Bitter component (3-methoxy-4-hydroxyphenol 1-O-beta-D-(6apos;-O-galloyl)-glucopyranoside) as well as preparation method and application thereof

Abstract

The invention discloses a bitter component (3-methoxy-4-hydroxyphenol 1-O-beta-D-(6 '-O-galloyl)-glucopyranoside) as well as a preparation method and application of the bitter component. The structural formula of the 3-methoxy-4-hydroxyphenol 1-O-beta-D-(6 '-O-galloyl)-glucopyranoside compound is as shown in a formula (I): # imgabs0. The invention also relates to the application of the bitter component (3-methoxy-4-hydroxyphenol 1-O-beta-D-(6'-O-galloyl)-glucopyranoside) in preparation of an antioxidant or antibacterial active preparation. The invention also relates to a preparation method of the 3-methoxy-4-hydroxyphenol 1-O-beta-D-(6 '-O-galloyl)-glucopyranoside compound and a preparation method of the 3-methoxy-4-hydroxyphenol 1-O-beta-D-(6'-O-galloyl)-glucopyranoside compound. The invention provides the efficient method for separating the bitter component (3-methoxy-4-hydroxyphenol 1-O-beta-D-(6 '-O-galloyl)-glucopyranoside) of the Yunnan golden camellia, the research period is shortened, and the research efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the field of chemical technology, and in particular to a bitter ingredient 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside and a preparation method and application thereof. Background Art

[0002] Camellia fascicularis, belonging to the genus Camellia of the family Theaceae, is a deciduous shrub known for its golden petals. It is known as the "Giant Panda of the Plant World" and the "Queen of Tea". Camellia fascicularis is not only a rare species in the camellia family, but also has a history of being used as both medicine and food in China for hundreds of years. Camellia fascicularis has the effect of treating dysentery, blood in the stool, tumors, hypertension and many other diseases. Yunnan Camellia fascicularis contains rich amino acids, minerals, and bioactive ingredients such as tea polyphenols, saponins, flavonoids, and tea polysaccharides. It has been confirmed by pharmacological and clinical experiments that it has multiple pharmacological effects such as antioxidant, anti-tumor, antibacterial, hypolipidemic, hypoglycemic, and cholesterol-lowering.

[0003] Despite the potential health benefits of Yunnan Camellia chrysantha, the bitter chemical components in its flowers and leaves have not been fully studied and reported. As a unique and extremely small population of the species, Yunnan Camellia chrysantha has great potential for exploring its functional activities. Therefore, the separation and identification of the bitter component 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-pyranoglucopyranoside in Yunnan Camellia chrysantha can not only provide a scientific basis for the deep processing and application of Camellia chrysantha, but also lay the foundation for further drug development and health food research.

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

[0005] The present invention aims to provide a bitter ingredient 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside with antioxidant and antibacterial activities, and a preparation method and application thereof.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows: In the first aspect, the present application provides a bitter ingredient 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside.

[0007] In a second aspect, the present application provides a method for preparing a bitter ingredient, 3-methoxy-4-hydroxyphenol 1-O-β-D-(6′-O-galloyl)-glucopyranoside.

[0008] In a third aspect, the present application provides a Yunnan Camellia chrysantha extract.

[0009] In a fourth aspect, the present application provides a bitter ingredient 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside for use in the preparation of antioxidant or antibacterial active preparations.

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

[0011]

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

[0013] a. Take a dried sample of Yunnan Golden Flower tea leaves and crush it to about 40 mesh;

[0014] b. Use 95% industrial methanol to extract under condensation reflux at 50°C for 3 times, each time for 3 hours, 2 hours, and 1 hour respectively;

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

[0016] d. Add distilled water to the methanol extract and mix thoroughly, add an equal volume of industrial ethyl acetate to fully extract three times, and obtain an ethyl acetate phase extract by low-pressure rotary evaporation at 40°C;

[0017] e. Use macroporous resin D101 to mix the sample and load it onto the column, elute it with a water / methanol gradient, and combine the same parts for TLC detection;

[0018] f. The combined extracts were mixed with silica gel, subjected to silica gel column chromatography, chloroform / methanol gradient elution, thin layer chromatography TLC detection, and the same parts were combined to obtain 9 components Fr. (AI);

[0019] g. The specific component Fr.F was chromatographed on an MCI companion column and segmented by TLC with a methanol / water gradient to obtain multiple components Fr.(FA-FI);

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

[0021] i.Fr.FAd was finally purified by semi-preparative high performance liquid chromatography (PHPLC) to obtain the compound (3-Methoxy-4-hydroxyphenol1-O-β-D-(6'-O-galloyl)-glucopyranoside).

