A flavonoid compound with antibacterial activity and a preparation method and application thereof

Abstract

This invention discloses a flavonoid compound with antibacterial activity, its preparation method, and its application. It is prepared from *Hylocereus undatus* twigs through pretreatment, extract extraction, silica gel column chromatography, and high-performance liquid chromatography separation. The compound is named: 4'-hydroxy-8-methoxy-6-(4-methyl-1... H 4'-pyrrole-2-yl)-flavonoid, its English name is: 4'-hydroxy-8-methoxy-6-(4-methyl-1 H (-pyrrol-2-yl)-flavone, which has the following structure: The antibacterial active flavonoids of this invention are obtained from *Hypericum esculentum* twigs through extraction, silica gel column chromatography, and high-performance liquid chromatography. The isopentenyl flavonoids, after activity testing, show good antibacterial activity and a sweet aftertaste. Using this compound in cigarette tipping paper can eliminate or reduce the possibility of harmful bacteria growth and reproduction in the cigarette tipping paper and improve its flavor.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry technology, specifically relating to a flavonoid compound extracted and isolated for the first time from the plant *Jackfruit* of the Moraceae family, and its application in cigarette tipping paper. Background Technology

[0002] Chinmillar originated in Malaysia and Mexico, and is also cultivated in Hainan, Fujian, Guangxi, and Yunnan provinces of China. A typical tropical fruit tree, Chinmillar thrives in warm, humid environments, growing best at altitudes below 1000 meters in fertile, well-drained soil. It is relatively weak in cold tolerance. The ripe flesh of Chinmillar is intensely sweet and fragrant, rich in nutrients, and has a pleasant flavor. The fruit can be eaten fresh, as the flesh is soft; it can also be processed into jams, preserves, juices, jellies, and canned goods. The roasted or boiled seeds are edible, tasting like chestnuts, and are rich in protein, fat, and carbohydrates. Furthermore, modern medical research has confirmed that Chinmillar has anti-edema, antibacterial, and anti-inflammatory effects. Consuming Chinmillar can enhance the hydrolysis of fibrin in the body, dissolving fibrin and blood clots obstructing tissues and blood vessels, thereby promoting local blood and fluid circulation, facilitating the absorption and reduction of inflammation and edema, and providing some adjunctive treatment for cerebral thrombosis and other diseases caused by thrombosis.

[0003] The main active ingredient in Chymina oleifera is isopentenyl flavonoids. Isopentenyl flavonoids are a branch of flavonoids, characterized by the presence of isopentenyl side chains on the flavonoid skeleton. Isopentenyl flavonoids exhibit a wide range of biological activities, particularly in promoting stem cell differentiation, antibacterial, anti-inflammatory and immunomodulatory effects, cardiovascular protection, improvement of metabolic diseases, improvement of osteoporosis, neuroprotection, anti-tumor, anti-aging, and reproductive effects. A significant correlation exists between the chemical structure and biological activity of isopentenyl flavonoids. Therefore, discovering more isopentenyl flavonoid compounds is particularly important for studying their structure-activity relationships. This invention isolates a novel isopentenyl flavonoid compound from Chymina oleifera, a compound that has not been previously reported. Notably, this compound, in addition to its significant antibacterial activity, also possesses a sweet aftertaste. When used as antibacterial tipping paper for cigarettes, it can inhibit harmful microorganisms in cigarette tipping paper and improve the taste of cigarettes; in addition, the compound of the present invention forms a 4-methyl-1-yl group with an isopentenyl side chain, which is discovered for the first time in a natural product. H Flavonoids with a pyrrole ring exhibiting high novelty in their structure. Summary of the Invention

[0004] The first objective of this invention is to provide a flavonoid compound with antibacterial activity; the second objective is to provide a method for preparing the aforementioned flavonoid compound with antibacterial activity; and the third objective is to provide applications of the aforementioned flavonoid compound with antibacterial activity.

[0005] The first objective of this invention is achieved by using the medicinal plant *Hylocereus undatus* (…). Artocarpus champeden The compound was prepared from (Lour.) Spren twigs via pretreatment, extract extraction, silica gel column chromatography, and high-performance liquid chromatography. The compound was named: 4'-hydroxy-8-methoxy-6-(4-methyl-1 H 4'-hydroxy-8-methoxy-6-(4-methyl-1-yl)-flavonoid, also known as pyrrole-2-yl)-flavonoid. H -pyrrol-2-yl)-flavone has the following structure:

[0006] .

[0007] The second objective of this invention is achieved by using the medicinal plant *Hylocereus undatus* branches as raw material, and preparing it through pretreatment, extract extraction, silica gel column chromatography, and high-performance liquid chromatography separation, specifically including the following steps:

[0008] A. Pretreatment: The raw material is pulverized using finely ground plant twigs to obtain material a;

[0009] B. Extraction of extract: Add 2 to 6 times the mass of organic extraction solvent to material a, and extract by soaking at room temperature 2 to 5 times, with each extraction time being 12 to 20 hours. Combine the extracts and filter to obtain sample extract b.

