Magnolol rhamnoside, and preparation method and application thereof

Abstract

The present application relates to a magnolol rhamnoside, a preparation method and application thereof, and belongs to the technical field of glycoengineering. The magnolol rhamnoside, also known as magnolol-2-O-alpha-rhamnoside, is a novel derivative compound of natural product magnolol through glycosylation modification. In the present application, the water solubility and tumor cell growth inhibition activity of the magnolol rhamnoside are determined for the first time, and the results show that the magnolol rhamnoside has higher water solubility and tumor cell growth inhibition activity than magnolol, and has important application value for the research and development of new anti-tumor drugs. In the present application, a recombinant Escherichia coli co-expressing an rfbBDAC operon for synthesizing dTDP-Rha and a rhamnosyltransferase Ss-RhaT is constructed, and the magnolol rhamnoside is prepared by microbial fermentation conversion method. The recombinant Escherichia coli is used to efficiently synthesize the magnolol rhamnoside by fermentation method with magnolol added in the culture medium as a substrate.

Description

Technical Field

[0001] This invention relates to a magnolol rhamnoside, its preparation method and application, belonging to the field of glycoengineering technology. Background Technology

[0002] Magnolol is a novel lignan natural product discovered in the bark of the magnolia officinalis tree (Magnolia officinalis), a plant in the Magnoliaceae family. It possesses a wide range of biological activities, including anti-inflammatory, anti-tumor, cardiovascular protective, anti-angiogenic, hypoglycemic, antioxidant, neuroprotective, gastrointestinal protective, and antibacterial effects, thus exhibiting significant medicinal potential. Chinese patent document CN103127039A (application number 201110375026.0) describes the application of magnoolol in the preparation of antitumor drugs, where the tumor cells include liver cancer cells and cervical cancer cells. Chinese patent document CN106924225A (application number 201511012983.1) describes magnoolol's ability to reverse multidrug resistance in tumor cells, making it a potential reversal agent for multidrug resistance in tumors; magnoolol also increases the sensitivity of multidrug-resistant tumor cells to antitumor drugs, making it a potential chemosensitizer; and a method for inhibiting the proliferation of multidrug-resistant tumor cells using a pharmaceutical composition combining antitumor drugs and magnoolol is also provided.

[0003] However, magnolol has extremely low water solubility, only 0.02 mM at room temperature. Furthermore, animal experiments have shown that this compound is rapidly metabolized in vivo; after oral administration, over 76% of magnolol and its metabolites were detectable in the feces and urine of mice within just 24 hours. These drawbacks result in low bioavailability of magnolol, severely limiting its application in clinical drug development. Therefore, preparing magnolol derivatives to improve water solubility and pharmacological activity can provide important fundamental support for the subsequent clinical application of magnolol.

[0004] Chinese patent document CN113321684A (application number 202011173725.2) discloses a magnolol derivative, its preparation method, and its uses. The magnolol derivative is a compound shown in the following formula or a salt thereof. This compound or its salt can effectively inhibit tumor growth, particularly showing good inhibitory effects on non-small cell lung cancer, glioma, colon cancer, breast cancer, renal cell carcinoma, and chondrosarcoma.

[0005]

[0006] Chinese patent document CN110343033A (application number 201810283433.0) discloses a series of magnolol derivatives, their preparation methods, and uses. These magnolol derivatives are mainly substituted derivatives at the 5-position of magnolol, and their structural formulas are shown below. The document also provides preparation methods and uses for these magnolol derivatives. Cytotoxicity tests have demonstrated that these magnolol derivatives possess excellent antitumor activity and have broad application prospects in the preparation of drugs for treating cancer.