[0022] Furthermore, 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 the present application provides a Yunnan Camellia chrysantha extract, which contains 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-pyranoglucoside as a bitter component.

[0024] The fourth aspect of the present application provides a bitter ingredient 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside (3-Methoxy-4-hydroxyphenol 1-O-β-D-(6′-O-galloyl)-glucopyranoside) for the preparation of antioxidant or antibacterial active preparations.

[0025] Beneficial effects: The present invention successfully separates a new bitter monomer compound 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside from Yunnan golden camellia for the first time, providing new chemical composition information for the research and application of Yunnan golden camellia. The present invention discloses an application of a bitter component 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside in the preparation of an antioxidant or antibacterial active preparation. The invention provides an efficient separation method of 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-pyranoglucoside, a bitter component of Yunnan golden camellia, which shortens the research cycle and improves the research efficiency.

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

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

[0028] (2) The present invention adopts a series of advanced extraction, separation and purification technologies, including condensation reflux extraction, ethyl acetate extraction, macroporous resin and small silica gel column chromatography, and semi-preparative high performance liquid chromatography (PHPLC) purification, which improves the separation efficiency and purity. The separated compounds are accurately identified by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry to ensure the accuracy of the compound structure.

[0029] (3) The present invention promotes drug development and health food research: The research results of the present invention provide new research directions and possibilities for the application of Camellia chrysantha in the field of drug development and health food. The present invention contributes to the protection and rational use of this rare plant resource and promotes the protection of biodiversity by studying the bitter component 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-pyranoglucoside in Camellia chrysantha. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 The figure is a flow chart of separation and purification of the compounds of the present invention.

[0032] Figure 2 HPLC profiles were prepared for the compounds of the invention.

[0033] Figure 3 The compound of the present invention 1 HNMR spectrum.

[0034] Figure 4 The compound of the present invention 13 C NMR spectrum. DETAILED DESCRIPTION

[0035] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0036] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

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

[0038] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and 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 the present application.

[0039] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0040] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

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

[0042] In a first aspect, an embodiment of the present application provides a bitter ingredient 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside. The structural formula of the 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside compound is shown in formula (I):

[0043]

[0044] A second aspect of the present application embodiment provides a

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

[0046] a. Take a dried sample of Yunnan Golden Flower tea leaves and crush it to about 40 mesh;

[0047] b. Use 95% industrial methanol to extract under condensation reflux at 50°C for 3 times, each time for 3 hours, 2 hours, and 1 hour respectively;

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

[0049] d. Add distilled water to the methanol extract and mix thoroughly, add an equal volume of industrial ethyl acetate to fully extract three times, and obtain an ethyl acetate phase extract by low-pressure rotary evaporation at 40°C;

[0050] e. Use macroporous resin D101 to mix the sample and load it onto the column, elute it with a water / methanol gradient, and combine the same parts for TLC detection;

[0051] f. The combined extracts were mixed with silica gel, subjected to silica gel column chromatography, chloroform / methanol gradient elution, thin layer chromatography TLC detection, and the same parts were combined to obtain 9 components Fr. (AI);

[0052] g. The specific component Fr.F was chromatographed on an MCI companion column and segmented by TLC with a methanol / water gradient to obtain multiple components Fr.(FA-FI);

[0053] h.Fr.FA was further purified and impurized using Sephadex LH-20 to obtain four components 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] A third aspect of an embodiment of the present application provides a Yunnan Camellia chrysantha extract, which contains 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-pyranoglucoside as a bitter component.

[0057] A fourth aspect of the present application provides a

[0058] Application of 3-Methoxy-4-hydroxyphenol 1-O-β-D-(6′-O-galloyl)-glucopyranoside compound in preparing antioxidant or antibacterial active preparations.

[0059] Example 1

[0060] like Figure 2 and Figure 3 As shown, a bitter ingredient 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] + , 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] The present invention discloses a method for preparing a bitter ingredient 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside, comprising the following steps:

[0067] a. Take a dried sample of Yunnan Golden Flower tea leaves and crush it to about 40 mesh;

[0068] b. Use 95% industrial methanol to extract under condensation reflux at 50°C for 3 times, each time for 3 hours, 2 hours, and 1 hour respectively;

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

[0070] d. Add distilled water to the methanol extract and mix thoroughly, add an equal volume of industrial ethyl acetate to fully extract three times, and obtain an ethyl acetate phase extract by low-pressure rotary evaporation at 40°C;

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

[0072] f. The combined extracts were mixed with silica gel, subjected to silica gel column chromatography, chloroform / methanol gradient elution, thin layer chromatography TLC detection, and the same parts were combined to obtain 9 components Fr. (AI);

[0073] g. The specific component Fr.F was chromatographed on an MCI companion column and segmented by TLC with a methanol / water gradient to obtain multiple components Fr.(FA-FI);

[0074] h.Fr.FA was further purified and impurized using Sephadex LH-20 to obtain four components 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 invention discloses a Yunnan camellia chrysantha extract, which contains 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside as a bitter component.

