A method for preparing black goji berry seed extract with antioxidant activity and identifying its main active ingredients

Abstract

This invention discloses a method for preparing an extract of black goji berry seeds with antioxidant activity and identifying its main active ingredients. The specific steps include: using dried black goji berry seed powder as raw material, and 70% acetone solution as the extraction solvent, under a material-to-liquid ratio of 1:10, ultrasonic-assisted extraction is performed three times at a power of 100 W for 30 min. The extracts are combined, centrifuged at 8000 rpm for 10 min, and the supernatant is evaporated to remove the solvent. The extract is then freeze-dried to obtain the black goji berry seed extract. The antioxidant activity of the black goji berry seed extract was tested using 2,2-diphenyl-1-trinitrohydrazine (DPPH). The antioxidant components in the extract were further identified using offline DPPH-UPLC, and the antioxidant active ingredients in the extract were identified using ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS / MS). This method is simple to operate and has a high extraction rate, providing a new approach for the development and utilization of antioxidant active ingredients in black goji berries and for the comprehensive utilization of black goji berries.

Description

Technical Field

[0001] This invention relates to a method for preparing an extract with antioxidant activity from black goji berry seeds and for identifying the main active ingredients therein, belonging to the technical field of extraction and identification of active ingredients of natural products of traditional Chinese medicine. Background Technology

[0002] Black goji berry (Lycium ruthenicum Murr.) is a plant belonging to the genus Lycium in the Solanaceae family, mainly distributed in Xinjiang, Ningxia, Tibet, Gansu, and Qinghai. Its fruit is sweet and juicy, and is often used as a traditional medicine and nutritional food. Studies have shown that black goji berries are rich in various bioactive components, possessing antioxidant, anti-fatigue, anti-radiation, and hypoglycemic effects. (Zhang Gong, Chen Shasha, Zhou Wu, et al. Research progress on the efficacy of black goji berries [J]. West China Pharmaceutical Journal, 2019, 34(06):638-642.). However, during the production and processing of black goji berry products, the byproduct, black goji berry seeds, is not effectively utilized, resulting in significant resource waste and environmental pollution. Identifying the chemical components in black goji berry seeds can provide valuable research data for the rational development and utilization of black goji berry resources.

[0003] Free radicals in the body are in a dynamic balance between continuous generation and elimination. However, when too many free radicals are generated or eliminated too slowly, a series of oxidative stress reactions will occur, which can induce various diseases such as neurodegenerative diseases, cardiovascular diseases, diabetes and cancer (Cai Xiaohua, Li Xiaowei, Li Guokun, et al. Protective effect of Moringa leaf water extract on H2O2-induced oxidative damage to HepG2 cells [J]. Modern Food Science and Technology, 2021, 37(10):62-69+111.). There are reports on the preparation method of black goji berry extract and its antioxidant activity. Chinese patent (CN113841894A) discloses the preparation method of black goji berry anthocyanin extract and freeze-dried powder. The extract has strong antioxidant and anti-aging health care effects. Chinese patent (CN102879500A) discloses a method for detecting antioxidant components in black goji berry juice using DPPH-HPLC. This method is simple and convenient to operate and can realize the evaluation of the effectiveness of antioxidant components in black goji berry juice. Chinese patent (CN110079383A) discloses a method for extracting oil from black goji berries and its preparation. The method involves extracting the oil from black goji berry seeds using ultrasound-assisted organic reagents, achieving an extraction rate of 88.79% and an acid value of 0.74 mg / g. However, there are few reports on the preparation and application of antioxidants from black goji berry seeds. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing antioxidant active extracts from black goji berry seeds and for rapid screening of active ingredients.

[0005] The technical solution adopted in this invention is as follows:

[0006] A method for preparing an extract of black goji berry seeds with antioxidant activity and identifying its main active ingredients, the method comprising the following steps:

[0007] 1) Dry the black goji berry seeds, grind them, and sieve them to obtain black goji berry seed powder;

[0008] 2) The dried black goji berry seed powder obtained in step 1) is used as raw material and mixed with the extraction solvent at a material-to-liquid ratio of 1g:5-40mL. Then, it is ultrasonically extracted for 10-50min. The extraction is repeated 3 times. The supernatants are combined, centrifuged, and the solvent is removed by rotary evaporation. Finally, the supernatant is freeze-dried in a freeze dryer to obtain black goji berry seed extract.

