Peony polyphenol extract as well as preparation method and application thereof

Abstract

The invention discloses a peony polyphenol extract as well as a preparation method and application thereof, and relates to the technical field of biological medicines. Dried flowers of paeonia ostii are used as raw materials, peony polyphenol powder is obtained through an ethanol extraction-pore resin purification method, and the polyphenol content of the peony polyphenol powder is larger than 80% through determination of a folin-phenol method. Paeonia suffruticosa polyphenol is rich in more than 30 kinds of main active ingredients, and comprises flavonoids, phenolic acids and polyphenol derivatives, and flavonoids and phenolic acid compounds account for 81.25% of the total number of identified ingredients. The peony polyphenol plays a role in resisting cervical cancer through double ways of inducing HeLa cell apoptosis and activating HeLa cell autophagy, and the problem of drug resistance of a single-target drug can be effectively solved; the compound has a good application value in anti-cancer drugs. Meanwhile, the peony polyphenol shows excellent antioxidant activity in various free radical models, remarkably inhibits ROS (reactive oxygen species) generation at a cellular level, relieves oxidative stress, has a cell protection effect, and can be used as a natural antioxidant and a cell protection agent.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a new use of peony polyphenol, and especially to a preparation method of peony polyphenol and application of the peony polyphenol in preparation of an anti-cervical cancer drug. BACKGROUND

[0002] Cervical cancer is one of the malignant tumors with high incidence in the female reproductive system worldwide, and its incidence and mortality rate rank in the front row among female malignant tumors, seriously threatening the life and health of women. At present, the main means for treating cervical cancer in clinic include surgical resection, radiotherapy, chemotherapy and targeted therapy, but traditional chemotherapeutic drugs (such as cisplatin and paclitaxel) have problems such as great toxic and side effects (such as bone marrow suppression, gastrointestinal reactions, liver and kidney function damage), single target, easy drug resistance, etc., resulting in low treatment compliance of patients and limited prognosis effect.

[0003] Plant polyphenols are a class of secondary metabolites widely existing in plant bodies, and have a polyhydroxy structure. Their biological activities have been confirmed by a large number of studies, including antioxidant, anti-inflammatory, antibacterial and antitumor activities. Compared with chemically synthesized drugs, plant polyphenols have the significant advantages of natural source, low toxicity and multi-target regulation, and have become an important direction for the research and development of natural anticancer active ingredients. For example, green tea polyphenol (EGCG) can play an antitumor effect by inhibiting the proliferation of lung cancer and breast cancer cells, and grape seed polyphenol and pomegranate polyphenol have also been confirmed to regulate the oxidative stress and apoptosis process of tumor cells.

[0004] Paeonia suffruticosa Andr. is a deciduous shrub of Ranunculaceae Paeonia, and is a rare woody ornamental plant unique to China, with a natural growth history of thousands of years and a history of artificial cultivation of more than two thousand years, and is known as the "King of Flowers" and "National Beauty". Paeonia suffruticosa is a treasure, and has important medicinal and edible values in addition to its high ornamental value. The medical bamboo slips unearthed in Gansu Wuwei of the Eastern Han Dynasty have recorded the treatment of blood stasis disease by Paeonia suffruticosa. Traditional medical classics such as Shennong's Herbal Classic, Pearl Casket, Qianjin Fang (Hua Tuo), Treatise on Febrile Diseases, Dian Nan Ben Cao, Tang Ben Cao, Compendium of Materia Medica, Treatise on the Classics of Materia Medica, Chongqing Hall Notes, and Compendium of Materia Medica have demonstrated the efficacy of Paeonia suffruticosa in unblocking the heart, liver and kidney channels, regulating blood and qi, activating meridians, removing turbid evil, storing essence and suppressing evil, nourishing the five viscera and prolonging life.

[0005] Among them, Paeonia ostii T. Hong et JX Zhang is an important oil peony variety. Its seeds can be used for oil extraction, and the resulting seed meal and dried peony flowers are rich in polyphenols, polysaccharides, and other active ingredients. Existing research shows that peony polyphenols have good antioxidant, hypoglycemic, and anti-inflammatory activities. However, research on their inhibitory effects on human cervical cancer HeLa cells, especially their specific molecular mechanisms and clinical application potential, in the field of oncology remains lacking. At the same time, the high-value utilization pathways of oil peony by-products (seed meal and withered flowers) are not yet clear, leading to serious waste of peony resources.

[0006] Against this backdrop, there is an urgent need in this field to develop an active ingredient for cervical cancer that is naturally derived, has low toxicity, and has a clear mechanism of action, while simultaneously achieving efficient utilization of peony resources, thus providing new ideas for the development of novel anti-cervical cancer drugs. Summary of the Invention

[0007] To address the aforementioned problems in the existing technology, the technical problem to be solved by the present invention is to provide a method for preparing peony polyphenol extract, thereby obtaining the peony polyphenol extract. Another technical problem to be solved by the present invention is to provide the application of peony polyphenol extract in the preparation of anticancer drugs, antioxidants, and cell protectants.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0009] The specific steps for preparing peony polyphenol extract are as follows:

[0010] 1) Crush the dried flowers of Danfeng peony into powder and set aside;

[0011] 2) Add an ethanol-water solution to the raw material powder, reflux in a constant temperature water bath several times to extract, and filter through gauze to remove the residue to obtain the extract;

[0012] 3) Purify the extract using macroporous resin;

[0013] 4) The purified eluent was concentrated under reduced pressure until no alcohol odor was detected, and the concentrate was freeze-dried to obtain a light yellow peony polyphenol powder.

[0014] In step 1), the dried flowers of Danfeng peony are pulverized and passed through a 40-mesh sieve for later use.

[0015] In step 2), the volume concentration of the ethanol-water solution is 70%, and the extraction is carried out twice under constant temperature water bath at 60℃ for 2 hours each time. The two extracts are then combined.

[0016] In step 3), the macroporous resin is type D101 macroporous resin.

[0017] In step 3), the specific process is as follows:

[0018] Resin pretreatment: D101 macroporous resin was soaked in 95% ethanol for 24 hours, and then rinsed with 95% ethanol and distilled water in sequence until there was no alcohol odor.