[0078] Example 4

[0079] The invention discloses an application of a bitter ingredient 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside in the preparation of an antioxidant or antibacterial active preparation.

[0080] Test Example 1

[0081] 1. Instruments and Materials

[0082] BrukerAV 500MHz nuclear magnetic resonance instrument (Bruker, Germany), XEVO G2-XS Q-TOF high-resolution mass spectrometer (Waters, USA), NP7000 semi-preparative liquid phase (Jiangsu Hanbang Technology Co., Ltd., China), AX224ZH\E electronic balance (Ohaus Instrument (Changzhou) Co., Ltd., China), 101-2ES electric heating blast 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-purpose UV analyzer (Shanghai Jiapeng Technology Co., Ltd., China), column chromatography process reagents are all industrial grade.

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

[0084] 2 Extraction and separation

[0085] Take 10.7kg of Yunnan Golden Flower tea dry sample, crush to about 40 mesh, and then use 95% industrial methanol at 50℃ for condensation reflux extraction for 3 times, each time for 3h, 2h, and 1h respectively. Combine the extracts, use a rotary evaporator at 50℃ to recover industrial methanol, and obtain 868.3g of Yunnan Golden Flower tea methanol extract. Add 5L of distilled water to mix it thoroughly, add an equal volume of industrial ethyl acetate to fully extract 3 times, and concentrate at 40℃ to obtain 177.8g of ethyl acetate phase extract. 175.0g of extract is mixed with 267.0g of macroporous resin D101 and loaded on the column. 水 / 甲醇=1:0~0:1 gradient elution, thin layer chromatography (TLC) detection and combination of the same parts. Combined extract 45.0g with 70.0g silica gel (200~300 mesh) sample, 320.0g silica gel (200~300 mesh) for silica gel column chromatography, V 氯仿：甲醇 =1:0~0:1 gradient elution, TLC detection and merging of the same parts to obtain 9 components (Fr.A~I).

[0086] Fr.F with V 氯仿：甲醇 =5:1 eluted part (6.0 g), MCI sample column chromatography, V 甲醇 / 水 =40% to 100% TLC segmentation was performed to obtain 9 components (Fr.A to Fr.I). The FA segment was analyzed by Sephadex LH-20 (V 甲醇 / 水 =95:5) to further purify and remove impurities to obtain 4 components (Fr.FAa~FAd). Fr.FAd was finally purified by semi-preparative high performance liquid chromatography (PHPLC) to obtain a monomer compound. Figure 1 The figure is a flow chart of separation of compounds of the present invention. Figure 2 This is a semi-preparative HPLC diagram of the compound of the present invention.

[0087] The Fr.A group to the Fr.I group were screened for in vitro antioxidant and antibacterial activities, and the Fr.F group was selected based on the results. The relevant screening data of the Fr.F group are attached as shown in Tables 1 and 2. The antioxidant activity of the components in Yunnan Golden Camellia is shown in Table 1: The antibacterial activity of the components in Yunnan Golden Camellia is 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] + , 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 a 96-well plate with a total volume of 210 μL per well. Equal volumes of ABTS solution (7 mM) and potassium persulfate solution (5 mM) were mixed evenly 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 an absorbance value of approximately 0.7 ± 0.02 at 734 nm. Then, 180 μL of ABTS working solution was added to each well, followed by 30 μL of samples of different concentrations (0.5, 0.1, 0.05, 0.01 mg / mL), dissolved and diluted with DMSO. After mixing evenly, the samples were incubated at room temperature in the dark for 6 min. Then, the absorbance value of each well was 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 per test sample + The free radical cation scavenging rate (%) was calculated as follows (1):

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

[0099] ABTS test results The antioxidant activity of compound 1 is shown in Table 3:

[0100] Table 3

[0101]

[0102] "-" means the experiment was not performed.

[0103] Compounds at 10-500 μg / mL concentration, ABTS + In the free radical scavenging experiment, it showed significant activity that was superior to the positive control (ascorbic acid). This result shows that the compound may have a more excellent effect in scavenging free radicals and resisting oxidative stress. This discovery has brought new candidate substances to the field of antioxidants, which will help to further explore its mechanism of action and potential applications.