[0007]

[0008] Glycosylation is a common structural modification in nature, found in many secondary metabolites produced by plants and microorganisms. Glycosylation modifies the physicochemical properties, biological activities, pharmacokinetic properties, and absorption and metabolism in the human body of natural products. Rhamnylation is a common glycosylation modification found in plant and bacterial secondary metabolites, significantly improving the physicochemical properties and biological activities of compounds. For example, solanine rhamnoside can target tumors and exert cytotoxicity through the interaction of its rhamnose residues with rhamnose lectin receptors on the surface of certain tumor cells (Wang, Y., J. Gao, G. Gu, G. Li, C. Cui, B. Sun, and H. Lou, In Situ RBL Receptor Visualization and Its Mediated Anticancer Activity for Solasodine Rhamnosides. ChemBioChem, 2011.12(16):p.2418-2420); emodin rhamnoside showed strong growth inhibitory effects on human non-small cell lung cancer cells A549, liver cancer cells HepG2, ovarian cancer cells OVCAR-3, cervical cancer cells HeLa, erythroleukemia cells K562, and gastric cancer cells SGC-790 (Xing, J., G. Song, J. Deng, L. Jiang, P. Xiong, B. Yang, and S. Liu, Antitumor Effects and Mechanism). (of Novel Emodin Rhamnoside Derivatives against Human Cancer Cells In Vitro. PLOS ONE, 2015.10(12):p.e0144781); Rhamnose glycoside derivatives of betulinic acid can selectively inhibit the growth of human colon cancer cells (Sylla, B., S. Lavoie, J. Legault, C. Gauthier, and A. Pichette, Synthesis, cytotoxicity and anti-inflammatory activity of rhamnose-containing ursolic and betulinic acids aponins. RSC Advances, 2019.9(68):p.39743-39757). Therefore, rhamnose glycosylation modification has important application value for the drug development of small molecule compounds.

[0009] There are currently no reports on the preparation of magnolol rhamnoside derivatives, their water solubility, and their inhibitory activity on tumor cell growth. Summary of the Invention

[0010] To address the shortcomings of existing technologies, this invention provides magnolol rhamnoside, its preparation method, and its applications. Compared to magnolol, the obtained magnolol rhamnoside exhibits significantly improved water solubility and inhibitory activity against human tumor cell growth, and can be used in the development of anti-tumor drugs.

[0011] The technical solution of the present invention is as follows:

[0012] A magnolol rhamnoside, chemically named magnolol-2-O-α-rhamnoside, has the molecular formula C2. 24 H 28 O6, chemical structural formula as follows:

[0013]

[0014] One method for preparing the above-mentioned magnolol rhamnoside involves using dTDP-Rha as a rhamnose donor, magnolol as a glycosyl acceptor, and rhamnosyltransferase as an enzyme catalyst to prepare magnolol rhamnoside via an enzyme-catalyzed reaction.

[0015] According to a preferred embodiment of the present invention, the rhamnosyltransferase is a rhamnosyltransferase Ss-RhaT derived from Saccharothrix syringae, the nucleotide sequence of which is shown in SEQ ID NO.2 and the amino acid sequence of which is shown in SEQ ID NO.3.

[0016] A method for preparing the above-mentioned magnolol rhamnoside involves using magnolol as a glycosyl acceptor and recombinant Escherichia coli BL21-Rfb-RhaT, which co-expresses the rfbBDAC operon synthesized by dTDP-Rha and the rhamnosyltransferase Ss-RhaT, as a fermentation strain to prepare magnolol rhamnoside through fermentation.

[0017] According to a preferred embodiment of the present invention, the rfbBDAC operon is derived from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO. 1.

[0018] According to a preferred embodiment of the present invention, the plasmid vector for the rfbBDAC operon is pACYCDuet-1.

[0019] According to a preferred embodiment of the present invention, the plasmid vector for expressing the rhamnosyltransferase Ss-RhaT is pET-22b(+).

[0020] A preferred embodiment of the present invention provides a method for preparing magnolol rhamnoside, which specifically includes the following steps:

[0021] Recombinant E. coli BL21-Rfb-RhaT, co-expressing the rfbBDAC operon synthesized from dTDP-Rha and the rhamnosyltransferase Ss-RhaT, was cultured to an OD500 concentration. 600 To obtain a fermentation broth containing magnolol rhamnoside, add IPTG to a final concentration of 0.5-2.0 mM and honokiol to a final concentration of 0.5-5.0 mM, and continue culturing at 16-37℃ for 6-72 hours.

