A fisetin-4'-o-alpha-d-glucopyranoside for resisting lung cancer and a preparation method and application thereof

Abstract

The application discloses a fisetin-4'-O-alpha-D-glucopyranoside for resisting lung cancer and a preparation method and application thereof, and the preparation method comprises the following steps: taking sucrose as a glycosyl donor, taking fisetin as a glycosyl acceptor, and performing an enzymatic transglycosylation reaction under the catalysis of sucrose phosphorylase to direct the glucose group generated by decomposing the sucrose to be transferred to a hydroxyl group at a C4' position of the fisetin, so that a target product is obtained; and the sucrose phosphorylase is derived from Bifidobacterium adolescentis. The application retains the original core biological activities of the fisetin, such as antioxidation and anti-inflammation, provides high-quality raw materials for the application of the fisetin in the fields of innovative drug development for resisting lung cancer and preparation of medical intermediates, and expands the clinical transformation potential of fisetin compounds.

Description

Technical Field

[0001] This invention relates to an anti-lung cancer flavin-4'-O-α-D-glucopyranoside, its preparation method and application, belonging to the field of biomedical technology. Background Technology

[0002] Fisetin (3,3′,4′,7-tetrahydroxyflavone) is a flavonoid compound naturally found in various fruits and vegetables, such as strawberries, apples, grapes, onions, and cucumbers. In recent years, due to its wide range of biological activities, fisetin has become a hot topic in natural product research and drug development. Fisetin possesses various biological activities, including antioxidant, anti-inflammatory, anticancer, neuroprotective, and anti-aging effects. In anti-tumor activity, fisetin has shown the ability to induce tumor cell apoptosis, inhibit tumor cell migration, and suppress angiogenesis. Regarding metabolic disorders, fisetin helps improve diabetic nephropathy and fatty liver degeneration caused by a high-fat diet. The anti-inflammatory and antioxidant effects of fisetin hold promise for treating inflammatory diseases and reducing oxidative stress. Furthermore, fisetin also exhibits positive effects in neuroprotection and anti-aging.

[0003] However, fuzzin faces significant application bottlenecks. Its susceptibility to intestinal metabolism and poor water solubility result in low oral bioavailability, severely limiting its clinical value. Glycosylation offers an effective strategy to overcome these limitations. By introducing glucose groups at specific hydroxyl sites in fuzzin for glycosylation modification, the water solubility and in vivo stability of free fuzzin can be specifically improved. This modification strategy not only provides a research direction for exploring the structure-activity relationship and derivatization potential of fuzzin as a novel pharmaceutical intermediate but also lays a theoretical foundation and provides experimental evidence for its rational application in innovative drug development.

[0004] As glycosylated derivatives of natural products, rutin glycosides are complex in molecular structure, making precise site modification difficult to achieve in chemical synthesis and resulting in low product purity. Currently, the mainstream synthetic method is enzymatic synthesis, the core mechanism of which lies in utilizing glycosyltransferases (such as UGT78D1) with UDP-glucose (UDP-Glc) as the glycosyl donor: through the transglycosylation activity of the enzyme, UDP-Glc is directionally transferred to the C3 / C7 hydroxyl site of rutin, ultimately generating the target glycoside product.

[0005] The main drawbacks of existing technologies are:

[0006] (1) The problem of regioselectivity of enzymes: Ficus flavin contains multiple phenolic hydroxyl groups that can be glycosylated, and glycosyltransferases from different sources usually have different regioselectivity. Lack of regioselectivity will lead to complex products (generating multiple monosaccharide-, disaccharide-, and even polysaccharide-, isomers).

[0007] (2) Glycosyl donor preference: Glycosyltransferases that use UDP-glucose as a glycosyl donor are an important class of enzymes that catalyze glycosylation reactions, but UDP-Glc is expensive.

[0008] (3) Many naturally derived glycosyltransferases have high activity for their natural substrates (such as flavonoids and flavonol analogs), but may have low activity for molecules with a specific hydroxyl pattern, such as rutin. Summary of the Invention

[0009] To address the problems existing in the prior art, the present invention provides an anti-lung cancer flavin-4'-O-α-D-glucopyranoside, its preparation method, and its application.

[0010] To achieve the above objectives, the present invention employs a lung cancer-resistant flavin-4'-O-α-D-glucopyranoside, which has the following structural formula:

[0011] .

