Use of mutton tallow in the preparation of a functional supplement against esophageal cancer cells

Abstract

The present application relates to the application of sheep oil in the preparation of anti-esophageal cancer cell function supplements, and the sheep oil is used as a nutritional supplement to reduce the esophageal cancer cell activity, maintain the body weight of the esophageal cancer patient, stabilize the nutrition and metabolism index, reduce the inhibition of radiotherapy on the hematopoietic system, improve the immune function of the esophageal cancer patient, reduce the inflammation reaction related to radiotherapy and protect the life quality of the esophageal cancer patient.The present application first proves the positive role of the sheep oil as a nutritional supplement in the treatment of esophageal squamous cell carcinoma patients, especially for the locally advanced unresectable esophageal squamous cell carcinoma patients, and provides a function supplement with lower cost and better effect for the future treatment of esophageal squamous cell carcinoma patients, which has important significance.

Description

Technical Field

[0001] This invention belongs to the field of edible oil technology, specifically relating to the application of mutton fat in the preparation of functional supplements for anti-esophageal cancer cells. Background Technology

[0002] Esophageal cancer is a common malignant tumor worldwide, with esophageal squamous cell carcinoma (ESCC) being the most prevalent. For unresectable locally advanced ESCC, radical radiotherapy or concurrent chemoradiotherapy is the standard treatment, but it often causes side effects such as radiation esophagitis and pain on swallowing, leading to insufficient nutrient intake and weight loss in patients, which in turn affects the treatment effect and quality of life.

[0003] Nutritional support plays an increasingly prominent role in cancer treatment. As an essential nutrient, fatty acids have also attracted much attention for their role in anti-tumor activity. Recent studies have found that certain saturated fatty acids, such as odd-chain saturated fatty acids (OCSFAs), exhibit anti-tumor activity. Our previous studies have confirmed that mutton tail oil, which is rich in heptadecanoic acid (C17:0), can inhibit the proliferation of lung cancer cells both in vivo and in vitro, and also shows potential inhibitory effects on HepG2 liver cancer cells, HeLa cervical cancer cells, and SW480 colon cancer cells. Therefore, further exploration of the application of mutton tail oil in the treatment of esophageal cancer has important potential value. Summary of the Invention

[0004] The purpose of this invention is to provide the application of mutton fat in the preparation of functional supplements for treating esophageal cancer cells in order to solve the above-mentioned problems.

[0005] The present invention achieves the above objectives through the following technical solutions: This invention provides the application of mutton fat in the preparation of functional supplements for treating esophageal cancer cells.

[0006] As a further optimization of the present invention, mutton fat is used to reduce the activity of esophageal cancer cells; mutton fat is used as a nutritional supplement to maintain the weight of esophageal cancer patients, stabilize nutritional and metabolic indicators, reduce the inhibition of the hematopoietic system by radiotherapy, improve the immune function of esophageal cancer patients, reduce radiotherapy-related inflammatory responses, and protect the quality of life of esophageal cancer patients.

[0007] As a further optimization of the present invention, the esophageal cancer patient is a patient with unresectable locally advanced esophageal squamous cell carcinoma, and the dosage of the mutton fat as a nutritional supplement is 0.5-3g per day.

[0008] As a further optimization of the present invention, the esophageal cancer cells are esophageal squamous cell carcinoma cells, specifically the ECA-109 cell line.

[0009] As a further optimization of the present invention, the mutton fat contains fatty acids C16:0, C18:0, C18:1, and C17:0, and the dosage of the mutton fat as a nutritional supplement is 0.5-3g per person per day.

[0010] As a further optimization of the present invention, the stable nutritional and metabolic indicators are maintained to keep the plasma glycated albumin, albumin and hemoglobin levels of esophageal cancer patients stable.

[0011] As a further optimization of the present invention, the reduction of the inhibition of the hematopoietic system by radiotherapy is to reduce the decrease in white blood cells and keep the red blood cell and lymphocyte counts stable.

[0012] As a further optimization of the present invention, improving the immune function of esophageal cancer patients involves increasing CD4 levels in esophageal cancer patients. + / CD8 + T cell ratio.

[0013] As a further optimization of the present invention, the reduction of radiotherapy-related inflammatory response is achieved by significantly inhibiting the IL-1 level in esophageal cancer patients after radiotherapy.

[0014] As a further optimization of the present invention, the protection of the quality of life of esophageal cancer patients includes maintaining the stability of the functional status score of esophageal cancer patients, reducing pain, and maintaining appetite.

