Application of durian kernel extract in preparation of anti-cervical cancer medicine and medicine prepared from durian kernel extract

Abstract

The invention discloses application of a durian kernel extract to preparation of an anti-cervical cancer medicine and the medicine, and belongs to the technical field of biological medicines. According to the method, dried durian kernels are used as raw materials, non-active lipid interferents are removed through specific petroleum ether ultrasonic degreasing pretreatment, then methanol ultrasonic extraction is adopted, and high-activity extracts of flavonoids and phenolic acid compounds are obtained. The obtained durian kernel extract (DSE) can effectively inhibit the activity of cervical cancer HeLa cells in vitro, can significantly inhibit the growth of subcutaneous transplantation tumors in nude mice in vivo, and shows good biological safety. In conclusion, the extract has clear anti-cervical cancer activity, and provides candidate resources and research basis for developing novel anti-cervical cancer drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of a durian seed extract in the preparation of an anti-cervical cancer drug and the drug thereof. Background Technology

[0002] Cervical cancer is the fourth most common malignant tumor among women worldwide and a leading cause of cancer death among women in developing countries. According to statistics from the World Health Organization (WHO), more than 600,000 new cases of cervical cancer are diagnosed globally each year, with over 300,000 deaths. The main pathogenesis of cervical cancer is closely related to persistent infection with high-risk human papillomavirus (HPV), especially HPV types 16 and 18. Although the widespread use of preventative HPV vaccines has reduced the incidence rate among young women to some extent, the treatment situation remains challenging for those who have already developed cancer or are ineligible for vaccination.

[0003] Currently, the standard clinical treatment for cervical cancer mainly includes surgical resection, radiotherapy, and chemotherapy. For advanced or recurrent metastatic cervical cancer, combination chemotherapy based on cisplatin, paclitaxel, and fluorouracil is the primary approach. However, these chemically synthesized drugs generally have serious toxic side effects: cisplatin has nephrotoxicity, ototoxicity, and emetogenicity; paclitaxel easily causes bone marrow suppression and peripheral neuropathy. More seriously, with the prolongation of chemotherapy cycles, tumor cells often develop multidrug resistance (MDR), leading to treatment failure. Therefore, the search for novel anticancer drugs with high efficacy, low toxicity, and unique mechanisms of action, especially the discovery of active ingredients from traditional medicinal and edible plants, has become an important strategic direction for antitumor drug development.

[0004] Durian (Durio zibethinus Murr.) is a tropical fruit belonging to the genus Durian in the Malvaceae family, primarily produced in Southeast Asia. Its fruit is rich in various bioactive compounds, such as polyphenols, flavonoids, and oligomeric proanthocyanidins, which possess antioxidant, anti-inflammatory, and antitumor activities. Currently, durian seeds are often considered waste, but studies have shown that their extracts contain high levels of phytochemicals and can be used to treat metabolic syndrome, diabetes, and other diseases. However, the effects of durian seed extract (Durio zibethinus Murr. seed extract, DSE) on cervical cancer HeLa cells remain unclear. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of the prior art and to provide a use of durian seed extract in the preparation of an anti-cervical cancer drug and the drug thereof.

[0006] The specific technical solution adopted in this invention is as follows:

[0007] In a first aspect, the present invention provides the use of durian seed extract in the preparation of an anti-cervical cancer drug.

[0008] Preferably, the purpose is to inhibit the proliferation of human cervical cancer cells or induce apoptosis in human cervical cancer cells.

[0009] Secondly, the present invention provides an anti-cervical cancer drug based on durian seed extract, the drug comprising an effective dose of durian seed extract.

[0010] Preferably, the effective dose of the durian seed extract in the drug is 30-60 mg / kg.

[0011] As a preferred embodiment, the preparation method of the durian seed extract is as follows: petroleum ether is added to the pretreated durian seeds, and ultrasonic extraction is performed at 40-45°C; the petroleum ether extract is separated and the defatted residue is retained; methanol is added to the defatted residue, and ultrasonic extraction is performed at 40-45°C; the methanol extract is separated, the methanol solvent is recovered, and the durian seed extract is obtained.