[0012] In a second aspect, the present invention also provides a method for preparing the anti-lung cancer flavin-4'-O-α-D-glucopyranoside, wherein sucrose is used as a glycosyl donor and flavin is used as a glycosyl acceptor, and an enzymatic transglycosylation reaction is carried out under the catalysis of sucrose phosphorylase to directionally transfer the glucose group generated from sucrose decomposition to the C4' hydroxyl group of flavin, thereby obtaining the target product; wherein the sucrose phosphorylase is derived from Bifidobacterium adolescentis.

[0013] Preferably, the amino acid sequence of the sucrose phosphorylase is SEQ ID No. 1, and the encoding gene sequence is SEQ ID No. 2.

[0014] Preferably, the sucrose phosphorylase is constructed by expressing it through the following steps:

[0015] (1) The recombinant expression plasmid containing the encoding gene was transformed into Escherichia coli BL21 (DE3) and inoculated into LB medium containing 100 mg / mL kanamycin at a volume fraction of 1%. The medium was cultured at 37°C for 12 hours at a speed of 220 r / min. The amino acid sequence of the encoding gene is shown in SEQ ID No.1.

[0016] (2) Take 2 mL of the culture medium from step (1) and add it to 200 mL of TB medium. Incubate at 37°C in a shaker until the OD of the bacterial culture is diluted 10 times. 600When the concentration is 0.20~0.24, add 0.5mM IPTG and induce fermentation at 25℃ for 16 hours; centrifuge the fermentation suspension at 0℃ and 8000r / min for 12~15min, wash with 20mM pH 6.0 MES buffer and centrifuge again, add 20mL of 20mM pH 6.0 MES buffer, sonicate in an ice water bath for 15min, centrifuge and collect the supernatant to obtain sucrose phosphorylase solution.

[0017] Preferably, each liter of LB medium contains the following components: 3-8g yeast extract, 6-14g tryptone, and 6-14g NaCl, using deionized water as the solvent;

[0018] The composition of each liter of TB culture medium is as follows: using deionized water as the solvent, it contains 5g glycerol, 13-14g tryptone, 25-30g yeast extract, 1-3g KH2PO4, and 15-20g K2HPO4·3H2O.

[0019] Preferably, the conditions for the enzymatic transglycosylation reaction are: a molar ratio of sucrose to rutin of 20:1, a reaction system containing 200 mM sucrose and 10 mM rutin, and a sucrose phosphorylase concentration of 3-6 U / mL.

[0020] The flavin glycoside was prepared by reacting continuously for 24 hours in MES buffer at 50°C and pH 7.0 with constant temperature shaking at 220 rpm.

[0021] Preferably, a purification step is also included:

[0022] The enzyme reaction solution was heated in a water bath at 100℃ for 5 min to terminate the reaction. Anhydrous ethanol was added to bring the ethanol concentration to 70%. The supernatant was concentrated by rotary evaporation and then extracted with ethyl acetate. The solution was concentrated again and purified by silica gel column chromatography. The solution was then loaded onto the sample using a dry method with a mobile phase of ethyl acetate:methanol = 10:1.

[0023] The eluent containing the target product was collected and purified by dextran gel column G15. The eluent was first washed with pure water until no monosaccharide residue was found, and then eluted with 30% ethanol. The eluent was collected and concentrated to obtain high-purity lacquinone-4'-O-α-D-glucopyranoside.

[0024] A third aspect of the invention also provides the use of flavin-4'-O-α-D-glucopyranoside as described above or flavin-4'-O-α-D-glucopyranoside prepared by the method described above in the preparation of anti-lung cancer drugs.

[0025] Preferably, the dosage form of the drug includes tablets, capsules, injections, suspensions, or gels.

[0026] Preferably, the drug exerts its anti-lung cancer effect by inducing lung cancer cell apoptosis, inhibiting lung cancer cell migration, or angiogenesis.

