A bitter ingredient (3-methoxy-4-hydroxyphenol 1-O-beta-D-(6'-O-galloyl)-glucopyranoside) and its preparation method and application

Abstract

This invention discloses a bitter component (3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside), its preparation method, and its application. The structural formula of the 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside compound is shown in Formula (I). The application of this bitter component (3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside) in the preparation of antioxidant or antibacterial active agents is also provided. This invention provides a highly efficient method for isolating the bitter component (3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside) from *Camellia chrysantha*, shortening the research cycle and improving research efficiency.

Description

Technical Field

[0001] This invention relates to the field of chemical technology, specifically to a bitter component, 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside, its preparation method, and its applications. Background Technology

[0002] Yunnan golden camellia (Camellia fascicularis), belonging to the genus Camellia in the family Theaceae, is a deciduous shrub renowned for its golden petals, earning it the titles of "giant panda of the plant kingdom" and "queen of the tea family." Not only is golden camellia a rare species within the camellia family, but it also boasts a centuries-long history of medicinal and culinary use in China. It is believed to have therapeutic effects on various ailments, including dysentery, hematochezia, tumors, and hypertension. Yunnan golden camellia is rich in amino acids, minerals, and bioactive components such as tea polyphenols, saponins, flavonoids, and tea polysaccharides. Pharmacological and clinical trials have confirmed its multiple pharmacological effects, including antioxidant, antitumor, antibacterial, lipid-lowering, blood sugar-lowering, and cholesterol-lowering properties.

[0003] Despite the numerous potential health benefits of *Camellia yunnanensis*, the bitter chemical components in its flowers and leaves have not been fully studied and reported. As a very small, endemic population of this species, *Camellia yunnanensis* holds immense potential for exploring its functional activities. Therefore, the isolation and identification of the bitter component 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside from *Camellia yunnanensis* will not only provide a scientific basis for the deep processing and application of *Camellia yunnanensis*, but also lay the foundation for further drug development and health food research.

[0004] Currently, there is a lack of a bitter component with antioxidant and antibacterial activities, 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside, and its preparation method. Summary of the Invention

[0005] The purpose of this invention is to provide a bitter component with antioxidant and antibacterial activities, 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside, its preparation method and application.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: In a first aspect, the present application provides a bitter component 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside.

[0007] Secondly, this application provides a method for preparing the bitter component 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside.

[0008] Thirdly, this application provides an extract of Camellia yunnanensis.

[0009] Fourthly, this application provides the use of the bitter component 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside in the preparation of antioxidant or antibacterial active preparations.

[0010] The first aspect of this application provides a bitter component, 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside, the structural formula of which is shown in formula (I):

[0011]

[0012] A second aspect of this application provides a method for preparing the bitter component 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside, comprising the following steps:

[0013] a. Take a dry sample of Yunnan golden flower tea leaves and grind it to approximately 40 mesh;

[0014] b. Extracted three times by reflux at 50°C using 95% industrial methanol, with each extraction lasting 3 hours, 2 hours, and 1 hour respectively;

[0015] c. Combine the extracts and use a rotary evaporator at 50°C and low pressure to recover industrial methanol, obtaining a methanol extract.

[0016] d. Add distilled water to the methanol extract and mix thoroughly. Add an equal volume of industrial ethyl acetate and extract thoroughly three times. Then, evaporate the extract at low pressure at 40°C to obtain the ethyl acetate phase extract.

[0017] e. Use macroporous resin D101 to load the sample onto the column, elute with a water / methanol gradient, and detect the same fractions by thin-layer chromatography (TLC).

[0018] f. Combined extracts were mixed with silica gel, subjected to silica gel column chromatography, eluted with chloroform / methanol gradient, and detected by thin-layer chromatography (TLC) to obtain 9 fractions Fr.(AI) by combining identical fractions;

[0019] g. A specific component Fr.F was subjected to MCI column chromatography with a methanol / water gradient, and TLC was performed to obtain multiple components Fr.(FA-FI);

[0020] h.Fr.FA was further purified using Sephadex LH-20 to obtain four fractions Fr.(FAa-FAd);

[0021] i.Fr.FAd was finally purified by semi-preparative high performance liquid chromatography (PHPLC) to obtain compound (3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside).

