Citrus fruit white skin layer blue stain substance derivative ASB-D and preparation method and application thereof

Abstract

The invention relates to the technical field of natural compound separation, and discloses a citrus fruit white skin layer'blue stain 'substance derivative ASB-D which is identified as 2, 4-dihydroxy-6-[(O-beta-D-glucosyl) group] phenyl (5, 6-dihydroxy-7-[(O-beta-D-glucosyl) group] benzo [d] thiazole-2-group) ketone and is a yellow compound more stable than ABS (Acrylonitrile Butadiene Styrene). The compound has a certain anti-oxidation function, and can be used for development of anti-oxidation products and prediction of an ABS (Acrylonitrile Butadiene Styrene) structure.

Description

Technical Field

[0001] The invention relates to the technical field of natural compound separation, and in particular to a citrus fruit white peel "blue change" substance derivative ASB-D and a preparation method and application thereof. Background Art

[0002] Since the 21st century, China's citrus industry has developed rapidly. With the rapid development of China's citrus industry, the white skin of fruits has been found to be "blue". "Blue change" is also called "browning change", "blue-brown change", "mildew" and "internal mold". Initially, the white skin tissue near the orange peel of the fruit changes to light brown-blue. When the disease is mild, it is limited to the white skin near the orange peel. When the disease is more serious, the brown-blue color extends to the entire white skin. The normal light yellow or light yellow white skin turns light brown-blue or dark brown-blue, and turns to black-blue in severe cases. "Blue change" not only affects the appearance of the fruit, but also causes the flavor of the fruit to deteriorate or even produce obvious odors when the disease is serious, which seriously affects the commerciality of the fruit and causes consumers to have strong concerns about its food safety. It has become a major concern for the citrus industry.

[0003] The problem of "blue discoloration" of the white cortex of citrus fruits was first reported in the United States in 1944, and was observed in oranges, tangerines, and grapefruits in Arizona, Florida, California, and Texas. In China, the "blue discoloration" problem has occurred in varieties such as Gonggan, Wogan, Mashuiju, Maogugan, Qianjinju, and Shatangju. Researchers believe that the "blue discoloration" of fruits is not a pathological change caused by pathogens, but a physiological lesion caused by abiotic stress. The "blue discoloration" substance (abedo bluing substance, ABS) was initially thought to be a natural water-soluble dye similar to anthocyanins, but no related structural identification reports have been reported. In recent years, researchers have found that there is no characteristic absorption peak of anthocyanins in the extract of the "blue discoloration" white cortex, and no related substances have been found by LC-MS detection.

[0004] Prior art CN114989631A discloses a method for separating and purifying ABS from the white peel of citrus fruit, which only separates detectable ABS but does not obtain a pure product. In addition, the chemical structure of ABS is unstable and easy to fade. The content of ABS in the fruit is extremely low, making it difficult to prepare a large amount of pure ABS. As a result, single crystal induction tests based on pure products are difficult to carry out effectively, and its structure cannot be analyzed by X-RAD. Therefore, it has been difficult to directly identify its structure. Summary of the invention

[0005] The present invention prepares ABS-D (purity ≥ 99%), a derivative of ABS, and identifies the chemical structure of ABS-D, thereby laying a solid foundation for further identifying the ABS structure and analyzing the "blue change" mutagenic mechanism.

[0006] The technical solution of the present invention is as follows:

[0007] The present invention provides a citrus fruit white skin "blue change" substance derivative ASB-D, the chemical structure of which is as follows:

[0008]

[0009] The present invention also provides a method for preparing the above ABS-D, comprising the following steps:

[0010] S1: preparing a dry powder of the "blue-turned" white cortex of the fruit, extracting the dry powder with a hydrochloric acid ethanol solution to obtain an extract, adjusting the pH of the extract to 6.5-7.5, standing and filtering, and obtaining an ABS crude extract;

[0011] S2: dissolving the ABS crude extract with formic acid, injecting into an HLB column, eluting impurities with water and 30-40% methanol in sequence, and eluting ABS with 0.04-0.2% hydrochloric acid methanol to obtain sample solution 1;

[0012] S3: injecting the sample solution 1 into the MCX column, eluting impurities with 1% hydrochloric acid and methanol, and eluting ABS with 1-10% ammonia methanol to obtain sample solution 2;

[0013] S4: subjecting the sample solution 2 to Sephadex LH-20 column chromatography, using 25-35% methanol as the eluent, collecting the blue band fractions, and concentrating;

[0014] S5: Purify the obtained concentrated solution by semi-preparative liquid chromatography, collect the effluent of ABS absorption peak at 570nm, and dry to obtain pure ABS;

[0015] S6: dissolving the pure ABS in a hydrochloric acid solution, heating in a water bath, purifying the solution using a semi-preparative liquid chromatograph, collecting the effluent of the ABS-D absorption peak at 410 nm, concentrating and drying to obtain pure ABS-D.

[0016] As an embodiment, the preparation method of the dry powder of the "blue-turned" white skin layer of the fruit is to freeze the fresh sample of the "blue-turned" white skin layer with liquid nitrogen and then grind it into powder with a pulverizer.