[0022] A further preferred embodiment of the method for constructing the recombinant Escherichia coli BL21-Rfb-RhaT is as follows:

[0023] (1) Using Escherichia coli BL21(DE3) genomic DNA as a template, primers F-Rfb and R-Rfb were designed, and the rfbBDAC operon sequence was obtained by PCR amplification. After ligating it with the plasmid vector pACYCDuet-1, the recombinant expression vector pAC-RfbBDAC was obtained.

[0024] The sequences of primers F-Rfb and R-Rfb are as follows:

[0025] F-Rfb:5'-GGGAATTC CATATG GTGAAGATACTTGTTACTGGTGGCG-3', the underline indicates the NdeI restriction site.

[0026] R-Rfb:5'-CGG GGTACC TTAAACTTTCTCAAAAAGTTCTCTGAATG-3', the underline indicates the Kpn I restriction site;

[0027] (2) Using the genomic DNA of Syringa syringae as a template, primers F-RhaT and R-RhaT were designed. The rhamnosyltransferase Ss-RhaT gene sequence was obtained by PCR amplification. After ligating it with the plasmid vector pET-22b(+), the recombinant expression vector pET22b-Ss-rhaT was obtained.

[0028] The sequences of primers F-RhaT and R-RhaT are as follows:

[0029] F-RhaT:5'-GGAATTC CATATG AAGATCCTGTTTTCGAGCCTCG-3', the underline indicates the Nde I restriction site.

[0030] R-RhaT:5'-CCG CTCGAG GGCGTACTGCGGCAGGG-3', the underline indicates the Xho I restriction site;

[0031] (3) The recombinant expression vector pAC-RfbBDAC and the recombinant expression vector pET22b-Ss-rhaT were co-transformed into Escherichia coli BL21(DE3), and positive transformants were screened to obtain recombinant Escherichia coli BL21-Rfb-RhaT.

[0032] More preferably, the bacterial cell concentration OD 600 It is 1.2.

[0033] More preferably, the final concentration of IPTG is 1.0 mM.

[0034] More preferably, the final concentration of magnolol is 1.0 mM.

[0035] For further optimization, the cells were cultured at 23°C for another 24 hours.

[0036] The above-mentioned application of magnolol rhamnoside in the preparation of antitumor drugs.

[0037] According to a preferred embodiment of the invention, the tumor includes: breast cancer, oral epithelial carcinoma, and colon cancer.

[0038] An antitumor drug containing a pharmaceutically effective dose of the above-mentioned magnolol rhamnoside.

[0039] According to a preferred embodiment of the invention, the tumor includes: breast cancer, oral epithelial carcinoma, and colon cancer.

[0040] Beneficial effects:

[0041] 1. The honokiol rhamnoside of this invention, also known as honokiol-2-O-α-rhamnoside, is a novel derivative compound derived from the natural product honokiol through glycosylation modification. This invention is the first to determine the water solubility and tumor cell growth inhibitory activity of honokiol rhamnoside. The results show that this honokiol rhamnoside compound has higher water solubility and tumor cell growth inhibitory activity than honokiol, and has important application value in the development of new anti-tumor drugs.

[0042] 2. This invention constructs a recombinant Escherichia coli that co-expresses the rfbBDAC operon (derivative of dTDP-Rha, deoxythymidine diphosphate rhamnose) and the rhamnosyltransferase Ss-RhaT. Magnoliofurol rhamnoside is prepared by microbial fermentation. The recombinant Escherichia coli uses magnolol added to the culture medium as a substrate to achieve efficient synthesis of magnolofurol rhamnoside through fermentation. Attached Figure Description

[0043] Figure 1 The HPLC chromatogram of magnolol rhamnoside is shown.

[0044] Figure 2This is the mass spectrum of magnolol rhamnoside;

[0045] Figure 3 The 1H NMR spectrum of magnolol rhamnoside;

[0046] Figure 4 The carbon NMR spectrum of magnolol rhamnoside;

[0047] Figure 5 The hydrogen-hydrogen correlation (COSY) spectrum of magnolol rhamnoside;

[0048] Figure 6 The carbon-hydrogen direct correlation (HSQC) spectrum of magnolol rhamnoside;

[0049] Figure 7 The HMBC (hydrogen-carbohydrate long-range correlation) spectrum of magnolol rhamnoside;

[0050] Figure 8 The results show the assay results of magnolol rhamnoside and magnolol on the toxicity of six human cells. Detailed Implementation

[0051] The technical solution of the present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the materials and reagents used in the following embodiments are all commercially available biological and chemical experimental materials.

