collected after passing through a 40-mesh sieve.

[0037] Similarly, before heat treatment of camellia oil meal, it is also possible to pre-crush the camellia oil meal to obtain camellia oil meal powder, and then heat treat the camellia oil meal powder.

[0038] In some embodiments, the extraction method of camellia seed meal extract includes the following steps: heat-treating camellia seed meal powder at 150-170℃ for 5-7 hours, extracting it in an extraction reagent and separating the extract, and concentrating it under reduced pressure to obtain an extract; dissolving the extract in chloroform and extracting it multiple times with a saturated sodium bicarbonate aqueous solution, and combining the extracts; acidifying the pH of the extract to 2-3 at 0±2℃, and extracting it multiple times with chloroform, and combining the extracts; concentrating the extract under reduced pressure and drying it to obtain camellia seed meal extract.

[0039] In some embodiments, during the extraction process in the extraction reagent, the extraction reagent used includes at least one of 80-95 vol.% aqueous ethanol solution and methanol. By extracting the camellia seed meal powder with alcohol, the active ingredients in it can be transferred into the alcohol solution, thereby completing the extraction.

[0040] In some embodiments, during the extraction process in the extraction reagent, camellia seed meal powder is mixed with the extraction reagent at a volume ratio of 1:(15-25), and extracted at 20-30°C for 36-60 hours, followed by filtration to separate the extract. In practice, during the alcohol extraction process, commonly used methods in the art can be employed to improve the efficiency of the extraction process, such as stirring and mixing.

[0041] In some embodiments, when the extract is concentrated under reduced pressure to obtain an extract paste, the solvent of the extraction reagent present in the extract can be recovered, thereby achieving the purpose of recycling the extraction reagent, reducing costs in the extraction process of camellia seed oil extract and improving environmental friendliness. Similarly, chloroform used can be recovered when the extract is concentrated under reduced pressure. In addition, when performing reduced pressure concentration, commonly used concentration methods in the art, such as rotary evaporation under reduced pressure, can be used to precipitate the dissolved substances in the extract as necessary.

[0042] In some embodiments, when using a saturated sodium bicarbonate solution for multiple extractions, the number of extractions is controlled to be 2-5 times to ensure sufficient extraction of the active ingredients in the chloroform solution. In some embodiments, when using chloroform to extract the acidified extract, the number of extractions is also controlled to be 2-5 times.

[0043] This invention also provides the application of the camellia seed meal extract extracted in any of the above embodiments in the preparation of an anti-colon cancer drug. In fact, the prepared drug can be an oral formulation, and the active ingredient in the prepared anti-colon cancer drug includes at least the camellia seed meal extract extracted according to this invention.

[0044] Example 1

[0045] This embodiment 1 provides a method for extracting camellia seed meal extract, including the following steps:

[0046] S0. After cold pressing camellia (a subtropical evergreen shrub) planted in Wuyuan County, Jiangxi Province, to extract camellia oil, the remaining agricultural by-products—camellia oil meal—are cleaned of rice straw and other debris. The meal is then cut into ping-pong ball-sized pieces, spread evenly on a tray, and baked at 160℃ in air for 6 hours until the surface is golden brown and has a caramel aroma. After naturally cooling to room temperature, the camellia oil meal pieces are pulverized using a grinding mill and passed through a 40-mesh sieve to obtain camellia oil meal powder.

[0047] S1. Take 1 kg of camellia seed meal powder and add it into 95 vol.% ethanol at a volume ratio of 1:20. Soak it at room temperature for 48 h to obtain a mixed dispersion system. After removing the residue by vacuum filtration, the mixed dispersion system is obtained as an extract. After removing the solvent by vacuum rotary distillation, the extract is obtained as an extract.

[0048] S2. Dissolve the extract in chloroform to obtain a dissolved organic phase. Transfer the dissolved organic phase to a separatory funnel and add saturated sodium bicarbonate solution. Extract the dissolved organic phase at a volume ratio of 1:1. Repeat the extraction three times and combine the supernatants to obtain the extract.