[0027] The principle of this invention:

[0028] For the first time, sucrase phosphorylase derived from Bifidobacterium adolescentis was used to synthesize rutin glycosyltransferase. Compared with other enzymes such as UDP-glucose transferase, sucrase phosphorylase has higher catalytic efficiency, and the substrate sucrose is cheaper and more readily available. Its catalytic reaction follows a double substitution mechanism, specifically as follows: First, the sucrose molecule, acting as a glycosyl donor, binds to the enzyme's active site. The enzyme catalyzes the cleavage of the glycosidic bond between the glucose and fructose groups in the sucrose molecule, releasing fructose. Simultaneously, the glucose group covalently bonds with the carboxyl residues of the enzyme's active site to form a stable glucose-enzyme covalent intermediate. Subsequently, the intermediate is attacked by a nucleophile in the system, resulting in a secondary displacement reaction: if the nucleophile is water, a hydrolysis reaction occurs to generate glucose; if it is inorganic phosphate, a phosphorylation reaction occurs to generate glucose-1-phosphate. In this invention, the lacquer flavonoid molecule acts as a glycosyl acceptor, and its C4' hydroxyl group specifically acts as a nucleophile to attack the glucose group in the glucosyl-enzyme intermediate, causing the glucose group to detach from the enzyme active site and directionally transfer to the C4' hydroxyl group of lacquer flavonoid, forming an α-O-glycosidic bond, and finally efficiently synthesizing the target product lacquer flavonoid-4'-O-α-D-glucopyranoside.

[0029] The above reaction uses sucrose and rutin as raw materials, and relies on the highly regioselective recognition ability of sucrose phosphorylase on the C4' hydroxyl group of rutin to achieve precise site-specific modification of the glucose group. This modification strategy significantly improves the water solubility of the product by introducing a hydrophilic glucose group into the rutin molecule. At the same time, the steric hindrance effect of the glucose group protects rutin from metabolic degradation by intestinal flora, thereby improving its in vivo absorption efficiency and metabolic stability, and ultimately greatly improving its bioavailability. In addition, the enzymatic reaction of this invention does not require harsh conditions such as high temperature and high pressure, and the sucrose donor is widely available and inexpensive. The reaction product is single and has no redundant isomers, which significantly simplifies the subsequent separation and purification process. This provides an efficient, economical, and environmentally friendly technical route for the industrial-scale production of rutin-4'-O-α-D-glucopyranoside, and lays an important foundation for the drug development, clinical translation, and structure-activity relationship study of this type of glycoside derivative, possessing significant industrial application value and scientific research value.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] (1) This invention utilizes sucrose phosphorylase derived from Bifidobacterium adolescentis to catalyze the transglycosylation reaction. This enzyme can specifically recognize the C4' hydroxyl group of fuzzin and directionally transfer the glucose group generated from sucrose decomposition to the target site, achieving the precise synthesis of fuzzin-4'-O-α-D-glucopyranoside. The product does not generate glycosylated isomers with other hydroxyl sites or polysaccharide byproducts, greatly simplifying the subsequent separation and purification process and reducing purification costs. At the same time, this enzyme has good catalytic activity for fuzzin, significantly improving the conversion rate of the target product and providing core technical support for large-scale preparation.

[0032] (2) This invention abandons the expensive UDP-glucose used in the prior art and selects sucrose, which is widely available and inexpensive, as the glycosyl donor, effectively reducing the cost of raw materials. The enzymatic reaction can be carried out efficiently under mild conditions of room temperature and pressure, without the need for harsh processes such as high temperature and high pressure. This not only reduces energy consumption and investment in special equipment, but also reduces the probability of impurity generation during the reaction, thus controlling production costs from the source. The characteristics of this process meet the actual needs of industrial production, laying a solid foundation for the scale-up production of lacquinone-4'-O-α-D-glucopyranoside and has good prospects for industrial application.

[0033] (3) The lacquer xanthophyll-4'-O-α-D-glucopyranoside synthesized in this invention improves the poor water solubility and susceptibility to intestinal metabolism of free lacquer xanthophyll by directional introduction of glucose groups, thereby enhancing the product's in vivo stability and absorption efficiency, and thus improving bioavailability. Its water solubility at 30°C increased from 13.6±1.3 mg / L to 109.8±6.3 mg / L, and the water solubility of lacquer xanthophyll-4'-O-α-D-glucopyranoside was about 8 times higher than that of lacquer xanthophyll. The product retains the original antioxidant, anti-inflammatory and other core biological activities of lacquer xanthophyll, providing high-quality raw materials for its application in the development of anti-lung cancer drugs and the preparation of pharmaceutical intermediates, and expanding the clinical translation potential of lacquer xanthophyll compounds. Attached Figure Description

[0034] Figure 1 This is a high-performance liquid chromatogram of the reaction solution of sucrose phosphorylase catalyzing the rutin transglycosylation reaction in this invention;

[0035] Figure 2 This is the mass spectrum of flavin-4'-O-α-D-glucopyranoside after separation and purification according to the present invention;

