Naphtho [2, 3-b] furan-5, 8-diketone compound with antioxidant activity as well as preparation method and application of naphtho [2, 3-b] furan-5, 8-diketone compound

Abstract

The invention belongs to the technical field of food chemistry, and particularly relates to a naphtho [2, 3-b] furan-5, 8-diketone compound with antioxidant activity as well as a preparation method and application of the naphtho [2, 3-b] furan-5, 8-diketone compound. The structural formula of the naphtho [2, 3-b] furan-5, 8-diketone compound with the antioxidant activity is as follows: # imgabs0 #. The naphtho [2, 3-b] furan-5, 8-diketone compound with the antioxidant activity, disclosed by the invention, is separated from a medicinal and edible plant Lonicera japonica, and an experimental result shows that the compound has relatively strong antioxidant activity. Meanwhile, the compound can be used as an additive for flavors and fragrances, can effectively prevent rancidity of the flavors and fragrances, and prolongs the shelf life of the flavors and fragrances.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant chemistry and specifically relates to a naphtho[2,3- b ]Furan-5,8-dione compounds and their preparation methods and applications. Background Art

[0002] Honeysuckle, also known as honeysuckle (Latin: Japanese honeysuckle Thunb), a perennial semi-evergreen woody plant of the Caprifoliaceae family, is a medicinal and edible plant. Because it blooms white at first and then turns yellow, it is named honeysuckle. Honeysuckle is a kind of honeysuckle that has been praised as a good medicine for clearing heat and detoxifying since ancient times. It is sweet, cold and fragrant. It clears heat without hurting the stomach. The fragrance is penetrating and can dispel evil. Honeysuckle can not only dissipate wind and heat, but also clear blood toxins. It is used for various febrile diseases, such as fever, rash, spots, heat toxic sores, sore throat, etc., and the effect is remarkable. With the progress of society and the development of science and technology, people's research on the comprehensive development and utilization of honeysuckle is becoming more and more in-depth. From the original medicinal value, it has developed into the development and production of health care foods, beauty and skin care products, and then developed into tourism and ecological agricultural engineering construction. Domestic and foreign studies have shown that the main chemical components in honeysuckle are: organic acids, phenylpropanoids, sterols, quinones, triterpenoid saponins, iridoid ether terpenoid glycosides and terpenoid volatile oils.

[0003] Natural antioxidants are an important type of food additives. Studies have shown that natural antioxidants in food not only have the effect of delaying the oxidation and deterioration of food, but also have multiple functions such as protecting cell membranes, expelling free radicals in cells, preventing heart disease and cancer, improving brain power, and delaying aging. The main types of natural substances with antioxidant properties found so far are: tannins, vitamins, anthraquinones, nitrogen-containing compounds, phytic acid, phenol, phenylpropanoids, coumarins, cumene, olefinic acid, etc. Replacing synthetic antioxidants with natural antioxidants is a development trend in the food industry. Natural plants are a very potential natural antioxidant resource. Finding new antioxidants that can remove free radicals in the body from natural plants will also be the development direction of modern medicine and health care industries. The present invention isolated a naphtho[2,3- b ] Furan-5,8-dione compounds, activity studies have shown that the compound has good antioxidant activity; the compound is safe, non-toxic, has significant antioxidant activity, and can be used to prevent oxidation and deterioration of tobacco flavors and spices and extend the shelf life of flavors and spices. There has been no related report on this compound so far. Summary of the invention

[0004] The first object of the present invention is to provide a naphtho[2,3- b ] furan-5,8-dione compounds; the second purpose is to provide the antioxidant activity of naphtho[2,3- b] Preparation method of furan-5,8-dione compounds; The third purpose is to provide the antioxidant activity of naphtho[2,3- b ]Application of furan-5,8-dione compounds.

[0005] The first object of the present invention is achieved by using the Yunnan ethnic medicinal plant honeysuckle ( Honeysuckle japonica Thunb) branches were used as raw materials and prepared through pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation. It was named: 3-methyl-7-(2'-oxopropyl)naphtho[2,3- b ]furan-5,8-dione; English name: 3-methyl-7-(2'-oxopropyl) naphtho[2,3- b ]furan-5,8-dione, whose molecular formula is: C 16 H 12 O 4 , having the following structure: .