[0015] The beneficial effects of this invention are as follows: 1) This invention is the first to demonstrate the positive role of mutton fat as a nutritional supplement in the treatment of patients with esophageal squamous cell carcinoma, especially for patients with locally advanced unresectable esophageal squamous cell carcinoma. It provides a functional supplement with lower cost and better effect for the future treatment of patients with esophageal squamous cell carcinoma, which is of great significance.

[0016] 2) This invention has verified that long-term supplementation with mutton tail oil rich in C17:0 can effectively alleviate weight loss caused by radiotherapy in patients with esophageal squamous cell carcinoma, maintain the stability of key nutritional indicators such as plasma albumin and hemoglobin, significantly reduce the inhibition of bone marrow hematopoietic function during radiotherapy, improve immune balance, and inhibit harmful excessive inflammatory response. The KPS index level effectively indicates that the nutritional intervention of mutton tail oil significantly maintains the patient's physical and cognitive functions, effectively controls pain and loss of appetite, thereby maintaining the overall quality of life during radiotherapy. Therefore, mutton oil can be used as a nutritional supplement to improve the nutritional status, immune function, and quality of life of patients with esophageal cancer. Attached Figure Description

[0017] Figure 1 This is a peak diagram of fatty acid composition of mutton fat.

[0018] Figure 2 The figure shows the effect of mutton fat on the cell viability of the ECA-109 cell line.

[0019] Figure 3 The figure shows the effect of C17:0 on the cell viability of the ECA-109 cell line.

[0020] Figure 4 C17:0 IC50 for ECA-109 cells 50 calculate.

[0021] Figure 5 A flowchart for clinical research participants. Detailed Implementation

[0022] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0023] I. Materials Unless otherwise specified, all experimental methods described below are conventional biochemical methods. Unless otherwise specified, all experimental materials used below were purchased from conventional biochemical reagent companies. ECA-109 (esophageal cancer cell line) was purchased commercially. Other experimental materials are listed in Table 1. All experiments were performed in triplicate, and the results were averaged. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0024] Table 1: Experimental Materials Sheep fat: Sheep tail fat (STF) provided by Fuyang Tianxiang Food Technology Co., Ltd. is heated and rendered, filtered with gauze to remove bacteria, and stored in portions at 4°C. The physicochemical properties of these oils, such as acid value and iodine value, meet the national food safety standards for animal fats. After being stored in portions at 4°C for 6 months and 12 months, the properties were still stable.

[0025] II. Detection of fatty acid components in mutton fat Take 0.06g of oil into a 15mL centrifuge tube, add 8mL of n-hexane, and after it is fully dissolved, add 2mL of saturated KOH-CH3OH solution. Finally, mix well, let stand at 40℃ for 30min, and after the layers are separated, take the upper layer into a sample bottle for instrumental analysis.

[0026] GC-MS detection conditions: SP-2560 highly polar capillary gas chromatographic column (100m × 0.25mm, 0.2µm), carrier gas: nitrogen; vaporization chamber temperature: 280℃; split injection, split ratio 20:1; injection port and detector temperatures both 250℃, column inlet pressure 278.6kPa; column temperature 100℃ for 5 min, ramped to 220℃ at a rate of 4℃ / min and held for 10 min, ramped to 240℃ at a rate of 4℃ / min and held for 20 min; injection volume 1µL; ionization method: EI source, single quadrupole detector; electron energy: 70eV; ion source temperature and interface temperature both 250℃.

[0027] like Figure 1 As shown: The test results indicate that the fatty acids in mutton fat are mainly composed of C16:0, C18:0, and C18:1, as well as C17:0, a fatty acid unique to ruminants. It also contains some branched-chain fatty acids. The test results of mutton fat from different breeds of sheep are generally the same. The mutton fat was dissolved in DMSO for subsequent experimental analysis.

[0028] III. The Effect of Sheep Fat on the Viability of Esophageal Cancer Cells ECA-109 cells (esophageal cancer cell line) in logarithmic growth phase were used to prepare cell suspensions, and 100 μL were seeded into 96-well plates per well. After cell attachment, the cells were starved with serum for 12 h, followed by drug treatment with different concentrations of C17:0-rich sheep tail oil prepared from complete culture medium at concentrations of 0, 1, 2, 4, 6, and 8 mg / mL. The cells were then incubated for another 48 h. Figure 2 As shown, the MTT assay results indicate that DMSO in this concentration range has no significant effect on the viability of esophageal cancer cells, while mutton tail oil has a significant inhibitory effect on ECA-109 esophageal cancer cells.