[0036] Figure 3 This is the one-dimensional 1H NMR spectrum of lacquer xanthocyanin-4'-O-α-D-glucopyranoside isolated and purified according to the present invention;

[0037] Figure 4 This is the one-dimensional carbon spectrum of laccasein-4'-O-α-D-glucopyranoside isolated and purified according to the present invention;

[0038] Figure 5 This is a two-dimensional HMBC spectrum of laccasein-4'-O-α-D-glucopyranoside isolated and purified according to the present invention;

[0039] Figure 6 This is a two-dimensional COSY spectrum of flavin-4'-O-α-D-glucopyranoside isolated and purified according to the present invention;

[0040] Figure 7 This is a two-dimensional HSQC spectrum of laccasein-4'-O-α-D-glucopyranoside isolated and purified according to the present invention;

[0041] Figure 8 This invention provides the isolated and purified flavin glycosides. 1 H and 13 The chemical shift diagram of C confirms the structure as rutin-4'-O-α-D-glucopyranoside;

[0042] Figure 9 This is a schematic diagram illustrating the effect of temperature on the sucrose phosphorylase of the present invention;

[0043] Figure 10 This is a schematic diagram illustrating the effect of pH on the sucrose phosphorylase of the present invention;

[0044] Figure 11 This is a schematic diagram illustrating the cell migration rate of human lung cancer cells NCl-H520 by the flavin glycosides isolated and purified according to the present invention.

[0045] Figure 12 This is a schematic diagram illustrating the effect of the purified rutin glycosides isolated in this invention on the cell proliferation of human lung cancer cells NCl-H520. Detailed Implementation

[0046] The following embodiments are further illustrations of the present invention and serve as explanations of the technical content of the present invention. However, the essence of the present invention is not limited to the embodiments described below. Those skilled in the art can and should know that any simple changes or substitutions based on the spirit of the present invention should fall within the protection scope claimed by the present invention.

[0047] Example 1

[0048] A method for expressing and constructing a sucrose phosphorylase, comprising the following steps:

[0049] (1) The amino acid sequence of sucrose phosphorylase BaSP (GenBank: AZH70705.1) from Bifidobacterium adolescentis was found by NCBI. Using pE-T28a(+) as a vector, the recombinant expression plasmid pET-28a(+)-BaSP was directly synthesized. The recombinant plasmid was transformed into E. coli BL21(DE3) and inoculated into LB medium containing 100 mg / mL kanamycin at a volume fraction of 1%. The medium was cultured at 37°C for 12 hours at a speed of 220 r / min.

[0050] The amino acid sequence of sucrose phosphorylase is SEQ ID No. 1, and its encoding gene sequence is SEQ ID No. 2.

[0051] SEQ ID No. 1:

[0052] MKNKVQLITYADRLGDGTIKSMTDILRTRFDGVYDGVHILPFFTPFDGADAGFDPIDHTKVDERLGSWDDVAELSKTHNIMVDAIVNHMSWESKQFQDVLAKGEESEYYPMFLTMSSVFPNGATEE DLAGIYRPRPGLPFTHYKFAGKTRLVWVSFTPQQVDIDTDSDKGWEYLMSIFDQMAASHVSYIRLDAVGYGAKEAGTSCFMTPKTFKLISRLREEGVKRGLEILIEVHSYYKKQVEIASKVDRVYD FALPPLLLHALSTGHVEPVAHWTDIRPNNAVTVLDTHDGIGVIDIGSDQLDRSLKGLVPDEDVDNLVNTIHANTHGESQAATGAAASNLDLYQVNSTYYSALGCNDQHYIAARAVQFFLPGVPQVY YVGALAGKNDMELLRKTNNGRDINRHYYSTAEIDENLKRPVVKALNALAKFRNELDAFDGTFSYTTDDDTSISFTWRGETSQATLTFEPKRGLGVDNTTPVAMLEWEDSAGDHRSDDLIANPPVVA;

[0053] SEQ ID No. 2:

[0054]

[0055] (2) Take 2 mL of the culture medium from step (1) and add it to 200 mL of TB medium. Incubate at 37°C in a shaker until the OD of the bacterial culture is diluted 10 times. 600 When the concentration is 0.20~0.24, add 0.5mM IPTG (isopropyl-β-D-thiogalactoside) and induce fermentation at 25℃ for 16 hours; centrifuge the fermentation suspension at 0℃ and 8000r / min for 13 min, wash with 20mM pH 6.0 MES buffer and centrifuge again, add 20mL of 20mM pH 6.0 MES buffer (2-(N-morpholino)ethanesulfonic acid buffer), sonicate in an ice water bath for 15 min, centrifuge and collect the supernatant to obtain sucrose phosphorylase solution.