[0017] Preferably, the volume concentration of the hydrochloric acid ethanol solution is 0.5-2%, and the solid-liquid ratio of the extraction is 1:5-25.

[0018] Preferably, the extraction is performed twice.

[0019] As an embodiment, a 5% volume concentration of NaHCO 3 Or ammonia water to adjust the pH of the extract.

[0020] As an embodiment, the ABS crude extract is dissolved and then filtered through a 0.22 μm filter membrane.

[0021] As an embodiment, in step S4, the flow rate is 0.4-0.6 mL / min, and one fraction is collected every 10 minutes.

[0022] As an embodiment, the chromatographic conditions of the semi-preparative liquid chromatograph are: C18 column, mobile phase A is acetonitrile, phase B is water, and the gradient elution conditions are as follows: 0-14min, 10%-22.5% A; 14-17min, 22.5%-100% A; 17-18min, 100%-10% A; 18-23min, 10% A; flow rate 4mL / min; detection wavelengths are 570 and 410nm respectively.

[0023] Preferably, in step S6 of the present invention, the temperature of the water bath is below 45° C., and the water bath time is 30-120 min.

[0024] Preferably, the concentration of the hydrochloric acid solution is 1-5%.

[0025] The ABS-D prepared by the invention has antioxidant activity and can be used for preparing antioxidant products.

[0026] Beneficial effects of the present invention:

[0027] Since there are many difficulties in the identification of ABS structure that are difficult to solve for the time being, the present invention derives ABS into another more stable yellow compound ABS-D under acidic and heating conditions, and separates and obtains pure ABS-D (purity ≥ 99%), which is identified as 2,4-dihydroxy-6-[(O-β-D-glucose)]phenyl(5,6-dihydroxy-7-[(O-β-D-glucose)]benzo[d]thiazol-2-yl)methanone. It has been verified that the ABS-D prepared by the present invention has antioxidant activity and can be used for the development of antioxidant active products. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : HPLC analysis of ABS at different purification stages, including: (A) hydrochloric acid ethanol extract of dry powder; (B) ABS crude extract; (C) HLB; (D) MCX.

[0029] Figure 2: UV-Vis and HPLC analysis during the separation and purification of ABS; wherein, (A) UV-Vis spectrum of hydrochloric acid ethanol extract of dry powder; (B) HPLC analysis (570nm) of hydrochloric acid ethanol extract of dry powder; (C) HPLC analysis (240nm) of pure ABS and its UV-Vis spectrum.

[0030] Figure 3 : From top to bottom, they are UPLC-QTOF-MS analysis of pure Gonggan ABS, UPLC-QTOF-MS analysis of sample solution 1 of Mashuiju and Wogan after HLB solid phase extraction pretreatment.

[0031] Figure 4 : HPLC analysis (240 nm) of the conversion of ABS to ABS-D.

[0032] Figure 5 : Structural information of compound ABS-D, including: (A) chemical structure of ABS-D; (B) UV-Vis spectrum of ABS-D; (C) primary mass spectrum of ABS-D; (D) secondary mass spectrum of ABS-D.

[0033] Figure 6 : Compound ABS-D 1 H NMR spectrum (600 MHz, CD 3 OD).

[0034] Figure 7 : Compound ABS-D 1 H NMR spectrum (600 MHz, DMSO-d 6 ).

[0035] Figure 8 : Compound ABS-D 13 C NMR spectrum (150 MHz, DMSO-d 6 ).

[0036] Fig. 9 : HSQC spectrum of compound ABS-D (DMSO-d 6 ).

[0037] Fig.10 : HMBC spectrum of compound ABS-D (DMSO-d 6 ).

[0038] Fig.11 : ROESY spectrum of compound ABS-D (DMSO-d 6 ).

[0039] Fig.12 : COSY, key HMBC and ROESY correlations of compound ABS-D.

[0040] Fig.13 : 13 Linear correlation between the calculated and experimental values ​​of ABS-D in C NMR chemical shift.

[0041] Fig.14 : DPPH (A) and ABTS (B) free radical scavenging activity of compound ABS-D. DETAILED DESCRIPTION

[0042] In the present invention, ABS is firstly separated and purified from the white peel of citrus fruits, and pure ABS (purity ≥ 99%) is obtained by separation and purification from the white peel of Gonggan "blue-discoloration" fruits, proving that ABS is also the substance that causes the "blue-discoloration" of the white peel of Wogan and Mashuiju fruits.