[0022] Further, in step (e), the volume ratio of water to methanol is 1:0 to 0:1; in step (f), the volume ratio of chloroform to methanol is 1:0 to 0:1.

[0023] The third aspect of this application provides a Yunnan golden camellia extract, which contains 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside as a bitter component.

[0024] The fourth aspect of this application provides the use of the bitter component 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside in the preparation of antioxidant or antibacterial active preparations.

[0025] Beneficial Effects: This invention successfully isolated a novel bitter monomer compound, 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside, from *Camellia yunnanensis* for the first time, providing new chemical composition information for the research and application of *Camellia yunnanensis*. The invention also relates to the application of this bitter component, 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside, in the preparation of antioxidant or antibacterial active agents. This invention provides an efficient method for separating 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside, a bitter component of Camellia chrysanthemi, which shortens the research cycle and improves research efficiency.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] (1) Enhancing the utilization value of Yunnan golden camellia: By isolating and identifying the bitter component 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside, a scientific basis can be provided for the deep processing of Yunnan golden camellia, which helps to improve the taste of Yunnan golden camellia and increase its market acceptance and utilization value. In-depth study of pharmacological effects: The present invention conducted antioxidant and antibacterial activity tests on the isolated bitter monomer compound, providing experimental data and theoretical basis for further research on the pharmacological effects of Yunnan golden camellia.

[0028] (2) This invention employs a series of advanced extraction, separation, and purification techniques, including reflux extraction, ethyl acetate extraction, macroporous resin and small silica gel column chromatography, and semi-preparative high-performance liquid chromatography (PHPLC) purification, which improves separation efficiency and purity. Nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry were used to accurately identify the separated compounds, ensuring the accuracy of their structures.

[0029] (3) This invention promotes drug development and health food research: The research results of this invention provide new research directions and possibilities for the application of Camellia chrysantha in the fields of drug development and health food. Through the study of the bitter component 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside in Camellia chrysantha, this invention helps to protect and rationally utilize this rare plant resource and promote the protection of biodiversity. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a flowchart illustrating the compound separation and purification process of the present invention.

[0032] Figure 2 High-performance liquid chromatography (HPLC) diagrams of the compounds of this invention.

[0033] Figure 3 The compounds of the present invention 1 HNMR image.

[0034] Figure 4 The compounds of the present invention 13 C NMR spectrum. Detailed Implementation

[0035] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0036] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0037] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0038] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0039] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0040] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a mass unit known in the chemical industry, such as μg, mg, g, or kg.

[0041] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0042] The first aspect of this application provides a bitter component, 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside, the structural formula of which is shown in Formula (I):

[0043]

[0044] A second aspect of the embodiments of this application provides a

[0045] The preparation method of the compound 3-Methoxy-4-hydroxyphenol 1-O-β-D-(6′-O-galloyl)-glucopyranoside includes the following steps:

[0046] a. Take a dry sample of Yunnan golden flower tea leaves and grind it to approximately 40 mesh;

[0047] b. Extracted three times by reflux at 50°C using 95% industrial methanol, with each extraction lasting 3 hours, 2 hours, and 1 hour respectively;

[0048] c. Combine the extracts and use a rotary evaporator at 50°C and low pressure to recover industrial methanol, obtaining a methanol extract.

[0049] d. Add distilled water to the methanol extract and mix thoroughly. Add an equal volume of industrial ethyl acetate and extract thoroughly three times. Then, evaporate the extract at low pressure at 40°C to obtain the ethyl acetate phase extract.

[0050] e. Use macroporous resin D101 to load the sample onto the column, elute with a water / methanol gradient, and detect the same fractions by thin-layer chromatography (TLC).