[0056] SDS-PAGE electrophoresis analysis of the samples showed that the molecular weight of the expressed sucrose phosphorylase was approximately 57.3 kDa, consistent with the predicted value.

[0057] The composition of each liter of LB medium is as follows: using deionized water as the solvent, it contains 5g yeast extract, 10g tryptone, and 10g NaCl; the composition of each liter of TB medium is as follows: using deionized water as the solvent, it contains 5g glycerol, 13.33g tryptone, 26.67g yeast extract, 2.31g KH2PO4, and 16.43g K2HPO4·3H2O.

[0058] The enzyme activity of the above sucrose phosphorylase was determined by the DNS (3,5-dinitrosalicylic acid) method. The amount of monosaccharide generated in the system was used as the detection index. The enzyme activity unit (U / ml) was defined as: the amount of enzyme required to catalyze the generation of 1 μmol of monosaccharide from sucrose per minute in 1 ml of reaction system at 55℃.

[0059] The specific steps are as follows:

[0060] Reaction system preparation: Mix 0.05g sucrose, 50μl enzyme solution and 50μl water, add 900μl MES buffer, and react at 55℃ for 0.5 hours (the control group uses inactivated enzyme solution).

[0061] Detection procedure: Take 50 μl of reaction solution, add 200 μl of water and 750 μl of DNS reagent, boil in a water bath for 5 minutes, then dilute with 5 ml of water, and measure the absorbance (OD) at 520 nm using a UV spectrophotometer. 520 );

[0062] Calculation method: Crude enzyme activity (U / ml) = OD 520 Value * 10 / 0.04267.

[0063] This method quantifies the amount of monosaccharide produced by colorimetry, which can rapidly characterize the catalytic activity of sucrose phosphorylase and provide a basis for optimizing the amount of enzyme in enzymatic reactions.

[0064] Example 2

[0065] A method for preparing an anti-lung cancer flavin-4'-O-α-D-glucopyranoside includes the following steps:

[0066] First, using sucrose as the glycosyl donor and rutin as the glycosyl acceptor, with a sucrose to rutin molar ratio of 20:1, the reaction system contained 200 mM sucrose and 10 mM rutin, and the amount of sucrose phosphorylase was 3~6 U / mL (5 U / mL in this example). The reaction was carried out for 24 hours under constant temperature shaking conditions of 220 rpm in MES buffer at 50℃ and pH 7.0 to obtain the rutin glycoside reaction solution.

[0067] The reaction solution was then heated in a 100°C water bath for 5 minutes to terminate the reaction. Anhydrous ethanol was added to a final concentration of 70%. The supernatant was concentrated by rotary evaporation and extracted with ethyl acetate. The extract was concentrated again, and the concentrate was purified by silica gel column chromatography. The solution was then loaded onto a dry mobile phase of ethyl acetate:methanol = 10:1. The eluent containing the product, obtained by TLC thin-layer chromatography, was concentrated and passed through a G15 dextran gel column. The column was first washed with pure water until no monosaccharide residue remained, and then eluted with 30% ethanol as the mobile phase to obtain the purified laccasein-4'-O-α-D-glucopyranoside. The yield was 18%.

[0068] The flavin glycoside obtained in Example 2 has the following structural formula:

[0069] ;

[0070] Figure 1 The image shows a high-performance liquid chromatogram of the reaction solution of sucrose phosphorylase catalyzing the transglycosylation of rutin. Analysis shows that sucrose phosphorylase catalyzes the highly specific transglycosylation of rutin.

[0071] The mass spectrum of the above-mentioned flavin-4'-O-α-D-glucopyranoside is shown below. Figure 2 As shown, the molecular formula is C 21 H 20 O 11 The mass spectrometry results showed negative ion mass spectrometry, indicating the loss of one hydrogen ion, with a molecular weight of 447, consistent with the molecular weight of flavin-4'-O-α-D-glucopyranoside.

[0072] Figure 3This is the one-dimensional hydrogen spectrum of lacquer-4'-O-α-D-glucopyranoside after separation and purification according to the present invention. Analysis shows that lacquer-4'-O-α-D-glucopyranoside has 11 hydrogen atoms with different chemical shifts, which is consistent with the structural formula.