[0052] The Saccharothrix syringae strain used in the examples was purchased from the China General Microbiological Culture Collection Center (http: / / www.cgmcc.net / ) as Saccharothrix syringae CGMCC 4.1716.

[0053] Example 1: Construction of recombinant Escherichia coli

[0054] (1) Using Escherichia coli BL21(DE3) genomic DNA as a template, primers F-Rfb and R-Rfb were designed based on the rfbBDAC operon sequence synthesized by Escherichia coli dTDP-Rha in the NCBI database (GenBank accession No.NC_012892.2), and the rfbBDAC operon sequence was obtained by PCR amplification.

[0055] The sequences of primers F-Rfb and R-Rfb are as follows:

[0056] F-Rfb:5'-GGGAATTC CATATG GTGAAGATACTTGTTACTGGTGGCG-3', the underline indicates the NdeI restriction site.

[0057] R-Rfb:5'-CGG GGTACC TTAAACTTTCTCAAAAAGTTCTCTGAATG-3', the underscore indicates the Kpn I restriction site.

[0058] The PCR amplification system is as follows (total volume 100 μL):

[0059] 20 μL of 5×PCR buffer, 8 μL of 2.5 mM dNTPs, 2 μL each of 10 μM primers, 2 μL of 100 ng / μL template, 2 μL of 5×TransStart FastPfu Fly DNA polymerase (2.5 U / μL), and 64 μL of ultrapure water.

[0060] The conditions for PCR amplification are as follows:

[0061] Pre-denaturation at 95°C for 10 minutes; denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 90 seconds, for 30 cycles; extension at 72°C for 10 minutes.

[0062] The rfbBDAC operon sequence and plasmid vector pACYCDuet-1 obtained by PCR amplification were double-digested with Nde I and KpnI, respectively. The rfbBDAC operon sequence and the digested fragments of pACYCDuet-1 were recovered and ligated with T4 ligase at 16℃ for 4 h. The ligation product was transformed into E. coli DH5α competent cells. The transformation product was plated on LB agar plates containing 50 mg / L chloramphenicol and incubated overnight at 37℃. Single colonies from the overnight incubation plates were picked and inoculated into 5 mL of LB liquid medium and incubated at 37℃ for 8-10 h. The plasmid was extracted and sequenced. The size of the rfbBDAC operon sequence was determined to be 3472 bp, and the nucleotide sequence is shown in SEQ ID NO.1. The recombinant expression vector pAC-RfbBDAC was obtained.

[0063] (2) Using the genomic DNA of Syringa syringae as a template, primers F-RhaT and R-RhaT were designed based on the gene sequence of the rhamnosyltransferase Ss-RhaT (GenBank No.WP_033433114) predicted in the genomic DNA of Syringa syringae in the NCBI database. The gene sequence of rhamnosyltransferase Ss-RhaT was obtained by PCR amplification.

[0064] The sequences of primers F-RhaT and R-RhaT are as follows:

[0065] F-RhaT:5'-GGAATTC CATATG AAGATCCTGTTTTCGAGCCTCG-3', the underline indicates the Nde I restriction site.

[0066] R-RhaT:5'-CCG CTCGAG GGCGTACTGCGGCAGGG-3', the underline indicates the Xho I restriction site.

[0067] The PCR amplification system is as follows (total volume 100 μL):

[0068] 20 μL of 5×PCR buffer, 8 μL of 2.5 mM dNTPs, 2 μL each of 10 μM primers, 2 μL of 100 ng / μL template, 2 μL of 5×TransStart FastPfu Fly DNA polymerase (2.5 U / μL), and 64 μL of ultrapure water.

[0069] The conditions for PCR amplification are as follows:

[0070] Pre-denaturation at 95°C for 10 minutes; denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 90 seconds, for 30 cycles; extension at 72°C for 10 minutes.