[0006] The second object of the present invention is achieved by using honeysuckle branches, a plant with medicinal and edible properties, as raw materials, through pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation, and specifically comprising the following steps: A. Pretreatment: crush the raw material honeysuckle branches and pass through a 20-50 mesh sieve to obtain material a; B. Extraction of extract: Add 2 to 6 times the mass of material a to the organic extraction solvent, soak and extract 2 to 5 times at room temperature, each extraction time is 12 to 20 hours, combine the extracts and filter to obtain the sample extract b; C. MCI decolorization: The sample extract is decolorized on an MCI column, the effluent is collected and concentrated under reduced pressure to obtain extract c; D. Silica gel column chromatography: add 200-250 mesh silica gel in an amount of 3-10 times the weight of extract c to the extract c and load the column, perform gradient elution with a chloroform-methanol solution in a volume ratio of 20:1-1:1, monitor by TLC, and combine the same fractions; E. High performance liquid chromatography separation: The eluate obtained by eluting with a chloroform-methanol solution in a ratio of 9:1 was separated and purified by high performance liquid chromatography to obtain the target antioxidant active diketone compounds.

[0007] The specific operations are as follows: A. Sample extraction and purification: Dry the honeysuckle branches, crush them into 20-50 mesh, and then extract them with solvent for 2-5 times. The solvent for each extraction is 2-6 times that of the raw material. The extraction time is 12-20 hours. Filter out the precipitate to obtain the sample extract.

[0008] B. The obtained extract is decolorized on an MCI column, and the eluate is collected and concentrated under reduced pressure to obtain an extract, which is used for column chromatography separation.

[0009] C. Silica gel column chromatography: The obtained extract is diluted with 1.5 - 3 times of acetone or methanol, and then mixed with silica gel at 0.8 - 2.0 times the weight of the extract. The silica gel for mixing is 80 - 100 mesh. The mixed sample is subjected to silica gel column chromatography for rough separation. The silica gel for packing the column is 200 - 250 mesh, and the weight of the silica gel used is 3 - 10 times the weight of the extract; The chloroform and methanol mixed organic solvents with a volume ratio of 20:1 - 1:1 are used for gradient elution. The gradient eluates of each gradient are collected and concentrated. After monitoring by TLC, the same parts are combined to obtain 6 components (A - F); D. High - performance liquid chromatography separation: The component B in step C (the part eluted with a 9:1 mixed organic solvent of chloroform - methanol) is separated and purified by high - performance liquid chromatography to obtain the described diketone compound.

[0010] Furthermore, preferably, the solvent in step A is an aqueous acetone solution with a volume concentration of 70% - 100%, an aqueous ethanol solution with a volume concentration of 90% - 100%, or an aqueous methanol solution with a volume concentration of 90% - 100%.

[0011] Furthermore, preferably, before the extract in step C is subjected to silica gel column chromatography, it is first diluted with acetone or methanol at 1.5 - 3 times the weight of the extract, and then mixed with 80 - 100 mesh silica gel at 0.8 - 2.0 times the weight of the extract, and then loaded onto the column.

[0012] Furthermore, preferably, in step C, during gradient elution, the volume ratios of the chloroform and methanol mixed organic solvents used are 20:1, 9:1, 8:2, 7:3, 6:4, and 1:1 in sequence. (Each time, it is necessary to elute until no components flow out, that is, there is no residue in the distillation flask after evaporating the solvent, and then change to the next gradient).

[0013] Furthermore, preferably, the high - performance liquid chromatography separation and purification in step E uses an aqueous methanol solution with a volume concentration of 55% - 62% as the mobile phase, the flow rate is 12 mL / min, a Zorbax PrepHT GF reversed - phase preparative column with dimensions of 21.2×250 mm, 5 μm is used as the stationary phase, the detection wavelength of the ultraviolet detector is 363 nm, 0.5 - 1.0 mL is injected each time, and the chromatographic peaks at 28 - 45 min are collected. After multiple accumulations and evaporation to dryness, the pure compound can be obtained.

[0014] The prepared antioxidant - active naphtho[2,3 - b furan - 5,8 - dione compounds are identified by the following method: HRESI-MS showed that its quasi-molecular ion peak was 291.0623 [M+Na] + (calculated value 291.0628), combined with 1 H NMR and DEPT spectra to determine its molecular formula as C 16 H 12 O 4 , and the degree of unsaturation was 11.

[0015] The infrared spectrum showed resonance absorption peaks of carbonyl groups (1725, 1668) and aromatic rings (1643, 1465, 1376 cm -1 ). The maximum absorption at 215, 278, and 346 nm in the ultraviolet spectrum also indicated the possible presence of an aromatic ring structure in the compound.