[0019] Sample loading: The extract was slowly passed through a pretreated macroporous resin column at a flow rate of 2 BV / h.

[0020] Elution: First, elute with 2 BV distilled water at a flow rate of 2 BV / h, then elute with 2 BV 30% ethanol at a flow rate of 2 BV / h, and finally elute with 3 BV 70% ethanol at a flow rate of 1.5 BV / h. Collect the 70% ethanol eluent.

[0021] In step 4), the 70% ethanol eluent is placed in a rotary evaporator under reduced pressure and concentrated at 50°C and -0.08 MPa until there is no alcohol odor, thus obtaining a concentrated solution.

[0022] In step 4), the concentrate is transferred to a freeze dryer and freeze-dried at -50°C and 0.1 Pa for 48 hours to obtain a light yellow peony polyphenol powder.

[0023] The peony polyphenol powder obtained by the method described above is a peony polyphenol extract preparation method.

[0024] The application of the peony polyphenol powder in the preparation of anti-cervical cancer drugs.

[0025] The application of the peony polyphenol powder in the preparation of antioxidants and / or cell protectants.

[0026] Beneficial effects: Compared with the prior art, the outstanding advantages of the present invention are as follows:

[0027] 1) This application uses dried flowers of Paeonia ostii T. Hong et JX Zhang as raw material, and obtains peony polyphenol powder by ethanol extraction-porous resin purification method. The polyphenol content is determined to be >80% by Folin-phenol method.

[0028] 2) Through identification, the peony polyphenols prepared in this application are rich in more than 30 major active ingredients, covering three major categories: flavonoids, phenolic acids and polyphenol derivatives. Among them, flavonoids and phenolic acids account for 81.25% of the total identified ingredients, which have extremely high utilization value.

[0029] 3) The embodiments of this application demonstrate that peony polyphenols exert their anti-cervical cancer effect through a dual pathway of "inducing HeLa cell apoptosis + activating HeLa cell autophagy." The specific molecular mechanisms are as follows: Inhibition of the mTOR / 4E-BP signaling pathway: reducing the protein expression levels of phosphorylated mTOR (p-mTOR) and phosphorylated 4E-BP (p-4E-BP), blocking cell growth and protein synthesis pathways; Activation of autophagy: upregulating the expression of autophagy marker proteins LC3 A / B, promoting the formation of autophagosomes, and accelerating the degradation of damaged mitochondria and misfolded proteins; Activation of the apoptosis cascade: upregulating the expression of pro-apoptotic proteins PARP (polyadenosine diphosphate ribose polymerase) and Cleaved Caspase-3 (activated caspase-3), inducing programmed cell death in HeLa cells. Therefore, the prepared peony polyphenols can effectively overcome the drug resistance problem of single-target drugs and have significant application value in anticancer drugs.

[0030] 4) The embodiments of this application demonstrate that peony polyphenols exhibit excellent antioxidant activity in various free radical models, and their antioxidant capacity is comparable to that of traditional tea polyphenols, and even slightly superior in some indicators. This provides experimental support for the development of peony polyphenols as a natural antioxidant / functional antioxidant ingredient in the food, health product, or pharmaceutical fields.

[0031] 5) The embodiments of this application demonstrate that peony polyphenols can significantly inhibit ROS generation at the cellular level, reduce oxidative stress, and have a cell-protective effect. This further enhances the application value of peony polyphenols as a functional cell protectant. Attached Figure Description

[0032] Figure 1 This is a graph showing the inhibitory effect of peony polyphenols on the proliferation of HeLa cells; in the graph, the horizontal axis represents the concentration of peony polyphenols, and the vertical axis represents the cell viability; different lowercase letters indicate significant differences, P < 0.05;

[0033] Figure 2 This is a diagram showing the morphological changes of HeLa cells after treatment with peony polyphenols; in the diagram, 1: control group, 2: 25.00 μg·mL -1 Group 3: 50.00 μg·mL -1 Group 4: 75.00 μg·mL -1 Group;

[0034] Figure 3 This is an Annexin V / PI staining image of HeLa cell nuclear morphology changes induced by peony polyphenols; in the image, 1: control group, 2: 25.00 μg / mL. -1 Group 3: 50.00 μg·mL -1 Group 4: 75.00 μg·mL -1Group 1, green fluorescence is Annexin V-FITC, red fluorescence is PI;

[0035] Figure 4 This is a flow cytometry result of the effect of peony polyphenols on HeLa cell apoptosis; in the figure, 1: control group, 2: 25.00 μg·mL -1 Group 3: 50.00 μg·mL -1 Group 4: 75.00 μg·mL -1 The lower right quadrant represents early apoptotic cells, and the upper right quadrant represents late apoptotic cells; different lowercase letters indicate significant differences, P < 0.05;

[0036] Figure 5 This is a graph showing the effect of peony polyphenols on autophagy in HeLa cells; in the graph, the horizontal axis represents the concentration of peony polyphenols, and the vertical axis represents the average fluorescence intensity of MDC; different lowercase letters indicate significant differences, P < 0.05;

[0037] Figure 6 This is a graph showing the effect of the combined use of 3-MA and peony polyphenols on autophagy in HeLa cells; in the graph, A: control group, B: 3-MA group, C: peony polyphenol group, D: 3-MA + peony polyphenol group, and the vertical axis represents the average fluorescence intensity of MDC; different lowercase letters indicate significant differences, P < 0.05.

[0038] Figure 7 This is a flow cytometry result of the combined use of 3-MA and peony polyphenols to induce apoptosis in HeLa cells; in the figure, 1: control group, 2: 3-MA group, 3: 3-MA + peony polyphenol group, 4: peony polyphenol group; different lowercase letters indicate significant differences, P < 0.05;

[0039] Figure 8 This is a Western blot result of the effect of peony polyphenols on the expression of autophagy and apoptosis-related proteins in HeLa cells; in the figure, lane 1: control group, lane 2: 25.00 μg / mL -1 Group, Lane 3: 50.00 μg·mL -1 Group, Lane 4: 75.00 μg·mL -1 Group. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art. Alternatively, they may be carried out according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0041] The main reagents and instruments used in the following examples are: ethanol (analytical grade, Sangon Biotech (Shanghai) Co., Ltd.), D101 macroporous resin (Nankai University Chemical Plant, Tianjin), Folin-phenol reagent (Beyotime Biotechnology Co., Ltd., Shanghai), gallic acid standard (purity ≥98%, Sigma-Aldrich), food-grade liquid CO2 (purity ≥99.9%), and supercritical fluid extraction apparatus (HA421-40-96, Jiangsu Huaan Scientific Instruments Co., Ltd.).