[0009] 3) The black goji berry seed extract obtained in step 2) was subjected to DPPH antioxidant activity test;

[0010] 4) The black goji berry seed extract obtained in step 2) was analyzed by UPLC to obtain the UPLC chromatogram; the antioxidant active components in the black goji berry seed extract obtained in step 2) were analyzed by offline DPPH-UPLC technology to determine the target peak with antioxidant activity.

[0011] 5) The antioxidant active ingredients in the extract were further identified and their molecular structures were determined using UPLC-QTOF-MS / MS.

[0012] Further, in step 2), the extraction solvent is a mixture of acetone and water, with the volume fraction of acetone in the mixture being 70%; the ratio of black goji berry seed powder to extraction solvent is 1g:10mL, the ultrasonic extraction temperature is 30℃, and the ultrasonic extraction time is 30min.

[0013] Further, in step 3), the DPPH scavenging effect of black goji berry seed extract on DPPH free radicals was tested. When the extract was mixed with 0.2 mM DPPH free radical solution at a volume ratio of 1:1 in the solution concentration range of 31.25-1000.00 μg / mL, the ability of black goji berry seed extract to scavenge DPPH free radicals increased with increasing concentration, and the scavenging rate increased from 32.85% to 92.38%.

[0014] Further, the ultra-high performance liquid chromatography (UHPLC) analysis conditions for the black goji berry seed extract described in step 4) are as follows: mobile phase A is 0.3% formic acid aqueous solution, mobile phase B is acetonitrile, and the mobile phase flow rate is 0.3 mL / min; gradient elution is used, specifically: 0–18 min, 95%–56% A; 18–20 min, 56%–10% A; 20–30 min, 10%–95% A; column temperature: 35℃; detection wavelength: 280 nm; injection volume: 3 μL; injection concentration: 12 mg / mL.

[0015] Further, in step 4), the offline DPPH-UPLC coupled technique is used with a sample injection concentration of 1 mg / mL. The testing process is as follows: the black goji berry seed extract obtained in step 2) is dissolved in a 70% methanol aqueous solution and divided into two equal parts. One part of the sample solution is mixed with a DPPH free radical solution and then detected by UPLC to obtain the chromatogram of the sample group. The other part of the sample solution is mixed with a 70% methanol aqueous solution and then detected by UPLC to obtain the chromatogram of the control group. The chromatograms of the sample group and the control group are compared. The compounds represented by the peaks with reduced or disappeared peak areas in the sample group chromatogram are the compounds with antioxidant activity.

[0016] Further, the UPLC-QTOF-MS / MS mass spectrometry conditions described in step 5) are as follows: ion source: ESI; mass spectrometry method: capillary voltage: 4500V; nebulizer gas (N2) pressure: 0.3bar; dry gas N2 flow rate: 4.0L / min; dry gas temperature: 220℃; data acquisition mode: positive and negative ion mode; molecular weight range: m / z 50~1500.

[0017] Furthermore, step 5) identified three main antioxidant active ingredients, namely N 1 -caffeoyl,N 10 -dihydrocaffeoyl spermidine; N 1 -dihydrocaffeoyl,N 10 -caffeoyl spermidine and N,N-dicaffeoyl-spermidine, denoted as compound 1, compound 2, and compound 3 respectively, have the following structural formulas:

[0018]

[0019] The present invention has the following advantages:

[0020] 1) Black goji berry seeds, as a plant by-product of food processing, contain a large number of bioactive substances. Extracting antioxidant active ingredients from black goji berry seeds avoids the waste of black goji berry seed resources and provides a scientific basis for further development and utilization of black goji berry resources.

[0021] 2) This invention provides a method for preparing black goji berry seed extract with antioxidant activity. The method is simple to operate and has a high extraction rate.