[0029] IV. Effects of C17:0 on the viability of esophageal cancer cells To further verify whether C17:0 is the key active ingredient inhibiting the viability of esophageal cancer cells, different concentrations of C17:0 solutions (0, 10, 50, 100, 150, 200, 250, 300, 350 μM) prepared with complete culture medium were added during the cell treatment stage, and the cells were cultured for 48 h. Figure 3 As shown, the MTT assay results indicated that ethanol at the corresponding concentrations had no significant toxicity to the cells, while C17:0 exhibited a significant dose-dependent toxic effect on ECA-109 cells. At a concentration of 200 μM, C17:0 showed the strongest inhibitory effect, reducing cell viability to 19.37%. Figure 4 As shown, its half-maximal inhibitory concentration (IC50) 50 The value was 111.69 μM.

[0030] V. Effects of mutton fat capsules on nutritional status, immune function, and quality of life in patients with unresectable locally advanced esophageal squamous cell carcinoma (ESCC). Preparation of mutton fat capsules: Prepare the capsule material solution according to the formula ratio of gelatin:glycerin:water = 0.5:1:1.2. Soak the gelatin in water to make it swell. After the gelatin melts, heat it to a certain temperature, add glycerin, and stir to mix evenly. Connect the gelatin tank to the automatic rotating capsule machine. Put the contents of the capsule (i.e., mutton fat) into the hopper. After the machine's various parameters are adjusted to normal, start the pressing method to make capsules. Set the soft capsule filling to 1g / capsule, set at 25℃ for 2 hours, dry at 30℃ for 24 hours, and package the finished soft capsules in oral high-density polyethylene bottles and store them in a sealed container.

[0031] The experimental method is as follows: (1) Research Design This study is a single-center, randomized, placebo-controlled, single-blind exploratory clinical trial. All patients were informed of the study's purpose and potential adverse reactions and signed written informed consent forms before participation. This prospective cohort study has been approved by the Human Ethics Review Committee of the First People's Hospital of Hefei, Anhui Province, China (Approval No.: 2022-KY-019) and registered with the Chinese Clinical Trial Registry (Approval No.: ChiCTR2600118094).

[0032] (2) Study population Between June 2022 and December 2024, patients with unresectable locally advanced ESCC were admitted to the Department of Oncology, Binhu Campus of Hefei First People's Hospital.

[0033] Inclusion criteria were: 1) histologically confirmed ESCC; 2) assessed by a multidisciplinary team (MDT) as unresectable or refusing surgery and suitable for radical radiotherapy; 3) age > 18 years; 4) expected survival > 6 months; 5) basically normal electrocardiogram and no unhealed external wounds; 6) renal function: creatinine (Cr) ≤ 2.0 × upper limit of normal (UNL); liver function: AST and ALT ≤ 2.5 × UNL; 7) voluntary participation, good compliance, ability to cooperate with trial observation and signing of written informed consent.

[0034] Exclusion criteria: 1) distant metastasis; 2) prior anti-tumor therapy or surgical treatment for esophageal cancer; 3) concurrent active malignant tumors; 4) allergy to STF or its products; 5) pregnant women, breastfeeding women, or women of childbearing age who are not using contraception; 6) poorly controlled neurological or mental illness, or poor treatment adherence, inability to cooperate, or inability to report treatment responses due to mental disorders; 7) concurrent participation in other clinical trials; 8) other circumstances deemed unsuitable for participation by the investigator.

[0035] Exclusion criteria: 1) Failure to comply with trial requirements or serious violation of the study protocol; 2) Cases withdrawn due to adverse events that cannot be evaluated for efficacy, but whose adverse events will be included in the statistical analysis; 3) Missing primary endpoints or severely incomplete case report form (CRF) data.

[0036] Research participant flowchart as follows Figure 5 As shown in Table 2, a total of 45 patients completed the study, including 21 in the STF group and 24 in the control group. Data were recorded for both groups before and after radiotherapy, specifically one day before the start of radiotherapy and seven days after the last radiotherapy. The two groups were equal in terms of age, sex, tumor stage, and baseline characteristics (p>0.05). Table 2 Characteristics of esophageal cancer patients Note: Data are expressed as mean ± standard deviation. a Or n (%), p-value based on paired t-test b There were no significant differences between the two groups in baseline characteristics such as age and sex; BMI: Body Mass Index; NRS 2002: Nutritional Risk Screening 2002; Current stage per AJCC 8th cTNM: Oncology Stage; AJCC: American Joint Committee on Cancer.