[0043] The present invention first prepares a pure ABS product of the white peel layer of citrus fruit, comprising the following steps:

[0044] S1: preparing a dry powder of the "blue-turned" white cortex of the fruit, extracting the dry powder with a hydrochloric acid ethanol solution to obtain an extract, adjusting the pH of the extract to 6.5-7.5, standing and filtering, and obtaining an ABS crude extract;

[0045] S2: dissolving the ABS crude extract with formic acid, injecting into an HLB column, eluting impurities with water and 30-40% methanol in sequence, and eluting ABS with 0.04-0.2% hydrochloric acid methanol to obtain sample solution 1;

[0046] S3: injecting the sample solution 1 into the MCX column, eluting impurities with 1% hydrochloric acid and methanol, and eluting ABS with 1-10% ammonia methanol to obtain sample solution 2;

[0047] S4: subjecting the sample solution 2 to Sephadex LH-20 column chromatography, using 25-35% methanol as the eluent, collecting the blue band fractions, and concentrating;

[0048] S5: Purify the concentrated solution by semi-preparative liquid chromatography, collect the effluent of ABS absorption peak at 570 nm, and dry to obtain pure ABS.

[0049] The "blue-changed" white skin layer samples of the fruit of the present invention are preferably "blue-changed" fresh samples of the white skin layer of citrus fruits such as Wogan, Gonggan, and Mashuiju. Subsequent experiments have shown that the ABS substances of the above-mentioned different citrus fruits are the same chemical substance. In the present invention, the preparation method of the "blue-changed" white skin layer dry powder of the fruit is to freeze the "blue-changed" white skin layer fresh sample with liquid nitrogen, and then grind it into powder with a pulverizer to facilitate the separation and extraction of ABS.

[0050] In the process of concentrating the crude ABS extract by a rotary evaporator, we found that if the rotary evaporation temperature is too high, the crude extract will fade quickly, and the ABS may have changed; if the rotary evaporation temperature is too low, the solvent cannot be completely removed, which is not conducive to subsequent separation and purification. Therefore, we can only concentrate and enrich ABS in other ways. In the previous extraction test, we found that neutral or alkaline ethanol cannot effectively extract ABS, while under acidic conditions, the solubility of ABS in ethanol will increase significantly. Based on this finding, the present invention extracts the dry powder with hydrochloric acid ethanol solution to obtain an extract, adds 5% sodium bicarbonate solution or ammonia water to the extract, adjusts the pH of the extract to 6.5-7.5, and stands and filters to obtain ABS crude extract. Preferably, by adjusting the solvent pH to 7, the solubility difference of ABS at different pH values ​​is utilized to precipitate ABS from ethanol, thereby achieving the purpose of enrichment.

[0051] When the present invention uses organic solvents of different polarities and concentrations to extract the ABS of the white cortex, it is found that the ABS can be effectively extracted only under acidic conditions, and then the ABS extraction method is optimized: 0.5-2% hydrochloric acid ethanol (v / v) extraction, preferably 1% hydrochloric acid ethanol (v / v) extraction; extraction time 0.5-4h, preferably 2h; solid-liquid ratio 1:5-25, preferably 1:15 (w / v). Under the above conditions, the extraction rate of ABS is relatively high. As a preferred embodiment, the extraction times are twice.

[0052] To determine whether ABS changes before and after extraction, we performed HPLC analysis on the crude ABS extract and its corresponding blue precipitate. ABS was detected simultaneously at 210nm and 570nm, where 570nm was used as the characteristic absorption wavelength of ABS to eliminate the interference of impurities and accurately locate ABS; 210nm was used as the terminal absorption wavelength to detect impurities as much as possible and was used to characterize the separation of ABS. Figure 1 It can be seen from A and B that at 570nm, the peak time and type of each ABS before and after enrichment did not change; at 210nm, there were multiple shoulder peaks before enrichment, and the shoulder peaks were significantly reduced after enrichment. The above results show that the enrichment method of the present invention can well retain ABS and has a certain purification effect on ABS.

[0053] The present invention uses formic acid to dissolve the ABS crude extract, and solid phase extraction is performed after passing through a 0.22 μm filter membrane. The volume concentration of the formic acid is preferably 20%. Selecting a suitable adsorbent and a correct elution procedure are the key to solid phase extraction. When further separating and purifying the ABS crude extract, chromatographic fillers such as silica gel, macroporous resin, and polyamide have been tried. However, ABS is very easy to tail or form dead adsorption on these fillers, resulting in very low ABS recovery rate and separation effect. HLB is not easy to form dead adsorption with ABS. The present invention uses an HLB solid phase extraction column to effectively remove residual salt and some hydrophilic impurities in the sample, and adding a trace amount of hydrochloric acid to the eluent can effectively inhibit the dead adsorption of ABS, thereby increasing its recovery rate to 95.2%. Therefore, the present invention uses HLB as an adsorbent, water, and 30-40% methanol (v / v) as elution solvents, and removes a portion of impurities as much as possible on the basis of retaining ABS. Subsequently, ABS is eluted with 0.04-0.2% hydrochloric acid methanol (v / v). As an embodiment, water, 35% methanol, and 0.04% hydrochloric acid methanol are used for elution in an amount of 6 ml respectively. The purification effect is as follows Figure 1 As shown in C, it can be seen that HLB removes a large amount of impurities while retaining ABS.

[0054] The present invention selects MCX as an adsorbent to further purify ABS. As an embodiment, 1% hydrochloric acid (v / v) and methanol are used as eluents to remove impurities, and then 1-10% ammonia methanol is used to elute ABS. As an embodiment, the elution amount of 1% hydrochloric acid and methanol is 6 ml respectively, and the elution amount of 5% ammonia methanol is 12 mL. Figure 1 D It can be seen that MCX can further remove a large number of impurities, and all of them are sharp single peaks at 210nm.

