A method for obtaining an extract of a dysphania glomerata callus and application thereof

Abstract

The application discloses a method for obtaining an extract of dysosma versipellis callus and application of the extract. The method comprises the following steps: inducing and culturing dysosma versipellis explants to obtain callus; subculturing the obtained callus to obtain proliferated callus; and extracting the obtained proliferated callus by ethanol reflux to obtain the extract of dysosma versipellis callus. The method can efficiently extract active ingredients of dysosma versipellis callus, and the obtained extract has a significant anti-breast cancer effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for obtaining and applying an extract from callus tissue of Podophyllum hexandrum. Background Technology

[0002] Octagonal lotus ( Dysosma versipellis (Hance) M. Cheng ex TS Ying) is a species of Podophyllum in the family Berberidaceae. Dysosma As a perennial herb, *Podophyllum hexandrum* is an endangered medicinal plant with high medicinal value. Its roots and rhizomes are used medicinally, traditionally possessing properties such as clearing heat and detoxifying, resolving phlegm and dissipating nodules, removing blood stasis and relieving pain, and anti-tumor effects. *Podophyllum hexandrum* is rich in various pharmacologically active secondary metabolites, including podophyllotoxin (PTOX). Podophyllotoxin is an important precursor to anti-tumor drugs such as etoposide and teniposide, possessing high medicinal and development value. With the continuous deepening of research on related drugs and natural active ingredients, the demand for *Podophyllum hexandrum* continues to increase. However, its wild resources are limited, and its long growth cycle restricts the sustainable utilization of these resources.

[0003] Podophyllum callus is a regenerated tissue obtained through in vitro plant tissue culture. It features a relatively short growth cycle, can be continuously cultured under controlled conditions, and can produce pharmacologically active secondary metabolites such as podophyllotoxin. Therefore, Podophyllum callus has potential application value as an alternative source of medicinal resources for Podophyllum. Existing studies have shown that by optimizing culture conditions and adding precursors such as phenylalanine, the accumulation of some active ingredients in Podophyllum callus can be regulated. However, currently, there is a lack of systematic research on whether Podophyllum callus can form a medicinally valuable alternative resource in terms of chemical composition and pharmacological activity, especially regarding the compositional characteristics of its various active ingredients and their correlation with antitumor efficacy.

[0004] Therefore, establishing a stable and efficient system for the culture and extraction of active ingredients from *Podophyllum hexandrum* callus, and further evaluating the composition and pharmacological effects of the main active ingredients in the obtained callus extract, is of great significance for expanding alternative medicinal resources of *Podophyllum hexandrum* and alleviating the pressure on the utilization of endangered medicinal plant resources. Summary of the Invention

[0005] The purpose of this invention is to provide an extract from the callus tissue of *Podophyllum hexandrum*, its method of obtaining it, and its application. By rationally setting up the culture system and extraction system, the active components of lignans (podophyllotoxin, deoxypodophyllotoxin) and flavonols (quercetin) in the callus tissue of *Podophyllum hexandrum* are efficiently extracted. The obtained extract has significant anti-tumor effects.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for obtaining an extract from callus tissue of Podophyllum hexandrum, comprising the following steps: S1. Inducing and culturing the explants of Podophyllum hexandrum to obtain callus tissue; S2. Subculture the obtained callus tissue to obtain proliferating callus tissue; S3. Extract the proliferating callus tissue obtained in step S2 by reflux with ethanol to obtain the Podophyllum hexandrum callus tissue extract. The culture medium used for the induction culture is formulated as follows: based on MS basal medium containing vitamin B5, it contains 30-35 g / L sucrose, 0.1-0.5 mg / L NAA, and 0.2-0.6 mg / L TDZ; As a specific embodiment of the present invention, the culture medium used for the induction culture is formulated as follows: based on MS (containing vitamin B5) medium, containing 30 g / L sucrose, 0.1 mg / L NAA, and 0.2 mg / L TDZ.

[0007] The culture medium used for the subculture was formulated as follows: based on MS basal medium containing vitamin B5, containing 30-35 g / L sucrose, 0.1-0.5 mg / L NAA, 0.2-1.0 mg / L 2,4-D and 0.2-0.6 mg / L TDZ.

[0008] As a specific embodiment of the present invention, the culture medium used for the subculture is formulated as follows: based on MS (containing vitamin B5) medium, containing 30 g / L sucrose, 1.0 mg / L 2,4-D, and 0.2 mg / L TDZ.