[0010] C. MCI decolorization: The sample extract was decolorized by MCI column, the eluent was collected and concentrated under reduced pressure to obtain extract c;

[0011] D. Silica gel column chromatography:

[0012] 1) Add 3 to 10 times the weight of extract c to pack a column with 200 to 250 mesh silica gel, and perform gradient elution with a chloroform-methanol solution with a volume ratio of 20:1 to 1:1. Monitor the elution by TLC and combine the identical fractions.

[0013] 2) Add 3 to 10 times the weight of the extract in 200 to 250 mesh silica gel to the eluent obtained by elution with a chloroform-methanol solution in a 9:1 ratio. Then, perform gradient elution with a chloroform-acetone solution in a volume ratio of 1:0 to 1:2. Combine the identical fractions by TLC monitoring.

[0014] E. High performance liquid chromatography separation: The eluent obtained by elution with a chloroform-acetone solution in a 7:3 ratio was purified by high performance liquid chromatography to obtain isopentenyl flavonoids with antibacterial activity.

[0015] The structures of the isopentenyl flavonoids prepared in the above steps can be identified by the following methods:

[0016] High-resolution mass spectrometry (HRESI-MS) analysis was performed first, and HRESI-MS showed that its quasi-molecular ion peak was at 370.1053 [M+Na]. + (Calculated value 370.1050), combined with its nuclear magnetic resonance... 1 H NMR, 13 C and DEPT spectra determined its molecular formula to be C. 21 H 17 NO4 has an unsaturation degree of 14.

[0017] The infrared spectrum of the compound shows hydroxyl (3418), carbonyl (1668), and aromatic rings (1616, 1549, 1457 cm⁻¹). -1 The resonance absorption peaks of the compound and the maximum absorption at 376, 275, and 215 nm in the ultraviolet spectrum also indicate the possible presence of an aromatic ring structure in the compound.

[0018] compound 1 H and 13 C NMR data (as shown in Table 1) Figure 1 and Figure 2 The data shows that it contains 21 carbons and 17 hydrogens, including one 1,2,3,5-tetrasubstituted benzene ring (C-5~C-10, H-5, H-7), one 1,4-disubstituted benzene ring (C-1'~C-6', H2-2', 6 and H2-3', 5'), and one... α,β - Unsaturated carbonyl group (C-2, C-3, C-4, H-3), a (4''-methylpyrrole-2-yl) segment (C-2''~C-6'', H-3'', H-5'', H3-6'' and -NH), a methoxy group (δ C 56.2 q and δ H 3.83s) and a phenolic hydroxyl group (δ) H10.22s). Based on the typical signals of two benzene rings, α,β-unsaturated carbonyl groups, and double bonds, plus the unsaturation degree of the two benzene rings being 8; the unsaturation degree of the α,β-unsaturated carbonyl group being 2; and the unsaturation degree of the pyrrole ring being 3, the compound should have one more ring. Further analysis of its NMR data revealed the presence of two oxidized aromatic aprotic substituted carbons (C-9 and C-2), indicating that C-9 and C-2 are connected by oxygen atoms to form a pyranone ring, suggesting that compound 1 is a flavonoid. Based on the HMBC correlations (e.g., H-3 and C-4, C-10, C-1′, H-5 and C-4, C-9, C-10, and H-2′ and C-2), the compound can be further analyzed. Figure 3 This further confirms that the compound has a flavonoid structure.

[0019] Table 1. Compounds of the present invention 1 H NMR and 13 C10 NMR data (CDCl3)

[0020]

[0021] Once the parent compound is determined, the remaining substituents, such as hydroxyl and methoxy, can be considered as substituents on the flavonoid. The presence of the (3-methylpyrrole-2-yl) structural fragment is also confirmed by the HMBC correlations between H-3′′ and C-2′′, C-4′′, C-5′′, C-6′′; H-5′′ and C-2′′, C-3′′, C-4′′, C-6′′; -NH and C-2′′, C-3′′, C-4′′, C-5′′; and H-6′′ and C-3′′, C-4′′, C-5′′. Furthermore, further analysis reveals the presence of a methoxy proton signal (δ). H The HMBC correlations between 3.83s and C-8 indicate that the methoxy group is located at C-8. The HMBC correlations between H-5, H-7, and C-2′′, and between -NH, H-3′′, and C-6, indicate that the (3-methylpyrrole-2-yl) structural segment is attached at C-6. The phenolic hydroxyl proton (δ) H The HMBC correlations of C-4′, C-2′, and C-6′ indicate that the phenolic hydroxyl group is substituted at the C-8 position. Thus, the structure of compound 1 is determined. This compound is named: 4'-hydroxy-8-methoxy-6-(4-methyl-1 H 4'-hydroxy-8-methoxy-6-(4-methyl-1-yl)-flavonoid, also known as pyrrole-2-yl)-flavonoid. H -pyrrol-2-yl)-flavone.