[0016] The 1 H and 13 C NMR spectra (as shown in Table 1, Figure 1 and Figure 2 ) showed that it contained 16 carbons and 12 hydrogens, including a 1,2,4,5-tetrasubstituted benzene ring (C-1, C-2, C-3, C-4, C-9, C-10, H-1, H-4), two carbonyl groups (C-5 and C-8), two pairs of double bonds (C-6, C-7, C-14, C-15, H-6 and H-15), a 2'-oxopropyl group (-CH 2 COCH 3 , C-11~C-13, H 2 -11 and H 3 -13), and a methyl group (C-16 and H 3 -16). Further analysis of its nuclear magnetic resonance data showed that a group of double bonds and two carbonyl groups should be connected to the benzene ring to form 1,4-naphthoquinone, and the other group of double bonds should be connected to the benzene ring to form a furan ring to satisfy the 11 degrees of unsaturation in the compound. In addition, the speculation of the presence of 1,4-naphthoquinone in the compound could be confirmed by the HMBC correlations of H-6 and C-5 / C-8 / C-10, H-4 and C-5 / C-9 / C-10, and H-1 and C-8 / C-9 / C-10 ( Figure 3 ), and the speculation that the other group of double bonds was connected to the benzene to form a furan ring could also be confirmed by the HMBC correlations of H-4 and C-2 / C-14, H-15 and C-2 / C-3 / C-14 ( Figure 3 ). The compound could be determined to have a naphtho[2,3- b furan-5,8-dione structure.

[0017] Table 1. 1 H NMR and 13 C NMR data of the compound of the present invention (CDCl 3 ) After the parent body of the compound is determined, the positions of the remaining substituents (methyl and 2'-oxopropyl) can also be determined by their HMBC correlations. The substitution of 2'-oxopropyl at the C-7 position can be confirmed by the HMBC correlations of H 2 -11 with C-6 / C-7 / C-8, and H-6 with C-11. The substitution of methyl at the C-14 can be determined by the HMBC correlations of H 3 -16 with C-3 / C-14 / C-15, and H-15 with C-16. Thus, the structure of the compound is accurately confirmed. It is named: 3-methyl-7-(2'-oxopropyl)naphtho[2,3- b furan-5,8-dione; and its English name is: 3-methyl-7-(2'-oxopropyl)naphtho[2,3- b furan-5,8-dione.

[0018] Infrared, ultraviolet and mass spectrometry data of the compound: Ultraviolet spectrum (in methanol), λ max (log ε ) 215(4.06), 278(3.78), 346(3.62); Infrared spectrum (KBr tablet): ν max 3162, 2957, 2849, 1725, 1668, 1643, 1465, 1376, 1239, 1165, 859 cm -1 ; 1 H and 13 C NMR data (500 and 125 MHz, (C 5 D 5 N), see Table 1; Positive ion mode HRESIMS m / z 291.0623 [M+Na] + (calculated value 291.0628, C 16 H 12 NaO 4 ).

[0019] The third object of the present invention is achieved as follows. The application of the antioxidant naphtho[2,3- b furan-5,8-dione compounds in the preparation of antioxidants.

[0020] The antioxidant activity of the compound was first tested. Using vitamin C as the positive control, the antioxidant activity of the compound was tested, and the antioxidant activity was expressed by the ability to scavenge DPPH free radicals; with 50 μg / mL as the primary screening concentration, its activity of scavenging lipid free radical DPPH was determined. Take a Costar 96-well plate, add 190 μL / well of freshly prepared DPPH ethanol solution (6.5×10 5 mol / L), add 10 μL / well of the sample to be tested, add 10 μL of normal saline to the blank well, mix well, seal the plate with a sealing film, and let it stand in the dark at room temperature for 30 minutes. Measure the absorbance value of each well on a UV2401 spectrophotometer, and the measurement wavelength is 517 nm; the scavenging rate of the sample on lipid free radical DPPH is calculated according to the following formula: DPPH scavenging rate (%) = (A 空白 -A 样品） / A 空白 ×100% A 空白 : absorbance value of the blank control group; A 样品 : absorbance value of the group added with the sample.

[0021] The sample was detected in parallel 5 times. The measurement results showed that the IC 50 value of the compound was 5.63 μg / mL. It was better than vitamin C (IC 50 value was 6.22 μg / mL). The compound of the present invention showed good antioxidant activity, and its value as a natural antioxidant was very prominent.