[0009] The conditions for the induction culture are: temperature 25±2℃, dark culture, and culture time 18-40 days; The conditions for the subculture were: temperature 25±2℃, light 16 h / d, for a total of 28-30 days.

[0010] The culture medium used for the subculture also contains an anti-browning agent; the anti-browning agent is activated carbon with a concentration of 0.4-0.6 g / L.

[0011] The explants of *Podophyllum hexandrum* are leaves or stem segments. Specifically, the explants are sterile leaves of *Podophyllum hexandrum*, cut into pieces measuring 1.0 cm × 1.0 cm.

[0012] The explants of *Podophyllum hexandrum* were disinfected before inoculation. The disinfection process included: disinfecting the explants with 2% NaClO for 8-10 min, 0.1% HgCl2 for 4-5 min, and 75% alcohol for 30-35 s.

[0013] As a specific embodiment of the present invention, the disinfection treatment includes: disinfection with 2% NaClO for 10 min and disinfection with 75% alcohol for 30 s as a base, combined with disinfection with 0.1% HgCl2 for 4 min.

[0014] The ethanol reflux extraction uses a 75% (v / v) aqueous ethanol solution; The ethanol reflux extraction includes: adding the obtained proliferating callus powder to a 75% ethanol aqueous solution at a material-to-liquid ratio of 1:10~20 g / mL, soaking at room temperature for 20~25 min, transferring to a water bath reflux extraction device, heating until the system is kept in a gentle boiling reflux state, extracting 2~4 times, each extraction for 1~1.5 h; combining the extracts, removing the residue by vacuum filtration to obtain the extract.

[0015] Step S3 further includes a concentration and drying step of the extract; the concentration and drying includes: transferring the obtained extract to a rotary evaporator, reducing the pressure to recover ethanol under water bath temperature of 50~65℃ and vacuum degree of 0.06~0.09MPa, concentrating to a concentrate with a relative density of 1.10~1.20 at 50℃; continuing to freeze-dry under vacuum, pulverizing, to obtain the Podophyllum hexandrum callus extract.

[0016] Secondly, the present invention also provides an extract of *Podophyllum hexandrum* callus tissue obtained by the method described above. The *Podophyllum hexandrum* extract comprises: lignan compounds podophyllotoxin and deoxypodophyllotoxin, and flavonol compound quercetin, with an active substance concentration reaching 100 μg / ml.

[0017] The structure of the lignan compound podophyllotoxin is shown in Formula I:

[0018] The structure of the deoxypodophyllotoxin is shown in Formula II:

[0019] The structure of the flavonol compound quercetin is shown in Formula III:

[0020] Thirdly, the present invention also provides the application of the *Podophyllum hexandrum* callus extract in the preparation of anticancer drugs. The cancer may be breast cancer.

[0021] In one specific embodiment of the present invention, the *Podophyllum hexandrum* callus extract is used in combination with an inducer. The concentration of the *Podophyllum hexandrum* callus extract does not exceed 100 μg / mL; the inducer is phenylalanine (Phe) and methyl jasmonic acid (MeJA); the concentration of phenylalanine is 100 μM Phe; and the concentration of methyl jasmonic acid is 50 μM.

[0022] Compared with the prior art, the beneficial effects achieved by the present invention are: This invention optimizes the culture conditions of *Podophyllum hexandrum* explants and the extraction conditions of callus tissue, thereby creating a synergistic effect among the conditions. This results in the efficient extraction of lignans (podophyllotoxin, deoxypodophyllotoxin) and flavonols (quercetin) from *Podophyllum hexandrum* callus tissue, significantly enhancing its anti-breast cancer effect. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The flowchart of the research process for the Podophyllum hexandrum callus culture system provided by this invention.

[0025] Figure 2 The following is a comparison of the effects of different treatments on the culture system of *Podophyllum hexandrum* callus provided by this invention; Figure A shows the survival rate (red line) and contamination rate (yellow line) of leaf explants under six combined disinfection treatments; B shows the survival rate and contamination rate of stem explants under six different disinfection schemes; C shows the survival and contamination of root explants under six disinfection combinations; D shows the survival rate and contamination rate of tuber explants under different disinfection conditions; E shows the morphological observation of callus induced by four types of explants (leaf, stem, root, and tuber); F shows the phenotypic comparison of browning degree in callus culture after treatment with three representative anti-browning agents (a: AC (activated carbon); b: AgNO3; c: PVP (polyvinylpyrrolidone); d: no anti-browning agent); G shows the growth status and induction performance of callus under the optimal culture medium formula selected through orthogonal experimental design (ai represents callus induced by 1-9 types of culture medium).