[0022] Literature review revealed that the compound of this invention is the first natural product to contain an isopentenyl group forming a 4-methyl-1-yl group. HFlavonoids with a pyrrole ring exhibit high novelty in their structure.

[0023] Infrared, ultraviolet, and mass spectrometry data of the compound: ultraviolet spectrum (methanol), λ max (log ε 376 (3.72), 275 (3.93), 215 (4.26) nm; Infrared spectrum (potassium bromide tablets): ν max 3418,3360, 3049, 2987, 2842, 1668, 1616, 1549, 1457, 1362, 1254, 1170, 1062, 870cm -1 ; 1 H and 13 C10 NMR data (500 and 125 MHz, (C5D5N), see Table-1; positive ion mode ESIMS) m / z 370[M+Na] + HRESIMS in positive ion mode m / z 370.1053[M+Na] + (Calculated value 370.1050, C) 21 H 17 NNaO4).

[0024] The third objective of this invention, the use of the isopentenyl flavonoids as antibacterial agents and their application in the preparation of antibacterial cigarette tipping paper, is achieved as follows:

[0025] The in vitro antibacterial activity of the compounds of this invention was tested using the agar diffusion method. First, the test bacteria were evenly spread on ordinary agar medium (beef extract, peptone, sodium chloride, serum, and agar). Then, tablets (5 mm in diameter) soaked in the test compound (isoprenyl flavonoid compound dissolved in 10 mL DMSO and diluted with water to a 50 μg / mL solution) were placed on the bacterial-containing medium and incubated at 25°C for 24-72 hours. The size of the inhibition zone was then observed. The results showed that the compounds of this invention exhibited strong activity against Staphylococcus aureus, Escherichia coli, Escherichia coli, Bacillus subtilis, and Proteus mirabilis; the inhibition rate exceeded 95%.

[0026] The application of the compound of the present invention in antibacterial tipping paper was further tested. The compound was applied at 50... µA concentration of g / mL was added to cigarette tipping paper. Following the testing methods of the People's Republic of China's "Hygienic Standard for Disposable Sanitary Products" GB15979-2002, cigarette tipping paper with the added compound was tested for total bacterial count, coliform bacteria, Staphylococcus aureus, Pseudomonas aeruginosa, hemolytic streptococci, and total fungal count. The results showed that the total bacterial count of the tipping paper with the added compound was significantly reduced. This compound exhibited significant inhibitory effects on several tested bacteria, with inhibition rates of over 93.8% against Escherichia coli and Staphylococcus aureus. Adding this compound to cigarette tipping paper significantly inhibits harmful bacteria. Since the tipping paper comes into direct contact with the mouth, the use of this compound in cigarette tipping paper prevents microbial contamination of cigarettes during smoking and delivery, effectively improving the hygiene and safety of cigarettes. Furthermore, this compound has a sweet aftertaste, good harmony with the characteristic aroma of tobacco, and excellent persistence; it also improves the flavor of cigarette tipping paper.

[0027] The advantages of this invention are:

[0028] (1) The compound of the present invention is the isopentenyl flavonoid of the present invention, which is isolated from the medicinal plant *Cinnamomum camphora*. *Cinnamomum camphora* has a large biological yield of twigs, the raw material source is very wide, the cost is low, and the compound is easy to isolate and prepare.

[0029] (2) The preparation method of this invention is a combination of solvent extraction, conventional column chromatography and high performance liquid chromatography. The compound preparation process is simple, the obtained compound has high purity, and subsequent industrial production is easy to achieve.

[0030] (3) The compound of this invention is non-toxic to animals, safe to use, and exhibits good antibacterial activity, with an inhibition rate of over 93% against Escherichia coli, Staphylococcus aureus, etc. When applied to cigarette tipping paper, it can inhibit the microorganisms contaminating the cigarette tipping paper. Since cigarette tipping paper comes into direct contact with the mouth, the use of this compound in cigarette tipping paper can prevent the cigarette from being contaminated by microorganisms during smoking and delivery, effectively improving the hygiene and safety of cigarettes.

[0031] (4) The compound of the present invention has a sweet aftertaste, good coordination with the characteristic aroma of tobacco, and good persistence; it also has the effect of improving the flavor of cigarette tipping paper.

[0032] (5) The compound of the present invention forms a 4-methyl-1-yl group with an isopentenyl side chain, which is the first time such a compound has been found in a natural product. H Flavonoids with a pyrrole ring exhibit high structural novelty and are of significant scientific importance for refining the structure-activity relationship of flavonoids. Attached Figure Description

[0033] Figure 1 The carbon NMR spectrum of the antibacterial active flavonoids of this invention ( 13 (C NMR).