[0012] Furthermore, the mass ratio of the petroleum ether to the durian seed is (15~20):1.

[0013] Furthermore, the mass ratio of methanol to degreasing residue is (15~20):1.

[0014] Preferably, the drug is in the form of tablets, powders, capsules, oral liquids, or sustained-release formulations.

[0015] Preferably, the drug also includes pharmaceutically acceptable excipients.

[0016] Furthermore, the excipients include one or more of fillers, binders, lubricants, disintegrants, antioxidants, or buffers.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] (1) This invention addresses the problems in existing technologies, such as the serious waste of durian seed resources, the crude extraction process leading to the active ingredients being encapsulated by oil and difficult to dissolve, and the unclear anti-tumor efficacy. It provides an innovative "degreasing-enrichment" coupled extraction process. This process uses dried durian seeds from Thailand as raw materials. Through a specific petroleum ether ultrasonic degreasing pretreatment, inactive lipid interfering substances are removed. Subsequently, methanol ultrasonic extraction is used to obtain highly active extracts of flavonoids and phenolic acids.

[0019] (2) Experimental studies have shown that the obtained durian seed extract (DSE) can effectively inhibit the viability of cervical cancer HeLa cells in vitro and significantly inhibit the growth of subcutaneous xenografts in nude mice in vivo, exhibiting good biocompatibility. Its anti-tumor mechanism is related to the activation of the caspase-3 apoptosis pathway. In summary, this extract has clear anti-cervical cancer activity, providing candidate resources and a research basis for the development of novel anti-cervical cancer drugs. Attached Figure Description

[0020] Figure 1 This is a flowchart illustrating the preparation process of durian seed extract in Example 1;

[0021] Figure 2 This is a comparison of the relative cell viability of different treatment groups in the HeLa cell viability experiment in Example 2;

[0022] Figure 3 The following are the results of the HeLa cell colony formation experiment in Example 2, where (a) is a colony map of different treatment groups; and (b) is a bar chart of the number of colonies in different treatment groups.

[0023] Figure 4 The following are the results of the HeLa cell migration experiment in Example 2, where (a) is a microscopic image of different treatment groups and (b) is a bar chart of cell counts for different treatment groups.

[0024] Figure 5 The results of HeLa cell apoptosis detection in Example 2 are shown, where (a) is a flow cytometry apoptosis graph of different treatment groups; and (b) is a bar chart of apoptotic cell count.

[0025] Figure 6 The results of mitochondrial membrane potential changes in HeLa cells treated with different concentrations of DSE in Example 2;

[0026] Figure 7 This is a graph showing the effect of different concentrations of DSE treatment on the expression level of Cleave-Caspase-3 protein in Example 2;

[0027] Figure 8 Figure (b) shows the changes in tumor size (a) and tumor mass in mice under different treatment groups in Example 3.

[0028] Figure 9 The graph shows the key liver and kidney function indicators of mice in different treatment groups in Example 3: alanine aminotransferase (a), aspartate aminotransferase (b), blood urea nitrogen (c), and creatinine (d).

[0029] Figure 10 The organ indices of the heart (a), liver (b), spleen (c), and kidney (d) of mice in different treatment groups in Example 3 are shown. Detailed Implementation

[0030] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.

[0031] Example 1

[0032] This embodiment provides a method for preparing durian seed extract, the process of which is as follows: Figure 1 As shown, the details are as follows:

[0033] (1) Take 85g of durian seeds from Thailand, crush them to 20 mesh, add 15 times the amount (by weight) of petroleum ether, and extract twice by ultrasonication (40℃) (to remove non-active lipid interfering substances), 1 hour each time. After extraction, separate the petroleum ether extract, recover the petroleum ether solvent, and retain the defatted residue after extraction.

[0034] (2) Add 15 times the amount (by mass) of methanol to the defatted residue and extract twice by ultrasound (40°C) (to extract highly active substances of flavonoids and phenolic acids), 1 hour each time. After extraction, separate the methanol extract, recover the methanol solvent, and obtain 16.82g of durian seed extract (DSE), with an extraction rate of 19.79%.