[0026] Figure 3 The results show the effects of the *Podophyllum hexandrum* extract provided by this invention on the proliferation of human breast cancer cell lines MCF-7 and MDA-MB-231; the figures show AE and α, and the concentrations of different extracts were detected by the CCK-8 assay. D. versipellisEffects of callus extract on the survival rate of breast cancer cells and normal cells; F, Clonogenic assay results comparing the long-term proliferation capacity of MCF-7 and MDA-MB-231 cells after treatment with BJL1 (5 μg / ml) or BJL2 (10 μg / ml) extracts; G, Apoptosis of MCF-7 cells treated with solvent control, BJL1 extract, or BJL2 extract by flow cytometry using Annexin V-FITC / PI staining, illustrated as a representative density map; H, Quantitative analysis of apoptotic cell population corresponding to the GI map; I, ​​Cell cycle distribution of MCF-7 cells after treatment with solvent control, BJL1 (5 μg / ml) extract, or BJL2 (10 μg / ml) extract; The figure shows a representative DNA content histogram obtained by flow cytometry, showing the percentage of cells in G0 / G1, S, and G2 / M phases; J, Western blotting. Blot analysis was performed on apoptosis-related proteins in MCF-7 cells treated with callus extract, including lysed CASPASE-3, CASPASE-3, BCL-2, and BAX; GAPDH was used as a loading control; the accompanying dot plot summarizes the relative protein expression levels in each biological replicate. Compared with the control group,* P <0.05,** P <0.01.

[0027] Figure 4 The results of in vivo anti-breast cancer activity of the extract of Podophyllum hexandrum provided by this invention include: A, BJL 1 (25 mg / kg), BJL 2 (50 mg / kg), BJL 3 (100 mg / kg); B, quantitative analysis of tumor volume in each group; C, quantitative analysis of tumor weight in each group.

[0028] Figure 5 To improve the efficacy of anti-breast cancer treatment with inducible agents; in the figure: A and B represent the results determined 30 days after induction. Dysosma versipellis The optimal concentrations of phenylalanine (Phe) and methyl jasmonic acid (MeJA) for podophyllotoxin production in callus tissue were determined. The highest accumulation of podophyllotoxin was observed with 100 μM Phe and 50 μM MeJA, respectively. CF represents the relative abundance of podophyllotoxin-7-glucoside, deoxypodophyllotoxin, quercetin, and kaempferol in callus tissue treated with 100 μM Phe or 50 μM MeJA for 30 days. GJ represents the effect of untreated, MeJA-induced, and Phe-induced callus extracts on the survival rate of MCF-7 breast cancer cells. J represents the dose-dependent effect of callus extracts on MCF-7 cell survival. KL represents the apoptosis of MCF-7 cells after treatment with 10 μg / mL untreated, Phe-induced, or MeJA-induced callus extracts, analyzed by flow cytometry. Compared with the control group, *P <0.05,** P <0.01. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, all reagents, materials, instruments, etc. used in the following examples are commercially available.

[0032] Reagents: Fetal bovine serum, penicillin-streptomycin, and DMEM medium were from Gibc Corporation, USA; CellCounting Kit-8 reagent kit, RIPA lysis buffer, and BCA protein assay kit were purchased from Beyotime Biotechnology Co., Ltd.

[0033] The formulation of the MS basal culture medium used in this invention is as follows: MS basal salts containing vitamin B5 (Coolaber, PM1031), Gamborg B5 basal salts containing vitamin (Coolaber, PM1321), and MS basal salts containing vitamin (Coolaber, PM519).

[0034] Instruments: Microplate reader (Varioskan LUX, Thermo Fisher Scientific, USA), flow cytometer (BDFACSCanto II, BD Biosciences, USA).

[0035] Cells: Human breast cancer cell lines MCF-7 and MDA-MB-231 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in Durbeco Modified Eagle Medium (DMEM; Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (100 U / mL penicillin and 100 μg / mL streptomycin) at 37°C in a humid environment containing 5% carbon dioxide.

[0036] Laboratory animals: BALB / c nude mice, female, 4-6 weeks old, Beijing Vital River Laboratory Animal Technology Co., Ltd., approval number: 2025B247.