[0034] Figure 2 The nuclear magnetic resonance hydrogen spectrum of the antibacterial active flavonoids of this invention ( 1 H NMR);

[0035] Figure 3 The key HMBC correlation diagram of the antibacterial active flavonoid compounds of this invention. Detailed Implementation

[0036] The present invention will be further described below with reference to embodiments and accompanying drawings, but this does not limit the present invention in any way. Any modifications or substitutions made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0037] The isopentenyl flavonoid compounds with antibacterial activity described in this invention are derived from the medicinal plant *Trichoderma harzianum* (also known as 'Turmeric'). Artocarpus champeden Using (Lour.)Spren) twigs as raw material, this product was prepared through pretreatment, extract extraction, silica gel column chromatography, and high-performance liquid chromatography separation, and named as: 4'-hydroxy-8-methoxy-6-(4-methyl-1 H 4'-hydroxy-8-methoxy-6-(4-methyl-1-yl)-flavonoid, also known as pyrrole-2-yl)-flavonoid. H -pyrrol-2-yl)-flavone has the following structure:

[0038]

[0039] The antibacterial flavonoids of this invention are prepared from the medicinal plant *Hylocereus undatus* twigs through pretreatment, extract extraction, silica gel column chromatography, and high-performance liquid chromatography separation, specifically including the following steps:

[0040] A. Pretreatment: The raw material, Yunnan ethnic medicinal plant *Trifolium repens*, is pulverized into fine branches to obtain material a;

[0041] B. Extraction of extract: Add 2 to 6 times the mass of organic extraction solvent to material a, and extract by soaking at room temperature 2 to 5 times, with each extraction time being 12 to 20 hours. Combine the extracts and filter to obtain sample extract b.

[0042] C. MCI decolorization: The sample extract was decolorized by MCI column, the eluent was collected and concentrated under reduced pressure to obtain extract c;

[0043] D. Silica gel column chromatography:

[0044] 1) Add 3 to 10 times the weight of extract c to pack a column with 200 to 250 mesh silica gel, and perform gradient elution with a chloroform-methanol solution with a volume ratio of 20:1 to 1:1. Monitor the elution by TLC and combine the identical fractions.

[0045] 2) Add 3 to 10 times the weight of the extract in 200 to 250 mesh silica gel to the eluent obtained by elution with a chloroform-methanol solution in a 9:1 ratio. Then, perform gradient elution with a chloroform-acetone solution in a volume ratio of 1:0 to 1:2. Combine the identical fractions by TLC monitoring.

[0046] E. High-performance liquid chromatography separation: The eluent obtained by elution with a chloroform-acetone solution in a 7:3 ratio is purified by high-performance liquid chromatography to obtain the target flavonoids with antibacterial activity.

[0047] The organic extraction solvent mentioned in step B is a 70%~100% methanol aqueous solution, a 70%~100% ethanol aqueous solution, or a 70%~100% acetone aqueous solution.

[0048] Step D, before loading the column, also includes a sample mixing step where the sample is dissolved in an organic solvent at 1.5 to 3 times its weight of material c, and then mixed with 80 to 100 mesh silica gel at 0.8 to 2.0 times its weight of material c.

[0049] The organic solvent is pure methanol or pure acetone.

[0050] The volume ratios of the chloroform-methanol solution mentioned in step 1) are 20:1, 9:1, 8:2, 7:3, 6:4 and 1:1.

[0051] In step E, high-performance liquid chromatography (HPLC) separation and purification used a 50-65% (v / v) methanol-water solution as the mobile phase at a flow rate of 12 mL / min, a 2.12 × 250 mm, 5 μm Zorbax PrepHT GF reversed-phase preparative column as the stationary phase, and a UV detector at a wavelength of 376 nm. Each injection was 0.5-1.0 mL, and chromatographic peaks were collected for 30-42 min. After multiple accumulations, the peaks were evaporated to dryness to obtain the target antibacterial flavonoid compounds.

[0052] The application of the isopentenyl flavonoid compound described in this invention is the application of the antibacterial flavonoid compound in the preparation of cosmetic additives.

[0053] The specific preparation method is as follows:

[0054] A. Sample Extraction and Purification: Dry the fine branches of *Hylocereus undatus* (a type of honeysuckle), pulverize to 20-50 mesh, and then extract with solvent 2-5 times, each time using 2-6 times the amount of solvent as the raw material, for 12-20 hours. Filter out the precipitate to obtain the sample extract, and concentrate under reduced pressure to obtain a paste. The prepared ethanol extract is partitioned in ethyl acetate and aqueous solution. The ethyl acetate layer is concentrated under reduced pressure to obtain a crude extract. After filtering out the precipitate, the crude extract is decolorized on an MCI column, and the eluent is collected and concentrated under reduced pressure to obtain a paste for column chromatography separation.