[0022] 3) This invention combines an offline DPPH-UPLC technique with UPLC-QTOF-MS / MS technique for the identification of antioxidant active ingredients in black goji berry seeds. This method is rapid, intuitive, simple and easy to implement, and has good reproducibility. Attached Figure Description

[0023] Figure 1 This is a sample image of black goji berry seeds from the present invention.

[0024] Figure 2 This is a comparison chart of the peak areas of three main compounds in the black goji berry seed extract of this invention under different extraction conditions.

[0025] Figure 3 This is a comparison of the scavenging abilities of black goji berry seed extract and vitamin C control agent against DPPH free radicals according to the present invention.

[0026] Figure 4 This is an offline DPPH-UPLC screening diagram of the black goji berry seed extract of the present invention.

[0027] Figure 5 This is a mass spectrum (negative ion mode) of three antioxidant active components in the black goji berry seed extract of this invention. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0029] 1. The specifications of the instruments and medicines involved in this invention are as follows:

[0030] XS205 DualRange analytical balance (Mettler Toledo, Switzerland); KH5200DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Waters Acquity UPLC ultra-high performance liquid chromatograph (Waters, USA); quadrupole time-of-flight mass spectrometer (Bruker Daltonics, Germany); Barnstead TII ultrapure water system (ThermoScientific, USA); R-215 rotary evaporator (Buchi, Switzerland).

[0031] Chromatographic grade acetonitrile and methanol were purchased from Merck KGaA GmbH & Co. KG, Germany; 2,2-diphenyl-1-trinitrohydrazine (DPPH) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; all other reagents were analytical grade.

[0032] The black goji berries used in the experiment were purchased from Beijing Tongrentang Health Pharmaceutical Co., Ltd.

[0033] 2. In the following examples, the antioxidant activity of the black goji berry extract was determined using ultra-high performance liquid chromatography (UHPLC). The peak areas of the three main compounds were used as a reference to compare the extraction efficiency of black goji berry seeds under different material-to-liquid ratios, different extraction reagents, and different extraction times.

[0034] The formula for calculating the yield of black goji berry seed extract is as follows:

[0035] Extract yield (%) = (Mass of extract / Mass of black goji berry seed powder) * 100%.

[0036] Example 1:

[0037] Accurately weigh 0.3g of black goji berry seed powder into a 50mL centrifuge tube. Use 70% acetone-water solution as the extraction solvent. The ratio of black goji berry seed powder to extraction solvent is 1:5 (unit: g / mL). Extract by ultrasonication at 30℃ for 30min with an ultrasonic power of 100W. Repeat the extraction three times. Combine the supernatants and centrifuge at 8000rpm for 10min. Remove the solvent by rotary evaporation at 40℃. Finally, freeze-dry the supernatant to obtain black goji berry seed extract.

[0038] Weigh 24 mg of black wolfberry seed extract, redissolve it in 2 mL of 50% methanol aqueous solution, filter through a 0.22 μm PTFE membrane, and prepare a 12 mg / mL sample solution for later use. The filtered extract was analyzed using UPLC (ultra-high performance liquid chromatography), with three main peaks analyzed in triplicate. The average peak area of ​​compound 1 was 776,872, the average peak area of ​​compound 2 was 2,779,701, and the average peak area of ​​compound 3 was 2,292,607.

[0039] The ultra-high performance liquid chromatography (UHPLC) analysis conditions were as follows: mobile phase A was 0.3% formic acid aqueous solution, mobile phase B was acetonitrile, and the mobile phase flow rate was 0.3 mL / min; gradient elution was used, specifically: 0–18 min, 95%–56% A; 18–20 min, 56%–10% A; 20–30 min, 10%–95% A; column temperature: 35℃; detection wavelength: 280 nm; injection volume: 3 μL; injection concentration: 12 mg / mL.