[0037] Example: Obtaining the extract of Podophyllum hexandrum The specific steps are as follows: Step 1: Preparation of subcultured Podophyllum callus 1) Take tender leaves from healthy Podophyllum hexandrum plants, rinse them under running tap water for 30 minutes, wash away surface impurities with dish soap, and place them in a sterile bottle under sterile conditions for later use.

[0038] 2) In a clean bench, the explants were surface-disinfected using the following method: 2% NaClO for 10 min + 0.1% HgCl2 for 4 min + 75% alcohol for 30 s. During the mercuric chloride treatment, 2-3 drops of Tween-20 were added, and the mixture was continuously shaken to ensure full contact with the explants. After each disinfectant treatment, the explants were rinsed 4-5 times with sterile water to remove any residual disinfectant. Finally, the explants were placed on sterile filter paper to absorb excess moisture before use.

[0039] 3) The leaf margins of the disinfected Podophyllum hexandrum explants from step 2) were removed with a sterile scalpel, and then inoculated into an induction medium and cultured in the dark at (25±2)℃ to obtain Podophyllum hexandrum callus.

[0040] The induction medium consisted of the following components: basal medium MS (containing vitamin B5), NAA (α-naphthaleneacetic acid) 0.1 mg / L, TDZ (thiazidone) 0.2 mg / L, sucrose 30 g / L, and agar 4.6 g / L.

[0041] 4) The Podophyllum callus obtained in step 3) was inoculated into the callus subculture culture medium and placed at (25±2)℃, with light for 16 h / d for a total of 30 days to obtain subcultured Podophyllum callus.

[0042] The subculture proliferation medium consists of the following components: basal medium MS (containing vitamin B5), 2,4-D 1.0 mg / L, TDZ 0.2 mg / L, sucrose 30 g / L, agar 5.0 g / L, and AC (activated carbon) 0.5 g / L.

[0043] Step 2: Preparation of Podophyllum hexandrum callus extract 1) Raw material pretreatment The subcultured Podophyllum callus obtained by artificial induction culture was dried in a drying oven at 40~60℃ until constant weight, and then pulverized to obtain Podophyllum callus dry powder.

[0044] 2) Ethanol reflux extraction Weigh out the dried powder of *Podophyllum hexandrum* callus tissue, add 75% ethanol aqueous solution at a mass-to-volume ratio of 1:15 (g / mL), soak at room temperature for 20 min, then transfer to a water bath reflux extraction device, heat until the system is kept in a gentle boiling reflux state, extract 3 times, 1 hour each time; combine the extracts, filter under reduced pressure to remove the residue, and obtain a clear *Podophyllum hexandrum* callus ethanol extract.

[0045] 3) Concentration and drying The above extract was transferred to a rotary evaporator, and ethanol was recovered under reduced pressure at a water bath temperature of 50-65℃ and a vacuum degree of 0.06-0.09MPa. The extract was concentrated to a concentration with a relative density of 1.10-1.20 at 50℃. After further vacuum freeze drying and pulverization, 75% ethanol extract powder of Podophyllum hexandrum callus was obtained.

[0046] It should be noted that the following experimental examples are only illustrative of the significant differences in the effects of different factors on tissue culture and callus induction, and do not represent that this invention only examines the following influencing factors, and should not be used to limit the scope of protection of this invention.

[0047] Experiment Example 1: The effect of different explant disinfection protocols on culture results This invention screens commonly used disinfectants in the field and ultimately determines sodium hypochlorite (NaClO), mercuric chloride (HgCl2), and ethanol as the disinfection combination. Based on this, the optimal disinfection time is screened and the optimized disinfection scheme is determined to be: 2% sodium hypochlorite soaking for 10 min, 0.1% mercuric chloride soaking for 4 min, and 75% ethanol soaking for 30 s.

[0048] Experimental Example 2: Callus Induction Efficiency of Different Explants The disinfection protocol obtained in Experiment 1 was used to test explant leaves, stems, roots, and rhizomes.

[0049] The results showed that the contamination rates of leaves, stems, roots, and rhizomes were 4.43±0.91%, 7.49±1.68%, 10.62±3.05%, and 18.62±2.75%, respectively, and the survival rates were 86.91±2.9%, 89.23±2.51%, 86.25±1.47%, and 74.84±4.97%, respectively. Figure 2 AD).