[0049] S3. After cooling the extract to 0℃, acidify the extract to pH 2-3 with 6mol / L cold hydrochloric acid. Then, extract the acidified extract with chloroform at a volume ratio of 1:1. Repeat the extraction three times and combine the organic layers to obtain the extract.

[0050] S4. After recovering the solvent by vacuum rotary distillation, a purified paste is obtained. The purified paste is dried in a vacuum drying oven at 50°C until the weight does not change, thus obtaining the camellia seed meal extract (TP-ACC).

[0051] Comparative Example 1

[0052] Comparative Example 1 provides a method for extracting camellia seed meal extract, which differs from Example 1 in that the baking at 160°C is not performed in step S0, and the camellia seed meal extract (ACC) is obtained in step S3.

[0053] Performance testing

[0054] Component analysis: The camellia seed extracts prepared in Example 1 and Comparative Example 1 were dissolved in methanol to prepare the test solutions. After filtration through a 0.22 μm organic filter membrane, the solutions were directly analyzed by UPLC-Q-TOF-MS. The analytes were separated on a C18 column (2.1 × 100 mm, 1.7 mmol / L) at 30 °C. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. The flow rate of the mobile phase was 0.3 mL / min. The gradient scheme was: 0–1 min, 5% mobile phase B; 1–10 min, 5%–40% mobile phase B; 10–14 min… n, 40%-54% mobile phase B; 14-17 min, 54%-60% mobile phase B; 17-27 min, 60%-95% mobile phase B; 27-29 min, 95% mobile phase B; 29-31 min, 95%-5% mobile phase B; 31-36 min, 5% mobile phase B; The mass spectrometer operates in negative ion mode, with an operating range of m / z = 50-1500, source temperature of 120℃, cone voltage of 40V, desolvation gas flow rate of 900L / h, desolvation temperature of 450℃, collector collision energy of 4.0V, and cone gas flow rate of 10L / h; The measurement results are as follows: Figure 2 As shown.

[0055] Cell culture: DMEM and FBS were mixed at a volume ratio of 9:1 to prepare complete cell culture medium. HCT-116 cells were revived from cryopreservation and passaged at logarithmic growth phase. The cells were then transferred to a 37°C incubator with 5% CO2 for incubation. When the cells reached about 80% confluence, the adherent cells were passaged. Following the principle of one-to-three passage, one-third of the cell suspension was aspirated into the culture flask, and 6-8 mL of complete culture medium was added. The flask was gently shaken to distribute the cells evenly at the bottom of the culture dish, and the flask was then incubated in the incubator.

[0056] Cytotoxicity assay: 1 mL (1.0 × 10⁻⁶) 5 HCT-116 cells (number / mL) were seeded in 96-well plates and cultured for 24 h. After adding different concentrations of Camellia oleifera pomace extract before and after heat treatment, the cells were cultured for another 24 h. Then, 20 μL of 5 mg / mL MTT (cytotoxicity assay kit purchased from Beijing Solarbio Science & Technology Co., Ltd.) solution was added to each well, and the cells were incubated for 4 h. The supernatant was removed, and 150 μL of LDMSO solution was added to each well. After shaking in the dark for 10 min, the absorbance was measured at 570 nm. The results are as follows: Figure 3 As shown.

[0057] Experimental Design: N-acetylcysteine ​​(NAC) plays a crucial role in interfering with free radical generation and is currently used as a direct scavenger of reactive oxygen species (ROS), an antioxidant in cancer biology and immuno-oncology, and is commonly used as an antioxidant in tumor cell, immune cell, and preclinical mouse xenograft studies. Oxaliplatin, a platinum-based drug, is widely used in clinical anticancer treatment, especially for colon cancer. This class of drugs can block the growth and proliferation of cancer cells by inducing DNA damage and inhibiting transcription and translation. Therefore, NAC was used as a pretreatment reagent, and oxaliplatin was used as a positive control.