[0040] The antioxidant active ingredients in the extract were identified using UPLC-QTOF-MS / MS. The structure testing procedure for the compounds corresponding to the three main peaks was as follows: ion source: ESI; mass spectrometry method: capillary voltage: 4500V; nebulizer gas (N2) pressure: 0.3bar; dry gas N2 flow rate: 4.0L / min; dry gas temperature: 220℃; data acquisition mode: positive and negative ion mode; molecular weight range: m / z 50~1500.

[0041] Three main antioxidant active ingredients were identified, namely N 1 -caffeoyl,N 10 -dihydrocaffeoylspermidine;N 1 -dihydrocaffeoyl,N 10 -caffeoyl spermidine and N,N-dicaffeoyl-spermidine, which will be referred to as compound 1, compound 2 and compound 3, respectively:

[0042] The structural formulas of the chemical components are as follows:

[0043]

[0044] Example 2:

[0045] The material-to-liquid ratio was changed to 1:20, while other conditions and operating procedures remained the same as in Example 1. Analysis of the extracts showed that the average peak area of ​​compound 1 was 812,900, the average peak area of ​​compound 2 was 2,914,076, and the average peak area of ​​compound 3 was 2,593,052.

[0046] Example 3:

[0047] The material-to-liquid ratio was changed to 1:30, while other conditions and operating procedures remained the same as in Example 1. Analysis of the extracts showed that the average peak area of ​​compound 1 was 828,604, the average peak area of ​​compound 2 was 2,949,719, and the average peak area of ​​compound 3 was 2,653,178.

[0048] Example 4:

[0049] The material-to-liquid ratio was changed to 1:40, while other conditions and operating procedures remained the same as in Example 1. Analysis of the extracts showed that the average peak area of ​​compound 1 was 555311, the average peak area of ​​compound 2 was 1979071, and the average peak area of ​​compound 3 was 1765027.

[0050] Example 5:

[0051] The material-to-liquid ratio was changed to 1:50, while other conditions and operating procedures remained the same as in Example 1. Analysis of the extracts showed that the average peak area of ​​compound 1 was 546,498, the average peak area of ​​compound 2 was 1,960,980, and the average peak area of ​​compound 3 was 1,865,906.

[0052] Example 6:

[0053] The material-to-liquid ratio was changed to 1:10, while other conditions and operating procedures remained the same as in Example 1. Analysis of the extracts showed that the average peak area of ​​compound 1 was 1,223,769, the average peak area of ​​compound 2 was 424,0091, and the average peak area of ​​compound 3 was 3,535,136.

[0054] Comparing Examples 1-6, it can be seen that when the material-to-liquid ratio is 1:10, the average peak area of ​​the three main compounds in the black goji berry seed extract is the highest.

[0055] Example 7:

[0056] The material-to-liquid ratio was 1:10, and the extraction solvent was 50% acetone-water solution (v / v). Other conditions and operating procedures were the same as in Example 1. Analysis of the extracts showed that the average peak area of ​​compound 1 was 460,809, the average peak area of ​​compound 2 was 1720,116, and the average peak area of ​​compound 3 was 1698,208.

[0057] Example 8:

[0058] The material-to-liquid ratio was 1:10, and the extraction solvent was 60% acetone-water solution (v / v). Other conditions and operating procedures were the same as in Example 1. Analysis of the extracts showed that the average peak area of ​​compound 1 was 971,331, the average peak area of ​​compound 2 was 3,518,585, and the average peak area of ​​compound 3 was 2,787,300.

[0059] Example 9:

[0060] The material-to-liquid ratio was 1:10, and the extraction solvent was 80% acetone-water solution (v / v). Other conditions and operating procedures were the same as in Example 1. Analysis of the extracts showed that the average peak area of ​​compound 1 was 940,162, the average peak area of ​​compound 2 was 3,340,498, and the average peak area of ​​compound 3 was 2,538,689.

[0061] Example 10:

[0062] The material-to-liquid ratio was 1:10, and the extraction solvent was 90% acetone-water solution (v / v). Other conditions and operating procedures were the same as in Example 1. Analysis of the extracts showed that the average peak area of ​​compound 1 was 760,109, the average peak area of ​​compound 2 was 2,746,813, and the average peak area of ​​compound 3 was 2,536,098.

