A free polyphenol rich in egcg of malus hallings fruit dregs, and an extraction process and application thereof

Abstract

The application belongs to the technical field of natural active ingredient extraction, and discloses a free polyphenol rich in EGCG, an extraction process of the free polyphenol and application of the free polyphenol. The free polyphenol is extracted from discarded fruit pomace of Malus hupehensis Rehd. by using a pre-cooled acetone aqueous solution multi-stage homogenization process. The process conditions are mild and controllable, and can effectively avoid the thermal oxidative damage of active polyphenol components, and the utilization rate of raw materials is high. The obtained free polyphenol is specifically enriched in characteristic active components such as (-) -epigallocatechin gallate and (+) -gallocatechin. Under the synergistic action of multiple components, the free polyphenol can inhibit the proliferation of HepG2 liver cancer cells and MDA-MB-231 triple-negative breast cancer cells, and plays a role through multiple mechanisms such as S-phase cell cycle arrest, induction of tumor cell apoptosis, and inhibition of tumor cell migration and invasion. The free polyphenol can be used for preparing functional food, health food and candidate drugs for inhibiting tumor cell proliferation, and realizes the resource-based high-value utilization of Malus hupehensis Rehd.

Description

Technical Field

[0001] This invention belongs to the field of natural active ingredient extraction technology, specifically relating to a free polyphenol from the pomace of the golden prickly pear rich in EGCG, its extraction process, and its application. Background Technology

[0002] prickly pear ( Rosa roxburghii Tratt is a perennial deciduous shrub whose fruit is rich in superoxide dismutase (SOD), vitamin C, flavonoids, and various bioactive substances. Its vitamin C content is among the highest of common fruits, with over 2000 mg of vitamin C per 100 grams of fresh fruit, earning it the reputation of "King of Vitamin C." Golden prickly pear (… Rosa sterilis SDshi Var. leioclada is an excellent cultivated variety of prickly pear. Its fruit is larger than that of ordinary prickly pear (the weight of a single fruit can reach 25-35 g), the flesh is thicker, and the average vitamin C content can reach 2500 mg / 100 g FW (that is, 2500 mg of vitamin C per 100 grams of fresh fruit). It also has softer thorns, is easy to peel, and has excellent suitability for fresh consumption and processing.

[0003] The deep processing of golden prickly pear juice produces a large amount of fruit pomace, a byproduct of which retains a significant amount of plant polyphenols and flavonoids in its cell wall components. Currently, this fruit pomace is mostly treated in a crude manner, either directly used as livestock feed or discarded altogether. This not only results in a huge waste of plant bioactive resources but also easily leads to solid waste environmental pollution problems.

[0004] Liver cancer and breast cancer are common malignant tumors in clinical practice. Abnormal proliferation, cell cycle disorder, invasion and metastasis of tumor cells not only directly threaten the body's life and health, but also induce chronic inflammation in the body by releasing inflammatory factors and aging-related secretory phenotype molecules, accelerating the aging of body cells and tissues. In turn, the aging of the body further increases the risk of liver cancer and breast cancer, forming a mutually reinforcing pathological vicious cycle. Therefore, the development of natural active substances that can inhibit tumor cell proliferation, cell cycle disorder and invasion and metastasis is of great practical significance for the research and development of related functional products.

[0005] Plant polyphenols can be broadly classified into two categories based on their form of existence in plant tissues: free polyphenols and bound polyphenols. These two types differ significantly in their material composition, transcellular absorption capacity, and bioactivity. Existing research on polyphenol extracts from prickly pear pomace largely focuses on evaluating the in vitro antioxidant and anti-aging activities of bound polyphenols. Systematic studies on the antitumor effects of free polyphenols, such as inhibiting tumor cell proliferation, cell cycle arrest, and anti-invasion and metastasis, are relatively scarce. Furthermore, existing research on prickly pear pomace primarily focuses on common prickly pear pomace pomace, with some processes emphasizing the extraction of bound polyphenols. There is a lack of specialized extraction processes and antitumor activity development for the highly bioavailable free polyphenols in golden prickly pear pomace pomace, hindering the resource utilization and high-value-added industrial development of golden prickly pear processing byproducts. Summary of the Invention

[0006] The purpose of this invention is to provide a free polyphenol from prickly pear pomace rich in EGCG, its extraction process, and its application. Using waste pomace from prickly pear processing as raw material, a mild extraction process for free polyphenols is established to obtain free polyphenols from prickly pear pomace pomace that specifically enriches active ingredients such as EGCG and (+)-gallic catechin. Furthermore, its application in inhibiting the proliferation of liver cancer cells and triple-negative breast cancer cells is developed, thereby realizing the high-value utilization of prickly pear processing by-products.

[0007] To achieve the above objectives, this invention provides an extraction process for free polyphenols from the pomace of *Pyracantha fortuneana* rich in EGCG, comprising the following steps: S1. Raw material pretreatment: The prickly pear fruit residue is dried at low temperature, crushed and sieved to obtain prickly pear fruit residue dry powder for later use. S2. Preparation of extraction solvent: Prepare an acetone aqueous solution with a volume fraction of 75-85% as the extraction solvent, and pre-cool it to 0-10℃ for later use. S3. Multi-stage homogenization extraction: Add the dry powder of golden prickly pear pomace to a centrifuge tube, and add pre-cooled extraction solvent at a mass-volume ratio of 1:8 to 1:12; after mixing evenly, perform homogenization treatment, then centrifuge and collect the supernatant; repeat the homogenization and centrifugation extraction operation on the residue until the supernatant is colorless and transparent, combine all the supernatants, evaporate under reduced pressure to remove the extraction solvent, and obtain free polyphenols from the golden prickly pear pomace pomace.