[0073] Figure 4 This is the one-dimensional carbon spectrum of flavin-4'-O-α-D-glucopyranoside isolated and purified according to the present invention. Analysis shows that flavin-4'-O-α-D-glucopyranoside has 21 carbon atoms with different chemical shifts, which is consistent with the structural formula. The chemical shift table of the proton and carbon spectra is shown in Table 1.

[0074] Figure 5 This is a two-dimensional HMBC spectrum of the isolated and purified flavin-4'-O-α-D-glucopyranoside. In the HMBC spectrum, there is a significant long-range correlation peak between the sugar end proton signal (δ~5.33, i.e. H-1”) and the carbon signal δ 145.55 (C-4') on the flavonoid nucleus, proving that the glucose group is connected to the phenolic hydroxyl group at the 4' position of the benzene ring of the nucleus through an α-O-glycosidic bond.

[0075] Figure 6 This is a two-dimensional COSY spectrum of flavin-4'-O-α-D-glucopyranoside isolated and purified according to the present invention.

[0076] Figure 7 This is a two-dimensional HSQC spectrum of flavin-4'-O-α-D-glucopyranoside isolated and purified according to the present invention.

[0077] The present invention isolates and purifies lacquinone-4'-O-α-D-glucopyranoside. 1 H and 13 The chemical shift table and schematic diagram of C are shown in Table 1 below. Figure 8 As shown, the structure was determined to be flavin-4'-O-α-D-glucopyranoside.

[0078] Table 1. Rhus flavin glycosides 1 H and 13 Chemical shift table of C

[0079]

[0080] Example 3

[0081] The optimal temperature for the sucrose phosphorylase involved in this invention is 50°C. Temperature affects the activity and stability of the enzyme. Therefore, this invention also investigated the effect of different temperatures on the synthesis of rutin-4'-O-α-D-glucopyranoside.

[0082] Experimental groups were set up (temperatures: 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃) and a blank control group (with inactivated enzyme added, all other conditions the same as the reaction group). 25U of enzyme was added to a 5mL (pH=6.0) MES buffer system of 10mM rutin and 10% (v / v) DMSO, and incubated together at different temperatures and 250rpm on a shaker. Samples were taken after 24h of reaction, boiled for 5min to inactivate the enzyme, centrifuged at 12000rpm for 5min, and the concentration of each component in the reaction solution was determined by HPLC.

[0083] HPLC detection conditions: Amide column;

[0084] Column temperature: normal temperature;

[0085] Mobile phase: Acetonitrile:KH2PO4 (50mM) = 65:35 (v / v), flow rate 0.5mL / min;

[0086] Detector: 360nm ultraviolet detector.

[0087] Based on the peak area of ​​rutin-4'-O-α-D-glucopyranoside, the relative enzyme activity and product concentration at different temperatures were calculated using the normalization method. Each experiment was repeated three times, and the results are as follows: Figure 9 As shown in the figure, the analysis shows that the optimal temperature for synthesizing laccasein-4'-O-α-D-glucopyranoside is 50℃, at which temperature the concentration of laccasein-4'-O-α-D-glucopyranoside synthesized by enzyme catalysis is the highest.

[0088] Example 4

[0089] The optimal storage pH for the sucrose phosphorylase involved in this experiment is 6.0, as pH affects enzyme activity and stability. This invention investigates the effects of different pH values ​​on the synthesis of rutin-4'-O-α-D-glucopyranoside.

[0090] Experimental groups (pH: 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0) and a blank control group (no enzyme added, all other conditions the same as the reaction group) were set up. 25 U of enzyme was added to a 5 mL MES buffer system of 10 mM rutin and 10% (v / v) DMSO at pH 6.0, and the mixture was incubated at 50°C and 250 rpm on a shaker. Samples were taken after 24 h of reaction, boiled for 5 min to inactivate the enzyme, centrifuged at 12000 rpm for 5 min, and the concentration of each component in the reaction solution was determined by HPLC.

[0091] HPLC detection conditions: Amide column; column temperature: room temperature; mobile phase: acetonitrile: 50 mL M KH2PO4 = 65–35 (v / v), flow rate: 0.5 mL / min; detector: UV detector 360 nm. Based on the peak area of ​​rutin-4'-O-α-D-glucopyranoside, the relative enzyme activity and product concentration at different pH values ​​were calculated using the normalization method. Each experiment was repeated three times, and the results are as follows: Figure 10 As shown in the figure, the analysis shows that the optimal pH for the synthesis of lacquer-4'-O-α-D-glucopyranoside is 7.0. At this pH, the concentration of lacquer-4'-O-α-D-glucopyranoside synthesized by enzyme catalysis is the highest.