[0063] Comparative examples 6-10 show that when the extraction solvent is a 70% (v / v) acetone aqueous solution, the average peak area of ​​the three main compounds in the black goji berry seed extract is the highest.

[0064] Example 11:

[0065] The material-to-liquid ratio was 1:10, the extraction solvent was 70% acetone-water solution (v / v), and the ultrasonic extraction time was 10 min. Other conditions and operating procedures were the same as in Example 1. Analysis of the extracts showed that the average peak area of ​​compound 1 was 670,426, the average peak area of ​​compound 2 was 2,411,931, and the average peak area of ​​compound 3 was 2,112,662.

[0066] Example 12:

[0067] The material-to-liquid ratio was 1:10, the extraction solvent was 70% acetone-water solution (v / v), and the ultrasonic extraction time was 20 min. Other conditions and operating procedures were the same as in Example 1. Analysis of the extracts showed that the average peak area of ​​compound 1 was 543,532, the average peak area of ​​compound 2 was 1,955,834, and the average peak area of ​​compound 3 was 1,690,357.

[0068] Example 13:

[0069] The material-to-liquid ratio was 1:10, the extraction solvent was 70% acetone-water solution (v / v), and the ultrasonic extraction time was 40 min. Other conditions and operating procedures were the same as in Example 1. Analysis of the extracts showed that the average peak area of ​​compound 1 was 554,934, the average peak area of ​​compound 2 was 1,973,948, and the average peak area of ​​compound 3 was 1,701,402.

[0070] Example 14:

[0071] The material-to-liquid ratio was 1:10, the extraction solvent was 70% acetone-water solution (v / v), and the ultrasonic extraction time was 50 min. Other conditions and operating procedures were the same as in Example 1. Analysis of the extracts showed that the average peak area of ​​compound 1 was 485,695, the average peak area of ​​compound 2 was 1,775,531, and the average peak area of ​​compound 3 was 1,491,404.

[0072] Comparing Examples 6 and 11-14, it can be seen that when the ultrasonic extraction time is 30 min, the average peak area of ​​the three main compounds in the black goji berry seed extract is the highest.

[0073] Example 15:

[0074] The material-to-liquid ratio was 1:10, the extraction solvent was 70% methanol-water solution (v / v), and the ultrasonic extraction time was 30 min. Other conditions and operating procedures were the same as in Example 1. Analysis of the extracts showed that the peak area of ​​compound 1 was 940439, the peak area of ​​compound 2 was 3201412, and the peak area of ​​compound 3 was 2783035.

[0075] Example 16:

[0076] The material-to-liquid ratio was 1:10, the extraction solvent was 70% ethanol-water solution (v / v), and the ultrasonic extraction time was 30 min. Other conditions and operating procedures were the same as in Example 1. Analysis of the extracts showed that the peak area of ​​compound 1 was 948,323, the peak area of ​​compound 2 was 3,027,802, and the peak area of ​​compound 3 was 2,798,661.

[0077] Example 17:

[0078] The material-to-liquid ratio was 1:10, the extraction solvent was 70% n-butanol-water solution, and the ultrasonic extraction time was 30 min. Other conditions and operating procedures were the same as in Example 1. Analysis of the extracts showed that the peak area of ​​compound 1 was 122108, the peak area of ​​compound 2 was 417509, and the peak area of ​​compound 3 was 536339.

[0079] Comparing Examples 6 and 15-17, it can be seen that when the extraction solvent is 70% acetone-water solution, the peak areas of the three main compounds of black goji berry seed extract are the highest, and the yield of black goji berry seed extract in Example 6 is 7.48%.

[0080] Example 18:

[0081] Accurately weigh 0.3 g of black goji berry peel powder into a 50 mL centrifuge tube. Other conditions and operating procedures are the same as in Example 6. Analysis of the extract showed that the average peak area of ​​compound 1 was 688085, the average peak area of ​​compound 2 was 1603462, and the average peak area of ​​compound 3 was 2470285. The yield of the black goji berry peel extract was 4.39%.