[0008] This invention employs a multi-stage homogenization extraction process using pre-cooled acetone aqueous solution to directionally extract free polyphenols from the pomace of *Pyracantha fortuneana*, fully enriching the free active components in the pomace. The overall process conditions are mild and controllable, effectively preventing the thermal oxidation of polyphenols and maximizing the preservation of the original biological activity of the free polyphenols. The prepared free polyphenols are specifically enriched with characteristic active substances such as (-)-epigallocatechin gallate (EGCG) and (+)-gallocatechin. Relying on the synergistic effect of core components such as EGCG and isoquercitrin, they can achieve tumor cell cycle arrest and induce mitochondrial-mediated early apoptosis of tumor cells, providing high-quality natural raw materials for the preparation of functional foods, health foods, or candidate drugs that inhibit the proliferation of liver cancer cells and / or breast cancer cells.

[0009] Preferably, the low-temperature drying temperature in step S1 is 40–50°C; the sieving is performed through a 40–60 mesh sieve. This achieves thorough dehydration of the fruit pomace while avoiding the oxidative degradation of polyphenolic active ingredients caused by high temperatures. The pulverization and sieving process yields fruit pomace dry powder of suitable particle size, which increases the contact area between the extraction solvent and the material, improving the dissolution efficiency of free polyphenols, and also prevents the material from being too fine, thus avoiding difficulties in subsequent centrifugal solid-liquid separation and ensuring the stable operation of the multi-stage homogenization extraction process.

[0010] Preferably, the homogenization speed in step S3 is 10,000–14,000 r / min, and the homogenization time is 2–5 min; this can fully break down the fruit pomace cell tissue, promote the full dissolution of intracellular free polyphenols, and at the same time avoid the mechanical friction heat generated by excessive speed and long processing time, thus reducing polyphenol oxidation damage; the centrifugation speed is 7,000–9,000 r / min, and the centrifugation time is 8–12 min, which can efficiently achieve solid-liquid two-phase separation, fully collect the supernatant of dissolved free polyphenols, reduce the residual loss of active polyphenols in the solid residue, and ensure the yield and activity quality of the extract.

[0011] Preferably, the temperature of the reduced pressure evaporation in step S3 is 40-60°C. This efficiently removes the acetone extraction solvent while avoiding the oxidation and decomposition of free polyphenols caused by high-temperature thermal effects. This maximizes the retention of characteristic active components such as EGCG and (+)-gallic catechin in the free polyphenols, ensuring that the biological activity of the obtained free polyphenols is not damaged.

[0012] To achieve the above objectives, this invention also provides a free polyphenol from the prickly pear pomace, rich in EGCG, obtained by the aforementioned extraction process. It contains EGCG, (+)-gallic acid, isoquercitrin, quercetin 3-O-sophoroside, quercetin-7-O-glucoside, catechin, and ellagic acid. Compared to free polyphenols from ordinary prickly pear pomace, the free polyphenols from the prickly pear pomace obtained by this invention specifically enrich highly bioavailable active components such as EGCG, (+)-gallic acid, and quercetin glycoside. It exhibits excellent cellular absorption performance, can achieve S-phase cell cycle arrest in tumor cells, and induce mitochondrial-mediated tumor cell apoptosis. It can be used as a functional food, health food, food additive, or a candidate drug raw material for inhibiting tumor cell proliferation.

[0013] This invention also provides the application of the above-mentioned EGCG-rich free polyphenols from the pomace of *Pyracantha fortuneana* in inhibiting the proliferation of liver cancer cells and / or breast cancer cells; wherein the liver cancer cells are HepG2 liver cancer cells; and the breast cancer cells are triple-negative breast cancer MDA-MB-231 cells. The free polyphenols from the pomace of this invention are rich in active components such as EGCG, isoquercitrin, and ellagic acid, which can arrest tumor cells in the S phase, inhibit the PI3K / AKT tumor survival signaling pathway, induce mitochondrial pathway-mediated early apoptosis of tumor cells, and simultaneously inhibit tumor cell migration and invasion. These polyphenols can be used to prepare functional foods, health foods, or candidate drugs that inhibit the proliferation of liver cancer cells and / or breast cancer cells, providing a novel natural raw material source for the development of related products.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention employs a multi-stage homogenization and directional extraction method using pre-cooled acetone aqueous solution to extract free polyphenols from the pomace of Prickly Pear. The process conditions are mild, and the operation is simple and controllable. The extraction solvent, acetone, can be recycled and reused, enabling the directional dissolution of free active polyphenols in the pomace. This avoids the shortcomings of traditional mixed extraction processes, which make it difficult to separate free / bound polyphenols downstream. It fully exploits the active resources of Prickly Pear processing by-products, resulting in high raw material utilization and good potential for industrial-scale production.

[0015] 2. The present invention extracts free polyphenols from the pomace of Prickly Pear, which specifically enriches characteristic active components such as EGCG, (+)-gallic catechin, and quercetin glycoside. The cell absorption rate of this free polyphenol can reach 42.31%, which has a higher cell internalization capacity and better bioavailability compared with the free polyphenols from ordinary pomace pomace.