[0055] Sephadex LH-20 is a filler that combines molecular sieve and reverse phase distribution chromatography, which can further separate ABS from impurities according to molecular weight. Considering that ABS is a water-soluble pigment, the present invention uses 25-35% methanol as the mobile phase and a flow rate of 0.4-0.6mL / min to separate ABS. As an embodiment, a fraction collector is used to collect the blue band, and 1 fraction is collected every 10 minutes. The collected liquid is concentrated using a rotary evaporator and a freeze dryer in turn.

[0056] The final purification was performed using a semi-preparative liquid chromatograph. The chromatographic conditions of the semi-preparative liquid chromatograph were: C18 column, mobile phase A was acetonitrile, phase B was water, and the gradient elution conditions were as follows: 0-14 min, 10%-22.5% A; 14-17 min, 22.5%-100% A; 17-18 min, 100%-10% A; 18-23 min, 10% A; flow rate 4 mL / min. The detection wavelength was 570 nm, and 2-4 ml of ABS solution was manually collected. The collected solution was subjected to rotary evaporation and freeze-dried to obtain pure ABS.

[0057] The present invention determines the ABS by NMR. 1 H NMR showed that the substance was tautomeristic, and it was difficult to solve this problem by trying different deuterated reagents. In addition, the chemical structure of ABS is unstable and easy to fade. The content of ABS in the fruit is extremely low (about 1.5×10 -9 ), it is difficult to prepare a large amount of pure ABS, which makes it difficult to carry out single crystal induction experiments effectively and unable to analyze its structure by X-RAD. Therefore, it has been difficult to directly identify its structure. In the early separation and purification process, we found that the blue ABS solution would gradually turn into light yellow under acidic conditions, and the conversion rate was affected by temperature and pH. Then, using hydrochloric acid as a solvent, the blue ABS solution was quickly changed into a yellow solution by heating in a water bath. As an embodiment, the concentration of the hydrochloric acid is 1-5%, the temperature of the water bath heating is below 45°C, preferably 35-42°C, and the water bath time is preferably within 120min, preferably 30-120min. HPLC shows that under acidic and heated conditions, ABS is derived into another more stable yellow compound (ABS-D). When the yellow substance is no longer generated, a semi-preparative liquid chromatograph is used for purification, and the purification method is as described above. Since the ABS-D solution is light yellow and only ABS-D has an absorption peak at 410 nm, 2-4 mL of the effluent of the ABS-D absorption peak was collected at 410 nm, concentrated and dried to obtain pure ABS-D (purity ≥ 99%), which was identified as 2,4-dihydroxy-6-[(O-β-D-glucosyl)]phenyl(5,6-dihydroxy-7-[(O-β-D-glucosyl)]benzo[d]thiazol-2-yl)methanone, which has certain antioxidant activity.

[0058] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0059] Example 1

[0060] Extraction of pure ABS from Gonggan

[0061] Take 9958.0g of fresh sample of the white skin of Gonggan "Blue Change", freeze it with liquid nitrogen and grind it into powder with a pulverizer. Then add 1% hydrochloric acid ethanol (v / v) at a solid-liquid ratio of 1:15 (w / v), and extract with magnetic stirring at room temperature for 2h. Repeat twice, and combine the two extracts after filtration. Slowly add 5% NaHCO 3 (v / v), adjust the pH of the extract to 7.0, let it stand until the blue flocs are fully precipitated, and filter to obtain the crude ABS extract.

[0062] The HLB solid phase extraction column (500 mg / 6 mL, Waters, Milford, USA) was activated and balanced. The crude ABS extract was dissolved in 20% formic acid (v / v), filtered through a 0.22 μm PES filter (Bickman Bio, Changde, China), and then the sample solution was adsorbed on the balanced HLB column. The impurities were eluted with 6 mL of water and 35% methanol (v / v), and the ABS was eluted with 6 mL of 0.04% hydrochloric acid and methanol (v / v) to obtain sample solution 1. Then, sample solution 1 was adsorbed on the balanced MCX column, and the impurities were eluted with 6 mL of 1% hydrochloric acid (v / v) and methanol, and the ABS was eluted with 12 mL of 5% ammonia and methanol (v / v) to obtain sample solution 2. ABS sample solution 2 was further separated by Sephadex LH-20 (Cytiva, Sweden) column chromatography (40×600 mm). The eluent was 30% methanol (v / v) at a flow rate of 0.5 mL / min. The blue band was collected using a BSZ-100 fraction collector (Qingpu Luxi Instrument, Shanghai, China), and one fraction was collected every 10 min. The collected solution was concentrated using a rotary evaporator and a freeze dryer (Boyikang Laboratory Instrument Co., Ltd., Beijing, China).