[0082] Comparing Examples 6 and 18, it can be seen that the peak areas and extraction yields of the three main compounds in the black goji berry seed extract are higher than those in the black goji berry peel extract.

[0083] Example 19: Determination of antioxidant activity of black goji berry seed extract by DPPH free radical scavenging experiment

[0084] The black wolfberry seed extract obtained in Example 6 was used as a solvent to prepare a series of sample solutions with extract concentrations of 31.25 μg / mL, 62.50 μg / mL, 125.00 μg / mL, 250.00 μg / mL, 500.00 μg / mL, and 1.000.00 μg / mL. DPPH powder was accurately weighed, dissolved in 75% ethanol solution, and diluted to a final volume in a 50 mL brown volumetric flask to prepare a 0.2 mM DPPH solution.

[0085] When determining the DPPH free radical scavenging effect of black goji berry seed extract, the following four sets of experiments were conducted in parallel:

[0086] Sample group: Use a pipette to pipette 100 μL of the above-prepared sample solution into a 96-well plate, then add 100 μL of 0.2 mM DPPH free radical ethanol solution to the wells, and let it stand at room temperature in the dark for 30 minutes. Then use an ELISA reader to measure the absorbance value of each well at 517 nm.

[0087] Positive control group: Using vitamin C as the control agent and 75% ethanol-water as the solvent, a series of positive control solutions with different vitamin C concentrations were prepared. 100 μL of the prepared positive control solution was pipetted into a 96-well plate, followed by the addition of 100 μL of 0.2 mM DPPH free radical ethanol solution. The plate was then incubated at room temperature in the dark for 30 minutes. The absorbance of each well was then measured at 517 nm using a microplate reader.

[0088] Blank group: Pipette 100 μL of 75% ethanol aqueous solution into a 96-well plate, then add 100 μL of 0.2 mM DPPH free radical ethanol solution to the wells, and let stand at room temperature in the dark for 30 minutes. Then use a microplate reader to measure the absorbance of each well at 517 nm.

[0089] Control group: Use a pipette to draw 100 μL of the above-prepared sample solution into a 96-well plate, then add 100 μL of 75% ethanol aqueous solution to the well, and let it stand at room temperature in the dark for 30 minutes. Then use an ELISA reader to measure the absorbance of each well at 517 nm.

[0090] In the four parallel experiments, A1 represents the absorbance measured in the sample group or positive control group; A2 represents the absorbance measured in the control group; and A0 represents the absorbance measured in the blank group. The formula for calculating the inhibitory activity is as follows:

[0091] DPPH free radical scavenging rate (%) = [1 - (A1 - A2) / A0] * 100%.

[0092] Following the above experimental method, black goji berry seed extract ( Figure 3(seed group) and vitamin C control drug ( Figure 3 The comparison chart of DPPH free radical scavenging rate results for the group with vitamin C is shown below. Figure 3 .

[0093] Example 20: Screening method for antioxidant active ingredients in black goji berry seed extract:

[0094] The black goji berry seed extract obtained in Example 6 was used to prepare a 1.0 mg / mL sample solution using 70% (v / v) methanol aqueous solution as the solvent. 0.4 g of DPPH was accurately weighed into a 100 mL brown volumetric flask and diluted with 70% (v / v) methanol aqueous solution to prepare a 4 mg / mL DPPH stock solution. This stock solution was then further diluted with 70% (v / v) methanol aqueous solution to prepare a 600 μg / mL DPPH working solution.

[0095] Sample group: 1.0 mL of sample solution was mixed with 1.0 mL of DPPH working solution (600 μg / mL) at a 1:1 volume ratio and reacted at room temperature in the dark for 30 min, followed by filtration through a 0.22 μm PTFE membrane. The sample group was analyzed using a Waters ACQUITY UPLC BEH C18 (2.1 × 100 mm, 1.7 μm) column. The HPLC conditions were as follows: mobile phase: 0.3 wt% formic acid aqueous solution (A) - acetonitrile (B); gradient elution mode (0–18 min, 95%–56% A; 18–20 min, 56%–10% A; 20–30 min, 10%–95% A); flow rate: 0.3 mL / min; column temperature: 35 °C; detection wavelength: 280 nm; injection volume: 10 μL.