[0016] 3. In vitro cell experiments confirmed that the free polyphenols in the prickly pear pomace of this invention have excellent anti-proliferative effects on MDA-MB-231 triple-negative breast cancer cells, with a selectivity index (SI) as high as 12.12. They have good targeted inhibition effect on tumor cells and good safety. They can arrest tumor cells in the S phase, induce mitochondrial-mediated early apoptosis of tumor cells, and significantly inhibit tumor cell migration and invasion. Compared with the free polyphenols in ordinary prickly pear pomace, the anti-tumor cell proliferation, invasion and metastasis effects are more prominent, providing a novel natural raw material for the preparation of functional foods, health foods or candidate drugs that inhibit the proliferation of liver cancer cells and triple-negative breast cancer cells. Attached Figure Description

[0017] To more clearly illustrate the embodiments of the present invention, the accompanying drawings related to the embodiments are briefly described below.

[0018] Figure 1 This is a bar chart comparing the polyphenol (A) and flavonoid (B) content of total polyphenols in the prickly pear pomace of two different species.

[0019] Figure 2 This is a total ion current chromatogram of free polyphenols in the prickly pear pomace.

[0020] Figure 3 The study investigated the inhibitory effects of two types of free and bound polyphenols from prickly pear pomace on the proliferation of HepG2 cells.

[0021] Figure 4 The study investigated the inhibitory effects of two types of free and bound polyphenols from prickly pear pomace on the proliferation of MDA-MB-231 cells.

[0022] Figure 5 The image shows a PI-stained flow cytometry cell cycle histogram of the effect of free polyphenols H-FP from prickly pear pomace on the cell cycle of MDA-MB-231 cells. (A) is the blank control group, (B) is the 0.5 mg / mL H-FP treatment group, (C) is the 1 mg / mL H-FP treatment group, (D) is the 2 mg / mL H-FP treatment group, (E) is the 3 mg / mL H-FP treatment group, and (F) is the 4 mg / mL H-FP treatment group.

[0023] Figure 6 This is a stacked bar chart showing the distribution of free polyphenols H-FP from prickly pear pomace on the cell cycle of MDA-MB-231 cells.

[0024] Figure 7 This is a flow cytometry scatter plot showing the effect of free polyphenols H-FP from prickly pear pomace on apoptosis in MDA-MB-231 cells.

[0025] Figure 8 This is a bar chart showing the percentage of apoptotic cells in relation to the effect of free polyphenols H-FP from prickly pear pomace on apoptosis in MDA-MB-231 cells.

[0026] Figure 9 The effect of free polyphenols H-FP from prickly pear pomace on the migration of MDA-MB-231 cells.

[0027] Figure 10 The effect of free polyphenols H-FP from prickly pear pomace on the invasion of MDA-MB-231 cells. Detailed Implementation

[0028] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are only for explaining the present invention and do not constitute any limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0029] The prickly pear pomace and ordinary prickly pear pomace used in this invention were provided by Guizhou Debao Agricultural Technology Co., Ltd.; human liver cancer cells (HepG2) were purchased from the ATCC Resource Center; and triple-negative breast cancer MDA-MB-231 cells were purchased from Suzhou Haixing Biotechnology Co., Ltd.

[0030] Example 1: Extraction process of free polyphenols from prickly pear pomace rich in EGCG, using prickly pear pomace as raw material, including the following steps: S1. Raw material pretreatment: Dry the prickly pear fruit residue in an oven at 45℃ for 12 hours, pulverize it into powder, and then sieve it through a 40-mesh sieve to obtain prickly pear fruit residue dry powder, which is then stored in a desiccator. S2. Preparation of extraction solvent: Prepare an 80% (v / v) acetone aqueous solution as the extraction solvent and pre-cool it to 0°C for later use. S3. Multi-stage homogenization extraction: Weigh 20 g of dried prickly pear pomace powder and place it in a centrifuge tube. Add pre-cooled extraction solvent at a material-to-liquid ratio of 1:10 (m / v). After thorough mixing, homogenize using a high-speed homogenizer at 12000 r / min for 3 minutes, then centrifuge at 8000 r / min for 10 minutes and recover the supernatant. Add pre-cooled extraction solvent to the residue and repeat the above homogenization and centrifugation steps three times until the supernatant is colorless and transparent. Combine all the supernatants and transfer them to a rotary evaporator. Evaporate under reduced pressure at 45℃ to remove the extraction solvent, obtaining free polyphenols from the prickly pear pomace (denoted as H-FP). Redissolve in ultrapure water and bring the volume to 50 mL to obtain an H-FP solution with a final concentration equivalent to 400 mg / mL of dry pomace. Store at -40℃ for later use.

[0031] To compare with free polyphenols, this invention also utilizes the residue from step S3, which is used to extract polyphenols bound to the prickly pear pomace, as a control sample. Specifically: Add a 4 mol / L NaOH solution to the residue from step S3 at a ratio of 1:4 (m / v); mix thoroughly by inverting and seal, then place on a magnetic stirring platform for alkaline hydrolysis digestion for 1 hour; after hydrolysis digestion, slowly add concentrated hydrochloric acid to adjust the pH of the system to 2.0; then add an appropriate volume of ethyl acetate for extraction, shake thoroughly, and centrifuge (8000 r / min); collect the ethyl acetate layer (supernatant), and repeat the extraction process until the collected liquid is no longer colorless; evaporate the combined ethyl acetate phases to dryness under reduced pressure at 45℃ to obtain prickly pear pomace-bound polyphenols (denoted as H-BP). Redissolve in ultrapure water and bring the volume to 50 mL to obtain an H-BP solution with a final concentration equivalent to 400 mg / mL of pomace dry weight, and store at -40℃ for later use.