[0050] Based on these results, callus induction efficiency was further used as an evaluation index to screen different explant types. The results showed that after disinfection, leaves and stems could normally induce callus formation, while roots and rhizomes could not. Figure 2 E). Therefore, the leaf was identified as D. versipellis The optimal explant for tissue culture.

[0051] Experiment Example 3: Effects of different anti-browning agents on callus induction efficiency Using commonly used experimental methods for evaluating anti-browning agents in this field, the results of the evaluation of three anti-browning agents (AC, PVP, and AgNO3) showed that 0.5 g / L AC had the best anti-browning effect, effectively reducing the browning rate and improving callus quality. Figure 2F). Leaf explants showed the highest callus induction rate (87.50±1.94%), and the induced callus tissue was dense and light green in color, making it suitable for subsequent batch culture and proliferation. Figure 2 G).

[0052] Experiment Example 4: Effect of callus induction medium on callus induction efficiency The results showed that adding 0.1 mg / L NAA and 0.2 mg / L TDZ to MS medium resulted in a callus induction rate of 94.59 ± 1.13% (Table 1).

[0053] Table 1. Investigation of callus induction culture medium

[0054] Experimental Example 5: Effect of callus proliferation medium on proliferation coefficient The results showed that the highest proliferation coefficient (2.18 ± 0.13) was obtained when 1.0 mg / L 2,4-D and 0.2 mg / L TDZ were added to MS medium.

[0055] Experimental Example 6: Pharmacodynamic Detection of Podophyllum hexandrum callus extract 1. Effects of Podophyllum hexandrum callus extract on the proliferation of MCF-7 and MDA-MB-231 cells (1) Cell culture and detection of anti-breast cancer activity Human breast cancer cell lines MCF-7 and MDA-MB-231 were cultured in Durbeco Modified Eagle Medium (DMEM; Gibco, USA) using 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (containing 100 U / mL penicillin and 100 μg / mL streptomycin). The culture conditions were 37°C in a humid environment with 5% carbon dioxide, and all experiments used cells in the logarithmic growth phase.

[0056] (2) Cell viability assay (CCK-8) Cell viability was determined using the CCK-8 assay. The specific steps were as follows: MCF-7 and MDA-MB-231 cells were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates and cultured overnight to allow them to adhere; subsequently, different concentrations of [missing information] were used to [missing information]. D. versipellisCells were treated with callus extract (at concentrations of 0-100 μg / ml, BJL1: 5 μg / ml; BJL2: 10 μg / ml) for 24, 48, or 72 hours; 10 μL of CCK-8 solution was added to each well at the specified time points, and the cells were incubated at 37°C for 1-2 hours; absorbance was measured at 450 nm using a microplate reader; cell viability was expressed as the percentage of live cells (relative to the untreated control group). All experiments were performed in triplicate and independently at least three times.

[0057] (3) Cloning experiment MCF-7 and MDA-MB-231 cells were seeded at a density of 500-1000 cells per well in 6-well plates and cultured for 24 hours to allow them to adhere. Cells were then treated with a specified concentration of callus extract (BJL1 or BJL2), and the medium containing the extract was changed every 3 days. After 10-14 days of culture, colonies were fixed with 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet solution for 30 minutes at room temperature. After washing with PBS, colonies containing more than 50 cells were counted using a phase-contrast microscope. The colony formation rate was calculated as: the number of colonies in the treatment group divided by the number of colonies in the control group.

[0058] (4) Apoptosis detection by flow cytometry Apoptosis was detected using the Annexin V-FITC / propidium iodide (PI) apoptosis detection kit. MCF-7 and MDA-MB-231 cells were co-cultured with callus extract for 48 hours, then cells were collected by trypsin digestion with EDTA-free enzymes, washed twice with cold PBS, and resuspended in 1× binding buffer at a concentration of 1×10⁻⁶. 6 Cells / mL. Then, 5 μL of Annexin V-FITC and 5 μL of PI were added to 100 μL of cell suspension, gently mixed, and incubated at room temperature in the dark for 15 minutes. After adding 400 μL of binding buffer, the stained cells were immediately analyzed by flow cytometry. Data were statistically analyzed using FlowJo software (version 10.8, Tree Star, USA), and early apoptotic cells (Annexin V-FITC) were quantified separately. + / PI - ) and late-stage apoptotic / necrotic cells (Annexin V-FITC) + / PI + ).