[0092] Example 5

[0093] The scratch healing assay was used to evaluate the effect of isolated and purified rutin glycosides on the cell migration rate of human lung cancer cells NCI-H520. The scratch healing assay involves artificially creating a "scratch" on a monolayer of adherent cells. Cells at the edge migrate into the void area, causing the void to shrink or even close. By acquiring and comparing images at multiple time points, the cell migration ability can be quantitatively assessed.

[0094] After counting, cells were seeded into 6-well plates and incubated at 37°C with 5% CO2 for 24 hours to allow cell adhesion and growth into a uniform monolayer. Using a 200 μL sterile pipette tip, cells were cross-shaped and gently rinsed three times with PBS to remove detached cell debris and ensure clean edges. After washing, serum-free culture medium was added to each well before drug administration. The experimental group consisted of three groups: a control group (no drug), and wells containing 100 μol of rutin glycoside (FG-100) and 100 μol of rutin (F-100). The 6-well plates were incubated at 37°C with 5% CO2 for 0 h and 24 h, and photographed afterward. The relative migration rate was (0 h area - 24 h area) / 0 h area. A lower relative migration rate indicates a stronger inhibitory effect of the drug on the migration of H520 lung cancer cells.

[0095] like Figure 11 As shown in the analysis, the control group H520 cells migrated significantly after 24 hours, while the H520 cells with added rutin and its glycosylation products showed almost no migration. Furthermore, rutin glycosides had a better inhibitory effect on the migration of lung cancer cells H520 than rutin alone. This indicates that 100 μmol of rutin glycosides has a good inhibitory effect on the migration of lung cancer cells H520, continuously inhibits the migration and colonization of residual cancer cells, reduces the risk of long-term recurrence and metastasis, and provides a candidate drug for adjuvant therapy after lung cancer surgery.

[0096] Example 6

[0097] To investigate the inhibitory effect of rutin glycosides on the proliferation of human lung cancer H520 cells, cell viability was determined using the MTT assay. The MTT assay, also known as the 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay, works on the principle that succinate dehydrogenase in the mitochondria of living cells reduces exogenous yellow MTT dye to insoluble blue-violet crystals called formazan. The amount of formazan produced is directly proportional to the number of living cells and their metabolic activity. In dead cells, this enzyme is inactivated and cannot reduce MTT. After dissolving the formazan crystals in dimethyl sulfoxide (DMSO), the absorbance (OD value) was measured at 570 nm using a microplate reader. A higher OD value indicates a greater number of living cells; a lower OD value indicates inhibited cell viability.

[0098] H520 cells in logarithmic growth phase were digested with trypsin to prepare a single-cell suspension. After counting and adjusting to an appropriate density, the cells were seeded into 96-well plates, with 100 μL of cell suspension (containing approximately 1 × 10⁶ cells) added to each well. 4 Cells were cultured at 37℃ in a 5% CO2 incubator for 24 hours to allow cell adhesion. After discarding the culture medium, blank control and 50 μmol, 100 μmol, and 200 μmol of rutin and rutin glycoside were added to each well, and cultured for another 24 hours. Then, 10 μL of MTT solution was added to each well, and the cells were cultured for 4 hours to allow formazan crystals to form. The supernatant was removed, and 150 μL of LDMSO was added to each well. The cells were shaken slowly for 10-15 minutes until the crystals were completely dissolved. The cells were then detected at 630 and 570 nm using a microplate reader.

[0099] like Figure 12 As shown, with the relative survival rate of the blank group being 1, its relative survival rate = (experimental group OD570 - experimental group OD630) / (control group OD570 - control group OD630). The relative survival rate of cells with added rutin glycosides decreased with increasing concentration. At a concentration of 200 μmol, its relative survival rate was only about 50% of that of the control group, indicating that rutin glycosides have a superior inhibitory effect on the proliferation of lung cancer cells H520. Its anti-metastasis and anti-growth effects can be developed into a local drug delivery formulation (such as an inhaler) or a combination adjuvant for lung squamous cell carcinoma, for postoperative recurrence and metastasis prevention, and can also serve as a candidate treatment strategy after drug resistance.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A flavin-4'-O-α-D-glucopyranoside for treating lung cancer, characterized in that, It has the following structural formula: 。 2. A method for preparing the anti-lung cancer flavin-4'-O-α-D-glucopyranoside as described in claim 1, characterized in that, Using sucrose as a glycosyl donor and rutin as a glycosyl acceptor, an enzymatic transglycosylation reaction is carried out under the catalysis of sucrose phosphorylase to directionally transfer the glucose groups produced by sucrose decomposition to the C4' hydroxyl group of rutin, thereby obtaining the target product; the sucrose phosphorylase is derived from Bifidobacterium adolescentis.