[0032] Extraction of total polyphenols from prickly pear pomace: Following the extraction process for free polyphenols (H-FP) and bound polyphenols (H-BP) from prickly pear pomace ...

[0033] Extraction of free polyphenols from common prickly pear pomace (comparative example) To compare with the free polyphenols in common prickly pear pomace, common prickly pear pomace was used as raw material, and the same extraction process parameters as in Example 1 were used to extract the free polyphenols in common prickly pear pomace (denoted as P-FP). The solution was reconstituted with ultrapure water and brought to a final volume of 50 mL to obtain a P-FP solution with a final concentration equivalent to 400 mg / mL of dry pomace. The solution was then frozen at -40°C for later use.

[0034] Similarly, following the same method for preparing control samples, common prickly pear pomace polyphenols (denoted as P-BP) were extracted using alkaline hydrolysis. P-FP and P-BP were then combined to obtain total polyphenols from common prickly pear pomace pomace (denoted as P-TP). After reconstitution and volume adjustment with ultrapure water, P-BP and P-TP solutions with final concentrations equivalent to 400 mg / mL of dry weight of common prickly pear pomace pomace were obtained.

[0035] Example 2: Determination of polyphenol and flavonoid content in total polyphenols (H-TP, P-TP) of two types of prickly pear pomace 1. Polyphenol content determination The quantitative analysis of polyphenols was based on the classic Folin-Ciocalteu colorimetric method. The specific procedure was as follows: 100 μL of H-TP solution, 100 μL of P-TP solution, and gallic acid standard solutions of different concentration gradients were added to a reaction tube, followed by the sequential addition of 400 μL of ultrapure water and 100 μL of Folin-Ciocalteu reagent. Since this reagent can induce redox reactions in phenols, the mixture was allowed to stand at room temperature for 6 minutes to complete the initial reaction. Subsequently, 1 mL of 7% (w / v) sodium carbonate solution was added to the reaction tube to neutralize the acidic environment, and 800 μL of ultrapure water was added to the specified volume. After vortex mixing, the reaction was carried out at room temperature in the dark for 1.5 hours, and the absorbance was then read at 760 nm. The polyphenol content was calculated based on the gallic acid (GAE) standard curve, and the results were expressed as milligrams of gallic acid equivalent per 100 grams of pomace raw material dry powder (mg GAE / 100 g DW). The results are shown below. Figure 1 As shown in (A).

[0036] 2. Determination of flavonoid content The quantification of flavonoids was performed using the SBC colorimetric method. Appropriate amounts of H-TP and P-TP solutions were measured and evaporated under nitrogen. The solutions were then redissolved in a 1:1 volume ratio tetrahydrofuran-ethanol mixture. 50 mM NaBH4, 74.6 mM AlCl3, glacial acetic acid, and 20 mM tetrachlorobenzenequinone were added dropwise to the solution sequentially. The mixture was heated at 100°C for 1 hour. After cooling, methanol, 16% vanillin colorimetric reagent, and concentrated hydrochloric acid were added. The optical density was measured at 490 nm. Catechin was used as a standard for quantification. The data were converted to milliequivalents of catechin per 100 g of dried fruit pomace powder (mg CE / 100 g DW). The results are as follows: Figure 1 As shown in (B).

[0037] Depend on Figure 1 (A) Figure 1(B) It can be seen that there is a significant difference in the accumulation level of active substances between the total polyphenols H-TP in the pomace of golden prickly pear and the total polyphenols P-TP in the pomace of common prickly pear. Among them, the total polyphenol content of H-TP is 3155.6±22.68 mg GAE / 100 g DW, and the total flavonoid content is 1981.92±58.88 mg CE / 100 g DW; significantly higher than the corresponding indicators of total polyphenols P-TP in the pomace of common prickly pear (the total polyphenol content of P-TP is 2161.01±18.95 mg GAE / 100 g DW, and the total flavonoid content is only 712.36±71.68 mg CE / 100 g DW), and the total polyphenol content of H-TP is about 1.5 times that of P-TP. Further component analysis revealed that the difference in active ingredient content between the two mainly stemmed from the free polyphenol components: the free polyphenol H-FP content in the pomace of *Pyracantha fortuneana* reached 2596.19 mg GAE / 100 g DW, accounting for approximately 80% of the total H-TP polyphenols, significantly higher than the 1635.48 mg GAE / 100 g DW of the free polyphenol P-FP in *Pyracantha fortuneana*. The distribution pattern of total flavonoids was completely consistent with that of total polyphenols, with the free flavonoid content in the pomace of *Pyracantha fortuneana* reaching as high as 1550.97 mg CE / 100 g DW, while the free flavonoid content in *Pyracantha fortuneana* was only 284.17 mg CE / 100 g DW. This confirms that the pomace of *Pyracantha fortuneana* possesses a stronger capacity for secondary metabolic accumulation of polyphenols and flavonoids, and the abundance of active substances in its free polyphenol components is far superior to that of *Pyracantha fortuneana*, providing a material basis for the excellent anti-tumor proliferation activity of the free polyphenols in the pomace of *Pyracantha fortuneana* in this invention.

[0038] Example 3: Determination of Phytochemical Components of Free Polyphenols in Two Types of Prickly Pear Fruit Pomace Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) was used to accurately identify and relatively quantify the polyphenol components in H-FP and P-FP solutions, respectively.