[0058] Experimental determination: 1 mL (1.0 × 10⁻⁶) 5 HCT-116 cells (number / mL) were seeded in 12-well plates and cultured for 24 hours. The following experimental groups were then tested:

[0059] The normal group was named CON group because 1 mL of complete culture medium was added to each cell well.

[0060] In the low-dose group, 1 mL of TP-ACC at a concentration of 100 μg / mL was added to the cell well plate;

[0061] In the medium-dose group, 1 mL of TP-ACC at a concentration of 300 μg / mL was added to the cell well plate;

[0062] In the high-dose group, 1 mL of TP-ACC at a concentration of 900 μg / mL was added to the cell well plate;

[0063] In the pretreatment group, 1 mL of 5 μmol / L NAC was added to the cell well plate for 2 h of pretreatment, followed by 1 mL of complete culture medium for continued culture.

[0064] For the low-dose pretreatment group, 1 mL of 5 μmol / L NAC was added to the cell well plate for 2 h of pretreatment, followed by 1 mL of 100 μg / mL TP-ACC.

[0065] In the pretreatment medium dose group, 1 mL of 5 μmol / L NAC was added to the cell well plate for 2 h of pretreatment, followed by 1 mL of 300 μg / mL TP-ACC.

[0066] For the high-dose pretreatment group, 1 mL of 5 μmol / L NAC was added to the cell well plate for 2 h of pretreatment, followed by 1 mL of 900 μg / mL TP-ACC.

[0067] In the positive control group, 1 mL of 15 μmol / L Oxaliplatin was added to the cell well plate.

[0068] After incubating the cells of the above experimental groups in an incubator for 24 hours, 200 μL of 0.25% trypsin was added to each well. After 2 minutes, culture medium containing fetal bovine serum was added to terminate the digestion of trypsin. The cells were then collected into centrifuge tubes, centrifuged at 1000 rpm for 5 minutes, and the cells were washed twice with PBS to obtain the digested HCT-116 cells. The following measurements were then performed:

[0069] 1. Intracellular ROS Measurement: The DCFH-DA reactive oxygen species detection kit purchased from Nanjing Jiancheng Biotechnology Research Institute was used to measure the ROS in each experimental group. The results are as follows: Figure 4 As shown;

[0070] 2. SOD and MDA assays: Cells in each experimental group were assayed using a detection kit purchased from Nanjing Jiancheng Biotechnology Research Institute. The results are as follows: Figure 5 and 6 As shown;

[0071] 3. Mitochondrial membrane potential measurement: The Rhodamine 123 kit purchased from Nanjing Jiancheng Biotechnology Institute was used to measure the mitochondrial membrane potential of cells in each experimental group. The results are as follows: Figure 7 As shown;

[0072] 4. Mitochondrial ROS Assay: The Mito SOX Red kit purchased from Shanghai Yisheng Biotechnology Co., Ltd. was used to measure the ROS levels in each experimental group of cells. The results are as follows: Figure 8 As shown;

[0073] 5. Apoptosis assay: Cells in each experimental group were stained with FITC and PI dyes using the V-FITC apoptosis detection kit purchased from Beyotime Biotechnology Co., Ltd. The apoptosis rate of each experimental group was then measured using flow cytometry. The results are as follows: Figure 9 As shown.

[0074] Data processing: The experimental data are expressed as mean ± standard error (Mean ± SEM). Differences between groups were analyzed by one-way ANOVA using SPSS 23.0 and plotted using the LSD test at a 5% confidence level. GraphPad Prism 7 was used for plotting.

[0075] Results analysis:

[0076] from Figure 2 As can be seen from the analysis, a total of 23 peaks were separated from the camellia seed extracts prepared in Example 1 and Comparative Example 1. Based on retention time (tR), molecular weight (MW), and molecular formula, a total of 38 components were identified. Among them, 21 components were identified in the TP-ACC prepared in Example 1 and 17 components were identified in the ACC prepared in Comparative Example 1. Meanwhile, the ACC mainly contains organic acids and fatty acids, while TP-ACC contains more phenols and organic acids, such as 4-acetoxyphenol, vanillic acid, and kaempferol, compared to the ACC.