[0022] Application of the described antioxidant naphtho[2,3- b furan-5,8-dione compounds in the preparation of fragrance and flavor additives.

[0023] Since most of the aroma components in fragrances and flavors contain unsaturated bonds, they are prone to oxidation and deterioration when exposed to air. Oxidation and deterioration will cause the color and aroma of fragrances and flavors to change. Reasonable use of antioxidants can significantly alleviate the oxidation and deterioration of fragrances and flavors and extend the shelf life of fragrances and flavors. Due to the relatively high toxicity of synthetic antioxidants such as BHA and BHT, great attention has been paid to the research and development of natural antioxidants at home and abroad. The present invention further evaluated the diketone compounds as fragrance antioxidants.

[0024] Take 10 g of fresh tobacco absolute, add 2.5 mg of the said compound, and stir well to dissolve it in a constant temperature water bath at 37 °C; after taking it out, let it stand at room temperature for 3 months. According to the "National Food Safety Standard - Edible Oil Products (GB15196 - 2015)", analyze and detect the acid value and peroxide value, and use the unadded tobacco absolute as a control. The results show that after adding the said compound, the acid value and peroxide value of the tobacco absolute decreased by 40.2% and 55.8% respectively. The results indicate that the compound of the present invention has a good effect of inhibiting the oxidative deterioration of tobacco absolute.

[0025] In addition, an artificial olfactory experiment was carried out with fresh tobacco absolute produced from the same raw materials and process as a control. The results show that after adding the compound of the present invention, there is no obvious change in the quality and clarity of the aroma of the tobacco absolute; while the quality and clarity of the aroma of the tobacco absolute without adding the compound of the present invention decreased. It shows that the diketone compound of the present invention has the effect of delaying the oxidative deterioration of essence and prolonging the shelf life of essence.

[0026] The advantages of the present invention are as follows: The compound of the present invention is naphtho[2,3 - b furan - 5,8 - dione, which is isolated from the branches of honeysuckle, a plant with both medicinal and edible uses. The branches of honeysuckle are the wastes of honeysuckle cultivation, with a large biological yield, very wide raw material sources, low cost, and easy separation and preparation.

[0027] The compound of the present invention has a good effect of prolonging the shelf life of essence. At the same time, it can be used as an antioxidant for cigarette essence. The results of cigarette sensory evaluation also show that adding this compound does not affect the smoking quality of cigarettes, and it is an ideal antioxidant for cigarette essence. Brief Description of the Drawings

[0028] Figure 1 The nuclear magnetic resonance carbon spectrum ( 13 C NMR) of the antioxidant active diketone compound of the present invention; Figure 2 The nuclear magnetic resonance hydrogen spectrum ( 1 H NMR) of the antioxidant active diketone compound of the present invention; Figure 3 The key HMBC correlation diagram of the antioxidant active diketone compound of the present invention. Detailed Embodiments

[0029] The present invention will be further described below in conjunction with the embodiments and drawings, but the present invention is not limited in any way. Any transformation or substitution based on the teachings of the present invention belongs to the protection scope of the present invention.

[0030] The naphtho[2,3-b]furan-5,8-dione compounds with antioxidant activity described in the present invention are prepared from the Yunnan medicinal and edible plant honeysuckle ( Japanese honeysuckle ) branches as raw materials, through pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation to prepare, named: 3-methyl-7-(2'-oxopropyl)naphtho[2,3-b]furan-5,8-dione; English name: 3-methyl-7-(2'-oxopropyl)naphtho[2,3- b ]furan-5,8-dione, whose molecular formula is: C 16 H 12 O 4 , having the following structure: .

[0031] The method for preparing the naphtho[2,3-b]furan-5,8-dione compounds with antioxidant activity of the present invention is to use the branches of honeysuckle, a plant with medicinal and edible properties, as raw materials, and to prepare the compounds through pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation, and specifically comprises the following steps: A. Pretreatment: crush the raw material honeysuckle branches through a 20-50 mesh sieve to obtain material a; B. Extraction of extract: Add 2 to 6 times the mass of material a to the material a, and soak and extract for 2 to 5 times at room temperature. Each extraction time is 12 to 20 hours. Combine the extracts and filter to obtain the sample extract b. C. MCI decolorization: The sample extract is decolorized on an MCI column, the effluent is collected and concentrated under reduced pressure to obtain extract c; D. Silica gel column chromatography: add 200-250 mesh silica gel in an amount of 3-10 times the weight of extract c to extract c and load the column, perform gradient elution with a chloroform-methanol solution in a volume ratio of 20:1-1:1, monitor by TLC, and combine the same parts; E. High performance liquid chromatography separation: The eluate obtained by eluting with a chloroform-methanol solution in a ratio of 9:1 is concentrated and then separated and purified by high performance liquid chromatography to obtain the target product, which is a diketone compound with antioxidant activity.