[0055] B. Initial silica gel column chromatography: Dilute the obtained extract with 1.5-3 times the amount of acetone or methanol, then mix with 0.8-2.0 times the weight of the extract using silica gel of 100-150 mesh. Perform coarse fractionation on a silica gel column using 200-250 mesh silica gel, with the weight of the silica gel being 3-10 times the weight of the extract. Elute using a gradient of chloroform and methanol mixed organic solvents at a volume ratio of 20:1 to 1:1. Collect and concentrate the eluents from each gradient, monitor by TLC, and combine identical fractions to obtain 6 fractions (AF).

[0056] C. Second silica gel column chromatography: Component b (chloroform-methanol 9:1 fraction) from step B is further subjected to silica gel column chromatography: the silica gel used is 200-250 mesh, and the weight of the silica gel is 3-10 times the weight of the extract. The column is eluted with a gradient of chloroform and acetone mixed organic solvents in a volume ratio of 1:0 to 1:2. The gradient eluents are collected and concentrated.

[0057] D. High-performance liquid chromatography separation: The component C (the part obtained by elution with a 7:3 mixture of chloroform and acetone organic solvent) from step C is separated and purified by high-performance liquid chromatography to obtain the flavonoids.

[0058] Furthermore, preferably, the solvent in step A is an aqueous solution of acetone with a volume concentration of 70-100%, an aqueous solution of ethanol with a volume concentration of 70-100%, or an aqueous solution of methanol with a volume concentration of 70-100%.

[0059] Furthermore, preferably, in step C, before silica gel column chromatography, the extract is first diluted with acetone or methanol at a weight of 1.5 to 3 times that of the extract, then mixed with 80 to 100 mesh silica gel at a weight of 0.8 to 2.0 times that of the extract, and then loaded onto the column.

[0060] Furthermore, preferably, in step C, the volume ratio of the chloroform and methanol mixed organic solvent used in the gradient elution is 20:1, 9:1, 8:2, 7:3, 6:4, and 1:1, respectively. (Elimination should continue until no component flows out, i.e., no component remains in the distillation flask after the solvent is evaporated, and then the next gradient is started).

[0061] Furthermore, preferably, the high-performance liquid chromatography (HPLC) separation and purification in step E uses a 50-65% (v / v) methanol-water solution as the mobile phase at a flow rate of 12 mL / min, a 2.12 × 250 mm, 5 μm Zorbax PrepHT GF reversed-phase preparative column as the stationary phase, a UV detector at a wavelength of 376 nm, and injects 0.5-1.0 mL each time. The chromatographic peaks are collected for 30-42 min, and after multiple accumulations, the sample is evaporated to dryness to obtain the pure compound.

[0062] The medicinal plant *Hylocereus undatus* used as the raw material in this invention is not limited by region or variety; any of these can be used to achieve this invention.

[0063] The invention will be further illustrated below using raw materials from different production areas in Yunnan:

[0064] Example 1

[0065] This embodiment provides a method for preparing the antibacterial active flavonoids of the present invention. The preparation method includes: extract extraction, silica gel column chromatography, and high performance liquid chromatography separation steps, using the medicinal plant *Hylocereus undatus* as the raw material. The specific operation is as follows:

[0066] The medicinal plant *Jianmila* used was produced in the Honghe River estuary of Yunnan Province. The raw material was a sample of finely crushed or cut branches of the medicinal plant *Jianmila*, which was extracted by soaking in a 70% methanol aqueous solution for 15 hours each time. The combined extracts were decolorized on an MCI column, and the eluent was concentrated under reduced pressure to obtain a paste. The paste was dissolved in methanol at twice its mass, then mixed with 90-mesh silica gel, packed into a 220-mesh silica gel column, and loaded onto the column. Gradient elution was performed using chloroform-methanol eluents at volume ratios of 20:1, 9:1, 8:2, 7:3, 6:4, and 1:1. The gradient eluents were collected, concentrated, and monitored by TLC. Identical fractions were combined to obtain 6 fractions AF. Among them, 218g of sample B (9:1) was added to 220-mesh silica gel at eight times its mass, packed into a column, and eluted with chloroform-acetone eluents at volume ratios of 1:0, 1:1, and 1:2. The gradient eluents were collected, concentrated, and monitored by TLC. Identical fractions were combined to obtain 3 fractions, of which 24.6g of the 7:3 fraction was further processed. 56% methanol was used as the mobile phase at a flow rate of 12ml / min, at a diameter of 2.12×250mm, and a focal length of 5 μm. µ A Zorbax PrepHT GF reversed-phase column was used as the stationary phase, and the UV detector was set at a wavelength of 376 nm. 0.8 mL of sample was injected each time, and the chromatographic peak was collected for 33.5 min. After multiple injections, the peak was evaporated to dryness to obtain the antibacterial isopentenyl flavonoid compound described in this invention.

[0067] The structures of the prepared isopentenyl flavonoids with antibacterial activity were identified by the following methods:

[0068] High-resolution mass spectrometry (HRESI-MS) analysis was performed first, and HRESI-MS showed that its quasi-molecular ion peak was at 370.1053 [M+Na]. + (Calculated value 370.1050), combined with its nuclear magnetic resonance... 1 H NMR, 13 C and DEPT spectra determined its molecular formula to be C. 21 H 17 NO4 has an unsaturation degree of 14.

