Method for ultrasonic-assisted extraction of lycium ruthenicum anthocyanin

Abstract

The invention provides a method for ultrasonic-assisted extraction of lycium ruthenicum anthocyanin, which comprises the following steps: (1) taking fresh lycium ruthenicum, removing pedicles, and grinding into homogenate in a precooled mortar; and (2) adding the lycium ruthenicum homogenate obtained in the step (1) into 75% of an extracting solution B containing 0.1% of hydrochloric acid according to a material-liquid ratio of 1: (30-70), and extracting for 10-60 minutes in a constant-temperature ultrasonic extractor under the conditions that the ultrasonic temperature is 20-60 DEG C and the ultrasonic extraction power is 160W-320W to obtain an anthocyanin extracting solution. According to the method, four factors which influence anthocyanin extraction, namely a material-liquid ratio, an ultrasonic extraction temperature, extraction time and extraction power, are optimized by utilizing a response surface analysis method, so that optimal ultrasonic extraction parameters are obtained; based on the parameters, the ultrasonic-assisted method for extracting the anthocyanin from the lycium ruthenicum, which is provided by the invention, is simple to operate, short in extraction time, high in extraction efficiency and low in cost.

Description

Technical Field

[0001] The invention relates to the technical field of plant secondary metabolite extraction, and in particular to a method for ultrasonic-assisted extraction of black wolfberry anthocyanins. Background Art

[0002] Anthocyanin is a water-soluble flavonoid natural pigment that is widely distributed in plants. Anthocyanin is a natural antioxidant with strong antioxidant effects. It can remove free radicals, slow down the aging process, and also has anti-inflammatory functions. Black wolfberry is a good source of anthocyanin substances. The anthocyanin substances in black wolfberry are of many types and high content. It is a natural source of anthocyanin with good quality and low price. Therefore, you can try to extract anthocyanin from black wolfberry as a source of anthocyanin.

[0003] However, the extraction method of anthocyanins is currently mainly organic solvent extraction, which has the disadvantages of long extraction time, low extraction yield, poor extraction stability, etc. In response to this, the field has begun to adopt ultrasound-assisted extraction, which does not affect the structure and activity of the extract, is simple to operate, has few impurities in the extraction liquid, short extraction time, and high extraction efficiency. However, how to reasonably optimize the ultrasound-assisted extraction parameters is the current focus.

[0004] Response surface method is a mathematical method that expresses the function by constructing a polynomial with a clear expression, taking the response of the system as a function of one or more factors, using graphical technology to display this functional relationship, and obtaining the regression relationship between the response and the influencing factors through regression analysis, so as to obtain the optimal extraction process parameters. Compared with the orthogonal method, the results can be obtained with a small number of test groups, and the optimal conditions obtained are not the set values, but within the range of the set conditions.

[0005] Based on this, in order to extract anthocyanins from black wolfberry rationally and efficiently, it is urgent to develop a method for optimizing ultrasound-assisted extraction of anthocyanins from black wolfberry based on response surface methodology. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for ultrasonic-assisted extraction of anthocyanins from black wolfberry, which optimizes ultrasonic parameters by using the response surface analysis method of the Box-Behnken Designs (BBD) central composite design model, and finally obtains an extraction method with simple operation, short extraction time, high extraction efficiency and low cost.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] A method for ultrasonically assisted extraction of anthocyanins from black wolfberry, comprising the following steps:

[0009] (1) Remove the stems of fresh black wolfberries and grind them into a homogenous slurry in a pre-cooled mortar;

[0010] (2) adding 75% ethyl extract containing 0.1% hydrochloric acid to the black wolfberry homogenate obtained in step (1) at a solid-liquid ratio of 1: (30-70), extracting in a constant temperature ultrasonic extractor for 10-60 minutes at an ultrasonic temperature of 20-60° C. and an ultrasonic extraction power of 160 W-320 W to obtain an anthocyanin extract.

[0011] As one of the preferred embodiments of the present invention, the material-liquid ratio, ultrasonic temperature, ultrasonic extraction power and ultrasonic extraction time in step (2) are all optimized and determined by response surface methodology.

[0012] As one of the preferred embodiments of the present invention, the solid-liquid ratio is specifically 1:61.2, the ultrasonic temperature is 38.3°C, the ultrasonic extraction power is 240W, and the ultrasonic extraction time is 16.65min.