[0032] The organic extraction solvent described in step B is a methanol aqueous solution with a mass concentration of 90% to 100%, an ethanol aqueous solution with a mass concentration of 90% to 100%, or an acetone aqueous solution with a mass concentration of 70% to 100%.

[0033] Step D also includes the step of dissolving material c in an organic solvent at 1.5 to 3 times the weight of material c and then adding 80 to 100 mesh silica gel at 0.8 to 2.0 times the weight of material c for sample mixing before loading onto the column.

[0034] The organic solvent is pure methanol or pure acetone.

[0035] The volume ratio of the chloroform-methanol solution described in step D is 20:1, 9:1, 8:2, 7:3, 6:4, and 1:1.

[0036] In step E, high-performance liquid chromatography separation and purification uses an aqueous methanol solution with a volume concentration of 55% - 62% as the mobile phase, a flow rate of 12 mL / min, a 21.2×250 mm, 5 μm Zorbax PrepHT GF reversed-phase preparation column as the stationary phase, a detection wavelength of 363 nm for the ultraviolet detector, an injection volume of 0.5 - 1.0 mL each time, collects the chromatographic peaks at 28 - 45 min, and after multiple accumulations, evaporates to dryness to obtain the target antioxidant active diketone compound.

[0037] The application described in the present invention is the application of the naphtho[2,3- b furan-5,8-dione compounds with antioxidant activity in the preparation of antioxidants.

[0038] The application described in the present invention is the application of the naphtho[2,3- b furan-5,8-dione compounds with antioxidant activity as flavor and fragrance additives.

[0039] The following further illustrates the present invention with specific examples: Example 1 This example provides a preparation method of the antioxidant compound described in the present invention. The preparation method includes: extract extraction, silica gel column chromatography, and high-performance liquid chromatography separation steps. Using the medicinal and edible homologous plant honeysuckle as the raw material, the specific operations are as follows: The used honeysuckle is produced in Yuxi, Yunnan. The raw material is a sample obtained by crushing or cutting the honeysuckle branches. It is soaked and extracted 4 times with a 90% methanol aqueous solution by mass, each time for 15 h. The combined extraction solution is decolorized on an MCI column, and the eluate is concentrated under reduced pressure to obtain an extract; the extract is dissolved in methanol with a mass twice that of the extract, then 90-mesh silica gel is added for sample mixing, and a 220-mesh silica gel column is packed. After sample mixing, it is loaded onto the column; gradient elution is carried out with chloroform-methanol eluents with volume ratios of 20:1, 9:1, 8:2, 7:3, 6:4, and 1:1 respectively. The gradient elution solution is collected, concentrated, and monitored by TLC. The same parts are combined to obtain 6 parts A - F. Among them, for the collected 222 g of sample B (9:1) part, 58% methanol is used as the mobile phase, the flow rate is 12 ml / min, a 21.2×250 mm, 5 µ μm Zorbax PrepHT GF reversed-phase preparation column is used as the stationary phase, the detection wavelength of the ultraviolet detector is 363 nm, the injection volume is 0.8 mL each time, the chromatographic peaks at 30.6 min are collected, and after multiple accumulations, it is evaporated to dryness to obtain the antioxidant active compound described in the present invention.

[0040] The compound structure was identified as follows: HRESI-MS showed that its quasi-molecular ion peak was 291.0623 [M+Na] + (calculated value 291.0628). Combining with 1 the H NMR and DEPT spectra, its molecular formula was determined to be C 16 H 12 O 4 , and the degree of unsaturation was 11.

[0041] The infrared spectrum showed resonance absorption peaks of carbonyl groups (1725, 1668) and aromatic rings (1643, 1465, 1376 cm -1 ). The maximum absorption of the ultraviolet spectrum at 215, 278, and 346 nm also indicated that there might be an aromatic ring structure in the compound.