[0042] The main raw material used in the following examples is: dried flowers of Paeonia ostii, collected from Jiangsu Province and identified by the Jiangsu Institute of Botany, Chinese Academy of Sciences.

[0043] Example 1: Preparation of Danfeng Peony Polyphenols

[0044] 1. Raw material pretreatment: Crush the dried flowers of Danfeng peony into powder, pass through a 40-mesh sieve, and take 200g of raw material powder for later use;

[0045] 2. Ethanol extraction: Add 2400 mL of 70% ethanol aqueous solution to the raw material powder, and reflux in a constant temperature water bath at 60℃ twice, 2 hours each time. Combine the two extracts and filter through gauze to remove the residue.

[0046] 3. Purification with macroporous resin:

[0047] Resin pretreatment: D101 macroporous resin was soaked in 95% ethanol for 24 hours, and then rinsed with 95% ethanol and distilled water in sequence until there was no alcohol odor.

[0048] Sample loading: Slowly pass the extract through a pretreated macroporous resin column (10 cm in diameter and 50 cm in height) at a flow rate of 2 BV / h (BV is column volume).

[0049] Elution: First, elute with 2 BV of distilled water at a flow rate of 2 BV / h, then elute with 2 BV of 30% ethanol at a flow rate of 2 BV / h, and finally elute with 3 BV of 70% ethanol at a flow rate of 1.5 BV / h. Collect the 70% ethanol eluent.

[0050] Concentration and drying: The 70% ethanol eluent was placed in a rotary evaporator under reduced pressure and concentrated at 50℃ and -0.08MPa until no alcohol odor was detected, to obtain a concentrated solution; the concentrated solution was transferred to a freeze dryer and freeze-dried at -50℃ and 0.1Pa for 48h to obtain a light yellow peony polyphenol powder;

[0051] 4. Content determination: The polyphenol content of peony polyphenol powder was determined by using the Folin-phenol method with gallic acid as the standard to draw a standard curve. The results showed that the polyphenol content was 83.24%.

[0052] Example 2 Identification of the main active ingredients in peony polyphenol extract

[0053] 1. Instruments, reagents, and raw materials

[0054] Instruments: 1% electronic balance, Shanghai Minqiao SL-202; KH-500DB CNC ultrasonic cleaner, Kunshan Hechuang Ultrasonic Instrument Co., Ltd.; rotary evaporator, Gongyi Yuhua YRE-2000E; circulating water vacuum pump, Zhengzhou Great Wall SHB-III; pure water system Elixir Essential 5UV / ultrapure water system Synergy UV pure water and ultrapure water system, Merck Millipore, Germany; liquid chromatography-mass spectrometry (LC-MS) system Agilent 1290 Infinity II UPLC-DAD-6546 ESI-QTOF MS; including the following components: G7120A, 1290 High Speed ​​Pump; G7129B, 1290 Vialsampler; G7116B, 1290 MCT; G7117A, 1290 DAD Fs; G6546A, 6546 LCMS / Q-TOF; MassHunter 11.0 workstation.

[0055] Reagents: Ethanol, Shanghai Reagent Company, Taicang, China; Methanol, Tedia Company Inc. (pesticide residue grade); Formic acid and Acetonitrile, ROE (>99%, LCMS grade); Deionized purified water (18Ω).

[0056] Raw material: Pale yellow peony polyphenol powder prepared in Example 1.

[0057] 2. Chromatographic-mass spectrometry conditions

[0058] Chromatographic conditions: Column: Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm). Mobile phase: Phase A (0.1% formic acid aqueous solution), Phase B (acetonitrile). Gradient elution program: 0-5 min 5% B; 5-30 min 5%-30% B; 30-50 min 30%-60% B; 50-60 min 60%-95% B. Column temperature: 30℃; Flow rate: 0.8 mL / min; Injection volume: 10 μL.

[0059] Mass spectrometry conditions: Ion source: Electrospray ionization source (ESI+). Scan range: m / z 100-1500. Drying gas temperature: 350℃; Drying gas flow rate: 10 L / min. Nebulizer gas pressure: 40 psi; Capillary voltage: 4000 V. Collision energy: 15-40 V (gradient collisions).

[0060] Database: TCM-database.2016.03.01.cdb.

[0061] 3. Component Identification Methods

[0062] The compounds were identified by comparing retention time (RT), precise molecular weight, and fragment ion peaks in secondary mass spectrometry, combined with searches of traditional Chinese medicine ingredient databases and literature reports. A score ≥ 85 was considered a reliable identification result.