[0035] Example 2

[0036] This embodiment uses the durian seed extract prepared in Example 1 to conduct an in vitro experiment on the inhibition of cervical cancer, as detailed below:

[0037] (1) HeLa cell resuscitation and culture

[0038] Resuscitated human cervical cancer cells (HeLa) were seeded into DMEM high-glucose medium (containing 10% FBS and 1% penicillin antibiotics) and cultured in an incubator at 37°C and 5% CO2. Cell morphology was observed periodically using an Olympus microscope.

[0039] Once the cells had adhered to the plate at 90%, HeLa cells were digested with 0.25% EDTA and collected, and seeded in 96-well plates at a density of 2000 cells per well.

[0040] (2) HeLa cell viability assay

[0041] Different concentrations (0.16 mg / mL, 0.18 mg / mL, 0.2 mg / mL, and 0.22 mg / mL) of durian seed extract (DSE) were added to 96-well plates seeded with HeLa cells for 12, 24, and 48 hours, respectively. The control group received no durian seed extract. After treatment, the medium was replaced with fresh medium, and cell viability was further calculated by measuring the OD value at 450 nm using the CCK-8 (Cell Counting Kit-8) reagent. The results are shown below. Figure 2 As shown. The half-maximal inhibitory concentration (IC50) was calculated for different treatments. 50 Value. HeLa cells were continuously exposed to DSE for 12, 24, and 48 hours, and the IC50 value... 50 The values ​​were 0.43, 0.28, and 0.22 mg / mL, respectively. This indicates that durian seed extract can inhibit the activity of cervical cancer cells.

[0042] (3) HeLa cell colony formation experiment

[0043] HeLa cells, after being revived and cultured, were seeded into different 12-well plates and divided into low-dose, medium-dose, high-dose, and control groups. The low-dose, medium-dose, and high-dose groups were supplemented with durian seed extract (DSE) at concentrations of 0.1 mg / mL, 0.2 mg / mL, and 0.3 mg / mL, respectively, while the control group received no DSE.

[0044] After thorough mixing, the cervical cancer cell suspensions from each group were incubated for 6–10 days. Following incubation, cells were fixed with paraformaldehyde and stained with crystal violet. Colony images were taken and counted using ImageJ. The results are shown below. Figure 3 As shown.

[0045] Colony formation assays reflect the ability of a single cell to continuously divide and proliferate, forming a colony of progeny cells. These results suggest that DSE not only inhibits cell activity in the short term but also significantly weakens the proliferative potential and clonal origin of HeLa cells in the long term.

[0046] (4) HeLa cell migration experiment

[0047] The inhibitory effect of durian seed extract (DSE) on the in vitro migration ability of HeLa cells was detected using Transwell chambers (Corning, 8.0 μm pore size polycarbonate membrane). Before the experiment, cells in the logarithmic growth phase were starved in serum-free medium for 12 h to synchronize the cell cycle. Cells were digested, collected, and resuspended in serum-free DMEM medium, and the cell density was adjusted to 2 × 10⁶ cells / mL. 5Cells were cultured at concentrations of DSE (0, 0.1, 0.2, and 0.3 mg / mL) and mixed to prepare drug-containing cell suspensions. 200 μL of this cell suspension was seeded into the upper chamber of a Transwell cell culture medium, while 600 μL of complete culture medium containing 20% ​​FBS was added to the lower chamber as a chemokine. The constructed migration system was incubated at 37°C with 5% CO2 for 24 h. Afterward, the chamber was removed, and unmigrated cells on the inner side of the upper chamber membrane were carefully wiped away with a cotton swab. Cells that migrated to the lower membrane surface were fixed with 4% paraformaldehyde at room temperature for 20 min, then stained with 0.1% crystal violet for 20 min. Excess dye was removed by washing with PBS, and the cells were air-dried. Finally, the cells were observed under an inverted microscope, with five fields of view (×200) randomly selected from each well for photographing. The number of migrating cells was counted using ImageJ software. Results are as follows: Figure 4 As shown, with increasing drug concentration, DSE significantly inhibited the migration ability of HeLa cells.