[0013] As one of the preferred embodiments of the present invention, the method further comprises the step of determining the anthocyanin content in the black wolfberry extract by a pH differential method, and / or the step of determining the anthocyanin component of the black wolfberry by a high performance liquid chromatography method.

[0014] As one of the preferred methods of the present invention, the specific steps of determining the anthocyanin content in the black wolfberry extract by the pH differential method are: taking the obtained anthocyanin extract and centrifuging it at 4000r / min for 10min; taking the supernatant after centrifugation, diluting it in pH1.0 and pH4.5 buffer solutions respectively, and determining the absorbance value using a visible spectrophotometer.

[0015] As one of the preferred methods of the present invention, the specific steps of determining the anthocyanin components of black wolfberry by high performance liquid chromatography are: taking the obtained anthocyanin extract and centrifuging it at 4000r / min for 10min; taking the supernatant after centrifugation, hydrolyzing it in a boiling water bath for 30min, taking it out and cooling it on ice, and making up the volume again with the extract; centrifuging it at 6000r / min for 10min, taking the supernatant, filtering it with a 0.22μm aqueous filter membrane, and determining its components by high performance liquid chromatography.

[0016] As one of the preferred embodiments of the present invention, a Thermo Fisher U3000 high performance liquid chromatograph is used, and the chromatographic conditions are as follows: (a) chromatographic column: Cs column, 250 mm×4.6 mm×5 m; (b) mobile phase A is an aqueous solution containing 1% formic acid, and mobile phase B is an acetonitrile solution containing 1% formic acid; (c) detection wavelength: 530 nm; (d) column temperature: 35° C.; (e) injection volume: 20 μL;

[0017] The elution program was as follows: at 0 min, flow rate of 0.8 mL / min, 92% mobile phase A, 8% mobile phase B; at 2 min, flow rate of 0.8 mL / min, 88% mobile phase A, 12% mobile phase B; at 5 min, flow rate of 0.8 mL / min, 80% mobile phase A, 20% mobile phase B; at 10 min, flow rate of 0.8 mL / min, 75% mobile phase A, 25% mobile phase B; at 15 min, flow rate of 0.8 mL / min Flow rate, 70% mobile phase A, 30% mobile phase B; at 18 min, flow rate of 0.8 mL / min, 55% mobile phase A, 45% mobile phase B; at 20 min, flow rate of 0.8 mL / min, 20% mobile phase A, 880% mobile phase B; at 22 min, flow rate of 0.8 mL / min, 92% mobile phase A, 8% mobile phase B; at 30 min, flow rate of 0.8 mL / min, 92% mobile phase A, 8% mobile phase B.

[0018] As one of the preferred embodiments of the present invention, the main components of the anthocyanins in black wolfberry are delphinium pigment, cyanidin pigment and geranium pigment measured by high performance liquid chromatography.

[0019] The advantages of the present invention compared to the prior art are:

[0020] (1) The present invention uses the response surface analysis method of the Box-Behnken Designs central composite design model to optimize the four factors affecting anthocyanin extraction, namely, the solid-liquid ratio, the ultrasonic extraction temperature, the extraction time, and the extraction power, to obtain the optimal ultrasonic extraction parameters; based on the above optimal ultrasonic extraction parameters, the present invention obtains a method for ultrasonic-assisted extraction of anthocyanins from black wolfberry with simple operation, short extraction time, high extraction efficiency, and low cost;

[0021] (2) The present invention uses a 75% ethanol reagent containing 0.1% hydrochloric acid, combined with ultrasound to assist in the extraction of anthocyanins from black wolfberry, wherein 75% ethanol has a high extraction rate for anthocyanins from black wolfberry, and can extract more anthocyanins;

[0022] (3) The present invention uses the response surface analysis method of the Box-Behnken Designs central composite design model to explore the optimal ultrasonic extraction parameters. Only three variable factors, three levels and a small number of experimental groups (only 17 groups of experiments) are needed to obtain the optimization results and the best yield. While improving the extraction efficiency, it further reduces energy consumption and pollutant emissions, which has practical significance for industrial production.