[0063] 4. Experimental Results

[0064] LC-MS / MS analysis revealed more than 30 major active ingredients in the peony polyphenol extract prepared in Example 1, covering three major categories: flavonoids, phenolic acids, and polyphenol derivatives. Flavonoids and phenolic acids accounted for 81.25% of the total identified ingredients. Among them, hyperoside, quercetin derivatives, and caffeic acid all scored 100, indicating a very high degree of confidence in their structural confirmation. The main components identified include: 6-O-digalolic acid methyl-β-D-glucoside (II), 3,4,6-trigalolic acid methyl-β-D-glucoside, 2,3,4,6-tetragalolic acid methyl-β-D-glucoside, shikimic acid, cinnamic acid, β-phenylacrylic acid, caffeic acid β-D-glucoside, caffeic acid, p-hydroxycinnamic acid, myricetin glucoside, m-methyl-p-hydroxycinnamic acid, ferulic acid, ethyl p-digalolic acid, ethyl cinnamate, ellagic acid, p-digalolic acid, o-benzylbenzoic acid, sodium ferulic acid, syringin-3-O-robenzin, syringin-3-O -β-D-glucoside, sese fruit flavonoids, quercetin, quercetin-2''-gallate, quercetin-5,7,4'-tri-O-β-D-glucopyranoside, quercetin-3-O-α-L-arabinopyranoside-(1-->6)-[2''-O-(E)-p-coumarate]-β-D-glucopyranoside, quercetin-3-O-[α-rhamnopyranoside-(1-->4)-α-rhamnopyranoside-(1-->6)-β-glucopyranoside], quercetin-3-O-(2''-O-α-rhamnoester-6''-O-malyl)-β-glucoside Glycosides, naringenin-4'-glucoside-7-rutin, myricetin-3-O-β-D-(6''-O-gallic acid)-glucopyranoside, kaempferol-3-O-chitosan trisaccharide, kaempferol-3-O-gluco-7,4'-OL-dirhamnopyranoside, kaempferol-3-arabinofuranoside, kaempferol 3-O-β-(6''-E-p-coumaryl glucopyranoside)-7-O-β-glucopyranoside, kaempferol 3-O-(2''-O-α-rhamnosyl-6''-O-malic acid)-β-glucoside, hyperoside, apigenin 7-O-β-galacturonoside Rutin, flavonoid-7-O-rhamnoside, flavonoid-7-O-diglucuronide, flavonoid-7-O-β-D-glucuronide butyl ester, flavonoid-7-O-β-D-(6'-p-hydroxycinnamoyloxy)-mannose, flavonoid-7-O-β-arabinofuranosyl-6,8-di-C-glucose, flavonoid-7-O-α-L-3-O-acetyrhamnosylpyranosyl-(1-6)-β-D-glucose, flavonoid-5-rhamnoside, flavonoid-4'-O-(2',6''-di-Op-coumaryl)-β-D-glucose, 5,7,3',5'-Tetrahydroxyflavanone-3-O-β-D-glucoside, 5,7,3',4'-Tetrahydroxy-8-methoxyflavone-3-O-β-D-galactopyranoside, 5,2',5'-Trihydroxy-6,7,8-Trimethoxyflavone, 3-O-methylquercetin-7-O-diglucoside-4'-O-glucoside, Malvaceae-3-arabinoside, Petunidin-3-glucoside, Petunidin, Pelargonin-3,5-diglucoside, Pelargonin, Delphinidin-3-arabinoside, Delphinidin-3-O-β-D-(6-(E)-p-coumaryl)galactopyranoside, Delphinidin-3'-O-(2''-O-galloyl-6''-O-acetyl-β-galactopyranoside), Delphinidin, Cyanidein.

[0065] Example 3: Verification of the in vitro antitumor effect of peony polyphenols on HeLa cells

[0066] 1. Materials and Reagents

[0067] Peony polyphenols: The pale yellow peony polyphenol powder prepared in Example 1 has a polyphenol content of 83.24%.

[0068] HeLa human cervical cancer cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. LC3 A / B, PARP, and Cleaved Caspase-3 antibodies were purchased from CST Biotechnology, USA. MTT, β-Actin antibody, fetal bovine serum (FBS), primary antibody diluent, secondary antibody diluent, penicillin-streptomycin mixture, ultrasensitive ECL chemiluminescence assay kit, BCA protein quantification kit, SDS-PAGE gel rapid preparation kit, RIPA cell lysis buffer, and horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody were purchased from Beyotime Biotechnology Co., Ltd. Dulbecco's modified Eagle medium (DMEM) and phosphate-buffered saline (PBS) were provided by Gibco, USA. Methanol (analytical grade) was supplied by Sangon Biotech (Shanghai) Co., Ltd.

[0069] 2. Instruments and Equipment

[0070] CO2 cell incubator (DH-160H1): Shanghai Santeng Instrument Co., Ltd.; Full-wavelength scanning microplate reader (Infinite M200 Pro): Tecan AG, Switzerland; Inverted fluorescence microscope (IX73): Olympus, Japan; Constant voltage and constant current electrophoresis system (EPS 300): Shanghai Tianneng Technology Co., Ltd.; Transfer apparatus (M-Blot T1000): Nanjing Zhongke Tongyi Technology Co., Ltd.; Gel electrophoresis imaging analysis system (Tanon-5200Multi): Shanghai Tianneng Technology Co., Ltd.

[0071] 3. Methods

[0072] 3.1 In vitro inhibition of tumor cell proliferation assay

[0073] Cells were seeded in 96-well plates and incubated in DMEM containing 10% FBS at 37 °C and 5% CO2. After seeding, cells were treated with a series of concentrations of peony polyphenol solution (0, 6.25, 12.50, 25.00, 50.00, 100.00, and 200.00 μg / mL). -1 Cells were treated separately under FBS-free conditions and incubated for 48 hours. At the end of the treatment period, 10 μL of MTT solution was added to each well, and incubation continued for another 4 hours. Then, 110 μL of supernatant was carefully discarded from each well, and 100 μL of DMSO was added to dissolve the formazan product. The mixture was shaken on a shaker for about 5 minutes to ensure complete dissolution. Finally, the absorbance (OD) of each well was read at 490 nm using a microplate reader.

[0074] 3.2 Annexin V-FITC / PI apoptosis staining assay

[0075] Cells in good growth condition were seeded onto slides in 6-well cell culture plates, and different concentrations of peony polyphenols (25.00, 50.00, and 75.00 μg / mL) were used according to the experimental groups. -1 The cells were treated with PBS-free medium and control group (no drug) for 24 h. After treatment, the cells were washed with PBS, followed by the addition of 195 μL binding buffer, 5 μL Annexin V-FITC staining solution, and 10 μL PI solution. The cells were incubated at 37 °C in the dark for 30 min. Finally, the cells were observed and photographed using an inverted fluorescence microscope.