[0051] f. Combined extracts were mixed with silica gel, subjected to silica gel column chromatography, eluted with chloroform / methanol gradient, and detected by thin-layer chromatography (TLC) to obtain 9 fractions Fr.(AI) by combining identical fractions;

[0052] g. A specific component Fr.F was subjected to MCI column chromatography with a methanol / water gradient, and TLC was performed to obtain multiple components Fr.(FA-FI);

[0053] h.Fr.FA was further purified using Sephadex LH-20 to obtain four fractions Fr.(FAa-FAd);

[0054] i.Fr.FAd was finally purified by semi-preparative high performance liquid chromatography (PHPLC) to obtain the compound 3-Methoxy-4-hydroxyphenol 1-O-β-D-(6′-O-galloyl)-glucopyranoside.

[0055] In some embodiments, in step (e), the volume ratio of water to methanol is 1:0 to 0:1; in step (f), the volume ratio of chloroform to methanol is 1:0 to 0:1.

[0056] The third aspect of this application provides a Yunnan golden camellia extract, which contains 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside as a bitter component.

[0057] The fourth aspect of the embodiments of this application provides a

[0058] Application of 3-Methoxy-4-hydroxyphenol and 1-O-β-D-(6′-O-galloyl)-glucopyranoside compounds in the preparation of antioxidant or antibacterial active agents.

[0059] Example 1

[0060] like Figure 2 and Figure 3 As shown, one bitter component of the present invention is 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside.

[0061] The structural formula of the compound 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside is shown in formula (I):

[0062]

[0063] 3-Methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside (compound): white powder, HR–ESI–MS m / z 477.1048 [M+Na+H] + The molecular formula is C 20 H 22 O 12 .

[0064] 1 H NMR (500MHz, Methanol-d4)δ H 7.10(2H,s,H-2”,6”),6.70(1H,d,J=2.7Hz,H-5),6.62(1H,d,J=8.6Hz, H-2),6.57(1H,dd,J=8.6,2.7Hz,H-6),4.73(1H,d,J=7.4Hz,H-1'),4.59 (1H,dd,J=11.9,2.1Hz,H-6a'),4.43(1H,dd,J=11.9,6.7Hz,H-6b'),3.9 2–3.76(1H,m,H-5'),3.71(3H,s,-OMe),3.50–3.39(3H,m,H-2',3',4'). 13 C NMR (126MHz, Methanol-d4)δ C 168.3(C-7”),152.7(C-1),149.2(C-3),146.6(C-3”,5”),143.1(C-4),139.9(C-4'),121.4(C-1'),116.1(C-5),110.2(C-2' ,6'),110.2(C-6),103.9(C-1'),103.9(C-2),77.9(C-3'),75.7(C-5'),74.9(C-2'),71.8(C-4'),64.9(C-6'),56.3(-OMe).

[0065] Example 2

[0066] A method for preparing the bitter component 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside according to the present invention includes the following steps:

[0067] a. Take a dry sample of Yunnan golden flower tea leaves and grind it to approximately 40 mesh;

[0068] b. Extracted three times by reflux at 50°C using 95% industrial methanol, with each extraction lasting 3 hours, 2 hours, and 1 hour respectively;

[0069] c. Combine the extracts and use a rotary evaporator at 50°C and low pressure to recover industrial methanol, obtaining a methanol extract.

[0070] d. Add distilled water to the methanol extract and mix thoroughly. Add an equal volume of industrial ethyl acetate and extract thoroughly three times. Then, evaporate the extract at low pressure at 40°C to obtain the ethyl acetate phase extract.

[0071] e. Use macroporous resin D101 to mix the sample and load it onto the column, elute with a water / methanol gradient, and detect the same fractions by TLC;

[0072] f. Combined extracts were mixed with silica gel, subjected to silica gel column chromatography, eluted with chloroform / methanol gradient, and detected by thin-layer chromatography (TLC) to obtain 9 fractions Fr.(AI) by combining identical fractions;

[0073] g. A specific component Fr.F was subjected to MCI column chromatography with a methanol / water gradient, and TLC was performed to obtain multiple components Fr.(FA-FI);

[0074] h.Fr.FA was further purified using Sephadex LH-20 to obtain four fractions Fr.(FAa-FAd);

[0075] i.Fr.FAd was finally purified by semi-preparative high performance liquid chromatography (PHPLC) to obtain the compound 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside.