[0039] The liquid phase elution program is configured as follows: 1) Positive ion mode mobile phase: Phase A is a 0.1% formic acid aqueous solution containing 5 mmol / L ammonium acetate, and Phase B is a 0.1% formic acid-acetonitrile system; 2) The mobile phase in negative ion mode is consistent with that in positive ion mode; 3) Gradient elution settings: Initially, from 0 to 1.5 min, phase B was maintained at 2%; from 1.5 to 5.0 min, the phase B concentration linearly increased to 50% and remained at this concentration until min 9.0; subsequently, within 1 min (9.0 to 10.0 min), phase B rapidly increased to 98% and remained at this concentration for 1 minute; from 10.0 to 11.0 min, phase B decreased stepwise back to 2%, and the system was equilibrated at this ratio until min 14. The system flow rate was kept constant at 0.35 μL / min, the column temperature was controlled at 40℃, and the single injection volume was set to 4 μL.

[0040] The total ion chromatograms of free polyphenols in the prickly pear pomace are as follows: Figure 2 As shown. To further clarify the types of compounds corresponding to the chromatographic peaks and the differences in the relative content of components, the chromatographic peak areas of the total ion current chromatogram were normalized, and qualitative and relative quantification were completed by comparing with the database; detailed qualitative and relative quantification results are shown in Table 1 (the data in the table are the relative quantitative values ​​of peak area in UPLC-MS / MS; a value of 10.01 indicates that the component is below the instrument detection limit).

[0041] Combination Figure 2 The total ion chromatogram and the component quantification results in Table 1 show that the metabolic focus of free polyphenols H-FP in the biosynthetic pathway of flavonoids is significantly shifted between that in common prickly pear pomace and that in common prickly pear pomace, P-FP. The component characteristics are significantly different. Compared with common prickly pear P-FP, the abundance of highly active polyphenol components such as catechin, (+)-gallic acid catechin, (-)-epigallocatechin gallate, isoquercitrin, and quercetin-3-O-sophoroside in H-FP is significantly increased. Moreover, (+)-gallic acid catechin, (-)-epigallocatechin gallate, and quercetin-3-O-sophoroside are below the instrument detection limit and almost undetectable in P-FP, which are the characteristic active components unique to H-FP. Among them, the (-)-epigallocatechin gallate ester highly enriched in H-FP has a special galloyl group, which can significantly enhance the electron-donating capacity of the polyphenol system and endow the product with bioactivity far exceeding that of ordinary prickly pear free polyphenols. At the same time, the glycosylation modification characteristics of the two prickly pear pomace free polyphenols are significantly different. The quercetin-like substances in H-FP mainly exist in glycosylated bound forms such as isoquercitrin, quercetin 3-O- derivatives, and quercetin 7-O- derivatives, while P-FP mainly consists of free quercetin aglycones with extremely poor water solubility and stability, containing only a small amount of 7-O- derivatives. The glycosylation modification at the C-3 position of quercetin can greatly improve the water solubility and chemical stability of polyphenol molecules, increasing the bioavailability to more than twice that of free aglycones.

[0042] In summary, the H-FP of this invention not only significantly outperforms ordinary prickly pear free polyphenol products in terms of overall polyphenol accumulation, but also exhibits differentiated characteristics in its metabolic pathway, centered on the enrichment of highly active EGCG components and efficient glycosylation modification. Through its unique component structure advantages, it significantly enhances the bioavailability and pharmacological targeting activity of the product, and possesses superior functionality and high-value development potential compared to ordinary prickly pear pomace free polyphenols.

[0043] Table 1. Main polyphenolic components of free polyphenols (H-FP, P-FP) in prickly pear pomace.

[0044] Example 4 Evaluation of the inhibitory effect of polyphenol extract on tumor cell proliferation and cytotoxicity This embodiment used human hepatocellular carcinoma HepG2 cells and triple-negative breast cancer MDA-MB-231 cells as cell models to evaluate the inhibitory activity of four polyphenol extracts (H-FP, P-FP, H-BP, and P-BP) on tumor cell proliferation. Simultaneously, the cytotoxic effects of the polyphenol extracts were investigated using the MTT assay for dose-response relationship. Further cell cycle, apoptosis, cell scratch, and Transwell cell invasion assays were conducted to explore the mechanisms by which the polyphenol extracts act on tumor cells. Specific experimental methods and results are as follows: 1. Cell Culture Cell resuscitation: Tumor cell lines (human hepatocellular carcinoma HepG2 cells and triple-negative breast cancer MDA-MB-231 cells) cryopreserved in liquid nitrogen were rapidly thawed by agitation in a 37°C water bath. Cell suspension was slowly added to sterile centrifuge tubes pre-filled with 2–3 mL of complete culture medium (500 mL DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin mixture). After gentle repositioning, the cells were centrifuged at low speed to remove DMSO-containing cryopreservation waste. The cell pellet was resuspended in fresh complete culture medium and seeded into T25 culture flasks, which were then incubated at 37°C in a 5% CO2 incubator for routine culture.

[0045] Cell cryopreservation: Select tumor cells in the logarithmic growth phase, with intact morphology and good survival rate. After routine trypsin digestion and centrifugation, collect the cell pellet, gently resuspend it in a special cell cryopreservation solution, aliquot it into cryovials, and label the cell line name, passage number and cryopreservation date. After a stepwise cooling process, transfer it to a liquid nitrogen tank for storage.

[0046] Cell passage: When cells in the T75 culture flasks achieve 80%–90% adherence and confluence, and the cells appear plump and well-formed under a microscope, passage is performed. Discard the original culture medium, gently wash the cell surface with sterile PBS buffer, aspirate any residual liquid, and add 2 mL of trypsin solution to completely cover the culture substrate. Digest at room temperature for 1–3 minutes. Once the cells are observed to shrink and round under a microscope, immediately add serum-containing complete culture medium to terminate the digestion reaction. Collect the cell suspension, centrifuge, resuspend the cell pellet, and seed the cells into brand-new T75 culture flasks according to their proliferation status and subsequent experimental requirements. Incubate in a constant temperature incubator for further culture.