[0042] The 1 H and 13 C NMR spectra of the compound (as shown in Table 1 Figure 1 and Figure 2 ) showed that it contained 16 carbons and 12 hydrogens, including a 1,2,4,5-tetrasubstituted benzene ring (C-1, C-2, C-3, C-4, C-9, C-10, H-1, H-4), two carbonyl groups (C-5 and C-8), two pairs of double bonds (C-6, C-7, C-14, C-15, H-6 and H-15), a 2'-oxopropyl group (-CH 2 COCH 3 , C-11~C-13, H 2 -11 and H 3 -13), and a methyl group (C-16 and H 3 -16). Further analyzing its nuclear magnetic resonance data, a group of double bonds and two carbonyl groups should be connected to the benzene ring to form 1,4-naphthoquinone, and the other group of double bonds should be connected to the benzene ring to form a furan ring to meet the 11 degrees of unsaturation in the compound. In addition, the speculation of the presence of 1,4-naphthoquinone in the compound could be confirmed by the HMBC correlations of H-6 and C-5 / C-8 / C-10, H-4 and C-5 / C-9 / C-10, and H-1 and C-8 / C-9 / C-10 ( Figure 3 ), and the speculation that the other group of double bonds was connected to the benzene to form a furan ring could also be confirmed by the HMBC correlations of H-4 and C-2 / C-14, H-15 and C-2 / C-3 / C-14 ( Figure 3 ). This compound could be determined to have a naphtho[2,3- b furan-5,8-dione structure.

[0043] After the parent body of the compound is determined, the positions of the remaining substituents (methyl and 2'-oxopropyl) can also be determined by their HMBC correlations. The substitution of 2'-oxopropyl at C-7 can be confirmed by the HMBC correlations of H 2 -11 with C-6 / C-7 / C-8, and H-6 with C-11. The substitution of methyl at C-14 can be determined by the HMBC correlations of H 3 -16 with C-3 / C-14 / C-15, and H-15 with C-16. Thus, the structure of the compound is accurately confirmed. It is named: 3-methyl-7-(2'-oxopropyl)naphtho[2,3- b furan-5,8-dione; and its English name is: 3-methyl-7-(2'-oxopropyl)naphtho[2,3- b furan-5,8-dione.

[0044] Example 2 This example provides a preparation method of the antioxidant compound of the present invention. The preparation method includes: extract extraction, silica gel column chromatography and high performance liquid chromatography separation steps. Using the medicinal and edible homologous plant honeysuckle as the raw material, the specific operations are as follows: The used honeysuckle is produced in Dali, Yunnan. The sample of the raw material honeysuckle branches after being crushed or cut into sections is soaked and extracted 5 times with an ethanol aqueous solution with a mass concentration of 98%, each time for 12 h. The combined extraction solution is decolorized on an MCI column, and the eluate is concentrated under reduced pressure to obtain an extract; the extract is dissolved in acetone with a volume 3 times that of the extract, then 100-mesh silica gel is added for sample mixing, and a 250-mesh silica gel column is packed. After sample mixing, it is loaded onto the column; gradient elution is carried out with chloroform-methanol eluents with volume ratios of 20:1, 9:1, 8:2, 7:3, 6:4 and 1:1 respectively. The gradient eluate is collected, concentrated, monitored by TLC, and the same parts are combined to obtain 6 parts A-F. For the collected sample B (9:1) part of 242 g, with 70% methanol as the mobile phase, the flow rate is 12 ml / min, a 21.2×250 mm, 5 µ μm Zorbax PrepHT GF reversed-phase preparative column is used as the stationary phase, the detection wavelength of the ultraviolet detector is 363 nm, 1.0 mL is injected each time, the chromatographic peak at 32.2 min is collected, and after multiple accumulations, it is evaporated to dryness to obtain the antioxidant compound of the present invention. After identification, the identification method is the same as that in Example 1, and the result shows that the molecular formula is C 16 H 12 O 4 .

