A pharmaceutical composition for treating cervical cancer and a preparation method thereof

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a pharmaceutical composition for treating cervical cancer and a preparation method thereof. The pharmaceutical composition for treating cervical cancer comprises sclareolide and polyporene, and the mass ratio of the sclareolide to the polyporene is 1:0.3-0.5. The present application finds through cervical cancer cell inhibition experiments that the sclareolide and the polyporene can synergistically inhibit the proliferation and activity of cervical cancer cells. Furthermore, it is found through the construction of a tumor transplantation model that the pharmaceutical composition of the present application can inhibit the growth of tumors, down-regulate the expression of RIPK1 and HPV E6 proteins, synergistically play an anti-cervical cancer role, and delay the progression of cervical cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a pharmaceutical composition for treating cervical cancer and its preparation method. Background Technology

[0002] Cervical cancer is the fourth most common malignant tumor among women worldwide and one of the malignant tumors with high incidence and mortality rates in the female reproductive system. Persistent infection with high-risk human papillomavirus (HPV) is the main contributing factor, seriously threatening the health and quality of life of women globally. HPV is a DNA virus that commonly invades human skin and mucous membranes, with HPV-16 and HPV-18 posing the highest cancer risk. Most HPV infections can be cleared by the body's immune system; however, if immunity is insufficient and persistent infection occurs, it can cause abnormal lesions in cervical epithelial cells, eventually progressing to cervical cancer.

[0003] Currently, clinical treatment primarily involves surgery, radiotherapy, chemotherapy, and traditional chemotherapy and immunotherapy. Surgery is mainly suitable for early-stage cervical cancer patients, removing lesions through procedures such as hysterectomy and cervical conization. However, recurrence is common post-surgery, and it can cause irreversible damage to the patient's reproductive function. Radiotherapy and chemotherapy are the main treatments for mid-to-late-stage cervical cancer. Commonly used chemotherapy drugs include platinum-based drugs such as cisplatin, carboplatin, and paclitaxel, as well as paclitaxel-based drugs. However, these drugs have significant toxic side effects, such as bone marrow suppression, gastrointestinal reactions, and liver and kidney damage, and long-term use can easily lead to drug resistance in tumor cells.

[0004] In recent years, natural plant extracts have become a research hotspot in anti-tumor drugs due to their unique advantages such as high safety and low toxicity. Currently, there are no reports on the use of a combination of perilla lactone and *Cibotium barometz* polysaccharide in the treatment of cervical cancer, nor are there studies on its anti-cancer mechanism. During our research on the molecular mechanisms of cervical cancer pathogenesis in our project (Project No.: 2025AHGXZK31376), we discovered that RNF85-mediated RIPK1 ubiquitination modification and abnormal activation of the IKK / NF-κB signaling pathway are closely related to the occurrence and development of cervical cancer. This discovery provides new research ideas and theoretical basis for the discovery of targeted therapeutic targets and clinical intervention for cervical cancer. Therefore, this invention provides a pharmaceutical composition for the treatment of cervical cancer and its preparation method. Summary of the Invention

[0005] The primary objective of this invention is to provide a pharmaceutical composition for treating cervical cancer; this composition has a significant anti-cervical cancer effect and few toxic side effects.

[0006] The second objective of this invention is to provide a method for preparing a pharmaceutical composition for treating cervical cancer, which is simple in process, convenient in operation, low in cost, and suitable for industrial production.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A pharmaceutical composition for treating cervical cancer, the composition comprising perillaldehyde and cane liver polysaccharide; wherein the mass ratio of perillaldehyde to cane liver polysaccharide in the composition is 1:(0.3-0.5).

[0008] Furthermore, the preparation of the *Cibotium barometz* polysaccharide includes the following steps: (1) Remove impurities from the whole plant of the dog liver vegetable, crush it, add water for extraction, filter to obtain the extract, repeat the extraction, combine the extracts, concentrate to obtain the concentrate; (2) Add anhydrous ethanol to the concentrate for alcohol precipitation, centrifuge to collect the precipitate, add the precipitate to water to obtain the precipitate solution; then add n-butanol-chloroform solution, shake, centrifuge, take the supernatant, and after dialysis and drying, obtain the cane liver polysaccharide.

[0009] Furthermore, in step (1), the amount of water added is 10-15 times the mass of the whole plant of *Gnaphalium affine*.

[0010] Furthermore, in step (1), the extraction temperature is 80-90℃ and the time is 1.5-2.5h.

[0011] Further, in step (1), the extraction is repeated 2-3 times; the concentrate is concentrated to 30-50% of its original volume.