[0076] 3.3 Annexin V-FITC / PI Double Staining Assay for Apoptosis

[0077] Cells in good growth condition were seeded into 6-well cell culture plates and treated with different concentrations of peony polyphenols (25.00, 50.00, and 75.00 μg / mL) according to experimental groups. -1The control group (without medication) and the control group (without medication) were treated for 48 h without FBS. After treatment, the suspended cells in the supernatant of each well were collected and placed in the corresponding centrifuge tubes. Then, 0.5 mL of trypsin digestion solution was added to the adherent cells remaining in the wells. When the cells detached and became round, 1 mL of culture medium was added to stop the digestion, and the cells were transferred to the corresponding centrifuge tubes. The combined cell suspension was centrifuged at 1,000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL of PBS. The cell suspension was centrifuged again at 1,000 rpm for 5 min, and the supernatant was discarded. 195 μL of binding buffer, 5 μL of Annexin V-FITC staining solution, and 10 μL of PI solution were added to the cell pellet. The cells were mixed and resuspended, and incubated at 37 ℃ in the dark for 15 min. Apoptosis was detected by flow cytometry.

[0078] 3.4 Autophagy Detection Experiment

[0079] Cells in the logarithmic growth phase were seeded in 6-well plates and incubated overnight to stabilize adhesion. Peony polyphenols were added to the experimental groups at final concentrations of 12.50, 25.00, and 50.00 μg / mL, respectively. -1 The control group was incubated with fresh complete culture medium for 24 h without FBS. After obtaining the cell pellet according to the treatment and collection method in 1.3.3, 1 mL of monodansylcadaverine (MDC) staining solution was added, and the cells were incubated at 37 °C in the dark for 30 min. The MDC staining solution was aspirated, and the cells were washed three times with Assay Buffer. Finally, autophagy was detected by flow cytometry.

[0080] 3.5 Western blot assay for proteins

[0081] After obtaining the cell pellet according to the processing and collection method in 1.3.3, extract and detect proteins according to the following steps: Lyse the cells thoroughly with lysis buffer and incubate on ice for several minutes. Centrifuge to remove cell debris, and take the supernatant as the total protein solution. Determine the protein concentration using the BCA method. Mix the protein sample with the loading buffer and heat in a 100 ℃ water bath or metal bath for 5 minutes to denature the protein. After cooling, aliquot and store at -80 ℃ for later use.

[0082] An appropriate amount of protein was loaded onto a 12% polyacrylamide separation gel electrophoresis (SDS-PAGE) sample for separation. After electrophoresis, the protein was transferred to a PVDF membrane. After transfer, the membrane was blocked at room temperature for 2 hours in blocking buffer containing 5% blocking agent (such as skim milk powder or BSA) to block non-specific binding sites. The membrane was then placed in a diluted primary antibody solution (LC3A / B, PARP, Cleaved Caspase-3, p-mTOR, p-4EBP) and incubated overnight at 4 °C. The next day, the membrane was washed three times with 1×TBST buffer for 15 minutes each time to remove unbound antibodies. Then, HRP-labeled secondary antibody at a concentration of 1:1000 was added and incubated at room temperature for approximately 2 hours, followed by three more washes with 1×TBST for 15 minutes each time. After washing, the membrane was developed in ECL Plus luminescent substrate according to the reagent instructions and photographed using an imaging system. Finally, the optical density of each band was measured using imaging software, and the relative gray values ​​were calculated for quantitative analysis.

[0083] 4. Data processing and statistical analysis

[0084] All data are expressed as mean ± standard deviation. Statistical analysis and graph creation were performed using GraphPad Prism 5.0 software. One-way ANOVA was used for multiple group comparisons; the least significant difference (LSD) method was used when homogeneity of variance was satisfied, and Welch correction was used for comparisons if variances were unequal. Unpaired Student's t-test was used for comparisons between two groups. All tests were two-tailed, with P < 0.05 considered statistically significant.

[0085] 5. Results and Analysis

[0086] 5.1 Inhibition of HeLa cell proliferation by peony polyphenols

[0087] Inhibition of excessive tumor cell proliferation is an important indicator for evaluating the antitumor activity of compounds. The MTT assay was used to detect the effect of different concentrations of peony polyphenols on the proliferation of HeLa cells. The results are as follows: Figure 1 As shown.

[0088] Depend on Figure 1 The results showed that the proliferation of HeLa cells was significantly inhibited with increasing concentrations of peony polyphenols, exhibiting a typical dose-dependent trend. When the peony polyphenol concentrations were 6.25, 12.50, 25.00, 50.00, 100.00, and 200.00 μg / mL, the cell viability gradually decreased, and the calculated cell proliferation inhibition rates were approximately 2.11, 11.73, 38.18, 50.43, 73.67, and 81.90%, respectively, with half-maximal inhibitory concentrations (IC50) of 81.90%. 50The concentration was 42.66 μg / mL.

[0089] The results showed that peony polyphenols could significantly inhibit the growth and proliferation of HeLa cells, and the inhibitory effect increased with increasing drug concentration.

[0090] 5.2 Effects of peony polyphenols on the morphology and apoptosis characteristics of HeLa cells

[0091] Cell morphology changes are an important and intuitive indicator of apoptosis induced by compounds. To further verify the effect of peony polyphenols on HeLa cells, HeLa cells were treated with different concentrations of peony polyphenols (25.00, 50.00, and 75.00 μg / mL) for 24 h, and the changes in cell morphology were observed using an inverted microscope.

[0092] The results showed that, compared with the control group, untreated cells were morphologically intact, adhered well, and were spindle-shaped or polygonal with abundant cytoplasm, tightly packed, and exhibiting vigorous growth. After treatment with peony polyphenols, the number of cells decreased significantly, and their morphology gradually changed, exhibiting cell shrinkage, wrinkling, rounding, and detachment. In the high-concentration group, the intercellular spaces increased significantly, adherent cells decreased significantly, and some cells appeared to float. Figure 2 These changes suggest that peony polyphenols can significantly affect the morphology and structure of HeLa cells.

[0093] Further observation of nuclear morphological changes was conducted using Annexin V / PI fluorescence staining. The results showed that the control group cells exhibited uniform nuclear staining and intact morphology, while cells treated with peony polyphenols showed typical apoptotic characteristics, including nuclear pyknosis, enhanced staining, and fragmentation into apoptotic bodies. Figure 3 Furthermore, as the treatment concentration increased, the proportion of apoptotic cells increased, and the fluorescence intensity gradually increased, indicating that peony polyphenols induced apoptosis in HeLa cells to some extent.