[0096] Control group: 1.0 mL of sample solution was mixed with 1.0 mL of 70% methanol aqueous solution at a volume ratio of 1:1, and reacted at room temperature in the dark for 30 min, followed by filtration through a 0.22 μm PTFE membrane. The control group was analyzed under the same liquid chromatography conditions as above.

[0097] By comparing the chromatogram of the sample with that of the control, the compounds represented by the peaks whose peak areas decrease or disappear are those with antioxidant activity. Figure 4 The results showed that compounds 1-3 were the main antioxidant active ingredients in black goji berry seeds.

[0098] Example 21: Identification of antioxidant active components in black goji berry seed extract:

[0099] The black goji berry seed extract obtained in Example 6 was prepared to a concentration of 20.0 mg / mL using a 50% (v / v) methanol aqueous solution. Three antioxidant active components in the black goji berry seed extract were identified using UPLC-QTOF-MS / MS technology.

[0100] The ultra-high performance liquid chromatography (UHPLC) conditions were as follows: mobile phase: 0.3 wt% formic acid aqueous solution (A)-acetonitrile (B); gradient elution mode (0–18 min, 95%–56% A; 18–20 min, 56%–10% A; 20–30 min, 10%–95% A); flow rate: 0.3 mL / min; column temperature: 35 ℃; detection wavelength: 280 nm; injection volume: 3 μL.

[0101] The high-resolution mass spectrometry conditions were as follows: ion source: ESI; mass spectrometry method: capillary voltage: 4500V; nebulizer gas (N2) pressure: 0.3 bar; drying gas (N2) flow rate: 4.0 L / min; drying gas temperature: 220℃; data acquisition mode: positive and negative ion mode; molecular weight range: m / z 50~1500; the mass spectrometer was calibrated using a calibration standard (sodium formate solution).

[0102] Compound 1: Molecular formula is C 25 H 33 N3O6. ESI-MS (negative ion mode) m / z: 470.2341, secondary fragments: 135.0469, 334.1805, 470.2338; ESI-MS (positive ion mode) m / z: 472.2265, secondary fragments: 220.0889, 310.2009, 472.2268. Based on molecular weight, molecular formula, secondary fragments, and retention time, compound 1 was identified as N3O6. 1 -caffeoyl,N 10 -dihydrocaffeoyl spermidine.

[0103] Compound 2: Molecular formula is C 25 H 33 N3O6. ESI-MS (negative ion mode) m / z: 470.2329, secondary fragments: 135.0465, 334.1798, 470.2329; ESI-MS (positive ion mode) m / z: 472.2265, secondary fragments: 222.1046, 310.2013, 472.2271. Based on molecular weight, molecular formula, secondary fragments, and retention time, compound 2 was identified as N3O6. 1 -dihydrocaffeoyl,N 10-caffeoyl spermidine.

[0104] Compound 3: Molecular formula is C 25 H 31 N3O6. ESI-MS (negative ion mode) m / z: 468.2168, secondary fragments: 135.0466, 161.0625, 306.1852, 332.1640, 468.2173; ESI-MS (positive ion mode) m / z: 470.2101, secondary fragments: 220.0881, 308.1844, 470.2094. Based on molecular weight, molecular formula, secondary fragments, and retention time, compound 3 was identified as N,N-dicaffeoyl-spermidine.

[0105] The contents described in this specification are merely an enumeration of the implementation forms of the inventive concept, and the scope of protection of this invention should not be regarded as limited to the specific forms described in the embodiments.