[0037] (3) Dietary intervention methods Patients were randomly assigned to two groups: the STF group received standard nutritional support and daily oral administration of 1 gram of STF soft capsules rich in heptadecanoic acid (provided and quality-controlled by the collaborating institutions Fuyang Tianxiang Food Technology Co., Ltd. and Anhui Habo Pharmaceutical Co., Ltd.), while the control group received standard nutritional support and placebo starch capsules with the same appearance. For patients receiving tube feeding, the contents of the capsules were drawn off with a syringe and mixed into their regular diet. All patients received intensity-modulated radiation therapy with a planned total dose of 50.4-60 Gy, delivered to the planned target area at doses of 1.8-2 Gy per fraction.

[0038] (4) Key outcome indicators Changes in body mass index (BMI) and Karnofsky Performance Status (KPS) before and after radiotherapy; secondary outcome measures: nutritional indicators: serum albumin (ALB) and hemoglobin (HB); routine blood parameters: white blood cell (WBC) and lymphocyte count; immune inflammatory markers: T cell subsets (CD4+). + / CD8 + The ratio of interleukin-1 (IL-1) and interleukin-1 (IL-1) levels were assessed using the European Cancer Research and Treatment Organization Quality of Life Questionnaire Core 30 (QLQ-C30).

[0039] (5) Statistical analysis Data were analyzed using SPSS 22.0 software. Non-normally distributed continuous variables were expressed as median and interquartile range. Within-group comparisons were performed using the Wilcoxon signed-rank test, and between-group comparisons were performed using the Mann-Whitney U test. Categorical variables were expressed as frequency and percentage, and Fisher's exact test was used for evaluation. A p-value < 0.05 was considered statistically significant.

[0040] 2. Experimental Results (1) The comparison of routine blood indicators, nutritional indicators and immune indicators before and after radiotherapy is shown in Table 3: Table 3 Comparison of routine blood parameters, nutritional parameters and immune parameters between the two groups of patients before and after radiotherapy ; ; ; ; ; Note: Data are presented as median (range), and p-values ​​and z-values ​​are based on the rank-sum test.

[0041] Experimental results: As shown in Table 3 above, after radiotherapy, the body mass index (BMI) of both groups of patients decreased, but the decrease in the STF group was significantly lower than that in the control group (p<0.011), indicating that nutritional supplementation in the STF group helped to reduce the weight loss caused by radiotherapy.

[0042] In the control group, plasma glycated albumin (PG-SGA) (p=0.018), albumin (ALB) (p=0.008), and hemoglobin (HB) (p=0.004) levels were significantly decreased after radiotherapy. In contrast, albumin (ALB) and hemoglobin (HB) levels remained relatively stable in the STF group (p>0.05), while plasma glycated albumin levels were significantly increased (p<0.05). This indicates that nutritional support plays a positive role in maintaining protein reserves and hemoglobin levels in patients undergoing STF treatment.

[0043] Regarding hematological parameters, the control group showed a significant decrease in white blood cell (WBC) (p=0.011), red blood cell (RBC) (p=0.007), and lymphocyte count (p<0.001); in contrast, the STF group showed only a slight decrease in white blood cell count (p=0.019), indicating that the nutritional support of STF protected the hematopoietic function of patients to some extent.

[0044] The data above indicate that STF supplementation helps reduce weight loss and maintain key nutritional and blood parameters during radiotherapy, which may reduce radiotherapy-related nutritional risks and hematological toxicity, and has a positive impact on the patient's overall tolerance and recovery.

[0045] Fatty acids may influence cancer progression by regulating immune cells and inflammatory responses. Therefore, this study further monitored related immune and inflammatory markers. The results showed that CD4 levels were significantly higher in the STF group after radiotherapy. + / CD8 + The T cell ratio was significantly lower than that in the control group (p<0.045), indicating that nutritional intervention in STF may improve the body's immune inflammatory state by regulating the distribution of T lymphocyte subsets, thereby having a positive impact on the recovery of cancer patients.

[0046] Regarding the inflammatory response, the level of IL-1 (interleukin-1) was significantly increased in the control group after radiotherapy (p<0.001), while no significant change was observed in the STF group (p=0.148). These results suggest that nutritional support in STF may help suppress or alleviate excessive inflammatory responses induced by radiotherapy. In summary, nutritional support for STF not only maintains body weight and protein reserves and protects hematopoietic function at the nutritional level, but also improves immune balance at the immune level and reduces inflammatory activation at the inflammatory level. Nutritional support for STF exerts a protective effect through multiple pathways of "nutrition-immunity-inflammation", providing patients with more comprehensive support.

[0047] (2) Quality of life (assessed using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30; QLQ-C30) is shown in Table 4: Table 4. Comparison of quality of life of patients in the control group before and after radiotherapy. ; ; ; ; ; ; ; Note: Data are presented as median (range), and p-values ​​and z-values ​​are based on rank-sum tests. KPS: Karnofsky Performance Status Scale; EORTC QLQ-C30: European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (30 core items).

