Quinoa bran oligopeptide with anti-liver cancer and anti-liver cancer metastasis functions and application of quinoa bran oligopeptide

Abstract

The invention discloses quinoa bran oligopeptide with functions of resisting liver cancer and liver cancer metastasis and application of the quinoa bran oligopeptide, and belongs to the technical field of bioactive peptide biology. In order to solve the problems of high toxicity, weak targeting property and the like of the existing anti-liver cancer and anti-liver cancer metastasis drugs, the oligopeptide with an obvious anti-liver cancer and anti-liver cancer metastasis function is separated and identified from quinoa bran peptide extracts for the first time, and the amino acid sequence of the oligopeptide is GFEWIAFK (GFK-8 for short). Experiments prove that the GFK-8 not only has an inhibiting effect on proliferation of liver cancer cells, but also can effectively inhibit lung metastasis of liver cancer in vivo at low concentration. The active peptide has the characteristics of small molecular weight, clear structure, strong activity, high safety and the like. Compared with the toxic and side effects of traditional anti-cancer drugs on liver function impairment, the GFK-8 shows the characteristic of lower toxicity. Therefore, the GFK-8 provides a key active ingredient basis for developing safer and more effective novel candidate drugs for resisting liver cancer and liver cancer metastasis, and has wide medical application prospects and market value.

Description

Technical Field

[0001] The present invention relates to the field of plant active peptide biotechnology, and in particular to a quinoa bran oligopeptide having the function of resisting liver cancer and liver cancer metastasis and an application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is the most common type of cancer worldwide, accounting for approximately 75-85% of primary liver malignancies. Due to the rapid and insidious onset of liver cancer, and the fact that most patients often have underlying liver disease resulting in atypical symptoms, most patients have missed the best time for radical treatment by the time they are diagnosed. Although radical treatments such as surgical resection and liver transplantation are currently feasible options, the low targeting and significant toxic side effects of therapeutic drugs, as well as the high recurrence and metastasis rates after surgery, severely restrict the therapeutic effect and become the main reason for the low survival rate of patients. Therefore, effectively inhibiting the occurrence, development, deterioration and metastasis of liver cancer has become a difficult problem that needs to be urgently solved in current clinical treatment. Discovering new active molecules that are both safe and effective in anti-liver cancer and liver cancer metastasis functions is of great significance for improving patient prognosis and increasing survival rates.

[0003] Quinoa (Chenopodium quinoa Willd.), a herbaceous plant in the Amaranthaceae family, is also known as quinoa, South American quinoa, and Indian wheat. It is not only rich in bioactive secondary metabolites (such as phenolic acids, saponins, and flavonoids), minerals, and vitamins, but also contains abundant high-quality protein (including several essential amino acids). This richness imparts multiple health benefits, including anti-inflammatory, antioxidant, cardiovascular protection, and potential anti-tumor properties, earning it the nickname "super grain." Quinoa bran, a key byproduct of quinoa processing, is also rich in protein and various active ingredients, representing a valuable resource for bioactive substances. In recent years, food-derived bioactive peptides have become a key area of ​​innovative drug development due to their significant advantages, including high safety, low toxicity, ease of absorption, and targeted properties.

[0004] Existing studies have reported that quinoa bran contains small molecule oligopeptides with anti-colon cancer, anti-blood pressure, anti-blood sugar, and anti-aging effects. However, there are no reports on its efficacy in resisting liver cancer and liver cancer metastasis. The present invention focuses on quinoa bran protein peptide resources and is committed to isolating and identifying novel oligopeptides with anti-liver cancer and liver cancer metastasis functions therefrom, aiming to provide candidate active molecules for the development of highly effective and low-toxic innovative drugs for anti-liver cancer and liver cancer metastasis. This not only provides an important reference for the high-value utilization of quinoa bran resources, but also lays a theoretical basis for the development of natural low-toxic drugs based on quinoa bran oligopeptides for anti-liver cancer and inhibition of liver cancer metastasis. Summary of the Invention

[0005] The present invention aims to provide a quinoa bran oligopeptide with anti-liver cancer and liver cancer metastasis properties, and to use the oligopeptide to alleviate liver cancer development and lung metastasis in liver cancer cells and BALB / c mice. The oligopeptide should have readily available raw materials, a low molecular weight, a simple structure, low toxicity, and high activity. Furthermore, low-concentration administration via intraperitoneal injection can achieve the desired effect of inhibiting liver cancer lung metastasis.