[0045] Example 3 This example provides a preparation method of the antioxidant compound of the present invention. The preparation method includes: extract extraction, silica gel column chromatography and high performance liquid chromatography separation steps. Using honeysuckle as the raw material, the specific operations are as follows: The honeysuckle used is produced in Hekou, Honghe, Yunnan. The raw material is a sample of crushed or cut honeysuckle branches, which is soaked and extracted twice with an aqueous acetone solution with a mass concentration of 70% for 12 hours each time. The combined extraction solution is decolorized on an MCI column, and the eluate is concentrated under reduced pressure to obtain an extract; the extract is dissolved in methanol at 1.5 times the mass of the extract, then silica gel of 80 mesh is added for sample mixing, and a column is packed with silica gel of 200 mesh. After sample mixing, it is loaded onto the column; gradient elution is carried out with chloroform-methanol eluents with volume ratios of 20:1, 9:1, 8:2, 7:3, 6:4, and 1:1 respectively. The gradient elution solution is collected, concentrated, monitored by TLC, and the same parts are combined to obtain 6 parts A-F. For 201 g of the collected sample B (9:1) part, 62% methanol is used as the mobile phase, the flow rate is 12 ml / min, and a ZorbaxPrepHT GF reversed-phase preparative column of 21.2×250 mm, 5 µ m is used as the stationary phase, the detection wavelength of the ultraviolet detector is 366 nm, 0.6 mL is injected each time, the chromatographic peak at 36.5 min is collected, and after multiple accumulations, it is evaporated to dryness to obtain the antioxidant active compound described in the present invention. After identification, the identification method is the same as that in Example 1, and the result shows that the molecular formula is C 16 H 12 O 4 。

[0046] Example 4 The antioxidant compound prepared in Example 1 was used for testing: Using vitamin-C as a positive control, the antioxidant activity of the compound was tested. The antioxidant activity was expressed by the ability to scavenge DPPH free radicals; the initial screening concentration was 50 μg / mL, and its activity of scavenging lipid free radical DPPH was measured. Take a costar 96-well plate, add 190 μL / well of freshly prepared DPPH ethanol solution (6.5×10 5 mol / L), add 10 μL / well of the sample to be tested, and add 10 μL of normal saline to the blank well. Mix well, seal the plate with a sealing film, and let it stand in the dark at room temperature for 30 minutes. Measure the absorbance value of each well on a UV2401 spectrophotometer at a measurement wavelength of 517 nm; the scavenging rate of the sample for lipid free radical DPPH is calculated according to the following formula: DPPH scavenging rate (%) = (A 空白 -A 样品 ) / A 空白 ×100% A 空白 : Absorbance value of the blank control group; A 样品 : Absorbance value of the group with the added sample.

[0047] The sample was detected in parallel 5 times. The measurement results showed that the IC 50 value of the compound was 5.63 μg / mL. It was superior to vitamin-C (IC50 With a value of 6.22 μg / mL, the compounds of the present invention exhibit good antioxidant activity.

[0048] In order to test the inhibitory effect of the compounds of the present invention on the oxidative deterioration of flavoring agents, the following experiments were carried out: (a) Tobacco absolute experiment Take 10 g of fresh tobacco absolute, add 2.5 mg of the said compound, and stir well to dissolve in a constant temperature water bath at 37°C; after taking out, place it at room temperature for 3 months. According to the "National Food Safety Standard - Edible Oil Products (GB15196 - 2015)", analyze and detect the acid value and peroxide value, and use the tobacco absolute without addition as a control. The results show that after adding the said compound, the acid value and peroxide value of the tobacco absolute decreased by 40.2% and 55.8% respectively. The results indicate that the compounds of the present invention have a good effect on inhibiting the oxidative deterioration of tobacco absolute.

[0049] In addition, an artificial olfactory experiment was also carried out with fresh tobacco absolute produced from the same raw materials and processes as a control. The results show that after adding the compounds of the present invention, the quality and clarity of the aroma of the tobacco absolute did not change significantly; while for the tobacco absolute without adding the compounds of the present invention, the quality and clarity of the aroma decreased.

[0050] (b) Experiment on cigarette bursting beads flavor Take 10 g of fresh cigarette bursting beads flavor, add 2.5 mg of the said compound, and stir well to dissolve in a constant temperature water bath at 37°C; after taking out, place it at room temperature for 3 months. According to the "National Food Safety Standard - Edible Oil Products (GB15196 - 2015)", analyze and detect the acid value and peroxide value, and use the tobacco absolute without addition as a control. The results show that after adding the said compound, the acid value and peroxide value of the cigarette bursting beads flavor decreased by 32.6% and 48.8% respectively. It shows that the compounds of the present invention have a good effect on inhibiting the oxidative deterioration of cigarette bursting beads flavor.

[0051] In addition, an artificial olfactory experiment was also carried out with cigarette bursting beads flavor produced from the same raw materials and processes as a control. The results show that after adding the compounds of the present invention, the quality and clarity of the aroma of the tobacco absolute did not change significantly; while for the tobacco absolute without adding the compounds of the present invention, the quality and clarity of the aroma decreased.