[0104] Test Example 2

[0105] Antimicrobial activity

[0106] Preparation of antimicrobial drug stock solution: Dissolve the drug in DMSO to a mass concentration of 0.5 mg / mL. Preparation of test bacterial solution: Thaw the frozen bacterial solution taken out from the -80°C low-temperature storage box at room temperature, sterilize the nutrient broth (NB) medium and culture it in a 37°C shaker overnight. Then take 2 mL of the overnight cultured bacterial solution and inoculate it into the NB medium and culture it at 37°C until A 600 =0.5, and then dilute 100 times with NB medium. Microbroth dilution method: Take a sterile 96-well plate, add 75μL NB medium dilution to wells A2-A11, add 75μL drug solution to wells A1-A2, take 75μL of the mixture from well A2 to well A3, take 75μL of the mixture from well A3 to well A4, take 75μL of the mixture to well A10, discard, then add 75μL 5% DMSO to well 12, add 75μL 5% DMSO to well A12, add 75μL bacterial solution to well A12, and mix well. After inoculation, incubate the 96-well plate at 37°C for 16h, observe the growth, and measure A with an enzyme reader. 600 , calculate the MIC.

[0107] Antibacterial test results The antibacterial activities of the compounds are shown in Table 4:

[0108] Table 4

[0109]

[0110] At a concentration of 500 μg / mL, the compound's inhibitory effect on Pseudomonas aeruginosa was comparable to that of the positive control (penicillin), but weaker than tetracycline, and it also had a certain effect on Escherichia coli and Staphylococcus aureus. This result suggests that the compound may have a more outstanding effect in inhibiting foodborne bacterial responses. This discovery has brought new candidate substances to the field of foodborne antimicrobial agents, which will help to further explore their mechanism of action and potential applications.

[0111] Prior to this invention, the research on the chemical components of Camellia chrysantha mainly focused on its known bioactive components, while relatively few studies were conducted on its bitter components. This invention aims to separate the bitter component 3-Methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside from Camellia chrysantha by advanced extraction, separation and analysis techniques, and 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 above shows and describes 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 by the above test examples, and the above test examples and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims

1. A bitter ingredient 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside, characterized in that: The bitter ingredient 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-pyranoglucoside has a structural formula as shown in formula (I):

2. The method for preparing the bitter ingredient (3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside) according to claim 1, characterized in that The steps include: a. Take a dried sample of Yunnan Golden Flower tea leaves and crush it to about 40 mesh; b. Use 95% industrial methanol to extract under condensation reflux at 50°C for 3 times, each time for 3 hours, 2 hours, and 1 hour respectively; c. Combine the extracts and use a rotary evaporator to recover industrial methanol at 50°C under low pressure to obtain a methanol extract; d. Add distilled water to the methanol extract and mix thoroughly, add an equal volume of industrial ethyl acetate to fully extract three times, and evaporate at low pressure at 40°C to obtain an ethyl acetate phase extract; e. Use macroporous resin D101 to mix the sample and load it onto the column, elute it with a water / methanol gradient, and combine the same parts for TLC detection; f. The combined extracts were mixed with silica gel, subjected to silica gel column chromatography, chloroform / methanol gradient elution, and thin layer chromatography TLC detection. The same parts were combined to obtain 9 components Fr.A-Fr.I; the Fr.A group-Fr.I group were screened for in vitro antioxidant and antibacterial activities, g. The specific component Fr.F was chromatographed on an MCI companion column and segmented by TLC with a methanol / water gradient to obtain multiple components Fr., namely FA-FI; h. Fr.FA was further purified and impurized using Sephadex LH-20 to obtain four components Fr., namely FAa-FAd; i.Fr.FAd was finally purified by semi-preparative high performance liquid chromatography (PHPLC) to obtain the compound 3-Methoxy-4-hydroxyphenol1-O-β-D-(6'-O-galloyl)-glucopyranoside.

3. The method for preparing the bitter ingredient 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-glucopyranoside according to claim 2, 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.

4. A Yunnan Camellia chrysantha extract, characterized in that The Yunnan golden camellia extract contains the (3-methoxy-4-hydroxyphenol 1-O-β-D-(6′-O-galloyl)-glucopyranoside) as claimed in claim 1 as a bitter component.

5. Use of the bitter ingredient (3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)-pyranoglucoside) according to claim 1 in the preparation of an antioxidant or antibacterial active preparation.

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