[0069] The infrared spectrum of the compound shows hydroxyl (3418), carbonyl (1668), and aromatic rings (1616, 1549, 1457 cm⁻¹). -1 The resonance absorption peaks of the compound and the maximum absorption at 376, 275, and 215 nm in the ultraviolet spectrum also indicate the possible presence of an aromatic ring structure in the compound.

[0070] compound 1 H and 13 C NMR data (as shown in Table 1) Figure 1 and Figure 2 The data shows that it contains 21 carbons and 17 hydrogens, including one 1,2,3,5-tetrasubstituted benzene ring (C-5~C-10, H-5, H-7), one 1,4-disubstituted benzene ring (C-1'~C-6', H2-2', 6 and H2-3', 5'), and one... α,β - Unsaturated carbonyl group (C-2, C-3, C-4, H-3), a (4''-methylpyrrole-2-yl) segment (C-2''~C-6'', H-3'', H-5'', H3-6'' and -NH), a methoxy group (δ C 56.2 q and δ H 3.83s) and a phenolic hydroxyl group (δ) H 10.22s). Based on the typical signals of two benzene rings, α,β-unsaturated carbonyl groups, and double bonds, plus the unsaturation degree of the two benzene rings being 8; the unsaturation degree of the α,β-unsaturated carbonyl group being 2; and the unsaturation degree of the pyrrole ring being 3, the compound should have one more ring. Further analysis of its NMR data revealed the presence of two oxidized aromatic aprotic substituted carbons (C-9 and C-2), indicating that C-9 and C-2 are connected by oxygen atoms to form a pyranone ring, suggesting that compound 1 is a flavonoid. Based on the HMBC correlations (e.g., H-3 and C-4, C-10, C-1′, H-5 and C-4, C-9, C-10, and H-2′ and C-2), the compound can be further analyzed. Figure 3 This further confirms that the compound has a flavonoid structure.

[0071] Once the parent compound is determined, the remaining substituents, such as hydroxyl and methoxy, can be considered as substituents on the flavonoid. The presence of the (3-methylpyrrole-2-yl) structural fragment is also confirmed by the HMBC correlations between H-3′′ and C-2′′, C-4′′, C-5′′, C-6′′; H-5′′ and C-2′′, C-3′′, C-4′′, C-6′′; -NH and C-2′′, C-3′′, C-4′′, C-5′′; and H-6′′ and C-3′′, C-4′′, C-5′′. Furthermore, further analysis reveals the presence of a methoxy proton signal (δ). H The HMBC correlations between 3.83s and C-8 indicate that the methoxy group is located at C-8. The HMBC correlations between H-5, H-7, and C-2′′, and between -NH, H-3′′, and C-6, indicate that the (3-methylpyrrole-2-yl) structural segment is attached at C-6. The phenolic hydroxyl proton (δ) H The HMBC correlations of C-4′, C-2′, and C-6′ indicate that the phenolic hydroxyl group is substituted at the C-8 position. Thus, the structure of compound 1 is determined. This compound is named: 4'-hydroxy-8-methoxy-6-(4-methyl-1 H 4'-hydroxy-8-methoxy-6-(4-methyl-1-yl)-flavonoid, also known as pyrrole-2-yl)-flavonoid. H -pyrrol-2-yl)-flavone.

[0072] Example 2

[0073] This embodiment provides a method for preparing the antibacterial active flavonoids of the present invention. The preparation method includes: extract extraction, silica gel column chromatography, and high performance liquid chromatography separation steps, using the medicinal plant *Hylocereus undatus* as the raw material. The specific operation is as follows:

[0074] The medicinal plant *Cinnamomum camphora* used was sourced from Yuanjiang County, Yuxi City, Yunnan Province. The raw material consisted of finely crushed or chopped branches of *Cinnamomum camphora*, extracted five times with 80% ethanol aqueous solution for 12 hours each time. The combined extracts were decolorized on an MCI column, and the eluent was concentrated under reduced pressure to obtain a paste. The paste was dissolved in acetone at three times its weight, then mixed with 100-mesh silica gel. The sample was packed into a 250-mesh silica gel column, mixed again, and loaded onto the column. Gradient elution was performed using chloroform-methanol eluents at volume ratios of 20:1, 9:1, 8:2, 7:3, 6:4, and 1:1. The gradient eluents were collected, concentrated, and monitored by TLC. Identical fractions were combined to obtain six fractions (AF). 262g of sample B (9:1) was collected and packed into a 250-mesh silica gel column (10 times the volume of the extract). Gradient elution was performed using chloroform-acetone eluents at volume ratios of 1:0, 1:1, and 1:2. The eluents were collected, concentrated, and monitored by TLC. Identical fractions were combined to obtain three fractions. 21.5g of the 7:3 fraction was then eluented using 56% methanol as the mobile phase at a flow rate of 12ml / min, at a column diameter of 2.12×250mm. µ A Zorbax PrepHT GF reversed-phase column was used as the stationary phase. The UV detector was set at 376 nm. 0.5–1.0 mL of sample was injected each time, and the chromatographic peak was collected for 33.5 min. After multiple injections and evaporation, the antibacterial active flavonoid compound of this invention was obtained. Identification was performed using the same method as in Example 1, and the resulting molecular formula was C1. 21 H 17 NO4.