[0059] (5) Cell cycle analysis MCF-7 and MDA-MB-231 cells were co-cultured with callus extract for 48 hours, and the cells were collected, washed twice with cold PBS, and fixed overnight at 4°C with 70% ice-cold ethanol. After fixing, the cells were washed with PBS and stained with PI / RNase staining buffer (BD Biosciences, USA) at room temperature in the dark for 30 minutes. DNA content was determined by flow cytometry (BD FACSCanto II), and the proportion of cells in G0 / G1, S, and G2 / M phases was analyzed using ModFit LT software (version 5.0, Verity Software House, USA).

[0060] (6) Western blot analysis MCF-7 cells were treated with callus extract (BJL1 5 μg / mL or BJL2 10 μg / mL) for 24 hours and lysed on ice for 30 minutes in RIPA lysis buffer (Beyotime, Shanghai, China) containing a mixture of protease and phosphatase inhibitors (Roche, Basel, Switzerland). The lysis buffer was centrifuged at 12,000×g for 15 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA protein assay kit (Beyotime). Equal amounts of protein (20–30 μg) were separated by 10% or 12% SDS-PAGE electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, USA). The membrane was blocked at room temperature for 1 hour in TBST solution containing 5% skim milk (Tris-buffered saline containing 0.1% Tween-20), followed by overnight incubation at 4°C with antibodies against cleavage caspase-3 (1:1000; CST #9664), caspase-3 (1:1000; CST #9662), Bcl-2 (1:1000; CST #4223), Bax (1:1000; CST #5023), and GAPDH (1:5000; CST #5174). After washing with TBST, the membrane was incubated at room temperature for 1 hour with HRP-labeled secondary antibody (1:5000; CST). Protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit (Beyotime) and imaged using a chemiluminescence imaging system (Tanon 5200, Shanghai, China). The band intensity was quantified using ImageJ software (National Institutes of Health, USA), with GAPDH used as an internal control.

[0061] like Figure 3 As shown, the extract from the callus of *Podophyllum hexandrum* significantly inhibited the proliferation of both cell lines in a dose-dependent manner, and this result was further confirmed by crystal violet staining. Figure 3AF). To elucidate its mechanism of action, flow cytometry was used to detect apoptosis and cell cycle distribution. The results showed that *Polygonum aviculare* callus extract effectively promoted apoptosis in breast cancer cell lines (AF). Figure 3 GH), cell cycle analysis showed that it mainly arrests cells in the G1 phase (G1 phase). Figure 3 I). Western blot analysis showed that the extract of *Polygonum aviculare* callus significantly upregulated the expression of apoptosis-related proteins, including caspase-3 and Baxobin. Figure 3 J).

[0062] 2. Whole animal experiments Female BALB / c nude mice (4-6 weeks old, weighing 18-20 grams) used in the experiment were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). They were housed in SPF-grade conditions with a 12-hour light / dark cycle and a temperature of 22-25°C, with free access to food and water. All animal experiments were approved by the institution's Animal Care and Use Committee. MCF-7 cells (5 × 10⁻⁶) were used. 6 Each cell was dissolved in 100 μL PBS and subcutaneously injected into the right flank of each mouse. When the tumor volume reached approximately 100 mm... 3 Mice were randomly divided into four groups (n=6 per group): (1) solvent control group (PBS), (2) low-dose callus extract group (25 mg / kg), (3) medium-dose callus extract group (50 mg / kg), and (4) high-dose callus extract group (100 mg / kg). The extract was administered orally once daily for 21 consecutive days. Body weight was recorded simultaneously to monitor systemic toxicity. At the end of the experiment, mice were sacrificed, tumor tissue was removed, weighed, and photographed. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the China Academy of Chinese Medical Sciences (Approval No.: 2025B247) and were conducted in strict accordance with the National Institutes of Health's Guidelines for the Care and Use of Laboratory Animals.

[0063] like Figure 4 As shown, the results indicate that callus extract can inhibit tumor growth in a dose-dependent manner, specifically by significantly reducing tumor volume and weight.