[0012] Further, in step (2), the amount of water added is 3-5 times the mass of the precipitate; after adding anhydrous ethanol, the alcohol content of the concentrate reaches 70 vol%-80 vol%; the alcohol precipitation temperature is 0-4℃ and the time is 10-12h.

[0013] Further, in step (2), the volume ratio of n-butanol to chloroform in the n-butanol-chloroform solution is 1:(4-6); the volume ratio of the n-butanol-chloroform solution to the precipitate solution is 1:(3-5).

[0014] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0015] A method for preparing a pharmaceutical composition for treating cervical cancer includes the following steps: Take perilla lactone and cane liver polysaccharide according to the above formula, mix them evenly, and add pharmaceutically acceptable excipients to obtain a drug composition for treating cervical cancer.

[0016] Compared with the prior art, the main advantages of the present invention are as follows: 1. This invention provides a pharmaceutical composition for treating cervical cancer. Cervical cancer cell inhibition experiments revealed that perilla lactone and *Cibotium barometz* polysaccharide synergistically inhibit the proliferation and activity of cervical cancer cells. Furthermore, through the construction of a xenograft model, it was found that the pharmaceutical composition of this invention can inhibit tumor growth, downregulate the expression of RIPK1 and HPV E6 proteins, synergistically exert anti-cervical cancer effects, and delay the progression of cervical cancer.

[0017] 2. The perilla lactone of this invention is derived from the natural plant perilla, and the cibotium barometz polysaccharide is a naturally sourced polysaccharide component. Both components are safe and non-toxic and will not affect the growth of normal cells, providing a safe and efficient natural drug composition for the treatment of cervical cancer. Attached Figure Description

[0018] Figure 1 The diagram shows the safety test results of the pharmaceutical composition of the present invention. Figure 2 The figure shows the results of RIPK1 and HPV E6 protein expression in nude mice with xenografts in each group of the present invention. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0020] Example 1 A pharmaceutical composition for treating cervical cancer, the composition comprising perillaldehyde and cane liver polysaccharide; wherein the mass ratio of perillaldehyde to cane liver polysaccharide is 1:0.3.

[0021] The preparation of the *Cibotium barometz* polysaccharide includes the following steps: (1) Remove impurities from the whole plant of *Gnaphalium affine*, crush it, pass it through a 50-mesh sieve, add deionized water, the amount of deionized water added is 12 times the mass of the whole plant of *Gnaphalium affine*; then extract it with water at 85℃ for 2 hours; filter to obtain the extract, repeat the extraction 3 times, combine the extracts and concentrate them to 40% of the original volume to obtain the concentrate. (2) Add anhydrous ethanol to the concentrate to make the alcohol content of the concentrate reach 80 vol%, and precipitate at 0℃ for 12 h; then centrifuge to collect the precipitate, dissolve the precipitate in deionized water, and add water in an amount that is 4 times the mass of the precipitate; obtain the precipitate solution; then add n-butanol-chloroform solution (the volume ratio of n-butanol to chloroform is 1:4) and shake for 25 min, wherein the volume ratio of the n-butanol-chloroform solution to the precipitate solution is 1:5, centrifuge, take the supernatant, dialyze and freeze dry to obtain the cane liver polysaccharide.

[0022] A method for preparing a pharmaceutical composition for treating cervical cancer includes the following steps: Take perilla lactone and cane liver polysaccharide according to the above formula ratio, mix them evenly, and add pharmaceutically acceptable excipients to obtain a drug composition for treating cervical cancer.

[0023] Example 2 A pharmaceutical composition for treating cervical cancer, the composition comprising perillaldehyde and cane liver polysaccharide; wherein the mass ratio of perillaldehyde to cane liver polysaccharide is 1:0.4.

[0024] The preparation of the *Cibotium barometz* polysaccharide includes the following steps: (1) Remove impurities from the whole plant of *Gnaphalium affine*, crush it, pass it through a 50-mesh sieve, add deionized water, the amount of deionized water added is 10 times the mass of the whole plant of *Gnaphalium affine*; then extract it with water at 90℃ for 1.5h; filter to obtain the extract, repeat the extraction 3 times, combine the extracts and concentrate them to 30% of the original volume to obtain the concentrate. (2) Add anhydrous ethanol to the concentrate to make the alcohol content of the concentrate reach 70 vol%, and precipitate at 0℃ for 10 h; then centrifuge to collect the precipitate, dissolve the precipitate in deionized water, and add water in an amount three times the mass of the precipitate; obtain the precipitate solution; then add n-butanol-chloroform solution (the volume ratio of n-butanol to chloroform is 1:4) and shake for 20 min, wherein the volume ratio of the n-butanol-chloroform solution to the precipitate solution is 1:3, centrifuge, take the supernatant, dialyze and freeze dry to obtain the *Gnaphalium affine* polysaccharide.