[0048] (5) Detection of HeLa cell apoptosis

[0049] Collect DSE-treated HeLa cell suspensions and resuspend in PBS. Then add Annexin V-APC and PI staining solution, and incubate in the dark for 15 minutes. After adjusting the voltages and compensations of the FSC, SSC, and fluorescence channels on a flow cytometer, analyze the data and plot apoptosis data using FlowJo. Results are as follows: Figure 5 As shown, DSE significantly promoted apoptosis in HeLa cells with increasing concentration.

[0050] (6) Detection experiment of changes in mitochondrial membrane potential in HeLa cells

[0051] HeLa cells were treated with different concentrations of DSE (0.03 mg / mL, 0.06 mg / mL, 0.09 mg / mL, 0.12 mg / mL, and 0.15 mg / mL) for 24 h, followed by incubation with JC-1 working solution at 37°C for 20 min. After washing off the working solution, complete culture medium was added, and cell fluorescence was observed under a microscope. Changes in mitochondrial membrane potential were measured using the fluorescent probe JC-1 after 24 h of DSE treatment with different concentrations. The results are as follows: Figure 6 As shown, when the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix, forming a polymer that produces red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix, and in this case, JC-1 exists as a monomer, producing green fluorescence. Red fluorescence labels live cells that maintain their mitochondrial membrane potential, while green fluorescence labels cells with lower mitochondrial membrane potentials.

[0052] The experimental results showed that as the concentration of DSE increased, the intensity of red fluorescence, representing live cells, gradually decreased, while the corresponding intensity of green fluorescence gradually increased. This demonstrates that durian seed extract (DSE) can induce a decrease in mitochondrial membrane potential in a dose-dependent manner, thereby initiating apoptosis in the mitochondrial pathway.

[0053] (7) Effects of different concentrations of DSE on the expression level of Cleave-Caspase-3 protein

[0054] HeLa cells were treated with different concentrations of DSE (0.1 mg / mL, 0.2 mg / mL, and 0.3 mg / mL), while the control group received no DSE. Cells were thoroughly lysed using RIPA lysis buffer. The cell lysates were centrifuged at high speed, and the supernatant was collected. Protein concentration was then determined using a BCA assay kit. The supernatant was mixed with 5× loading buffer and heated in a metal bath at 100°C for 10 minutes. Protein samples were separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. The PVDF membrane was blocked with milk and then incubated with the target primary antibody and corresponding secondary antibody. After immersion in ultrasensitive ECL luminescent buffer, images were obtained using an imager. The results are as follows: Figure 7 As shown.

[0055] Experimental results showed that as the concentration of DSE treatment increased, the expression level of the pro-apoptotic protein Cleave-Caspase-3 in HeLa cells gradually increased.

[0056] Example 3

[0057] This embodiment uses the durian seed extract prepared in Example 1 to conduct an in vitro experiment on the inhibition of cervical cancer, as detailed below:

[0058] (1) 21 BALB / C-nu nude mice were selected and acclimatized for one week.

[0059] (2) Culture HeLa cells to the logarithmic growth phase, then prepare a HeLa cell suspension and adjust the cell concentration to at least 2 × 10⁻⁶. 7 per mL.

[0060] (3) Inject 100 μL of the above HeLa cell suspension (containing 2×10⁻⁶ cells) into the right axilla of each nude mouse. 6 (Number of cells). Continuous observation was conducted after inoculation, with the appearance of a noticeable tumor in the axilla as the criterion for successful model establishment. Tumor volume V = H × W 2 / 2, where H is the length of the tumor and W is the width of the tumor.

[0061] (4) The durian seed extract (DSE) was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 250 mg / mL. The stock solution was then diluted with 0.5% sodium carboxymethyl cellulose aqueous solution to obtain working solutions with concentrations of 3 mg / mL and 6 mg / mL, respectively.