[0071] The rhamnosyltransferase Ss-RhaT gene sequence and plasmid vector pET-22b(+) obtained by PCR amplification were double-digested with Nde I and Xho I, respectively. The gene sequence and the digested fragments of the plasmid vector were recovered and ligated with T4 ligase at 16℃ for 4 h. The ligation product was transformed into E. coli DH5α competent cells. The transformation product was plated on LB agar plates containing 50 mg / L ampicillin and incubated overnight at 37℃. Single colonies from the overnight incubation plates were picked and inoculated into 5 mL of LB liquid medium and incubated at 37℃ for 8-10 h. The plasmid was extracted and sequenced. The size of the gene encoding rhamnosyltransferase Ss-RhaT was determined to be 1146 bp, and the nucleotide sequence is shown in SEQ ID NO.2. The encoded amino acid sequence is shown in SEQ ID NO.3. The recombinant expression vector pET22b-Ss-rhaT was obtained.

[0072] (3) The recombinant expression vector pAC-RfbBDAC and the recombinant expression vector pET22b-Ss-rhaT were co-transformed into Escherichia coli BL21(DE3). The transformation products were plated on LB agar plates containing 50 mg / L ampicillin and 50 mg / L chloramphenicol and incubated overnight at 37°C. Single colonies were picked from the overnight incubated plates and identified by sequencing to obtain positive transformants, which are the recombinant Escherichia coli BL21-Rfb-RhaT that co-expresses the rfbBDAC operon synthesized by dTDP-Rha and the rhamnosyltransferase Ss-RhaT.

[0073] Example 2: Synthesis of magnolol rhamnoside

[0074] The recombinant Escherichia coli BL21-Rfb-RhaT constructed in Example 1 was cultured in LB medium at 23°C until the bacterial concentration reached OD100. 600 When the concentration of 1.2 is 1, add 1 mM IPTG and 1 mM honokiol to the culture medium and continue culturing at 23°C for 24 hours to obtain the fermentation broth containing honokiol rhamnoside.

[0075] One L of the fermentation broth containing magnolol rhamnoside was extracted three times with 300 mL of ethyl acetate. The extracts were combined, dried under reduced pressure by distillation, and dissolved in 15 mL of chloroform / ethyl acetate (5:2, v / v). Chromatographic separation was performed using a 10 mm ID × 10 cm Silica gel 60F254 silica gel column (Merck, Germany). Elution was performed with chloroform / ethyl acetate (5:2, v / v), and 30 mL of eluent was collected continuously. The sample was then eluted with 30 mL of methanol. The methanol-eluted product was concentrated and filtered, then purified by HPLC, followed by mass spectrometry and NMR analysis to obtain a magnolol rhamnoside solution. The obtained magnolol rhamnoside solution was distilled under reduced pressure and lyophilized to powder, yielding a total of 183 mg of magnolol rhamnoside. The conversion rate of magnolol to magnolol rhamnoside was 86%.

[0076] The equipment and conditions used for HPLC separation and purification are as follows:

[0077] Agilent 1260 high-performance liquid chromatograph; Agilent G1314B ultraviolet detector, detection wavelength 295nm; Agilent TC-C 18 The analytical column (4.6 × 150 mm) was used; the column temperature was 25℃, and the mobile phase was acetonitrile and water. Elution was performed at a flow rate of 1 mL / min at the following intervals: 0 min (25% acetonitrile), 0–25 min (85% acetonitrile), 25–30 min (100% acetonitrile), and 30–35 min (25% acetonitrile). The eluted samples were collected. The HPLC chromatogram is shown below. Figure 1 As shown.

[0078] Mass spectrometry structure identification:

[0079] The collected magnolol rhamnoside eluent was analyzed by mass spectrometry, and the results are as follows: Figure 2 As shown, the characteristic molecular ion peak [MH] of the target product - The value is m / z 411.1787, [M+Cl]. - With an m / z of 447.1516, the molecular weight of the product is estimated to be approximately 412.1886, which is consistent with the molecular weight of magnolol monorhamnoside.