[0094] In summary, both microscopic and fluorescent staining observations indicate that peony polyphenols can not only inhibit the proliferation of HeLa cells, but also induce their biochemical apoptosis.

[0095] 5.3 Annexin V / PI double staining to detect cell apoptosis

[0096] To further verify the pro-apoptotic effect of peony polyphenols on HeLa cells, the apoptosis rate was quantitatively analyzed by Annexin V-FITC / PI double staining combined with flow cytometry. HeLa cells were treated with different concentrations of peony polyphenols (25.00, 50.00, and 75.00 μg / mL) for 48 h, and then cells were collected and stained for detection. The results are as follows: Figure 4 As shown.

[0097] Flow cytometry results showed that, compared with the control group, peony polyphenols significantly increased the proportion of early and late apoptosis in HeLa cells, and the apoptosis rate increased with increasing treatment concentration. In the control group, most cells were distributed in Annexin V... - / PI - Quadrants indicate good cell survival; after treatment with 25.00 μg / mL peony polyphenols, the number of early apoptotic cells increased slightly; when the concentration was increased to 50.00 μg / mL, the proportion of early apoptosis and some late apoptosis increased significantly; in the high concentration group of 75.00 μg / mL, the cell apoptosis rate increased significantly, and the proportion of early and late apoptotic cells was significantly higher than that in the control group (P < 0.05).

[0098] Experimental results showed that peony polyphenols significantly induced apoptosis in HeLa cells in a concentration-dependent manner. This indicates that the mechanism by which peony polyphenols inhibit cell proliferation is mainly through activation of intracellular apoptosis signaling pathways.

[0099] 5.4 Effects of peony polyphenols on autophagy in HeLa cells

[0100] Autophagy is a highly conserved self-degradation process in eukaryotic cells, playing a central role in maintaining cellular homeostasis, responding to metabolic stress, and regulating cell fate. Over-activated autophagy can lead to the extensive degradation of key intracellular proteins and organelles, thereby disrupting cellular structural integrity and functional reserves, ultimately driving cells towards apoptosis. To explore the possible mechanism by which peony polyphenols induce apoptosis in HeLa cells, this study used a monodansylcadaverine (MDC) fluorescent probe combined with flow cytometry to detect autophagy. The results are as follows: Figure 5 As shown.

[0101] The results showed that the fluorescence intensity of HeLa cells in the control group was low, indicating that autophagy was maintained at a normal level. After treatment with peony polyphenols, the fluorescence intensity of cells increased significantly, and showed a gradual upward trend with increasing drug concentration. Compared with the control group, the autophagy level in the 12.50 μg / mL group was slightly increased, while in the 25.00 μg / mL and 50.00 μg / mL groups, the fluorescence signal was significantly increased (P < 0.05), indicating a significant enhancement of autophagy.

[0102] These results indicate that peony polyphenols can activate autophagy in HeLa cells, triggering the degradation of damaged mitochondria, cytoplasm, and misfolded proteins, suggesting that they exert their pro-apoptotic effect through the mitochondrial-dependent apoptosis pathway. This result is consistent with the aforementioned Annexin V / PI double staining results, further demonstrating that peony polyphenol-induced apoptosis in HeLa cells is closely related to excessive activation of autophagy.

[0103] 5.5 Effects of peony polyphenols on HeLa cell apoptosis via autophagy

[0104] To further investigate the relationship between autophagy and apoptosis in cells treated with peony polyphenols, the effects of combined peony polyphenols and the autophagy inhibitor 3-methyladenine (3-MA) ​​on autophagy and apoptosis were detected using the MDC method, Annexin V-FITC / PI double staining combined with flow cytometry. The results are as follows: Figure 6 , Figure 7 As shown.

[0105] Figure 6 The results showed that the fluorescence intensity of HeLa cells in the control group and the 3-MA group was low, indicating that autophagy was maintained at a normal level. After treatment with 50.00 μg / mL peony polyphenol, the fluorescence intensity of the cells increased significantly. Compared with the 50.00 μg / mL group, the fluorescence signal was weakened after using 5.00 mM 3-MA in combination with 50.00 μg / mL peony polyphenol (P < 0.05), suggesting that autophagy was inhibited.

[0106] Figure 7 The results showed that, compared with the control group, treatment with 50.00 μg / mL peony polyphenols significantly increased the proportion of early and late apoptosis in HeLa cells; compared with the 50.00 μg / mL group, the apoptosis rate was significantly reduced after the combination of 5.00 mM 3-MA and 50.00 μg / mL peony polyphenols, indicating that apoptosis was inhibited (P < 0.05).

[0107] These results indicate that the combined use of peony polyphenols and 3-MA can inhibit the autophagy and apoptosis induced by peony polyphenols, suggesting that it may induce apoptosis in HeLa cells by activating autophagy.

[0108] 5.6 Effects of peony polyphenols on the expression of autophagy and apoptosis-related proteins in HeLa cells

[0109] To further elucidate the molecular mechanism by which peony polyphenols induce apoptosis in HeLa cells, Western blot was used to detect its effects on the expression of apoptosis-related proteins and signaling pathways. The results are as follows: Figure 8 As shown.

[0110] The results showed that, compared with the control group, the expression levels of autophagy marker protein LC3 A / B and pro-apoptotic marker proteins PARP and Cleaved Caspase-3 in HeLa cells were significantly increased after treatment with peony polyphenols, and these changes were concentration-dependent. Combined with the aforementioned results on the effects of peony polyphenols combined with 3-MA on autophagy and apoptosis, this further proves that peony polyphenols can promote cell apoptosis by inducing autophagy and activating the Caspase cascade reaction.

[0111] Furthermore, the results of signaling pathway-related protein detection showed that peony polyphenols could inhibit the phosphorylation levels of mTOR and 4E-BP proteins. Compared with the control group, the relative expression levels of both proteins were significantly reduced after peony polyphenol treatment, suggesting that peony polyphenols may enhance the sensitivity of cells to apoptosis signals by downregulating the activity of the mTOR / 4E-BP signaling axis and weakening its promoting effect on cell growth and protein synthesis.