[0064] 3. Inducer treatment enhances the efficacy of anti-breast cancer therapy. like Figure 5As shown, LC-MS / MS analysis revealed that both inducers, phenylalanine (Phe) and methyl jasmonate (MeJA), significantly promoted the accumulation of deoxypodophyllotoxin, PTOX, and quercetin, with 50 μM MeJA being the most effective condition (inducer stimulation time was 30 days). Pharmacodynamic experiments showed that callus extracts treated with 100 μM Phe or 50 μM MeJA exhibited stronger growth inhibition than the untreated group, with the 50 μM MeJA group showing the most significant effect. Treatment with 5 μg / mL and 10 μg / mL extracts further demonstrated that the induced callus extracts significantly promoted cell apoptosis, with the 50 μM MeJA condition again producing the strongest response. These results demonstrate that stimulating factors can enhance... D. versipellis The accumulation of bioactive components (such as PTOX) in callus tissue enhances its anti-breast cancer activity. In summary, this invention... D.versipellis Callus cultures exhibit anti-breast cancer activity: callus extract significantly inhibited breast cancer cell proliferation, induced apoptosis, and suppressed tumor growth in vivo. To further enhance pharmacological efficacy, we optimized the treatment regimen using phenylalanine (Phe) and methyl jasmonate (MeJA) as inducers. 50 μM MeJA significantly promoted the accumulation of PTOX-related metabolites and substantially enhanced the anti-breast cancer activity of callus extract. This study comprehensively constructed a system for evaluating drug alternatives and demonstrated... D.versipellis Callus culture is a sustainable, scalable alternative resource with greater pharmaceutical potential.

[0065] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for obtaining an extract from Podophyllum hexandrum callus, characterized in that, Includes the following steps: S1. Inducing and culturing the explants of Podophyllum hexandrum to obtain callus tissue; S2. Subculture the obtained callus tissue to obtain proliferating callus tissue; S3. Extract the proliferating callus tissue obtained in step S2 by reflux with ethanol to obtain the Podophyllum hexandrum callus tissue extract. The culture medium used for the induction culture is formulated as follows: based on MS basal medium containing vitamin B5, containing 30-35 g / L sucrose, 0.1-0.5 mg / L NAA, and 0.2-0.6 mg / L TDZ; The culture medium used for the subculture was formulated as follows: based on MS basal medium containing vitamin B5, containing 30-35 g / L sucrose, 0.1-0.5 mg / L NAA, 0.2-1.0 mg / L 2,4-D and 0.2-0.6 mg / L TDZ.

2. The acquisition method according to claim 1, characterized in that, The conditions for the induction culture are: temperature 25±2℃, dark culture, and culture time of 18-40 days; And / or, the subculture conditions are: temperature 25±2℃, light 16 h / d, for a total of 28-30 days.

3. The acquisition method according to claim 1, characterized in that, The culture medium used for the subculture also contains an anti-browning agent. And / or, the anti-browning agent is activated carbon with a concentration of 0.4-0.6 g / L.

4. The method for obtaining according to any one of claims 1-3, characterized in that, The explants of Podophyllum hexandrum are leaves or stem segments.

5. The method for obtaining according to any one of claims 1-3, characterized in that, The *Podophyllum hexandrum* explants were disinfected before inoculation. The disinfection process includes: disinfecting the explants of Podophyllum hexandrum sequentially with 2% NaClO for 8-10 min, 0.1% HgCl2 for 4-5 min, and 75% alcohol for 30-35 s.

6. The method for obtaining according to any one of claims 1-3, characterized in that, The ethanol reflux extraction uses a 75% (v / v) aqueous ethanol solution; The ethanol reflux extraction includes: adding the obtained proliferating callus powder to a 75% ethanol aqueous solution at a material-to-liquid ratio of 1:10~20 g / mL, soaking at room temperature for 20~25 min, transferring to a water bath reflux extraction device, heating until the system is kept in a gentle boiling reflux state, extracting 2~4 times, each extraction for 1~1.5 h; combining the extracts, removing the residue by vacuum filtration to obtain the extract.

7. The method for obtaining according to any one of claims 1-3, characterized in that, Step S3 also includes the steps of concentrating and drying the extract; The concentration and drying process includes: transferring the obtained extract to a rotary evaporator, reducing the pressure to recover ethanol under reduced pressure at a water bath temperature of 50-65℃ and a vacuum degree of 0.06-0.09MPa, and concentrating the extract to a concentration with a relative density of 1.10-1.20 at 50℃; continuing to freeze-dry under vacuum and pulverizing to obtain the *Podophyllum hexandrum* callus extract.

8. An extract of Podophyllum hexandrum callus obtained by the method of any one of claims 1-7.

9. The use of the Podophyllum hexandrum callus extract according to claim 8 in the preparation of anticancer drugs.

10. The application according to claim 9, characterized in that, The cancer in question is breast cancer.

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