[0063] The concentrated ABS was purified using a NP7000 semi-preparative liquid chromatograph (Hanbang Technology, Jiangsu, China). Chromatographic conditions: Waters BEH C18 column (10mm×250mm, 5μm, Waters, Milford, USA), mobile phase A is acetonitrile, phase B is water, gradient elution conditions are as follows: 0-14min, 10%-22.5% A; 14-17min, 22.5%-100% A; 17-18min, 100%-10% A; 18-23min, 10% A; flow rate 4mL / min, detection wavelength 570nm, manually collect 2mL ABS absorption peak effluent. The collected liquid was rotary evaporated and freeze-dried to obtain 3.7mg of pure ABS with a purity of ≥99%.

[0064] Example 2

[0065] Extraction of tangerine pure ABS

[0066] Take 20.0g of fresh sample of the white skin of the "blue change" of Ma Shui tangerine, freeze it with liquid nitrogen and grind it into powder with a pulverizer. Then add 0.7% hydrochloric acid ethanol (v / v) at a solid-liquid ratio of 1:25 (w / v), and extract with magnetic stirring at room temperature for 4h. Repeat twice, and combine the two extracts after filtration. Slowly add 5% NaHCO 3 (v / v), adjust the pH of the extract to 6.5, let it stand until the blue flocs are fully precipitated, and filter to obtain the crude ABS extract.

[0067] The HLB solid phase extraction column (500 mg / 6 mL, Waters, Milford, USA) was activated and balanced. The crude ABS extract was dissolved in 20% formic acid (v / v), filtered through a 0.22 μm PES filter (Bickman Bio, Changde, China), and then the sample solution was adsorbed on the balanced HLB column. The impurities were eluted with 6 mL of water and 30% methanol (v / v), and the ABS was eluted with 6 mL of 0.1% hydrochloric acid and methanol (v / v) to obtain sample solution 1. Then, sample solution 1 was adsorbed on the balanced MCX column, and the impurities were eluted with 6 mL of 1% hydrochloric acid (v / v) and methanol, and the ABS was eluted with 12 mL of 8% ammonia and methanol (v / v) to obtain sample solution 2. ABS sample solution 2 was further separated by Sephadex LH-20 (Cytiva, Sweden) column chromatography (40×600 mm). The eluent was 25% methanol (v / v) at a flow rate of 0.4 mL / min. The blue band was collected using a BSZ-100 fraction collector (Qingpu Luxi Instrument, Shanghai, China), and one fraction was collected every 10 min. The collected solution was concentrated using a rotary evaporator and a freeze dryer (Boyikang Laboratory Instrument Co., Ltd., Beijing, China).

[0068] The concentrated ABS was purified using a NP7000 semi-preparative liquid chromatograph (Hanbang Technology, Jiangsu, China). Chromatographic conditions: Waters BEH C18 column (10mm×250mm, 5μm, Waters, Milford, USA), mobile phase A is acetonitrile, phase B is water, gradient elution conditions are as follows: 0-14min, 10%-22.5% A; 14-17min, 22.5%-100% A; 17-18min, 100%-10% A; 18-23min, 10% A; flow rate 4mL / min, detection wavelength 570nm, manually collect 4mL ABS absorption peak effluent. The collected liquid was rotary evaporated and freeze-dried to obtain pure ABS with a purity of ≥99%.

[0069] Example 3

[0070] Extraction of pure ABS from Wogan

[0071] Take 20.0g of fresh sample of the white skin of the "blue change" mandarin orange, freeze it with liquid nitrogen and grind it into powder with a pulverizer. Then add 2% hydrochloric acid ethanol (v / v) at a solid-liquid ratio of 1:5 (w / v), extract with magnetic stirring for 1h at room temperature, repeat twice, and combine the two extracts after filtration. Slowly add 5% ammonia water (v / v) to the extract, adjust the pH of the extract to 7.5, let it stand, wait for the blue floccules to fully precipitate, and filter to obtain the ABS crude extract.

[0072] The HLB solid phase extraction column (500 mg / 6 mL, Waters, Milford, USA) was activated and balanced. The crude ABS extract was dissolved in 20% formic acid (v / v), filtered through a 0.22 μm PES filter (Bickman Bio, Changde, China), and then the sample solution was adsorbed on the balanced HLB column. The impurities were eluted with 6 mL of water and 40% methanol (v / v), and the ABS was eluted with 6 mL of 0.2% hydrochloric acid and methanol (v / v) to obtain sample solution 1. Then, sample solution 1 was adsorbed on the balanced MCX column, and the impurities were eluted with 6 mL of 1% hydrochloric acid (v / v) and methanol, and the ABS was eluted with 12 mL of 2% ammonia and methanol (v / v) to obtain sample solution 2. ABS sample solution 2 was further separated by Sephadex LH-20 (Cytiva, Sweden) column chromatography (40×600 mm). The eluent was 35% methanol (v / v) at a flow rate of 0.6 mL / min. The blue band was collected using a BSZ-100 fraction collector (Qingpu Luxi Instrument, Shanghai, China), and one fraction was collected every 10 min. The collected solution was concentrated using a rotary evaporator and a freeze dryer (Boyikang Laboratory Instrument Co., Ltd., Beijing, China).