[0112] Comprehensive analysis indicates that the inhibitory effect of peony polyphenols on HeLa cells is closely related to its regulation of the mTOR-4E-BP signaling pathway and its promotion of autophagy-induced apoptosis. Peony polyphenols induce apoptosis by reducing the phosphorylation levels of mTOR and 4E-BP, promoting the upregulation of LC3A / B, and activating cleaved caspase-3 and PARP. This result further demonstrates that peony polyphenols exert their antitumor effects through a multi-target synergistic regulatory pathway.

[0113] Example 4: Determination of the in vitro antioxidant activity of peony polyphenols

[0114] This embodiment aims to evaluate the free radical scavenging ability and oxygen free radical absorption ability of the peony polyphenols extracted and prepared in this embodiment 1, in order to verify its potential value as a natural antioxidant, and to compare it with commercially available tea polyphenols, so as to provide experimental evidence for its application in antioxidant, health care, functional foods or medicines.

[0115] 1. Materials

[0116] The peony polyphenol extract (dry weight or solution form) obtained in Example 1 was dissolved in a suitable solvent (water, buffer solution or methanol / ethanol mixture) and diluted to different concentration gradients as needed for later use.

[0117] Reference standard: Commercially available tea polyphenols.

[0118] Reagents: DPPH (1,1-diphenyl-2-picrylhydrazine) radical; ABTS (2,2′-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid)) diammonium salt + potassium persulfate (K2S2O8) to generate ABTS. + • Free radical cations; fluorescent probes (fluorescein sodium) and AAPH (2,2′-azobis(2-amidinopropane) dihydrochloride) as free radical donors for ORAC fluorescence assay; solvents such as methanol / ethanol, water, and buffers (e.g., phosphate-buffered saline PBS); conventional laboratory instruments such as microplates (96-well microplates / fluorescent plates), microplate readers (UV-Vis), fluorescent microplate readers (for ORAC), and pipettes.

[0119] 2. Methods

[0120] 2.1 Determination of DPPH free radical scavenging ability

[0121] Dissolve DPPH in methanol (or ethanol) to prepare a solution of 0.1 mmol·L⁻¹. -1 DPPH working solution. Peony polyphenols and control tea polyphenols were prepared at multiple gradient concentrations (5, 10, 25, 50, 100 µg·mL). -1 Prepare sample solutions, with three replicates for each concentration.

[0122] In a 96-well plate, add 100 µL of sample solution (or control solution) + 200 µL of DPPH working solution to each well, mix well, and incubate at room temperature in the dark for 30 min. Include a blank control (DPPH + solvent only), a negative control (sample + solvent, no DPPH), and a standard control (tea polyphenols + DPPH).

[0123] After the reaction was completed, the absorbance was measured at 517 nm using a spectrophotometer.

[0124] Calculate the DPPH scavenging rate:

[0125]

[0126] Plot a concentration-clearance curve and calculate the half-inhibitory concentration (IC50). 50 ).

[0127] 2.2 ABTS + • Free radical scavenging capacity determination

[0128] Prepare an ABTS stock solution and a potassium persulfate (K2S2O8) solution, mix them in equal volumes, and let them stand in the dark at room temperature for 12 h to generate ABTS. + • Free radical cation solution. Dilute the ABTS with a buffer solution (such as PBS). + • Prepare the solution to the working concentration so that its absorbance at 734 nm is approximately 0.70–0.80 (the specific value can be calibrated based on preliminary experiments).

[0129] Peony polyphenol extract / tea polyphenol standard were diluted to gradient concentrations (5, 10, 20, 40, 80, 160, 320 µg·mL). -1 Triple replicates were performed for each concentration. In a 96-cell plate, 50 µL of sample solution + 200 µL of BTS was added to each well. + • After mixing the working solution, incubate at room temperature in the dark for 6–10 min. Set up a blank (buffer solution + ABTS).+ •), Negative control (sample + buffer), Positive control (tea polyphenols + ABTS) + ·)Group.

[0130] The absorbance after the reaction was measured at a wavelength of 734 nm, and the ABTS scavenging rate was calculated.

[0131]

[0132] Calculate IC by plotting concentration versus clearance rate. 50 .

[0133] 2.3 ORAC (Oxygen Radical Absorbance Capacity) Determination

[0134] Fluorescent probe (fluorescein sodium) and AAPH were selected as free radical donors (generating peroxy radical ROO•). Reaction buffer (PBS, pH 7.4) and reagents were prepared.

[0135] Peony polyphenol extract and tea polyphenol standards were prepared using a series of gradient concentrations (5, 10, 25, 50, 100 µg·mL). -1 ), and prepare standard gradients to plot standard curves.

[0136] Add 150 µL of fluorescent probe working solution + sample solution (or 25 µL of standard / buffer blank) to each well of a 96-well fluorescent plate, mix well, and incubate in the dark at 37 °C for about 30 min. Add 25 µL of AAPH solution to initiate the free radical generation reaction; immediately place the plate in a fluorescence microplate reader and perform kinetic fluorescence measurement at an excitation wavelength of 485 nm and an emission wavelength of 520–525 nm, reading the fluorescence intensity once per minute for 35–60 min.

[0137] Record the fluorescence intensity-time curves for blank, standard, and sample, calculate the area under each curve (AUC), and then convert the antioxidant capacity of the sample to TEAC using the standard curve.

[0138] 3. Experimental Results

[0139] The peony polyphenols extracted in this application exhibited strong free radical scavenging and oxygen free radical absorption capabilities as determined by three in vitro antioxidant methods: DPPH, ABTS, and ORAC (Table 1). In the DPPH scavenging experiment, the scavenging rate of peony polyphenols significantly increased with increasing concentration, and its IC50 value was [missing information]. 50The concentration was approximately 38.7 ± 1.8 µg / mL, superior to the positive control tea polyphenols, indicating its high scavenging ability against DPPH free radicals. (In ABTS...) + • In the cation scavenging experiment, the IC50 of peony polyphenols 50 The concentration was approximately 28.4 ± 1.3 µg / mL, slightly better than the tea polyphenol control, indicating a good scavenging effect on both water-soluble and lipid-soluble free radicals. In the ORAC experiment, the antioxidant activity of peony polyphenols was expressed as Trolox equivalent (TEAC), with an ORAC value of approximately 920 ± 40 µmol TE / g extract, slightly higher than the tea polyphenol control, indicating that peony polyphenols possess strong antioxidant protective capabilities.