[0076] Example 3

[0077] The present invention discloses a Yunnan golden camellia extract, which contains 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside as a bitter component.

[0078] Example 4

[0079] The present invention relates to the application of a bitter component, 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside, in the preparation of antioxidant or antibacterial active agents.

[0080] Experimental Example 1

[0081] 1. Instruments and Materials

[0082] The following instruments were used in the column chromatography process: Bruker AV 500MHz nuclear magnetic resonance spectrometer (Bruker GmbH, Germany), XEVO G2-XS Q-TOF high-resolution mass spectrometer (Waters Inc., USA), NP7000 semi-preparative liquid chromatography system (Jiangsu Hanbang Technology Co., Ltd., China), AX224ZH\E electronic balance (Ohaus Instruments (Changzhou) Co., Ltd., China), 101-2ES electric heating drying oven (Beijing Yongguangming Medical Instrument Co., Ltd., China), N-1300 rotary evaporator, CA-111 cold trap (Shanghai Ailang Instrument Co., Ltd., China), SHZ-DⅢ circulating water vacuum pump (Gongyi Yuhua Instrument Co., Ltd., China), ZF-7 three-way ultraviolet analyzer (Shanghai Jiapeng Technology Co., Ltd., China). All reagents used in the column chromatography process were industrial grade.

[0083] The specimen of *Camellia fascicularis* (52860), identified by taxonomist Min Tianlu, is preserved in the herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences. The *Camellia fascicularis* tea leaves used in this experiment were obtained in December 2019 in the Daweishan Nature Reserve, Hekou County, Yunnan Province, China, and identified as *Camellia fascicularis* by Professor Xiang Jianying, a taxonomist from Southwest Forestry University.

[0084] 2 Extraction and Separation

[0085] 10.7 kg of dried Yunnan golden chrysanthemum tea leaves were pulverized to approximately 40 mesh and extracted three times with 95% industrial methanol under reflux at 50°C for 3 h, 2 h, and 1 h respectively. The extracts were combined, and the industrial methanol was recovered using a rotary evaporator at 50°C under low pressure, yielding 868.3 g of Yunnan golden chrysanthemum tea methanol extract. 5 L of distilled water was added and thoroughly mixed, followed by an equal volume of industrial ethyl acetate for extraction three times. The extract was then concentrated at 40°C under low pressure to obtain 177.8 g of ethyl acetate phase extract. 175.0 g of the extract was mixed with 267.0 g of macroporous resin D101 and loaded onto a column at V... 水 / 甲醇A gradient elution ratio of 1:0 to 0:1 was used, followed by thin-layer chromatography (TLC) to detect the combined fractions. 45.0 g of the combined extract was mixed with 70.0 g of silica gel (200-300 mesh) and subjected to silica gel column chromatography on 320.0 g of silica gel (200-300 mesh). 氯仿：甲醇 = 1:0 to 0:1 gradient elution, TLC detection combined the same fractions to obtain 9 fractions (Fr.A to I).

[0086] Fr.F with V 氯仿：甲醇 =5:1 elution fraction (6.0 g), MCI column chromatography with sample, with V 甲醇 / 水 =40%–100% TLC segmentation yielded 9 fractions (Fr. A–Fr. I). The FA segment was analyzed using Sephadex LH-20 (V 甲醇 / 水 =95:5) Further purification and impurity removal yielded four components (Fr.FAa~FAd), and Fr.FAd was finally purified by semi-preparative high performance liquid chromatography (PHPLC) to obtain the monomeric compound. Figure 1 This is a flowchart illustrating the compound separation process of the present invention. Figure 2 This is a high-performance liquid chromatography (HPLC) diagram of the semi-preparative compound of the present invention.