[0006] To achieve the above object, the present invention provides the following technical solutions: A quinoa bran oligopeptide with anti-liver cancer and liver cancer metastasis functions has an amino acid sequence of Gly-Phe-Glu-Trp-Ile-Ala-Phe-Lys (GFEWIAFK, abbreviated as GFK-8) and a molecular weight of 997.16 Da.

[0007] A method for screening quinoa bran oligopeptides with anti-liver cancer and liver cancer metastasis functions comprises the following steps: The first step: treating quinoa bran with acetone sulfate-ammonium precipitation and biomimetic digestion to obtain quinoa bran protein peptide extract; Step 2: After desalting and ultrafiltration of the quinoa bran protein peptide extract, a quinoa bran peptide extract with a diameter of less than 3 kDa is obtained, and the sequence of the peptide segments in the quinoa bran peptide extract with a diameter of less than 3 kDa is identified by LC-MS / MS liquid chromatography-mass spectrometry to construct a peptide database of quinoa bran peptide extracts with a diameter of less than 3 kDa, thereby obtaining a peptide extract containing the quinoa bran oligopeptide sequence with anti-liver cancer and liver cancer metastasis functions.

[0008] Step 3: Screen and identify quinoa bran oligopeptides with anti-liver cancer and liver cancer metastasis functions.

[0009] The quinoa bran oligopeptide with anti-liver cancer and liver cancer metastasis function of the present invention can inhibit the proliferation of liver cancer cells, and at the same time has a significant inhibitory effect on the migration and invasion ability of liver cancer cells at low concentrations.

[0010] The quinoa bran oligopeptide with anti-liver cancer and liver cancer metastasis function of the present invention can significantly inhibit the occurrence and development of liver cancer and lung metastasis in BALB / c mice after intraperitoneal injection.

[0011] The quinoa bran oligopeptide with anti-liver cancer and liver cancer metastasis function can significantly inhibit the proliferation and metastasis of liver cancer cells in vivo and in vitro, and has low toxicity, and has certain potential in the treatment of liver cancer.

[0012] A medicine with anti-liver cancer and liver cancer metastasis functions, comprising the quinoa bran oligopeptide with anti-liver cancer and liver cancer metastasis functions.

[0013] Compared with the existing patents, the beneficial effects of this invention are: This invention utilizes quinoa bran, an underdeveloped resource with significant resource cost advantages. For the first time, the quinoa bran oligopeptide GFK-8 was screened and identified from this resource. This peptide exhibits a clear dual anti-liver cancer effect: while inhibiting liver cancer cell proliferation, it can also specifically block liver cancer lung metastasis at low concentrations. Its natural, food-derived properties offer significant safety advantages, potentially mitigating the hepatotoxicity risks associated with traditional chemotherapy and targeted drugs. Based on these properties, GFK-8, a quinoa bran oligopeptide, can be developed as a standalone drug with anti-liver cancer and liver cancer metastasis properties, or as an adjunct ingredient in combination with traditional therapies (surgery / chemoradiotherapy / targeted therapy). This provides a novel, highly effective and safe candidate for addressing the development and progression of liver cancer and postoperative metastasis and recurrence, filling a technological gap in this field. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Effects of four oligopeptides on the proliferation of hepatocellular carcinoma cells; Figure 2 GFK-8 two-dimensional and three-dimensional conformation fitting structure diagram; Figure 3 Effects of GFK-8 on the migration of hepatocellular carcinoma cells Figure 4 The inhibitory effect of GFK-8 on liver cancer tumor development in vivo; Figure 5 Inhibitory effect of GFK-8 on lung metastasis of hepatocellular carcinoma in vivo. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, this patent is further described in detail below in conjunction with specific embodiments.

[0016] Experimental Methods: Quinoa bran was treated with acetone-ammonium sulfate precipitation, followed by biomimetic digestion using pepsin and trypsin, and then desalted using a 100Da dialysis bag. The quinoa bran protein peptide extract was then ultrafiltered using a 3kDa molecular weight ultrafiltration tube to prepare a quinoa bran peptide extract containing active oligopeptides. LC-MS / MS was then used to determine the molecular weight and sequence of each peptide fragment prepared from the quinoa bran peptide extract.