[0073] The concentrated ABS was purified using a NP7000 semi-preparative liquid chromatograph (Hanbang Technology, Jiangsu, China). Chromatographic conditions: Waters BEH C18 column (10mm×250mm, 5μm, Waters, Milford, USA), mobile phase A is acetonitrile, phase B is water, gradient elution conditions are as follows: 0-14min, 10%-22.5% A; 14-17min, 22.5%-100% A; 17-18min, 100%-10% A; 18-23min, 10% A; flow rate 4mL / min, detection wavelength 570nm, manually collect 3mL ABS absorption peak effluent. The collected liquid was rotary evaporated and freeze-dried to obtain pure ABS with a purity of ≥99%.

[0074] Example 4

[0075] HPLC Analysis of ABS

[0076] ABS in the samples was detected by 1260 Infinity HPLC (Agilent, Palo Alto, CA, USA) equipped with a DAD detector. Chromatographic conditions: Waters XBridgeTM C18 column (4.6 mm×250 mm, 5 μm, Waters, Milford, USA); mobile phase A was acetonitrile, B was 0.05% phosphoric acid (v / v), gradient elution conditions were 0-25 min, 5%-25% A; 25-30 min, 25%-100% A; 30-31 min, 100%-10% A; equilibrium time 7 min; flow rate 1 mL / min, injection volume 5 μL, column temperature 35°C, detection wavelengths 210 nm, 240 nm, 570 nm.

[0077] Example 5

[0078] UPLC-QTOF-MS / MS Analysis of ABS

[0079] ACQUITY-UPLC I-Class-G2-XS-QTOF (Waters, Milford, USA) was used to analyze the molecular weights of the three varieties of ABS in Examples 1-3. Chromatographic conditions: ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm, Waters, Milford, USA); mobile phase A was acetonitrile, B was 0.1% formic acid (v / v), gradient elution program: 0-8 min, 10%-30% A; 8-9 min, 30%-100% A; 9-10 min, 100%-10% A; 10-12 min, 10% A, flow rate 0.4 mL / min, column temperature 35°C, injection volume 1 μL. Mass spectrometry conditions: electrospray ion source (ESI), positive ion mode detection, capillary voltage 3 kV, cone voltage 40 V, collision voltage 20 V at low energy, collision voltage 40 V at high energy, desolvation temperature 250 °C, ion source temperature 100 °C, desolvation gas flow rate 600 L / h, cone gas flow rate 50 L / h.

[0080] from Figure 3 It can be seen that at around 2.4 min, the molecular ion peaks m / z of Gonggan, Mashuiju and Wogan ABS were 642.1135, 642.1134 and 642.1134, respectively, indicating that the ABS of the three varieties are the same chemical substance.

[0081] Example 6

[0082] Preparation of "blue change" substance derivative (ABS-D)

[0083] 3.0 mg ABS was dissolved in 1 mL 1% hydrochloric acid (v / v), and heated in a water bath at 45° C. The HPLC method of Example 4 was used to detect the change of ABS when the heating time was 0, 30, 60, 90, and 120 min, respectively. Figure 4 As shown in the figure, when the heating time is 30 to 120 minutes, HPLC detected a new substance (ABS-D) at about 10.8 minutes, and the peak area of ​​ABS-D gradually increased with the extension of heating time; correspondingly, the peak area of ​​ABS gradually decreased, indicating that ABS-D was transformed from ABS. When the heating time is 120 minutes, ABS is completely transformed into ABS-D, and no new absorption peak appears in the chromatogram, indicating that ABS-D has no other changes under the conditions of 1% hydrochloric acid and water bath heating, and ABS-D is a more stable substance than ABS.

[0084] In order to improve the purity of ABS-D as much as possible, ABS-D was purified by semi-preparative liquid chromatography as described in Example 1, and 3 mL of ABS-D absorption peak effluent was manually collected at 410 nm. The collected liquid was subjected to rotary evaporation and freeze-dried to finally obtain 2.9 mg of ABS-D with a purity of ≥99%.

[0085] Example 7

[0086] UV-Vis and NMR analysis of ABS-D

[0087] Methanol was used as solvent, and the UV-Vis spectra of the target compounds were scanned with a UV-Vis spectrophotometer (Puxi General Instrument Co., Ltd., Beijing, China) at a scanning wavelength of 210-800 nm. AVANCE III 600 MHz (Bruker, Germany) equipped with a CPTC I low-temperature probe was used to determine the target compounds. 1 H NMR, 13 C NMR, 1 H- 1 HCOSY, HSQC, HMBC, and ROESY were used to calculate the chemical shift of ABS-D and determine the chemical structure of ABS-D.