[0087] In vitro antioxidant and antibacterial activities of Fr.A-Fr.I groups were screened, and Fr.F group was selected based on the results. The relevant screening data for Fr.F group are shown in Tables 1 and 2 below. The antioxidant activities of the components in *Camellia yunnanensis* are shown in Table 1; the antibacterial activities of the components in *Camellia yunnanensis* are shown in Table 2.

[0088] Table 1

[0089]

[0090] Table 2

[0091]

[0092] 3. Structural Analysis

[0093] 3-Methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside (compound): white powder, HR–ESI–MS m / z 477.1048 [M+Na+H] + The molecular formula is C 20 H 22 O 12 .

[0094] 1 H NMR (500MHz, Methanol-d4)δ H 7.10(2H,s,H-2”,6”),6.70(1H,d,J=2.7Hz,H-5),6.62(1H,d,J=8.6Hz, H-2),6.57(1H,dd,J=8.6,2.7Hz,H-6),4.73(1H,d,J=7.4Hz,H-1'),4.59 (1H,dd,J=11.9,2.1Hz,H-6a'),4.43(1H,dd,J=11.9,6.7Hz,H-6b'),3.9 2–3.76(1H,m,H-5'),3.71(3H,s,-OMe),3.50–3.39(3H,m,H-2',3',4'). 13 C NMR (126MHz, Methanol-d4)δ C 168.3(C-7”),152.7(C-1),149.2(C-3),146.6(C-3”,5”),143.1(C-4),139.9(C-4'),121.4(C-1'),116.1(C-5),110.2(C-2' ,6'),110.2(C-6),103.9(C-1'),103.9(C-2),77.9(C-3'),75.7(C-5'),74.9(C-2'),71.8(C-4'),64.9(C-6'),56.3(-OMe).

[0095] 4 Antioxidant activity

[0096] The experiment used 96-well plates, with a total capacity of 210 μL per well. Equal volumes of ABTS solution (7 mM) and potassium persulfate solution (5 mM) were mixed thoroughly and reacted in the dark at room temperature for 12 h to obtain an ABTS radical cation stock solution. The mixture was then diluted with anhydrous methanol to achieve an absorbance of approximately 0.7 ± 0.02 at 734 nm. Next, 180 μL of ABTS working solution was added to each well, followed by 30 μL of sample at different concentrations (0.5, 0.1, 0.05, and 0.01 mg / mL), dissolved, and then diluted with DMSO. After thorough mixing, the samples were incubated in the dark at room temperature for 6 min. The absorbance of each well was then measured at 734 nm using a microplate reader, and the results were obtained from at least three independent experiments. Ascorbic acid was used as a positive control.

[0097] ABTS for each test sample + The free radical cation scavenging rate (%) was calculated as follows (1):

[0098] ABTS +Clearance rate (%) = [blank - (sample - control)] / blank × 100% (1)

[0099] The antioxidant activity of compound 1 as determined by ABTS testing is shown in Table 3.

[0100] Table 3

[0101]

[0102] "-" indicates that the experiment was not performed.

[0103] ABTS compounds at concentrations of 10-500 μg / mL + The compound exhibited significantly superior activity compared to the positive control (ascorbic acid) in free radical scavenging experiments. This result suggests that the compound may possess more effective efficacy in scavenging free radicals and resisting oxidative stress. This discovery provides a new candidate for antioxidants and contributes to a deeper understanding of their mechanisms of action and potential applications.

[0104] Experimental Example 2

[0105] Antibacterial activity

[0106] Preparation of antibacterial drug stock solution: The drug was dissolved in DMSO to a concentration of 0.5 mg / mL. Preparation of test bacterial solution: The frozen bacterial solution taken from the -80℃ cryopreservation chamber was thawed at room temperature, sterilized in nutrient broth (NB) medium, and cultured overnight in a shaker at 37℃. Then, 2 mL of the overnight cultured bacterial solution was inoculated into NB medium and cultured at 37℃ until A 600 =0.5, then dilute 100 times with NB medium. Microbroth dilution method: Take a sterile 96-well plate, add 75 μL of NB medium diluent to wells A2-A11, add 75 μL of drug solution to wells A1-A2, transfer 75 μL of the mixture from well A2 to well A3, transfer 75 μL of the mixture from well A3 to well A4, and transfer 75 μL of the mixture to well A10, discarding the latter. Then add 75 μL of 5% DMSO to well A12, add 75 μL of 5% DMSO to well A12, and add 75 μL of bacterial culture to well A12, mix well. After inoculation, incubate the 96-well plate at 37℃ for 16 h, observe the growth, and measure A using a microplate reader. 600 Calculate the MIC.