[0080] NMR structure identification:

[0081] 5 mg of the magnolol rhamnoside powder prepared above was dissolved in deuterated DMSO and subjected to nuclear magnetic resonance analysis. The combined 1H NMR spectrum was analyzed. 1 H NMR)( Figure 3 ), carbon spectrum ( 13 C NMR)( Figure 4 ), Hydrogen-hydrogen correlation spectrum (COSY) ( Figure 5 ), C-H direct correlation spectrum (HSQC) () Figure 6 ), Hydrogen long-range correlation spectrum (HMBC) Figure 7 The chemical shifts and coupling constants of carbon and hydrogen at each position were determined, and the structure of the newly synthesized product was identified as magnolol-2-O-α-rhamnoside. 1 A single peak signal of the rhamnose anomeric proton was observed at a chemical shift of 5.14 ppm in the 1H NMR spectrum. Combined with the fact that the rhamnose donor in *E. coli* is dTDP-β-rhamnose, and that the recombinant rhamnose transferase Ss-RhaT is a configuration-inverting enzyme, it is inferred that the rhamnose is linked to the magnolol molecule via an α-bond. Cross signals were observed between the proton hydrogen of rhamnose and the C-2 of magnolol in the HMBC spectrum, thus confirming that the rhamnose is linked to the C-2 of magnolol.

[0082] Example 3: Synthesis of magnolol rhamnoside

[0083] 0.5 μL of the recombinant expression vector pET22b-Ss-rhaT constructed in Example 1 was transformed into Escherichia coli BL21(DE3) competent cells. The transformation product was plated on LB agar plates containing 50 mg / L ampicillin and incubated overnight at 37°C. Positive clones were screened. Positive clones were inoculated into 20 mL of LB liquid medium containing 50 mg / L ampicillin and incubated at 37°C for 10 h. The bacterial culture was then inoculated at a 1% inoculation rate into 1 L of LB liquid medium containing 50 mg / L ampicillin and incubated at 37°C for 3 h. When OD... 600 When the growth rate reaches 0.6-0.8 (logarithmic growth phase), IPTG at a final concentration of 0.5 mM is added for induction, and the mixture is cultured on a shaker at 100 rpm at 16°C for 20 h. The recombinant rhamnosyltransferase Ss-RhaT is obtained after purification.

[0084] The synthesis of magnolol rhamnoside was carried out in 50 mM Tris-HCl buffer (pH 8.5) in a 100 μL volume. The reaction system also included the following components at final concentrations: 2 mM dTDP-Rha, 2 mM honokiol, 5 mM MgCl2, and 500 μg / mL purified rhamnosyltransferase Ss-RhaT. The prepared reaction system was incubated at 42 °C for 6 h to catalyze the reaction, yielding a reaction solution containing magnolol rhamnoside.

[0085] Example 4: Properties of magnolol rhamnoside

[0086] (1) Determination of the water solubility of magnolol rhamnoside

[0087] Excess magnolol rhamnoside powder or magnolol powder prepared in Example 2 was added to 1.5 mL Eppendorf tubes containing 1 mL of deionized water at room temperature (25±2℃). The Eppendorf tubes were then placed in an ultrasonic cleaner at 200 W to assist in the dissolution of the compounds for 1 h. The ultrasonically treated samples were centrifuged at 10,000 × g for 10 min, and the supernatant was then filtered through a 0.22 μm pore size filter membrane. The content of the compounds in the filtrate was quantitatively analyzed by HPLC.

[0088] The experimental results are shown in Table 1. The results show that the maximum solubility of magnolol in water is 49.62 μM, while the maximum solubility of magnolol rhamnoside is 1335.12 μM. The water solubility of magnolol rhamnoside is about 27 times higher than that of the aglycone magnolol.