[0048] Results: Quality of life (QOL) assessment showed that patients in the control group experienced significant deterioration in multiple functional domains, including KPS score (functional status score), overall quality of life (p=0.005), physical function (p<0.001), cognitive function (p=0.046), pain (p=0.007), and decreased appetite (p=0.013) (Table 6). In contrast, all QOL scores in the STF group remained stable before and after radiotherapy, with no significant decrease (Table 7).

[0049] Further analysis showed that the KPS score in the control group significantly decreased after radiotherapy (p<0.001), while it remained stable in the STF group, with a statistically significant difference between the two groups (p=0.013). This directly suggests that patients in the STF group maintained better physical strength and self-care ability, which is crucial for treatment tolerance and daily life. Regarding appetite, the appetite score in the control group significantly decreased after radiotherapy (p=0.013); while in the STF group, although the median score remained stable before and after radiotherapy, the highest value decreased slightly, with no statistically significant change within the group. The comparison between the two groups showed that the appetite score in the STF group was significantly better than that in the control group after radiotherapy (p=0.005). Stable appetite not only helps improve patients' nutritional intake but also provides a direct behavioral basis for maintaining nutritional indicators such as albumin and hemoglobin. Furthermore, the effective improvement in pain control further improved patient comfort.

[0050] IV. Conclusion In summary, this invention first determined the effects of mutton tail oil and its key active ingredient C17:0 on the viability of esophageal cancer cells (esophageal squamous cell carcinoma ECA-109) through an MTT assay. Then, through a randomized controlled clinical trial, it preliminarily explored the effects of supplementing mutton tail oil rich in heptadecanoic acid on the nutritional status, immune function, and quality of life of patients with locally advanced unresectable esophageal squamous cell carcinoma (ESCC). The study found that long-term supplementation with mutton tail oil rich in C17:0 effectively alleviated radiotherapy-induced weight loss in ESCC patients, maintained stable levels of key nutritional indicators such as plasma albumin and hemoglobin, significantly reduced the inhibition of bone marrow hematopoietic function during radiotherapy, improved immune balance, and suppressed harmful excessive inflammatory responses. KPS levels effectively indicated that nutritional intervention with mutton tail oil significantly maintained patients' physical and cognitive functions, effectively controlled pain and loss of appetite, thereby maintaining overall quality of life during radiotherapy. Therefore, mutton tail oil can be used as a nutritional supplement to improve the nutritional status, immune function, and quality of life of esophageal cancer patients.

[0051] The above description merely illustrates several embodiments of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. Application of mutton fat in the preparation of functional supplements for anti-esophageal cancer cells.

2. The application according to claim 1, characterized in that: Sheep fat is used to reduce the activity of esophageal cancer cells; as a nutritional supplement, it is used to maintain the weight of esophageal cancer patients, stabilize nutritional and metabolic indicators, reduce the inhibition of the hematopoietic system by radiotherapy, improve the immune function of esophageal cancer patients, reduce radiotherapy-related inflammatory responses, and protect the quality of life of esophageal cancer patients.

3. The application according to claim 2, characterized in that: The esophageal cancer patients mentioned are those with unresectable locally advanced esophageal squamous cell carcinoma.

4. The application according to claim 2, characterized in that: The esophageal cancer cells are esophageal squamous cell carcinoma cells, specifically the ECA-109 cell line.

5. The application according to claim 2, characterized in that: The mutton fat contains fatty acids C16:0, C18:0, C18:1, and C17:

0. The dosage of the mutton fat as a nutritional supplement is 0.5-3g per person per day.

6. The application according to claim 2, characterized in that: The stable nutritional and metabolic indicators are maintained to keep the plasma glycated albumin, albumin and hemoglobin levels stable in esophageal cancer patients.

7. The application according to claim 2, characterized in that: The reduction of radiotherapy's suppression of the hematopoietic system is achieved by reducing the decrease in white blood cells and maintaining stable red blood cell and lymphocyte counts.

8. The application according to claim 2, characterized in that: The improvement of immune function in esophageal cancer patients is achieved by increasing CD4 levels in esophageal cancer patients. + / CD8 + T cell ratio.

9. The application according to claim 2, characterized in that: The reduction of radiotherapy-related inflammatory response is achieved by significantly inhibiting IL-1 levels in esophageal cancer patients after radiotherapy.

10. The application according to claim 2, characterized in that: The goal of protecting the quality of life for esophageal cancer patients is to maintain stable functional status scores, reduce pain, and preserve appetite.

Images