[0017] Experimental results: A peptide database of quinoa bran peptide extracts less than 3 kDa containing oligopeptides with anti-liver cancer and liver cancer metastasis functions was constructed.

[0018] Experimental method: The PeptideRanker functional activity predictor was used to predict bioactive peptides in the peptide database of quinoa bran peptide extracts less than 3kDa. Then, Toixinpred and PeptideRanker were used to predict the safety and toxicity evaluation of active peptides with a score greater than 0.8. The screened active peptides were further functionally evaluated using the MLACP 2.0 anti-tumor activity prediction scorer.

[0019] Experimental results: Four oligopeptides with high credibility, strong biological activity, non-toxicity and non-sensitizing properties were obtained, including the quinoa bran oligopeptide sequence with anti-liver cancer and liver cancer metastasis functions (as shown in Table 1). No relevant research reports on each peptide component were found in the BIOPEP-UWM bioactive peptide database, indicating that they are new peptide components.

[0020]

[0021] Principle: The CCK8 reagent contains a water-soluble tetrazolium salt (WST-8). Under the action of electron carriers, the tetrazolium salt (WST-8) is reduced by mitochondrial dehydrogenases in living cells to a highly water-soluble yellow formazan product. The amount of formazan generated is proportional to the number of living cells, and cell viability can be analyzed by measuring absorbance.

[0022] Experimental method: 5×10 3 After the HepG2 cells were cultured overnight in a 37°C incubator, the four active peptides screened in Example 2 were added for 24 hours (0, 2.5, 5, 10, 15, and 20 μM), with three replicates for each concentration of the four active peptides. After the treatment time was up, 10 μL of CCK8 reagent was added to each well and incubated in a 37°C incubator for 2 hours. The absorbance was measured at a wavelength of 450 nm using a microplate reader to obtain the corresponding cell viability and calculate the IC 50 Then the effects of the four oligopeptides on the survival rate of HepG2 were determined.

[0023] Experimental results: Make a curve graph based on the number of cells in each well and the measured OD value (such as Figure 1 As shown in A), the IC values ​​of the four oligopeptides screened for the proliferation of human liver cancer cells HepG2 were calculated. 50 , the analysis showed that the peptide with the best effect was GFK-8 (such as Figure 1 B).

[0024] Experimental method: The screened oligopeptide peptide GFK-8 was subjected to structural fitting, and the secondary structure of the oligopeptide GFK-8 with anti-liver cancer function was drawn using Chemdraw, and then the three-dimensional structure with the lowest energy of GFK-8 was fitted using Chemdraw3D software.

[0025] Experimental results: The secondary structure of the quinoa bran oligopeptide GFK-8 with anti-liver cancer and liver cancer metastasis function was drawn (such as Figure 2 A) and the three-dimensional structure fitting diagram (as shown in Figure 2 B).

[0026] Experimental Methods: First, the CCK8 assay was used to examine the effect of low concentrations of GFK-8 (2.5 and 5 μM) on the proliferation of HepG2 liver cancer cells. Next, a line was drawn on the back of a 24-well plate with a marker. HepG2 cells in the logarithmic growth phase were trypsinized and resuspended in RPMI-1640 medium. The cells were counted under a microscope and seeded into 24-well plates (5 × 10 5 / well) and culture overnight. The next day, use a 200 μL sterile pipette tip to scratch the horizontal line on the back of the well plate perpendicular to the ruler. Then, wash the cells three times with PBS, remove the scratched cells, and add 500 μL serum-free culture medium. Treat with GFK-8 and take pictures at 0 hour and 24 hours. After measuring the migration distance, calculate the cell migration rate.

[0027] Experimental results: After 24 hours of treatment with GFK-8 at low concentrations (2.5, 5μM), the survival rate of HepG2 liver cancer cells was greater than 80%, and there was no significant effect on proliferation. Therefore, this concentration was selected as the subsequent GFK-8 treatment concentration. Cell scratch test showed that GFK-8 at low concentrations (such as Figure 3 A) can significantly inhibit the migration performance of liver cancer cells HepG2 (as shown in Figure 3 B), and there is an obvious dose-effect relationship between the inhibition rate and concentration (as shown in Figure 3 C).