[0089] Table 1. Results of water solubility determination of magnolol rhamnoside

[0090]

[0091] (2) Determination of the growth inhibitory activity of magnolol rhamnoside on human tumor cells

[0092] The inhibitory activities of magnolol and magnolol rhamnoside on the cell growth of one normal human cell line (MCF-10a) and five human tumor cell lines (human breast cancer cells MCF-7, human breast cancer cells MDA-MB-231, human oral epithelial cancer cells KB, human colon cancer cells SW480, and human prostate cancer cells PC-3) were determined using the MTT assay. The specific steps are as follows:

[0093] Cells were cultured to the logarithmic growth phase, digested with 0.25% trypsin, and the cells were collected and counted under an inverted microscope. The cell suspension to be tested was prepared at a concentration of approximately 5 × 10⁻⁶. 3100 μL of cells / mL was added to each well of a 96-well plate, with three replicates per group. The seeded 96-well plates were incubated at 37°C in a 5% CO2 incubator for 24 h to allow for full cell adhesion. The supernatant was aspirated from the culture medium, leaving the cells. 100 μL of serum-free fresh culture medium was then added to each well. Next, 100 μL of PBS solution (pH 7.4) containing either honokiol or honokiol rhamnoside was added, with final concentrations of 10 μM, 20 μM, 50 μM, 80 μM, 100 μM, and 200 μM, respectively. The control group was brought to a final volume of 200 μL with PBS solution. Incubate 96-well plates at 37°C in a cell culture incubator containing 5% CO2 for 24 or 48 hours. Then, aspirate the supernatant, retaining the cells. Add 90 μL of fresh culture medium and 10 μL of MTT solution to each well, and continue incubation for 4 hours. Add DMSO, mix well, and incubate for another 30 minutes. Shake for 2 minutes to remove air bubbles and ensure the formazan is homogeneous. Finally, record the absorbance (Abs) at 490 nm. 490 ), calculate cell viability.

[0094] Cell survival rate, such as Figure 8As shown, magnolol (MAG) showed no growth inhibitory activity against human normal breast cells MCF-10a and human breast cancer cells MCF-7 at the tested concentrations of 10-200 μM. However, it exhibited varying concentration-dependent cell growth inhibitory activities against four other tumor cell types (MDA-MB-231, KB, SW480, and PC-3). In contrast, magnolol rhamnoside (MAG-Rha) showed concentration-dependent cell growth inhibitory activity against all six cell types, but its inhibitory activity against tumor cells MDA-MB-231, KB, and SW480 was significantly higher than its inhibitory activity against human normal breast cells MCF-10a. 10-20 μM honokiol (MAG) showed no significant inhibitory activity on the growth of MDA-MB-231 tumor cells. When the concentration was increased to 50 μM, the cell survival rate was 90.3% (48 h). While 10 μM honokiol rhamnoside (MAG-Rha) showed some inhibitory activity on the growth of normal human breast cells MCF-10a with a cell survival rate of 89.2% (48 h), it more strongly inhibited the growth of MDA-MB-231 cells, reducing the cell survival rate to 42.9% (48 h). When the concentration of honokiol rhamnoside (MAG-Rha) was increased to 20 μM and 50 μM, the survival rates of MCF-10a cells were 88.2% (48 h) and 55% (48 h), respectively, while the survival rate of MDA-MB-231 cells further decreased to 22.5% (48 h) and 17.1% (48 h). 10-20 μM honokiol (MAG) had no significant inhibitory activity on tumor cells KB and SW480, while 20 μM honokiol rhamnoside (MAG-Rha) significantly inhibited the growth of both cell types, with KB cell survival rate of 69.4% (48 h) and SW480 cell survival rate of 78.3% (48 h).

Claims

1. A magnolol rhamnoside, characterized in that, The chemical name is magnolol-2- O - α - Rhamnose glycoside, molecular formula C 24 H 28 O6, chemical structural formula as follows: 。 2. A method for preparing magnolol rhamnoside according to claim 1, characterized in that, Magnolol rhamnoside was prepared by an enzymatic reaction using dTDP-Rha as the rhamnose donor, magnolol as the glycosyl acceptor, and rhamnosyltransferase as the enzyme catalyst; wherein the rhamnosyltransferase was derived from *Syringa syringae* (a type of fungus). Saccharothrix syringae The rhamnosyltransferase Ss-RhaT has the nucleotide sequence shown in SEQ ID NO. 2 and the amino acid sequence shown in SEQ ID NO.

3.