[0075] Example 3

[0076] This embodiment provides a method for preparing the antibacterial active flavonoids of the present invention. The preparation method includes: extract extraction, silica gel column chromatography, and high performance liquid chromatography separation steps, using the medicinal plant *Hylocereus undatus* as the raw material. The specific operation is as follows:

[0077] The medicinal plant *Cinnamomum camphora* used was produced in Menghai County, Xishuangbanna, Yunnan Province. The raw material was a sample of *Cinnamomum camphora* (finely crushed or cut into sections), extracted twice with 80% acetone aqueous solution for 12 hours each time. The combined extracts were decolorized on an MCI column, and the eluent was concentrated under reduced pressure to obtain an extract. The extract was dissolved in 1.5 times its weight of methanol, then mixed with 80-mesh silica gel, packed into a 200-mesh silica gel column, and loaded onto the column after mixing. Gradient elution was performed using chloroform-methanol eluents at volume ratios of 20:1, 9:1, 8:2, 7:3, 6:4, and 1:1. The gradient eluent was collected and concentrated. TLC monitoring was performed, and identical fractions were combined to obtain six fractions (AF). Of the collected sample B (9:1) fraction (210g), three times the volume of the extract was added to a 200-mesh silica gel column. Gradient elution was performed using chloroform-acetone eluents at volume ratios of 1:0, 1:1, and 1:2. The gradient eluent was collected and concentrated. TLC monitoring was performed, and identical fractions were combined to obtain three fractions. The 7:3 fraction (19.5g) was then used as the mobile phase with 56% methanol at a flow rate of 12 ml / min, a column diameter of 2.12 × 250 mm, and a column height of 5 μm. µ A Zorbax PrepHT GF reversed-phase column was used as the stationary phase. The UV detector was set at 376 nm. 0.5–1.0 mL of sample was injected each time, and the chromatographic peak was collected for 33.5 min. After multiple injections and evaporation, the antibacterial active flavonoid compound of this invention was obtained. Identification was performed using the same method as in Example 1, and the resulting molecular formula was C1. 21 H 17 NO4.

[0078] Example 4

[0079] Any compound prepared in Examples 1-3 was a yellow gel-like substance. The determination method was the same as in Example 1, confirming that the compounds prepared in Examples 1-3 were the 4'-hydroxy-8-methoxy-6-(4-methyl-1-hydroxy ...(4-methyl-1-(4-methyl-1-(4-methyl- H -pyrrole-2-yl)-flavonoids.

[0080] Example 5

[0081] Antibacterial activity tests were conducted on any of the isopentenyl flavonoids prepared in Examples 1-4. The test results are as follows:

[0082] In vitro antibacterial experiments were conducted using the agar diffusion method. First, the test bacteria were evenly spread on a standard agar medium (beef extract, peptone, sodium chloride, serum, and agar). Then, tablets (5 mm in diameter) soaked in the test compound (benzopyranolone compound dissolved in 10 mL DMSO and diluted with water to a 50 μg / mL solution) were placed on the bacterial-containing medium. The plates were then incubated at 25°C for 24-72 hours, after which the size of the inhibition zone was observed. The results showed that the compound of this invention exhibited strong activity against Staphylococcus aureus, Escherichia coli, Escherichia coli, Bacillus subtilis, and Proteus mirabilis; the inhibition rate exceeded 95%.

[0083] Example 6

[0084] Safety evaluation was conducted on any of the benzopyran lactone compounds prepared in Examples 1-4. The results are as follows: Mouse bone marrow micronucleus assay, Ames assay, and TK gene mutation assay all demonstrated that the compounds of the present invention have low toxicity to animals and are safe to use.

[0085] The in vitro antibacterial activity of the compounds of this invention was tested using the agar diffusion method. First, the test bacteria were evenly spread on ordinary agar medium (beef extract, peptone, sodium chloride, serum, and agar). Then, tablets (5 mm in diameter) soaked in the test compound (isoprenyl flavonoid compound dissolved in 10 mL DMSO and diluted with water to a 50 μg / mL solution) were placed on the bacterial-containing medium and incubated at 25°C for 24-72 hours. The size of the inhibition zone was then observed. The results showed that the compounds of this invention exhibited strong activity against Staphylococcus aureus, Escherichia coli, Escherichia coli, Bacillus subtilis, and Proteus mirabilis; the inhibition rate exceeded 95%.