[0025] A method for preparing a pharmaceutical composition for treating cervical cancer includes the following steps: Take perilla lactone and cane liver polysaccharide according to the above formula ratio, mix them evenly, and add pharmaceutically acceptable excipients to obtain a drug composition for treating cervical cancer.

[0026] Example 3 A pharmaceutical composition for treating cervical cancer, the composition comprising perillaldehyde and cane liver polysaccharide; wherein the mass ratio of perillaldehyde to cane liver polysaccharide is 1:0.5.

[0027] The preparation of the *Cibotium barometz* polysaccharide includes the following steps: (1) Remove impurities from the whole plant of *Gnaphalium affine*, crush it, pass it through a 50-mesh sieve, add deionized water, the amount of deionized water added is 15 times the mass of the whole plant of *Gnaphalium affine*; then extract it with water at 90℃ for 1.5h; filter to obtain the extract, repeat the extraction 3 times, combine the extracts and concentrate them to 50% of the original volume to obtain the concentrate. (2) Add anhydrous ethanol to the concentrate to make the alcohol content of the concentrate reach 80 vol%, and precipitate at 4℃ for 12 h; then centrifuge to collect the precipitate, dissolve the precipitate in deionized water, and add water in an amount 5 times the mass of the precipitate; obtain the precipitate solution; then add n-butanol-chloroform solution (the volume ratio of n-butanol to chloroform is 1:4) and shake for 30 min, wherein the volume ratio of the n-butanol-chloroform solution to the precipitate solution is 1:5, centrifuge, take the supernatant, dialyze and freeze dry to obtain the cane liver polysaccharide.

[0028] A method for preparing a pharmaceutical composition for treating cervical cancer includes the following steps: Take perilla lactone and cane liver polysaccharide according to the above formula ratio, mix them evenly, and add pharmaceutically acceptable excipients to obtain a drug composition for treating cervical cancer.

[0029] Comparative Example 1 Comparative Example 1 provides a pharmaceutical composition for treating cervical cancer, comprising only one component: canis liver polysaccharide; the rest is consistent with Example 1.

[0030] Comparative Example 2 Comparative Example 2 provides a pharmaceutical composition for treating cervical cancer, comprising only the component: perilla lactone; otherwise consistent with Example 1.

[0031] Experimental Example 1: In vitro cytotoxicity experiment L929 mouse fibroblasts in logarithmic growth phase were used as the cell line. L929 cells were digested with 0.25% trypsin and resuspended in MEM medium containing 10% fetal bovine serum. Cells were then cultured at 0.5 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 μg / mL into 96-well plates and incubated at 37°C for 24 h. The original culture medium was then discarded, and the following media were added: experimental group culture medium containing 100 μg / mL of the drug composition from Example 1 (perilla lactone: *Cynodonta scabra* polysaccharide = 1:0.3); negative control group culture medium containing 10% fetal bovine serum (MEM); and positive control group culture medium containing 10% DMSO (DMEM complete medium). Cells were then incubated at 37°C for 24 h. Cell morphological changes were then observed under a microscope and photographed. The results are shown below. Figure 1 .

[0032] Depend on Figure 1As can be seen, the cells in the positive control group were shrunken and round, showing a toxic reaction, while the cells in the experimental group and the normal control group had similar morphology, indicating that the combined use of perilla lactone and cane liver polysaccharide had no significant effect on normal cells and had good safety.

[0033] Experimental Example 2: Pharmacodynamic Experiment of Drug Composition on Human Cervical Cancer Cells SiHa This experiment used human cervical cancer cells (SiHa) as experimental cells to investigate the inhibitory effects of different ratios of perilla lactone and cane liver polysaccharide on cell proliferation.