[0052] (c) Cigarette addition experiment Use triacetin to prepare a 0.5 mg / mL solution of the tobacco absolute and rose absolute added with the said compound. Spray it evenly onto the filter tow at 5% of the weight of the filter tow to make a filter rod, and then make cigarettes by conventional cigarette rolling and tipping. Conduct sensory evaluation, and use the same cigarettes without adding the compound of the present invention as a control. The evaluation results show that the smoothness and sweetness of the cigarettes added with the compound of the present invention are improved, the salivation effect is obvious, and the smoking comfort is significantly improved.

[0053] Example 5 Conduct tests with the antioxidant compounds prepared in Example 2 and Example 3 respectively. The method is the same as that in Example 4. The results show that the antioxidant compounds of the present invention have good effects on inhibiting the oxidation and deterioration of tobacco absolute and have good effects on inhibiting the oxidation and deterioration of the pearl essence for cigarettes.

Claims

1. A naphtho[2,3- b ] Furan-5,8-dione compounds, characterized in that The product is prepared from the branches of the Yunnan medicinal and edible plant honeysuckle, through pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation. It is named: 3-methyl-7-(2'-oxopropyl)naphtho[2,3- b ]furan-5,8-dione; English name: 3-methyl-7-(2'-oxopropyl)naphtho[2,3- b ]furan-5,8-dione, whose molecular formula is: C 16 H 12 O4, has the following structure: 。 2. The antioxidant activity of naphtho[2,3- b ] A method for preparing furan-5,8-dione compounds, characterized in that: The method is prepared by using the twigs of honeysuckle, a plant with medicinal and edible properties, as raw materials through pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation, and specifically comprises the following steps: A. Pretreatment: crush the raw material honeysuckle branches through a 20-50 mesh sieve to obtain material a; B. Extraction of extract: Add 2 to 6 times the mass of material a to the material a, and soak and extract for 2 to 5 times at room temperature. Each extraction time is 12 to 20 hours. Combine the extracts and filter to obtain the sample extract b. C. MCI decolorization: The sample extract is decolorized on an MCI column, the effluent is collected and concentrated under reduced pressure to obtain extract c; D. Silica gel column chromatography: add 200-250 mesh silica gel in an amount of 3-10 times the weight of extract c to extract c and load the column, perform gradient elution with a chloroform-methanol solution in a volume ratio of 20:1-1:1, monitor by TLC, and combine the same parts; E. High performance liquid chromatography separation: The eluate obtained by eluting with a chloroform-methanol solution in a ratio of 9:1 is concentrated and then separated and purified by high performance liquid chromatography to obtain the target product, which is a diketone compound with antioxidant activity.

3. The preparation method according to claim 2, characterized in that: The organic extraction solvent described in step B is a methanol aqueous solution with a mass concentration of 90% to 100%, an ethanol aqueous solution with a mass concentration of 90% to 100%, or an acetone aqueous solution with a mass concentration of 70% to 100%.

4. The preparation method according to claim 2, characterized in that: Step D also includes the step of dissolving material c in an organic solvent at 1.5 to 3 times the weight of material c and then adding 80 to 100 mesh silica gel at 0.8 to 2.0 times the weight of material c for sample mixing before loading onto the column.

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 solutions described in step D are 20:1, 9:1, 8:2, 7:3, 6:4 and 1:

1.

7. The preparation method according to claim 2, characterized in that: In step E, the HPLC separation and purification was performed with a methanol aqueous solution with a volume concentration of 55% to 62% as the mobile phase, a flow rate of 12 mL / min, a 21.2×250 mm, 5 μm Zorbax PrepHT GF reverse phase preparative column as the stationary phase, and a UV detector with a detection wavelength of 363 nm. 0.5 to 1.0 mL was injected each time, and the chromatographic peaks of 28 to 45 min were collected. After multiple accumulations, the target naphtho[2,3- b ]Furan-5,8-dione compounds.

8. The antioxidant activity of naphtho[2,3- b ] The application of furan-5,8-dione compounds is characterized in that: The antioxidant activity of naphtho[2,3- b ]Application of furan-5,8-dione compounds in the preparation of antioxidants.

9. The antioxidant activity of naphtho[2,3- b ] The application of furan-5,8-dione compounds is characterized in that: The antioxidant activity of naphtho[2,3- b ]Application of furan-5,8-dione compounds in the preparation of flavor and fragrance additives.

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