[0047] 2. MTT cytotoxicity test The potential toxicity of four polyphenol extracts to HepG2 and MDA-MB-231 cells was detected using the MTT assay kit (Nanjing Jiancheng Bioengineering Institute), and the safe dosing concentration range for subsequent functional experiments was screened.

[0048] Cells were seeded in 96-well clear white plates and cultured routinely until cell adhesion was achieved for 24 h. The original culture medium was then discarded, and culture medium containing different concentration gradients of H-FP, P-FP, H-BP, and P-BP polyphenol extracts were added. A blank control group and a negative control group were also included. Incubation continued for another 24 h. After incubation, the culture medium was discarded, cells were washed with PBS buffer, and complete culture medium containing MTT reagent was added. Cells were stained at 37 ℃ in the dark for 4 h. The supernatant was discarded, and DMSO reagent was added to each well to dissolve the purple formazan crystals. The OD value of each well was measured at 490 nm, and cell viability curves were plotted. The half-maximal toxicity concentration (CC) of each polyphenol extract sample was calculated. 50 (mg / mL), according to CC 50 Establish a safe dosing range; the highest dosing concentration in subsequent functional studies should not exceed 10% of the lethal dose (CC). 10 This ensures that the dosage concentration is within a safe range without significant cytotoxicity.

[0049] 3. Evaluation of the antiproliferative activity of polyphenol extracts against tumor cells Tumor cells in logarithmic growth phase were collected after trypsin digestion. Cell counting was performed under a microscope, and the cell suspension was seeded into 96-well culture plates. The plates were incubated at 37°C for 4 hours until complete cell adhesion was achieved. The original culture medium was discarded and replaced with fresh medium containing a series of polyphenol extracts at varying concentrations for drug intervention. Incubation continued for 72 hours. The culture plates were then subjected to MTT assay. Cell viability was calculated based on the measured optical density values. A dose-response curve was fitted, and the half-maximal inhibitory concentration (MCC) was calculated. 50 (mg / mL) and the selectivity index (SI=CC)50 / EC 50 The result is as follows Figure 3 , Figure 4 As shown in Tables 2 and 3.

[0050] Table 2. Indicators of the antiproliferative activity of polyphenol extracts against human hepatocellular carcinoma HepG2 cells

[0051] Indicators of the antiproliferative activity of polyphenol extracts on triple-negative breast cancer MDA-MB-231 cells

[0052] Depend on Figure 3 As shown in Table 2, all four polyphenol extracts exhibited in vitro anti-proliferative activity against HepG2 liver cancer cells. The selectivity index (SI) of each polyphenol extract sample was greater than 2, indicating that the proliferation inhibition effect did not originate from non-specific cytotoxicity. Among them, the EC50 value of H-FP was 0.43±0.04 mg / mL, which was significantly lower than that of the other three polyphenol extracts, demonstrating the strongest in vitro anti-HepG2 cell proliferation activity; however, the SI value of H-FP was 3.96±0.04, which was the lowest among the four samples, suggesting that its targeting selectivity for HepG2 cells was weaker than that of the other three samples.

[0053] Depend on Figure 4 As shown in Table 3, within the non-toxic concentration range, CC 10 Within the cell line, the half-maximal inhibitory concentration (IC50) of H-FP against MDA-MB-231 cells was EC50. 50 The concentration was only 2.21 ± 0.18 mg / mL; more importantly, its selectivity index (SI) was as high as 12.12 ± 1.14, significantly better than other polyphenol extracts (SI values ​​ranged from 2.5 to 6.1), demonstrating excellent inhibitory ability on the proliferation of MDA-MB-231 cells. Compared with the HepG2 cell model (where the SI value of H-FP was only 3.96), H-FP has the dual advantages of "low cytotoxicity" and "high targeting anti-proliferative potential" on MDA-MB-231 cells.

[0054] 4. Effects of H-FP on the cell cycle of MDA-MB-231 cells Cell cycle analysis was performed using a cell cycle and apoptosis detection kit (Beyotime). MDA-MB-231 cells were seeded in 6-well plates. After 24 hours of stable cell attachment, the medium was replaced with complete culture medium containing different concentration gradients (0.5, 1, 2, 3, 4 mg / mL) of *Rhizophora rubra* pomace free polyphenol extract (H-FP). A blank control group (complete medium without H-FP) was also included. The treatment continued for another 24 hours. After the drug treatment, the supernatant from each well was collected for later use. Then, trypsin without EDTA was added to digest the adherent cells for 1–2 minutes. Digestion was terminated using the collected serum-containing supernatant, and the cell pellet was collected by centrifugation.

[0055] Cell pellet was resuspended in PBS buffer. While pipetting the cells, pre-chilled anhydrous ice-cold ethanol was slowly added dropwise. After mixing, the mixture was refrigerated at 4°C overnight for fixation. The next day, the fixative was removed by centrifugation. The cell pellet was washed with pre-chilled PBS buffer to remove residual ethanol, and the supernatant was discarded after another centrifugation. Propidium iodide (PI) staining agent and RNase A enzyme were added to the cell pellet according to the kit instructions. The cells were resuspended using a pipette. The samples were incubated in a dark room at 37°C for 30 minutes. Cell cycle fluorescence signals were acquired using flow cytometry, and cell population distribution data at different cell cycles were analyzed using ModFit LT 5.0 software. The results are shown below. Figure 5 , Figure 6 As shown.