[0107] The antibacterial activity of the compounds is shown in Table 4.

[0108] Table 4

[0109]

[0110] At a concentration of 500 μg / mL, the compound showed comparable inhibitory effects against *Pseudomonas aeruginosa* to the positive control (penicillin), but weaker than tetracycline. It also exhibited some activity against *Escherichia coli* and *Staphylococcus aureus*. These results suggest that the compound may possess superior efficacy in inhibiting foodborne bacterial responses. This discovery provides a new candidate substance for the field of foodborne antibacterial agents, contributing to a deeper understanding of its mechanism of action and potential applications.

[0111] Prior to this invention, research on the chemical composition of Camellia chrysantha mainly focused on its known bioactive components, while research on its bitter components was relatively limited. This invention aims to isolate the bitter component 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside from Camellia chrysantha in Yunnan using advanced extraction, separation, and analysis techniques, and to conduct in-depth research on its structure and bioactivity, in order to provide new perspectives and scientific support for the utilization and development of Camellia chrysantha.

[0112] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above-described experimental examples. The experimental examples and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. A bitter component, 3-methoxy-4-hydroxyphenol 1- O - β -D-(6'- O The method for preparing -galloyl)-glucopyranoside is characterized by: Includes the following steps: a. Take a dry sample of Yunnan golden flower tea leaves and grind it to 40 mesh; b. Extracted three times by reflux at 50 °C using 95% industrial methanol, with each extraction lasting 3 hours, 2 hours, and 1 hour respectively; c. Combine the extracts and use a rotary evaporator at 50 °C and low pressure to recover industrial methanol, obtaining a methanol extract. d. Add distilled water to the methanol extract and mix thoroughly. Add an equal volume of industrial ethyl acetate and extract thoroughly three times. Then, evaporate the extract at low pressure at 40 °C to obtain the ethyl acetate phase extract. e. Use macroporous resin D101 to load the sample onto the column, elute with a water / methanol gradient, and detect the same fractions by thin-layer chromatography (TLC). f. The identical portions from step (e) were mixed with silica gel and subjected to silica gel column chromatography with chloroform / methanol gradient elution. Thin-layer chromatography (TLC) was then performed to detect the same portions, resulting in nine fractions Fr. A-Fr. I. The Fr. A-Fr. I fractions were then screened for in vitro antioxidant and antibacterial activities. g. A specific component Fr. F was subjected to MCI column chromatography with a methanol / water gradient, and TLC was performed to obtain multiple components Fr.FA-Fr.FI; h.Fr. FA was further purified using Sephadex LH-20 to remove impurities, yielding four fractions: Fr.FAa-Fr.FAd. i.Fr. FAd was finally purified by semi-preparative high-performance liquid chromatography (PHPLC) to obtain compound 3-methoxy-4-hydroxyphenol 1- O - β -D-(6'- O 3-Methoxy-4-hydroxyphenol 1- (-galloyl)-glucopyranoside O - β -D-(6′- O -galloyl)-glucopyranoside; The bitter component 3-methoxy-4-hydroxyphenol 1- O - β -D-(6'- O The structural formula of (-galloyl)-glucopyranoside is shown in formula (I): （I） 。 2. The bitter component 3-methoxy-4-hydroxyphenol 1- according to claim 1 O - β -D-(6'- O A method for preparing (-galloyl)-glucopyranoside, characterized in that: In step (e), the volume ratio of water to methanol is 1:0 to 0:1; in step (f), the volume ratio of chloroform to methanol is 1:0 to 0:1.

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