3. The method for preparing flavin-4'-O-α-D-glucopyranoside for anti-lung cancer as described in claim 2, characterized in that, The amino acid sequence of the sucrose phosphorylase is SEQ ID No. 1, and the encoding gene sequence is SEQ ID No.

2.

4. The method for preparing flavin-4'-O-α-D-glucopyranoside for treating lung cancer as described in claim 2, characterized in that, The sucrose phosphorylase was expressed and constructed through the following steps: (1) The recombinant expression plasmid containing the encoding gene was transformed into Escherichia coli BL21 (DE3) and inoculated into LB medium containing 100 mg / mL kanamycin at a volume fraction of 1%. The medium was cultured at 37°C for 12 hours at a speed of 220 r / min. The amino acid sequence of the encoding gene is shown in SEQ ID No.

1. (2) Take 2 mL of the culture medium from step (1) and add it to 200 mL of TB medium. Incubate at 37°C in a shaker until the OD of the bacterial culture is diluted 10 times. 600 When the concentration is 0.20~0.24, add 0.5mM IPTG and induce fermentation at 25℃ for 16 hours; centrifuge the fermentation suspension at 0℃ and 8000r / min for 12~15min, wash with 20mM pH 6.0 MES buffer and centrifuge again, add 20mL of 20mM pH 6.0 MES buffer, sonicate in an ice water bath for 15min, centrifuge and collect the supernatant to obtain sucrose phosphorylase solution.

5. The method for preparing flavin-4'-O-α-D-glucopyranoside for treating lung cancer as described in claim 4, characterized in that, The composition of each liter of LB medium is as follows: using deionized water as the solvent, it contains 3-8g yeast extract, 6-14g tryptone, and 6-14g NaCl; The composition of each liter of TB culture medium is as follows: using deionized water as the solvent, it contains 5g glycerol, 13-14g tryptone, 25-30g yeast extract, 1-3g KH2PO4, and 15-20g K2HPO4·3H2O.

6. The method for preparing flavin-4'-O-α-D-glucopyranoside for treating lung cancer as described in claim 2, characterized in that, The conditions for the enzymatic transglycosylation reaction are as follows: the molar ratio of sucrose to rutin is 20:1, the reaction system contains 200 mM sucrose and 10 mM rutin, and the amount of sucrose phosphorylase is 3~6 U / mL. The flavin glycoside was prepared by reacting continuously for 24 hours in MES buffer at 50°C and pH 7.0 with constant temperature shaking at 220 rpm.

7. The method for preparing flavin-4'-O-α-D-glucopyranoside for lung cancer treatment as described in claim 2, characterized in that, It also includes a purification step: The enzyme reaction solution was heated in a water bath at 100℃ for 5 min to terminate the reaction. Anhydrous ethanol was added to bring the ethanol concentration to 70%. The supernatant was concentrated by rotary evaporation and then extracted with ethyl acetate. The solution was concentrated again and purified by silica gel column chromatography. The solution was then loaded onto the sample using a dry method with a mobile phase of ethyl acetate:methanol = 10:

1. The eluent containing the target product was collected and purified by dextran gel column G15. The eluent was first washed with pure water until no monosaccharide residue was found, and then eluted with 30% ethanol. The eluent was collected and concentrated to obtain high-purity lacquinone-4'-O-α-D-glucopyranoside.

8. The use of flavin-4'-O-α-D-glucopyranoside as described in claim 1 or flavin-4'-O-α-D-glucopyranoside prepared by any one of claims 2-7 in the preparation of anti-lung cancer drugs.

9. The application as described in claim 8, characterized in that, The dosage forms of the drug include tablets, capsules, injections, suspensions, or gels.

10. The application as described in claim 8, characterized in that, The drug exerts its anti-lung cancer effect by inducing apoptosis in lung cancer cells, inhibiting lung cancer cell migration, or inhibiting angiogenesis.

Images