[0086] This compound was added to cigarette tipping paper at a concentration of 50 μg / mL. Following the testing method of the People's Republic of China's "Hygienic Standard for Disposable Sanitary Products" GB15979-2002, a 2.0 × 3.0 mm sample of cigarette tipping paper containing this compound was taken and tested for total bacterial count, coliform bacteria, Staphylococcus aureus, Pseudomonas aeruginosa, hemolytic streptococci, and total fungal count. The results showed that the total bacterial count of the tipping paper containing this compound was significantly reduced. This compound exhibited significant inhibitory effects on several tested bacteria, with inhibition rates of over 93.8% against Escherichia coli and Staphylococcus aureus. Adding this compound to cigarette tipping paper significantly inhibited harmful bacteria. Furthermore, this compound has a sweet aftertaste, good harmony with the characteristic aroma of tobacco, and excellent persistence; it also improves the flavor of cigarette tipping paper.

Claims

1. A flavonoid compound with antibacterial activity, characterized in that, It was prepared from *Hylocereus undatus* twigs through pretreatment, extract extraction, silica gel column chromatography, and high-performance liquid chromatography, and named as: 4'-hydroxy-8-methoxy-6-(4-methyl-1 H 4'-hydroxy-8-methoxy-6-(4-methyl-1-yl)-flavonoid, also known as pyrrole-2-yl)-flavonoid. H -pyrrol-2-yl)-flavone has the following structure: 。 2. A method for preparing the antibacterial flavonoid compound according to claim 1, characterized in that, It is prepared from the Yunnan ethnic minority medicinal plant *Trifolium repens* branches through pretreatment, extract extraction, silica gel column chromatography, and high-performance liquid chromatography separation, specifically including the following steps: A. Pre-treatment: The raw material, Yunnan ethnic medicinal honey, is finely crushed or cut into sections to obtain material a; B. Extraction of extract: Add 2 to 6 times the mass of organic extraction solvent to material a, and extract by soaking at room temperature 2 to 5 times, with each extraction time being 12 to 20 hours. Combine the extracts and filter to obtain sample extract b. C. MCI decolorization: The sample extract was decolorized by MCI column, the eluent was collected and concentrated under reduced pressure to obtain extract c; D. Silica gel column chromatography: 1) Add 3 to 10 times the weight of extract c to pack a column with 200 to 250 mesh silica gel, and perform gradient elution with a chloroform-methanol solution with a volume ratio of 20:1 to 1:

1. Monitor the elution by TLC and combine the identical fractions. 2) The eluent obtained by eluting with a 9:1 chloroform-methanol solution was concentrated under reduced pressure to obtain component d. 3 to 10 times the weight of the obtained component d was packed into a column with 200 to 250 mesh silica gel and gradient eluted with a chloroform-acetone solution with a volume ratio of 1:0 to 1:

2. The same fractions were combined by TLC monitoring. E. High performance liquid chromatography separation: The eluent obtained by elution with a chloroform-acetone solution in a 7:3 ratio was separated and purified by high performance liquid chromatography to obtain the target flavonoids with antibacterial activity; The preparation method is characterized in that, in step E, high-performance liquid chromatography (HPLC) separation and purification uses a 50-65% (v / v) methanol-water solution as the mobile phase, a flow rate of 12 mL / min, and a latitude and longitude of 2.12 × 250 mm. μ A Zorbax PrepHT GF reversed-phase preparative column was used as the stationary phase, and the UV detector was set at a wavelength of 376 nm. Each injection was 0.5 to 1.0 mL, and chromatographic peaks were collected for 30 to 42 minutes. After multiple injections, the peaks were evaporated to dryness to obtain the antibacterial flavonoids.

3. The preparation method according to claim 2, characterized in that, The organic extraction solvent mentioned in step B is a 70%~100% methanol aqueous solution, a 70%~100% ethanol aqueous solution, or a 70%~100% acetone aqueous solution.

4. The preparation method according to claim 2, characterized in that, Step D, before loading the column, also includes a sample mixing step where the sample is dissolved in an organic solvent at 1.5 to 3 times its weight of extract c, and then mixed with 80 to 100 mesh silica gel at 0.8 to 2.0 times its weight of extract c.

5. The preparation method according to claim 4, characterized in that, The organic solvent is pure methanol or pure acetone.

6. The preparation method according to claim 2, characterized in that, The volume ratios of the chloroform-methanol solution mentioned in step 1) are 20:1, 9:1, 8:2, 7:3, 6:4 and 1:

1.

7. The application of the antibacterial flavonoid compound according to claim 1, characterized in that, The application of the aforementioned antibacterial active flavonoids in the preparation of antibacterial agents.

8. The application of the antibacterial flavonoid compound according to claim 1, characterized in that, The application of the aforementioned antibacterial active flavonoids in the preparation of antibacterial tipping paper for cigarettes.

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