[0140] Table 1 Test Results Comparison

[0141]

[0142] The results from the three in vitro methods show that peony polyphenols exhibit excellent antioxidant activity in various free radical models, with antioxidant capacity comparable to, and even slightly superior to, traditional tea polyphenols in some indicators. This provides experimental support for the development of peony polyphenols as a natural antioxidant / functional antioxidant component in the food, health product, or pharmaceutical fields.

[0143] Example 5: Determination of the inhibitory effect of peony polyphenols on intracellular oxygen free radical (ROS) levels.

[0144] To further verify the antioxidant effect of the peony polyphenols extracted and prepared in this application at the cellular level, this embodiment uses an adherent tumor cell model (human cervical cancer HeLa cells) and uses the fluorescent probe method (DCFH-DA) to detect the changes in intracellular reactive oxygen species (ROS) levels after drug treatment, so as to evaluate the inhibitory effect of peony polyphenols on oxidative stress-induced ROS generation.

[0145] 1. Materials

[0146] The peony polyphenol extract obtained in Example 1.

[0147] Control substance (positive control): Commercially available tea polyphenols.

[0148] ROS fluorescent probe: 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA); kit.

[0149] Cell culture reagents: serum-free culture medium, PBS, and conventional culture medium.

[0150] Instruments and equipment: CO2 incubator (37 ℃, 5% CO2), fluorescence microscope, flow cytometer, pipettes and other standard cell experimental equipment.

[0151] 2. Methods

[0152] Adherent HeLa cells were seeded in 6-well plates, the culture medium was aspirated, and different concentrations of peony polyphenols (12.5, 25, 50 µg / mL) were diluted with serum-free medium and added to each well. A control group (serum-free medium), a positive control group (H2O2), and a reference antioxidant group (tea polyphenols) were set up. The cells were incubated in a 37 ℃ CO2 incubator in the dark for 2 h to induce ROS changes.

[0153] After treatment, discard the drug reaction solution and wash the cells once with PBS to remove residual drug. Dilute DCFH-DA 1:1000 in serum-free medium to a final concentration of approximately 10 µM. Discard the washing solution, add an appropriate amount of DCFH-DA working solution to each well, and incubate in the dark at 37 °C for 20 min. After incubation, wash the cells twice with serum-free medium to remove any probes that have not yet entered the cells.

[0154] Flow cytometry assay: Cells were digested with trypsin to prepare a single-cell suspension, which was then resuspended in PBS. DCF fluorescence intensity was measured in a flow cytometer using a 488 nm excitation / 525–530 nm emission channel.

[0155] 3. Experimental Results

[0156] In a cell model using adherent HeLa tumor cells, intracellular ROS levels were detected using the DCFH-DA fluorescent probe to evaluate the inhibitory effect of peony polyphenols on oxidative stress. Table 2 shows that, compared with the positive ROS-induced group (H2O2 treatment group), cells pretreated with peony polyphenols (25 µg / mL and 50 µg / mL) exhibited significantly lower ROS levels (P < 0.05). The ROS levels in the reference antioxidant tea polyphenol group showed similar results.

[0157] Table 2 Statistical Table of Test Results

[0158]

[0159] The above results indicate that peony polyphenols can significantly inhibit ROS generation and alleviate oxidative stress at the cellular level, thus exhibiting cytoprotective effects. This further enhances the application value of peony polyphenols as a functional antioxidant / cytoprotective agent.

[0160] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing peony polyphenol extract, characterized in that, The specific steps are as follows: 1) Crush the dried flowers of Danfeng peony into powder and set aside; 2) Add an ethanol-water solution to the raw material powder, reflux in a constant temperature water bath several times to extract, and filter through gauze to remove the residue to obtain the extract; 3) Purify the extract using macroporous resin; 4) The purified eluent was concentrated under reduced pressure until no alcohol odor was detected, and the concentrate was freeze-dried to obtain a light yellow peony polyphenol powder.

2. The method for preparing peony polyphenol extract according to claim 1, characterized in that, In step 1), the dried flowers of Danfeng peony are pulverized and passed through a 40-mesh sieve for later use.

3. The method for preparing peony polyphenol extract according to claim 1, characterized in that, In step 2), the volume concentration of the ethanol-water solution is 70%, and the extraction is carried out twice under constant temperature water bath at 60℃ for 2 hours each time. The two extracts are then combined.

4. The method for preparing peony polyphenol extract according to claim 1, characterized in that, In step 3), the macroporous resin is type D101 macroporous resin.

5. The method for preparing peony polyphenol extract according to claim 1, characterized in that, In step 3), the specific process is as follows: Resin pretreatment: D101 macroporous resin was soaked in 95% ethanol for 24 hours, and then rinsed with 95% ethanol and distilled water in sequence until there was no alcohol odor. Sample loading: The extract was slowly passed through a pretreated macroporous resin column at a flow rate of 2 BV / h. Elution: First, elute with 2 BV of distilled water at a flow rate of 2 BV / h, then elute with 2 BV of 30% ethanol at a flow rate of 2 BV / h, and finally elute with 3 BV of 70% ethanol at a flow rate of 1.5 BV / h. Collect the 70% ethanol eluent.

6. The method for preparing peony polyphenol extract according to claim 1, characterized in that, In step 4), the 70% ethanol eluent is placed in a rotary evaporator under reduced pressure and concentrated at 50°C and -0.08 MPa until there is no alcohol odor, thus obtaining a concentrated solution.

7. The method for preparing peony polyphenol extract according to claim 1 or 6, characterized in that, In step 4), the concentrate is transferred to a freeze dryer and freeze-dried at -50°C and 0.1 Pa for 48 hours to obtain a light yellow peony polyphenol powder.

8. The peony polyphenol powder prepared by the method for preparing peony polyphenol extract according to any one of claims 1-7.

9. The use of the peony polyphenol powder according to claim 8 in the preparation of anti-cervical cancer drugs.

10. The use of the peony polyphenol powder according to claim 8 in the preparation of antioxidants and / or cell protectants.

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