Claims

1. A method for identifying the main active ingredients in a black goji berry seed extract with antioxidant activity, wherein, Extract preparation and active ingredient identification include the following steps: 1) Dry the black goji berry seeds, grind them, and sieve them to obtain black goji berry seed powder; 2) The dried black goji berry seed powder obtained in step 1) is used as raw material, mixed with the extraction solvent, and then extracted by ultrasonication. The extraction is repeated 3 times. The supernatants are combined, centrifuged, and the supernatant is evaporated by rotary evaporation to remove the solvent. Finally, it is placed in a freeze dryer to freeze dry and obtain black goji berry seed extract. 3) The black goji berry seed extract obtained in step 2) was subjected to 2,2-diphenyl-1-trinitrohydrazine DPPH antioxidant activity test. 4) The black goji berry seed extract obtained in step 2) was analyzed by ultra-high performance liquid chromatography (UPLC) to obtain the UPLC chromatogram; the antioxidant active components in the black goji berry seed extract obtained in step 2) were analyzed by offline DPPH-UPLC to determine the target peak with antioxidant activity. 5) The antioxidant active ingredients in the extract were further identified by ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS / MS) to determine the molecular structure corresponding to the target peak of antioxidant activity in step 4). Step 2) The extraction solvent is a mixture of acetone and water, with the volume fraction of acetone in the mixture being 70%; the ratio of black goji berry seed powder to extraction solvent is 1 g : 10 mL, the ultrasonic extraction temperature is 25-35℃, and the ultrasonic extraction time is 30 min. Step 5) Three main antioxidant active ingredients were identified, namely N 1 -caffeoyl,N 10 -dihydrocaffeoylspermidine;N 1 -dihydrocaffeoyl, N 10 -caffeoyl spermidine and N,N-dicaffeoyl-spermidine, which will be referred to as compound 1, compound 2 and compound 3, respectively: The structural formulas of the active ingredients are as follows: ； ； ； Step 4) The ultra-high performance liquid chromatography (UHPLC) analysis conditions for the black goji berry seed extract are as follows: The chromatographic column was a Waters ACQUITY UPLC BEH C18, 2.1 × 100 mm, 1.7 µm; Mobile phase A was 0.3% formic acid aqueous solution, and mobile phase B was acetonitrile. The mobile phase flow rate was 0.3 mL / min. Gradient elution was used, specifically: 0–18 min, 95%–56% A; 18–20 min, 56%–10% A; 20–30 min, 10%–95% A. Column temperature: 35℃; detection wavelength: 280 nm; injection volume: 3 μL; injection concentration: 12 mg / mL.

2. The method for identifying the main active ingredients of black goji berry seed extract with antioxidant activity as described in claim 1, characterized in that: Step 3) The antioxidant activity of black goji berry seed extract was tested using DPPH. When the extract was mixed with 0.2 mM DPPH free radical solution at a volume ratio of 1:1 in the solution concentration range of 31.25-1000.0 μg / mL, the ability of black goji berry seed extract to scavenge DPPH free radicals increased with increasing concentration, and the scavenging rate increased from 32.85% to 92.38%.

3. The method for identifying the main active ingredients of black goji berry seed extract with antioxidant activity as described in claim 1, characterized in that: Step 4) The offline DPPH-UPLC coupled method uses a sample injection concentration of 1 mg / mL. The testing process is as follows: The black goji berry seed extract obtained in step 2) is dissolved in a 70% methanol aqueous solution and divided into two equal parts. One part of the sample solution is mixed with a DPPH free radical solution and then detected by UPLC to obtain the chromatogram of the sample group. The other part of the sample solution is mixed with a 70% methanol aqueous solution and then detected by UPLC to obtain the chromatogram of the control group. The chromatograms of the sample group and the control group are compared. The compounds represented by the peaks with reduced or disappeared peak areas in the sample group chromatogram are the compounds with antioxidant activity.

4. The method for identifying the main active ingredients of black goji berry seed extract with antioxidant activity as described in claim 1, characterized in that: Step 5) The UPLC-QTOF-MS / MS mass spectrometry conditions are as follows: Ion source: ESI; Mass spectrometry method: Capillary voltage: 4500V; Nebulizer gas N2 pressure: 0.3 bar; Drying gas N2 flow rate: 4.0 L / min; Drying gas temperature: 220℃; Data acquisition mode: Positive and negative ion mode; Molecular weight range: m / z 50~1500.

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