[0077] from Figure 3 It can be seen from this that TP-ACC affects the IC50 of HCT-116 cells. 50 The concentration was 313 μg / mL, while the IC50 of ACC on HCT-116 cells was 313 μg / mL. 50 The concentration was 865 μg / mL, indicating that TP-ACC is more sensitive to HCT-166 cells than ACC, which shows that heat treatment during the extraction of camellia oil meal can effectively enhance the inhibitory effect on HCT-166 cells.

[0078] from Figure 4 The results show that adding different concentrations of TP-ACC can increase the ROS level of HCT-116 cells. After NAC pretreatment, the amount of ROS generated in HCT-116 cells was reduced after adding different concentrations of TP-ACC. The high-dose group showed a significant difference before and after NAC pretreatment, indicating that ROS directly participated in the regulatory role.

[0079] from Figure 5 As can be seen, compared with the control group, NAC pretreatment alone for 2 hours had no significant effect on SOD activity in HCT-116 cells, but the addition of oxaliplatin significantly reduced SOD activity. Simultaneously, the SOD activity of cells in the experimental groups with added TP-ACC was also significantly reduced, indicating that TP-ACC caused oxidative stress damage to HCT-116 cells. After NAC pretreatment for 2 hours followed by TP-ACC treatment, the SOD activity of cells increased, especially with a more significant increase in SOD activity observed with high concentrations of TP-ACC.

[0080] from Figure 6 The results showed that, compared with the control group, NAC pretreatment for 2 hours had no significant effect on the MDA content in HCT-116 cells, while the oxaliplatin group significantly increased the MDA content. Furthermore, TP-ACC significantly increased the content of the lipid peroxidation marker MDA because it promotes cellular oxidative stress, indicating that it caused lipid peroxidation damage to HCT-116 cells. After 2 hours of NAC pretreatment followed by 24 hours of treatment with a high concentration of heat-treated Camellia oleifera meal extract, the MDA level in HCT-116 cells significantly decreased.

[0081] The above results indicate that the anti-colon cancer effect of TP-ACC is related to reducing the activity of the antioxidant enzyme SOD and increasing the level of cellular lipid peroxides, and that this mechanism is related to the regulation of ROS.

[0082] from Figure 7 The results show that, compared with the control group, NAC pretreatment for 2 hours had no significant effect on the mitochondrial membrane potential of HCT-116 cells, while the oxaliplatin group significantly reduced the mitochondrial membrane potential. Meanwhile, all doses of TP-ACC significantly reduced the mitochondrial membrane potential of HCT-116 cells. Pretreatment with NAC for 2 hours followed by treatment with different concentrations of TP-ACC significantly increased the mitochondrial membrane potential of HCT-116 cells, indicating that NAC can mitigate the damage caused by TP-ACC to the mitochondria of HCT-116 cells.

[0083] from Figure 8 As can be seen, compared with the control group, the mitochondrial ROS levels of HCT-116 cells were significantly enhanced after treatment with different concentrations of TP-ACC. Figure 7 and Figure 8 This indicates that TP-ACC causes mitochondrial damage in HCT-116 cells by inducing changes in mitochondrial ROS content and MMP levels.

[0084] from Figure 9 The results showed that, compared with the control group, NAC pretreatment for 2 hours had no significant effect on the apoptosis of HCT-116 cells, while oxaliplatin in the positive control group significantly increased the apoptosis rate of HCT-116 cells. Low, medium, and high doses of TP-ACC significantly increased the apoptosis rate of HCT-116 cells. Furthermore, pretreatment of HCT-116 cells with NAC for 2 hours followed by the addition of medium and high doses of TP-ACC significantly reduced TP-ACC-induced apoptosis in HCT-116 cells, indicating that the pro-apoptotic effect of heat-treated Camellia oleifera seed extract on HCT-116 cells is related to ROS.