3. A method for preparing magnolol rhamnoside according to claim 1, characterized in that, It uses magnolol as a glycosyl acceptor to co-express dTDP-Rha synthesis. rfbBDAC The recombinant Escherichia coli BL21-Rfb-RhaT containing the operon and rhamnosyltransferase Ss-RhaT was used as a fermentation strain to prepare magnolol rhamnoside; wherein the nucleotide sequence of the rhamnosyltransferase Ss-RhaT is shown in SEQ ID NO. 2 and the amino acid sequence is shown in SEQ ID NO.

3.

4. The preparation method according to claim 3, characterized in that, The rfbBDAC The operon is derived from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO.

1.

5. The preparation method according to claim 3, characterized in that, The rfbBDAC The plasmid vector for the operon is pACYCDuet-1.

6. The preparation method according to claim 3, characterized in that, The plasmid vector for expressing the rhamnosyltransferase Ss-RhaT is pET-22b (+).

7. A method for preparing magnolol rhamnoside according to claim 1, characterized in that, Specifically, the steps include the following: Synthesized by co-expressing dTDP-Rha rfbBDAC Recombinant Escherichia coli BL21-Rfb-RhaT containing the operon and rhamnosyltransferase Ss-RhaT was cultured to an OD concentration. 600 To obtain a fermentation broth containing honokiol rhamnoside, IPTG with a final concentration of 0.5-2.0 mM and honokiol with a final concentration of 0.5-5.0 mM are added to the culture medium, and the mixture is cultured at 16-37 °C for 6-72 hours. The nucleotide sequence of the rhamnosyltransferase Ss-RhaT is shown in SEQ ID NO. 2, and the amino acid sequence is shown in SEQ ID NO.

3.

8. The preparation method according to claim 7, characterized in that, The method for constructing the recombinant Escherichia coli BL21-Rfb-RhaT is as follows: (1) Using Escherichia coli BL21 (DE3) genomic DNA as a template, primers F-Rfb and R-Rfb were designed, and PCR amplification was performed to obtain rfbBDAC The operon sequence was ligated to the plasmid vector pACYCDuet-1 to obtain the recombinant expression vector pAC-RfbBDAC; The sequences of primers F-Rfb and R-Rfb are as follows: F-Rfb:5'-GGGAATTC CATATG GTGAAGATACTTGTTACTGGTGGCG-3', underscore indicates Nde I restriction site, R-Rfb:5'-CGG GGTACC TTAAACTTTCTCAAAAAGTTCTCTGAATG-3', underscore indicates Kpn I. Enzyme cleavage site; (2) Using the genomic DNA of Syringa syringae as a template, primers F-RhaT and R-RhaT were designed. The rhamnosyltransferase Ss-RhaT gene sequence was obtained by PCR amplification. After ligating it with the plasmid vector pET-22b (+), the recombinant expression vector pET22b-Ss-rhaT was obtained. The sequences of primers F-RhaT and R-RhaT are as follows: F-RhaT:5'-GGAATTC CATATG AAGATCCTGTTTTCGAGCCTCG-3', underscore indicates Nde I restriction site, R-RhaT:5'-CCG CTCGAG GGCGTACTGCGGCAGGG-3', underscore indicates Xho I. Enzyme cleavage site; (3) The recombinant expression vector pAC-RfbBDAC and the recombinant expression vector pET22b-Ss-rhaT were co-transformed into Escherichia coli BL21 (DE3), and positive transformants were screened to obtain recombinant Escherichia coli BL21-Rfb-RhaT.

9. The preparation method according to claim 7, characterized in that, One or more of the following conditions must be met: i. The bacterial cell concentration OD 600 It is 1.2; ii. The final concentration of the IPTG is 1.0 mM; iii. The final concentration of magnolol is 1.0 mM; iv. Continue culturing at 23 °C for 24 hours.

10. The use of magnolol rhamnoside according to claim 1 in the preparation of antitumor drugs; wherein the tumor is: breast cancer, oral epithelial carcinoma, and colon cancer.

11. An antitumor drug, characterized in that, The tumor contains a pharmaceutically effective dose of magnolol rhamnoside as described in claim 1; the tumor is: breast cancer, oral epithelial carcinoma, or colon cancer.

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