[0088] Compound ABS-D is a light yellow powder. Its UV-Vis spectrum shows that its ultraviolet absorption is significantly purple-shifted compared to ABS ( Figure 5 B), indicating that the macroconjugated system in the structure is destroyed. HRESIMS shows that the quasi-molecular ion peak of ABS-D is m / z 660.1213 [M+H] + (C 26 H 30 NO 17 S,calc.660.1223)( Figure 5 C), indicating that the molecular formula of the compound is C26 H 29 NO 17 S, calculated unsaturation is 13. MS 2 In the spectrum, it can be observed that ABS-D has two consecutive removals of 162Da (C 6 H 10 O 5 ) to produce [M+HC 6 H 10 O 5 ] + and 336.0186[M+H-2C 6 H 10 O 5 ] + Characteristic ion fragments ( Figure 5 D), suggesting that ABS-D is a di-O-hexoside.

[0089] 1 H NMR (600 MHz, DMSO-d 6 ) shows two characteristic sugar terminal protons [δ H 4.80 (1H, d, J = 7.8 Hz) and 4.75 (1H, d, J = 7.8 Hz)] and in δ H 2.90~3.70 shows 10 protons on sugars, and at the same time 13 C NMR (150 MHz, DMSO-d 6 ) shows two characteristic sugar terminal carbon signals (δ C 105.4,100.5), and chemical shift δ C There are 10 oxygen-linked carbon signals on sugars between 60 and 80, which further confirms the presence of double O-hexose groups. The sugar proton signals and carbon signals were compared with the glucose data reported in the literature, and combined with the two-dimensional NMR data to confirm that the two sugar groups are β-D-pyranose glucose (Abdallah et al., 2016; Mohamed & Ibrahim, 2020; Shi et al., 2013). In addition to removing the sugar proton signals, 1 The H NMR spectrum combined with the HSQC spectrum showed four active hydrogen proton signals in the high field region ([δ H 9.79 (1H, s), 9.77 (1H, brs), 9.71 (1H, brs) and 9.38 (1H, s)], and three aromatic proton signals were also shown in the low-field region ([δ H7.25 (1H, s), 6.12 (1H, br s) and 6.05 (1H, br s)]. The aromatic proton signal is not proportional to the sugar proton signal. It is speculated that the aromatic proton is more active and easily deuterated. This phenomenon is particularly common in polyhydroxybenzophenone glycosides (Beelders et al., 2014; Pandey et al., 2013; Sun et al., 2014). 13 In addition to the two glucose carbon signals, the C NMR spectrum also gives 14 carbon signals, including a carbonyl carbon signal (δ C 187.6), 13 aromatic carbon signals. The HMBC spectrum consists of aromatic protons δ H 6.05(1H,brs) and δ C 160.5,157.3,108.0,94.0 correlation,δ H 6.12(1H,brs) and δ C 160.5,157.1,108.0,96.5 related; active hydrogen protons δ H 9.79(1H,s) and δ C 160.5,96.5,94.0 correlation,δ H 9.71(1H,s) and δ C 157.3,108.0,96.5 related; sugar end proton δ H 4.75 (1H, d, J = 7.8 Hz) and δ C 157.1, thus determining the structure of the A ring ( Fig.12 ). At the same time, according to the aromatic proton δ H 7.25(1H,s) and δ C HMBC correlations of 147.9, 146.1, 139.7, 122.7, and active hydrogen protons δ H 9.77(1H,s) and δ C 147.9,139.7,106.1 related, δ H 9.38(1H,s) and δ C 147.9,139.7,137.7 related; sugar terminal proton δ H 4.80 (1H, d, J = 7.8 Hz) and δ C 137.7 correlation to further determine the structure and chemical shift assignment of the C ring. H 6.12 (1H, brs) and δ H 6.05(1H,brs) and δ CThe weak HMBC correlation at 187.6 confirmed the position of the carbonyl group. Based on the molecular formula and unsaturation given by HRESIMS, combined with the NMR data of benzo[d]thiazol-2-yl(phenyl)methanone compounds reported in the literature (Spadaro et al., 2012), ABS-D was determined to be 2,4-dihydroxy-6-[(O-β-D-glucosyl)]phenyl(5,6-dihydroxy-7-[(O-β-D-glucosyl)]benzo[d]thiazol-2-yl)methanone( Figure 5 A).

[0090] Because the A ring and C ring in the structure are connected by multiple quaternary carbons and heteroatoms, in order to further confirm the structure of ABS-D, the density functional method was used to optimize the geometric structure of ABS-D at the B3-LYP / 6-31G(d) level, and its corresponding 13 C NMR chemical shift. 13 The correlation analysis between the C NMR theoretical value and the experimental value showed that there was a good linear relationship between the two (R 2 =0.995, Fig.13 ), indicating that the calculated data are in good agreement with the experimental data, which supports the correctness of the structure from a theoretical perspective.

[0091] Example 8

[0092] ABS-D Antioxidant Activity Assay

[0093] 1. DPPH free radical scavenging activity

[0094] DPPH free radical scavenging activity was determined by referring to the method of Kano et al. (2005) with slight modifications.