[0034] 2.1 The perilla lactone obtained in this invention was dissolved in medical dimethyl sulfoxide (DMSO) and diluted with DMEM medium to prepare perilla lactone solutions of 25 µg / mL, 50 µg / mL, 100 µg / mL, and 200 µg / mL for later use. Human cervical cancer cells (SiHa) in the logarithmic growth phase were taken and diluted with DMEM medium containing 10% fetal bovine serum at a concentration of 2 × 10⁻⁶ mg / mL. 4 Cells were seeded at a density of [number] cells / mL in 96-well plates and incubated at 37°C in a CO2 incubator for 24 hours, after which the old culture medium was discarded. Then, different concentrations of styrax lactone solution (25µg / mL, 50µg / mL, 100µg / mL, 200µg / mL) were added to each well for incubation. A control group without the drug composition was also included, treated at 37°C in a 5% CO2 incubator for 48 hours, followed by incubation with MTT solution (5mg / mL) for another 4 hours. After incubation, the supernatant was discarded, DMSO was added, and the absorbance (OD value) of each well was measured using a microplate reader at a wavelength of 570nm. The inhibition rate of the drug against human cervical cancer cells SiHa was calculated as: Inhibition rate = (1 - OD value of the drug composition co-culture group / OD value of the blank control group) × 100%. The results are shown in Table 1.

[0035] 2.2 Following the method described above, *Callicarpa scabra* polysaccharide was prepared into solutions of 25 µg / mL, 50 µg / mL, 100 µg / mL, and 200 µg / mL. A control group without *Callicarpa scabra* polysaccharide was also included. The inhibition rate of *Callicarpa scabra* polysaccharide against human cervical cancer cells (SiHa) was calculated. The results are shown in Table 1.

[0036] 2.3 The pharmaceutical compositions of Examples 1-3 of the present invention (see Table 1 below) were prepared into solutions of 25 µg / mL, 50 µg / mL, 100 µg / mL, and 200 µg / mL according to the method described above. A control group without the pharmaceutical compositions was also set up. The inhibition rate of each group of pharmaceutical compositions against human cervical cancer cells SiHa was calculated, and the results are shown in Table 1.

[0037] As shown in Table 1, both perilla lactone and cane liver polysaccharide can inhibit the proliferation of cervical cancer cells SiHa in a concentration-dependent manner, with perilla lactone showing stronger activity. The combined use of perilla lactone and cane liver polysaccharide can improve the inhibitory effect on cervical cancer cells, and the inhibitory effect is far better than that of perilla lactone or cane liver polysaccharide alone.

[0038] Experimental Example 3: Effect of the drug composition on the acute toxicity of BALB / c nude mice After a single administration of 250 mg / kg of the drug combination of Example 1 (perillin: cane liver polysaccharide = 1:0.3), Comparative Example 1 (cane liver polysaccharide), and Comparative Example 2 (perillin) to BALB / c nude mice, the mice were observed daily for 14 consecutive days, and the results are recorded in Table 2.

[0039] Acute toxicity refers to the poisoning or death caused by a single administration of a large concentration of exogenous drug to the test organism. Table 2 shows that after a single administration of a high concentration of drug, all vital signs of the BALB / c nude mice remained good, and no symptoms of poisoning or death were observed. This indicates that the pharmaceutical composition of the present invention has very low toxicity in nude mice.

[0040] Experimental Example 4: Pharmacodynamic Experiment of Drug Composition on Transplanted Tumor Nude Mice 4.1 Experimental Animals Healthy female BALB / c nude mice, weighing 18-22g, were selected as experimental animals and housed in an SPF-grade laboratory with an ambient temperature controlled at 25℃ and a relative humidity of 50-60%. The cycle consisted of alternating 12-hour light and 12-hour dark cycles. After 7 days of acclimatization, the mice were used for tumor implantation.

[0041] 4.2 Model Construction Under aseptic conditions, human cervical cancer cells (SiHa) in the logarithmic growth phase were used for experiments; the logarithmic growth phase human cervical cancer cells (SiHa) were diluted with serum-free DMEM medium to a concentration of 1×10⁻⁶. 7 A suspension of 0.2 mL / mL was disinfected with povidone-iodine and then inoculated subcutaneously into the right axilla of nude mice to establish a nude mouse model of xenograft tumor.

[0042] 4.3 Grouping and Dosing After tumor formation, the above-mentioned cell xenograft nude mouse model was divided into a model group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, and Comparative Example 2 group, with 10 mice in each group. Each treatment group was administered the corresponding composition of Example 1 (perilla lactone: Canis lucidum polysaccharide = 1:0.3), Example 2 (perilla lactone: Canis lucidum polysaccharide = 1:0.4), Example 3 (perilla lactone: Canis lucidum polysaccharide = 1:0.5), Comparative Example 1 (Canis lucidum polysaccharide), and Comparative Example 2 (perilla lactone) by gavage at a dose of 50 mg / kg. The model group was given an equal volume of physiological saline. The drugs were administered once every other day for 14 consecutive days.

[0043] 4.4 Calculation of tumor quality and tumor inhibition rate After the last administration, nude mice were euthanized and the tumors were removed and weighed. The tumor inhibition rate (%) was calculated as (1 - tumor weight in the treatment group / tumor mass in the model group) × 100%, and the results are shown in Table 3.