[0056] Depend on Figure 5 , Figure 6 It was found that H-FP could inhibit the cell cycle progression of MDA-MB-231 cells in the S phase, resulting in a significant decrease in the number of cells in the G1 and G2 / M phases. This trend was more pronounced in the high-concentration treatment group, showing a clear dose-dependent effect. The low-concentration group had a smaller impact on cell cycle distribution; however, as the treatment concentration increased to medium and high levels, the proportion of cells in the G1 and G2 / M phases decreased to varying degrees compared to the control group. Specifically, after treatment with 4 mg / mL H-FP, the proportion of cells in the G1 phase decreased from 53.22% in the control group to 40.55%, and the decrease in the proportion of cells in the G2 / M phase was particularly significant, sharply decreasing from 10.79% to 2.53%. At the same time, the proportion of cells in the S phase increased significantly from 35.99% in the control group to 54.89%. When cells are arrested in the S phase, DNA cannot be further replicated and transmitted, resulting in a decrease in the proportion of cells entering the G2 / M phase, affecting the effectiveness of cellular genetic material transmission, and exhibiting an anti-proliferative effect.

[0057] 5. Effects of H-FP on apoptosis in MDA-MB-231 cells The Annexin V-FITC / PI dual staining kit (Nanjing Jiancheng Biotechnology Institute) was used to quantitatively assess apoptosis levels. Cell plating, sample administration, and cell digestion and harvesting procedures were consistent with cell cycle analysis. Drug concentrations were set at 0.5, 2, and 4 mg / mL. After harvesting, the viable cell pellet was not fixed with ethanol; it was centrifuged, the supernatant discarded, and then binding buffer, Annexin V-FITC dye, and PI dye were added sequentially according to the kit's recommended ratio. After thorough mixing, the mixture was incubated at room temperature in the dark for 10 minutes. Flow cytometry was then used for analysis, with an excitation wavelength of 488 nm and an emission wavelength of 530 nm to capture the green fluorescence signal of FITC and the red fluorescence signal of PI, respectively. The data were analyzed using FlowJo 10.8.1 software to create an apoptosis quadrant scatter plot. The results are shown below. Figure 7 , Figure 8 As shown in the figure. Apoptosis analysis results showed that H-FP mainly inhibited the proliferation of MDA-MB-231 cells by inducing early apoptosis. With increasing H-FP concentration, the proportion of early apoptosis showed a significant dose-dependent effect, at 2.08%, 4.09%, 7.65%, and 25.4%, respectively; the highest concentration group (4.0 mg / mL) showed an induction effect approximately 6 times that of the low concentration group (0.5 mg / mL). Flow cytometry scatter plot ( Figure 7 The study clearly demonstrated a significant shift in the cell population from the Q4 quadrant (viable cell zone) to the Q3 quadrant (early apoptosis zone). These results indicate that induced programmed cell death is one of the core mechanisms by which H-FP exerts its anti-breast cancer effect. Apoptosis, as a gene-regulated autonomous cell death process, plays a crucial role in maintaining homeostasis and inhibiting tumorigenesis. The potent pro-apoptotic activity exhibited by H-FP is largely attributed to its highly enriched (-)-epigallocatechin gallate (EGCG). EGCG, as the core functional component of H-FP, effectively inhibits the malignant proliferation of MDA-MB-231 cells by strongly triggering multiple apoptotic pathways.

[0058] 6. Effects of H-FP on the migration of MDA-MB-231 cells The effect of H-FP, a free polyphenol extract from *Pyracantha fortuneana* pomace, on the migration of MDA-MB-231 cells was determined using a cell scratch assay. The cell scratch assay was performed in 6-well plates, with 6 × 10⁶ cells seeded per well. 5Tumor cells were collected; when the cell confluence reached 90% or higher, a vertical scratch was made in each well, and the cells were washed 2-3 times with PBS buffer and the washings were aspirated. A control group and a polyphenol extract treatment group were set up, with the polyphenol extract concentration consistent with the anti-proliferative activity experiment in this embodiment. The scratched areas were photographed under an inverted microscope at 0h, 24h, and 48h treatment time points. Within the set time points, the greater the cell migration distance (i.e., the larger the proportion of wound healing area), the stronger its migration ability. The experiment used ImageJ software to quantitatively analyze the scratch area, and the wound healing rate was calculated by comparing it with the initial (0h) state. The results are as follows: Figure 9 As shown.

[0059] Depend on Figure 9 Data confirmed that H-FP effectively inhibited the cell healing process at both 24 h and 48 h time points. Specifically, after 24 h of intervention with different concentrations of H-FP, the cell healing rate was limited to 42%, 25%, 18%, 10%, and 6% of the control group, respectively; by 48 h, the healing rates of each dose group were further reduced to 22%, 11%, 14%, 11%, and 7% of the control group. This result suggests that medium and high concentrations of H-FP can almost completely block the migration trajectory of MDA-MB-231 cells, exhibiting extremely excellent anti-migration activity.