[0095] Add 50 μL of ABS-D solution of different concentrations (25, 50, 100, 150, 200 μg / mL) to 950 μL DPPH (50 μg / mL) solution, vortex mix, stand at room temperature in the dark for 30 minutes, and measure the absorbance value at 517 nm using a UV spectrophotometer. Using VC as a positive control, the DPPH free radical scavenging rate is calculated as follows:

[0096] DPPH free radical scavenging rate (%) = [1-(A s -A c ) / A b ]×100

[0097] A s Represents the absorbance of the mixture of the test solution and DPPH solution, A c Represents the absorbance of the mixture of the test solution and anhydrous ethanol solution, Ab Represents the absorbance of the mixture of DPPH solution and sample solvent. Each group of experiments was repeated three times.

[0098] 2. ABTS free radical scavenging activity

[0099] The ABTS free radical scavenging activity was determined by referring to the method of Re et al. (1999) with some modifications.

[0100] Weigh 200 mg of ABTS and 68.8 mg of potassium persulfate, dilute to 100 mL with water, and place at room temperature in the dark for 24 h as the ABTS mother solution. Then dilute the ABTS mother solution with ethanol until the absorbance is 0.7 ± 0.02 (OD 734 ) as ABTS working solution. Add 75 μL of ABS-D solution of different concentrations (12.5, 25, 50, 100, 150, 200 μg / mL) to 925 μL ABTS working solution, vortex mix, place at room temperature away from light for 6 minutes, and measure its absorbance value at 734 nm using a UV spectrophotometer. VC is used as a positive control. The calculation formula is as follows:

[0101] ABTS free radical scavenging rate (%) = [(A b -A s ) / A b ]×100

[0102] A b Represents the absorbance of the mixture of ABTS working solution and sample solvent, A s Represents the absorbance of the mixture of the sample solution to be tested and the ABTS working solution. Each group of experiments was repeated three times.

[0103] The antioxidant capacity of ABS-D was evaluated by measuring the DPPH and ABTS free radical scavenging activities. Fig.14 As shown in the figure, with the increase of concentration, the scavenging rate of DPPH and ABTS free radicals of ABS-D gradually increased. At 200 μg / mL, the scavenging rates of DPPH and ABTS free radicals were 27.45±0.15% and 98.48±0.43%, respectively, indicating that ABS-D has a certain scavenging ability for both DPPH and ABTS free radicals.

[0104] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A citrus fruit white skin "blue change" substance derivative ASB-D, the chemical structure of which is as follows:

2. The method for preparing ABS-D according to claim 1, characterized in that: The following steps are involved: S1: preparing dry powder of the "blue-turned" white cortex of the fruit, extracting the dry powder with a hydrochloric acid ethanol solution to obtain an extract, adjusting the pH of the extract to 6.5-7.5, standing and filtering, and obtaining an ABS crude extract; S2: dissolving the ABS crude extract with formic acid, injecting into an HLB column, eluting impurities with water and 30-40% methanol in sequence, and eluting ABS with 0.04-0.2% hydrochloric acid methanol to obtain sample solution 1; S3: injecting the sample solution 1 into the MCX column, eluting impurities with 1% hydrochloric acid and methanol, and eluting ABS with 1-10% ammonia methanol to obtain sample solution 2; S4: subjecting the sample solution 2 to Sephadex LH-20 column chromatography, using 25-35% methanol as the eluent, collecting the blue band fractions, and concentrating; S5: purifying the concentrated solution by semi-preparative liquid chromatography, collecting the effluent of the ABS absorption peak at 570 nm, and drying to obtain pure ABS; S6: dissolving the pure ABS in a hydrochloric acid solution, heating in a water bath, purifying the solution using a semi-preparative liquid chromatograph, collecting the effluent of the ABS-D absorption peak at 410 nm, concentrating and drying to obtain pure ABS-D.

3. The method according to claim 2, characterized in that: The preparation method of the "blue-changed" white skin layer dry powder of the fruit is as follows: a fresh sample of the "blue-changed" white skin layer is frozen with liquid nitrogen, and then ground into powder with a pulverizer.

4. The method according to claim 2, characterized in that: The volume concentration of the hydrochloric acid ethanol solution is 0.5-2%, and the solid-liquid ratio of the extraction is 1:5-25.

5. The method according to claim 2, characterized in that: The pH of the extract was adjusted with 5% volume concentration of NaHCO3 or ammonia water.

6. The method according to claim 2, characterized in that: In step S4, the flow rate is 0.4-0.6 mL / min, and one fraction is collected every 10 min.

7. The method according to claim 2, characterized in that: In step S5 or S6, the chromatographic conditions of the semi-preparative liquid chromatograph are: C18 column, mobile phase A is acetonitrile, phase B is water, and the gradient elution conditions are as follows: 0-14 min, 10%-22.5% A; 14-17 min, 22.5%-100% A; 17-18 min, 100%-10% A; 18-23 min, 10% A; flow rate 4 mL / min; detection wavelengths are 570 and 410 nm, respectively.

8. The method according to claim 2, characterized in that: In step S6, the temperature of the water bath is below 45° C., and the water bath time is 30-120 min.

9. The method according to claim 2, characterized in that: The concentration of the hydrochloric acid solution is 1-5%.

10. Use of the ABS-D according to claim 1 in the preparation of antioxidant products.

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