[0062] (5) When the average tumor volume in nude mice reaches 100 mm 3 They were randomly divided into 3 groups, with 7 animals in each group:

[0063] DSE low-dose group: injected with working solution at a concentration of 3 mg / mL, calculated as 30 mg / kg;

[0064] High-dose DSE group: Injected working solution with a concentration of 6 mg / mL, calculated as 60 mg / kg;

[0065] Control group: Administered an equal volume of 0.5% sodium carboxymethyl cellulose aqueous solution.

[0066] All groups received continuous intraperitoneal injection, and tumor volume was measured and calculated periodically using calipers during the administration period. The maximum tumor volume was determined when it reached 1000 mm². 3 All mice were euthanized by carbon dioxide inhalation. Tumor tissue was isolated and weighed; serum was collected, and heart, liver, spleen, and kidney tissues were also collected and weighed.

[0067] (6) Key liver and kidney function indicators (ALT, AST, BUN, Cr) in serum were measured using a fully automated biochemical analyzer, and organ index was calculated. Organ index = (wet weight of organ / body weight) × 100%.

[0068] Figure 8 This example shows the changes in tumor size (a) and tumor mass (b) in mice from different treatment groups. According to... Figure 8 It can be seen that DSE inhibited the growth rate of HeLa cell tumors over time.

[0069] Figure 9 To display key liver and kidney function data of mice in different treatment groups, Figure 10 It represents the organ indices of the heart, liver, spleen, and kidneys.

[0070] The results showed that, regarding liver function, the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) remained stable in different groups of mice, with no dose-dependent increase observed. Simultaneously, there were no significant changes in liver organ indices. These two results consistently indicate that DSE did not cause significant toxic damage to the liver under experimental conditions.

[0071] Regarding renal function, blood urea nitrogen (BUN) levels remained stable across all groups, indicating that renal metabolic function was not significantly affected. However, the high-dose group (60 mg / kg) showed significantly elevated serum creatinine (Cr) levels and renal organ indices, suggesting that DSE may cause mild renal overload or structural adaptation changes at high doses. However, given the lack of a synchronous increase in BUN, this change may not yet constitute severe functional renal injury. Overall, DSE is highly safe at low doses, with no significant changes in any indicators. While high doses showed mild renal effects, normal BUN levels indicate non-serious damage. Further studies could optimize dosage or adjust the dosing regimen to further control potential risks and ensure a safe window for clinical application.

[0072] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. The use of a durian seed extract in the preparation of an anti-cervical cancer drug.

2. The use of the durian seed extract according to claim 1 in the preparation of an anti-cervical cancer drug, characterized in that, The stated purpose is to inhibit the proliferation of human cervical cancer cells or induce apoptosis in human cervical cancer cells.

3. An anti-cervical cancer drug based on durian seed extract, characterized in that, It contains an effective dose of durian seed extract.

4. The anti-cervical cancer drug based on durian seed extract according to claim 3, characterized in that, The effective dose of durian seed extract in the drug is 30-60 mg / kg.

5. The anti-cervical cancer drug based on durian seed extract according to claim 3, characterized in that, The preparation method of the durian seed extract is as follows: petroleum ether is added to the pretreated durian seeds, and ultrasonic extraction is performed at 40~45℃; the petroleum ether extract is separated and the defatted residue is retained; methanol is added to the defatted residue, and ultrasonic extraction is performed at 40~45℃; the methanol extract is separated, the methanol solvent is recovered, and the durian seed extract is obtained.

6. The anti-cervical cancer drug based on durian seed extract according to claim 5, characterized in that, The mass ratio of petroleum ether to durian kernel is (15~20):

1.

7. The anti-cervical cancer drug based on durian seed extract according to claim 5, characterized in that, The mass ratio of methanol to degreasing residue is (15~20):

1.

8. The anti-cervical cancer drug based on durian seed extract according to claim 3, characterized in that, The drug is in the form of tablets, powders, capsules, oral liquids, or sustained-release formulations.

9. The anti-cervical cancer drug based on durian seed extract according to claim 3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

10. The anti-cervical cancer drug based on durian seed extract according to claim 9, characterized in that, The excipients include one or more of fillers, binders, lubricants, disintegrants, antioxidants, or buffers.

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