[0060] In the field of tumor biology, cell migration is a crucial biological step for tumor cells to break through the extracellular matrix (ECM) and achieve distant dissemination; clinically, approximately 90% of cancer deaths are attributed to tumor metastasis. This invention reveals that H-FP's strong inhibitory effect on the migration of highly invasive MDA-MB-231 cells is primarily attributed, based on its highly enriched (-)-epigallocatechin gallate (EGCG). Simultaneously, the abundant ellagic acid (EA) in H-FP also plays a vital synergistic role in the anti-migration network. Therefore, the superior anti-migration activity exhibited by H-FP is essentially the result of the combined intervention of its core component EGCG (dominant) and ellagic acid (synergistic). This "multi-target synergistic effect" of natural polyphenols effectively blocks the metastatic potential of breast cancer cells.

[0061] 7. Effects of H-FP on MDA-MB-231 cell invasion The effect of H-FP, a free polyphenol extract from *Pyracantha fortuneana* pomace, on the invasion of MDA-MB-231 cells was determined using a Transwell cell invasion assay. Well-grown tumor cells were starved for 16 h using serum-free medium to eliminate the interference of serum growth factors on cell invasion ability. Cell suspension was added to the upper chamber of a pre-coated Substrate-coated Transwell plate; the lower chamber of a 24-well plate was filled with medium containing 20% ​​fetal bovine serum. A control group and a polyphenol extract experimental group were established, with the drug concentration consistent with the anti-proliferative activity experiment. Cells were cultured for 48 h. After culture, the Transwell chambers were washed with PBS buffer, cells were fixed with 4% paraformaldehyde at room temperature, and washed again with PBS buffer; staining was performed with 0.1% crystal violet at room temperature for 15–20 min, the chambers were washed, and residual cells inside the upper chamber were wiped away with cotton swabs; images were taken under an inverted microscope, and data were analyzed using ImageJ software.

[0062] Transwell invasion assays showed that H-FP exhibited a highly significant concentration-dependent inhibitory effect on the ability of MDA-MB-231 cells to penetrate the basement membrane, such as... Figure 10 As shown in the figure, quantitative analysis revealed that with increasing H-FP intervention concentration, the number of successfully penetrating cells decreased significantly in a stepwise manner from 170.2 in the control group, with the relative invasion rates of each dose group being only 64%, 42%, 37%, 17%, and 8% of the control group, respectively. Especially under high-concentration intervention, the invasive potential of cells was almost completely suppressed. Given the extremely high invasiveness and poor clinical prognosis of triple-negative breast cancer (TNBC), this indicates that H-FP has significant potential application value in blocking the TNBC metastasis cascade.

[0063] This invention uses the pomace, a byproduct of prickly pear processing, as raw material and employs a multi-stage homogenization extraction process with pre-cooled acetone aqueous solution to directionally prepare free polyphenols from the pomace. The process conditions are mild, maximizing the retention of characteristic active components such as EGCG, resulting in high raw material utilization and promising prospects for industrial application. The obtained free polyphenols from the pomace have unique components that can significantly inhibit the proliferation of HepG2 and MDA-MB-231 tumor cells. They exert their effects through multiple pathways, including inducing S-phase cell cycle arrest, promoting early apoptosis of tumor cells, and inhibiting tumor cell migration and invasion. This provides important technical support for the high-value utilization of pomace pomace byproducts and the development of raw materials for functional foods, health foods, and anti-tumor drug candidates.

[0064] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the protection scope of the present invention.

Claims

1. An extraction process for free polyphenols from prickly pear pomace rich in EGCG, characterized in that, Includes the following steps: S1. Raw material pretreatment: The prickly pear fruit residue is dried at low temperature, crushed and sieved to obtain prickly pear fruit residue dry powder for later use. S2. Preparation of extraction solvent: Prepare an acetone aqueous solution with a volume fraction of 75-85% as the extraction solvent, and pre-cool it to 0-10℃ for later use. S3. Multi-stage homogenization extraction: Add the dry powder of golden prickly pear pomace to a centrifuge tube, and add pre-cooled extraction solvent at a mass-volume ratio of 1:8 to 1:12; after mixing evenly, perform homogenization treatment, then centrifuge and collect the supernatant; repeat the homogenization and centrifugation extraction operation on the residue until the supernatant is colorless and transparent, combine all the supernatants, evaporate under reduced pressure to remove the extraction solvent, and obtain free polyphenols from the golden prickly pear pomace pomace.

2. The extraction process according to claim 1, characterized in that: The low-temperature drying temperature in step S1 is 40-50℃; the sieving is done through a 40-60 mesh sieve.

3. The extraction process according to claim 1, characterized in that: The homogenization speed in step S3 is 10000-14000 r / min, and the homogenization time is 2-5 min; the centrifugation speed is 7000-9000 r / min, and the centrifugation time is 8-12 min.

4. The extraction process according to claim 1, characterized in that: The temperature for vacuum evaporation in step S3 is 40–60°C.

5. A free polyphenol from the pomace of *Pyracantha fortuneana* rich in EGCG, characterized in that: The product is obtained by extraction process according to any one of claims 1 to 4, and contains (-)-epigallocatechin gallate, (+)-gallocatechin, isoquercitrin, quercetin 3-O-sophoroside, quercetin 7-O-glucoside, catechin and ellagic acid.

6. The use of the free polyphenols in the prickly pear pomace rich in EGCG as described in claim 5 in inhibiting the proliferation of liver cancer cells and / or breast cancer cells.

7. The application according to claim 6, characterized in that: The free polyphenols in the prickly pear pomace are used to prepare functional foods, health foods, or candidate drugs that inhibit the proliferation of liver cancer cells and / or breast cancer cells.

8. The application according to claim 6 or 7, characterized in that: The liver cancer cells are HepG2 liver cancer cells; the breast cancer cells are triple-negative breast cancer MDA-MB-231 cells.

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