[0044] Table 3 shows that the model group, which received no drug intervention, had the largest tumor weight. The tumor weight in each treatment group was significantly lower than that in the model group, indicating that the combination of perilla lactone and *Cibotium barometz* polysaccharide significantly inhibited the growth of human cervical cancer SiHa xenografts in the in vivo tumor-bearing nude mouse model. Specifically, the tumor weight in Examples 1-3 was lower than that in Comparative Examples 1 and 2, and the tumor inhibition rates in Comparative Examples 1 and 2 were lower than those in Example 1, demonstrating that the in vivo tumor-inhibiting effect of the combination of perilla lactone and *Cibotium barometz* polysaccharide in this invention is far superior to that of the comparative examples using either alone.

[0045] 4.5 Western blot experiment Tumor tissues from Examples 1-3 and Comparative Examples 1-2 were collected, and 1% protease inhibitors and phosphatase inhibitors were added to the tumor tissues. Lysis was performed on ice for 30 min; then centrifuged at 12000 rpm for 10 min at 4 °C. Proteins were extracted from each well using the BCA method. The protein samples were mixed with 5× Loading Buffer and denatured at 100 °C for 10 min. The protein samples from Examples 1-3 and Comparative Examples 1-2 were then separated by electrophoresis on an SDS-PAGE gel, transferred to a membrane, blocked with TBST buffer containing 5% skim milk for 1 h at room temperature, incubated overnight with GAPDH, RIPK1, and E6 antibodies, and then incubated with HRP-conjugated secondary antibody at room temperature before development. The expression of RIPK1 and E6 proteins in the cells was detected, and the results are as follows: Figure 2 As shown.

[0046] Depend on Figure 2It was found that the combined use of perilla lactone and cane liver polysaccharide could significantly downregulate the expression of RIPK1 and HPV E6 proteins in tumor tissues, suggesting that they may exert a synergistic anti-cervical cancer effect by inhibiting the RIPK1-mediated necroptosis pathway and reducing E6-mediated p53 degradation.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A pharmaceutical composition for treating cervical cancer, characterized in that, The composition comprises perilla lactone and cibotium barometz polysaccharide; the mass ratio of perilla lactone to cibotium barometz polysaccharide in the composition is 1:(0.3-0.5).

2. The pharmaceutical composition for treating cervical cancer according to claim 1, characterized in that, The preparation of the *Cibotium barometz* polysaccharide includes the following steps: (1) Remove impurities from the whole plant of the dog liver vegetable, crush it, add water for extraction, filter to obtain the extract, repeat the extraction, combine the extracts, concentrate to obtain the concentrate; (2) Add anhydrous ethanol to the concentrate for alcohol precipitation, centrifuge to collect the precipitate, add the precipitate to water to obtain the precipitate solution; then add n-butanol-chloroform solution, shake, centrifuge, take the supernatant, and after dialysis and drying, obtain the cane liver polysaccharide.

3. The pharmaceutical composition for treating cervical cancer according to claim 2, characterized in that, In step (1), the amount of water added is 10-15 times the weight of the whole herb of *Gnaphalium affine*.

4. The pharmaceutical composition for treating cervical cancer according to claim 2, characterized in that, In step (1), the extraction temperature is 80-90℃ and the time is 1.5-2.5h.

5. The pharmaceutical composition for treating cervical cancer according to claim 2, characterized in that, In step (1), the extraction is repeated 2-3 times; the concentrate is concentrated to 30-50% of its original volume.

6. The pharmaceutical composition for treating cervical cancer according to claim 2, characterized in that, In step (2), the amount of water added is 3-5 times the mass of the precipitate; after adding anhydrous ethanol, the alcohol content of the concentrate reaches 70 vol%-80 vol%; the alcohol precipitation temperature is 0-4℃ and the time is 10-12h.

7. The pharmaceutical composition for treating cervical cancer according to claim 2, characterized in that, In step (2), the volume ratio of n-butanol to chloroform in the n-butanol-chloroform solution is 1:(4-6); the volume ratio of the n-butanol-chloroform solution to the precipitate solution is 1:(3-5).

8. The pharmaceutical composition for treating cervical cancer according to claim 1, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

9. A method for preparing a pharmaceutical composition for treating cervical cancer according to any one of claims 1 to 8, characterized in that, Includes the following steps: Take perilla lactone and cane liver polysaccharide according to the above formula, mix them evenly, and add pharmaceutically acceptable excipients to obtain a drug composition for treating cervical cancer.

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