[0085] In summary, heat treatment of camellia seed oil meal before extraction effectively reduces the median lethal concentration (LD50) for HCT-116 cells, thus demonstrating excellent anti-colon cancer effects. Simultaneously, TP-ACC significantly increased ROS levels in HCT-116 cells in a dose-dependent manner, significantly decreased SOD activity, and significantly increased MDA levels, thereby promoting oxidative stress in HCT-116 cells. Since pretreatment with NAC can mitigate the damage to the antioxidant defense system of HCT-116 cells caused by TP-ACC to some extent, this further indicates that the mechanism of action of TP-ACC in colon cancer cells is related to ROS.

[0086] Furthermore, TP-ACC induced a decrease in mitochondrial membrane potential and an increase in mitochondrial ROS in HCT-116 cells in a dose-dependent manner, ultimately leading to apoptosis in HCT-116 cells. Pretreatment with NAC could mitigate these effects to some extent. In conclusion, this indicates that TP-ACC exerts its anti-colon cancer effect through a ROS-dependent mechanism and ultimately induces apoptosis in colon cancer cells, thus demonstrating a certain inhibitory effect against colon cancer.

[0087] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A method for extracting camellia seed cake extract for use in anti-colon cancer drugs, characterized in that, Includes the following steps: After heat-treating the camellia seed cake powder at 150-170℃ for 5-7 hours, it was extracted in an extraction reagent and the extract was separated. The extract was concentrated under reduced pressure to obtain an extract paste. The extract paste was dissolved in chloroform and extracted multiple times with saturated sodium bicarbonate aqueous solution. The extracts were then combined to obtain an extract. The pH of the extract was acidified to 2-3 at 0±2℃ and extracted multiple times with chloroform. The organic layers were then combined to obtain an extract. Camellia oleifera meal extract was obtained by concentrating the extract under reduced pressure and then drying it; wherein the extraction reagent was an 80-95 vol% ethanol aqueous solution.

2. The extraction method according to claim 1, characterized in that, When using a saturated sodium bicarbonate solution for multiple extractions, the number of extractions is 2-5 times; and / or, when using chloroform for multiple extractions, the number of extractions is 2-5 times.

3. The extraction method according to claim 1, characterized in that, When camellia seed cake powder is heat-treated at 150-170℃: the average particle size of the camellia seed cake powder is 0.40-0.50mm; and / or, the passing rate of the camellia seed cake powder through a 40-mesh sieve is 100%.

4. The extraction method according to claim 1, characterized in that, The process of extracting and separating the extract in the extracting reagent includes: mixing the heat-treated camellia seed meal powder with the extracting reagent at a volume ratio of 1:(15-25), extracting at 20-30℃ for 36-60 hours, and then filtering to separate the extract.

5. The extraction method according to claim 1, characterized in that, When the extract is obtained by vacuum concentration, the extraction reagent is recovered; and / or, when the extract is concentrated under vacuum, chloroform is recovered.

6. An extract of camellia seed cake obtained by the extraction method according to any one of claims 1 to 5.

7. The use of a camellia seed meal extract obtained by the extraction method according to any one of claims 1 to 5 in the preparation of an anti-colon cancer drug.

8. The application according to claim 7, characterized in that, The camellia seed oil extract promotes apoptosis by increasing ROS levels in colon cancer cells; and / or, the camellia seed oil extract promotes apoptosis by decreasing SOD activity in colon cancer cells; and / or, the camellia seed oil extract promotes apoptosis by increasing MDA levels in colon cancer cells; and / or, the camellia seed oil extract promotes apoptosis by decreasing mitochondrial membrane potential in colon cancer cells; and / or, the camellia seed oil extract promotes apoptosis by increasing mitochondrial ROS levels in colon cancer cells.

9. The application according to claim 8, characterized in that, The colon cancer cells were HCT-116 cells.

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