[0028] Experimental method: HepG2 cells with good growth status were taken and digested with trypsin. When the cell morphology changed significantly under the microscope, the digestion was stopped and the cells were suspended in PBS and the cell density was adjusted to 2×10 6 200 μL of HepG2 cells suspended in PBS buffer were then injected subcutaneously into the axilla of each mouse. Tumor formation was observed one week later. Twenty tumor-bearing mice were randomly divided into a control group and a GFK-8 group. GFK-8 was administered intraperitoneally every two days at a dose of 20 mg / kg for a total of seven treatments. The control group received an equal volume of PBS solution. Body weight and tumor diameter were recorded during the treatment. At the end of the treatment, the mice were sacrificed, and subcutaneous tumor tissue was removed, weighed, and stored.

[0029] Experimental results: Compared with the control group, which showed decreased activity, poor mental state, lethargy, decreased appetite, and dull hair, the GFK-8-treated group showed better condition, with normal feeding and behavioral activity. The tumor growth curve showed that GFK-8 could significantly inhibit tumor growth in a time-dependent manner ( Figure 4 A, B). The weight of the isolated tumor tissue was measured and the weight of the tumor in the GFK-8 treated group was significantly reduced (e.g. Figure 4 C). The effect of GFK-8 treatment on the body weight of mice showed that GFK-8 had no significant effect on the body weight of mice (e.g. Figure 4 D). The above results indicate that GFK-8 can inhibit the development and deterioration of liver cancer tumors with minimal toxicity in mice.

[0030] Experimental method: 20 female BALB / c mice were weighed and randomly divided into two groups according to their body weight: control group and GFK-8 group, with 10 mice in each group. The logarithmic phase HepG2 cells cultured as described above were taken and the cell concentration was adjusted to 2×10 6 A mouse model of liver cancer lung metastasis was established by injecting 200 μL of HepG2 cells into the tail vein of each mouse using a single-cell suspension of 0.2 mL of GFK-8. A 10 mg / kg dose of GFK-8 was administered intraperitoneally, while a control group received a corresponding volume of PBS every two days for a total of seven treatments. Within 24 hours of the seventh GFK-8 treatment, all mice were anesthetized and sacrificed. Whole lungs were removed for tumor metastasis analysis, observing the presence, size, and distribution of lung tumors. The number of lung nodules and lung enlargement were also counted.

[0031] Experimental results: GFK-8 treatment can significantly inhibit the number of nodules of lung metastasis of mouse liver cancer cells (such as Figure 5 As shown in A and B, it alleviates lung tissue enlargement and weight loss in mice with liver cancer (as shown in Figure 5 C, as shown in 5D).

[0032] The above-mentioned embodiments merely express several implementation methods of the present invention. Although the description thereof is relatively specific and detailed, it should not be understood as limiting the scope of protection of the invention.

Claims

1. A quinoa bran oligopeptide with anti-liver cancer and liver cancer metastasis function, characterized in that: The amino acid sequence is GFEWIAFK.

2. The method for preparing the quinoa bran oligopeptide having anti-liver cancer and liver cancer metastasis function according to claim 1, comprising the following steps: (1) After quinoa bran is precipitated with acetone-ammonium sulfate and biomimetic digested with pepsin and trypsin, the product is dialyzed and desalted using a 100Da molecular weight dialysis bag, and then ultrafiltered using a 3kDa molecular weight ultrafiltration tube. The powder obtained by vacuum freeze-drying is the quinoa bran peptide extract with a molecular weight less than 3kDa. (2) Identify peptides less than 3 kDa in quinoa bran peptide extracts, and further use the PeptideRanker functional activity prediction tool, Toixinpred safety and toxicity evaluation predictor and MLACP 2.0 anti-tumor activity prediction scorer to perform functional evaluation on the peptides. Based on the scores, four peptides containing the oligopeptide sequences with anti-liver cancer and liver cancer metastasis functions were obtained, and preliminary effect evaluation was performed to further screen out the quinoa bran oligopeptides with anti-liver cancer and liver cancer metastasis functions.

3. Use of the quinoa bran oligopeptide according to claim 1 in the preparation of drugs for resisting liver cancer and liver cancer metastasis.

4. A drug with anti-liver cancer and liver cancer metastasis function, characterized in that: The medicine contains the quinoa bran oligopeptide as claimed in claim 1.

Images