[0023] (4) The present invention uses high performance liquid chromatography (HPLC) to analyze anthocyanin components and determine the components contained therein, which facilitates subsequent separation optimization, saves time and improves efficiency;

[0024] (5) The present invention promotes food safety and has good economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the effect of extraction time on anthocyanin yield in Experimental Example 1;

[0026] Figure 2 This is the effect of extraction temperature change on anthocyanin yield in Experimental Example 1;

[0027] Figure 3 This is the effect of the change in the material-liquid ratio on the anthocyanin yield in Experimental Example 1;

[0028] Figure 4 This is the effect of the extraction power change on the anthocyanin yield in Experimental Example 1;

[0029] Figure 5 It is a three-dimensional response surface diagram of the effects of extraction time and material-liquid ratio on anthocyanin yield in Experimental Example 1;

[0030] Figure 6 It is a two-dimensional response surface diagram of the effects of extraction time and material-liquid ratio on anthocyanin yield in Experimental Example 1;

[0031] Figure 7 It is a three-dimensional response surface diagram of the effects of extraction time and extraction temperature on anthocyanin yield in Experimental Example 1;

[0032] Figure 8 It is a two-dimensional response surface diagram of the effects of extraction time and extraction temperature on anthocyanin yield in Experimental Example 1;

[0033] Fig. 9 It is a three-dimensional response surface diagram of the effects of solid-liquid ratio and extraction temperature on anthocyanin yield in Experimental Example 1;

[0034] Fig.10 It is a two-dimensional response surface diagram of the effect of solid-liquid ratio and extraction temperature on anthocyanin yield in Experimental Example 1;

[0035] Fig.11 This is the chromatogram of the mixed standard anthocyanin in Experimental Example 2 (in the figure, peaks 1 to 4 correspond to delphinium pigment, cyanidin, petunia pigment, geranium pigment, peony pigment, and malva pigment, respectively);

[0036] Fig.12 This is the chromatogram of anthocyanins from black wolfberry in Experimental Example 2 (in the figure, peaks 1, 2, and 4 correspond to delphinium pigment, cyanidin, and geranium pigment, respectively). DETAILED DESCRIPTION

[0037] The following is a detailed description of the embodiments of the present invention. The embodiments are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the experimental methods used in the following embodiments are conventional methods unless otherwise specified; the materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified.

[0038] Example 1

[0039] The method of ultrasonic-assisted extraction of anthocyanins from black wolfberry of this embodiment comprises the following steps:

[0040] (1) Obtaining raw materials:

[0041] Take fresh black wolfberry, remove the stems, and grind into a homogenous slurry in a pre-cooled mortar.

[0042] (2) Sample preparation:

[0043] The black wolfberry homogenate obtained in step (1) was added with 75% ethyl extract containing 0.1% hydrochloric acid at a solid-liquid ratio of 1:30, and extracted for 60 minutes in a constant temperature ultrasonic extractor (Ningbo Licheng Instrument Co., Ltd.) at an ultrasonic temperature of 20° C. and an ultrasonic extraction power of 160 W to obtain anthocyanin extract.

[0044] (3) Determination of anthocyanin content in black wolfberry extract by pH differential method:

[0045] Take 1 mL of the obtained anthocyanin extract and centrifuge it at 4000 r / min for 10 min; take the supernatant after centrifugation and dilute it to 4 mL in pH 1.0 hydrochloric acid-potassium chloride and pH 4.5 hydrochloric acid-sodium acetate buffer, respectively; use a visible spectrophotometer (Shanghai Meixi Instrument Co., Ltd.) and distilled water as a reference to measure the absorbance values ​​at wavelengths of 530 nm and 700 nm, respectively, and calculate the anthocyanin content.

[0046] Example 2

[0047] The method of ultrasonic-assisted extraction of anthocyanins from black wolfberry of this embodiment comprises the following steps:

[0048] (1) Obtaining raw materials:

[0049] Take fresh black wolfberry, remove the stems, and grind into a homogenous slurry in a pre-cooled mortar.

[0050] (2) Sample preparation:

[0051] The black wolfberry homogenate obtained in step (1) was added with 75% ethyl extract containing 0.1% hydrochloric acid at a solid-liquid ratio of 1:61.2, and extracted for 16.65 minutes in a constant temperature ultrasonic extractor (Ningbo Licheng Instrument Co., Ltd.) at an ultrasonic temperature of 38.3°C and an ultrasonic extraction power of 240W to obtain anthocyanin extract.

[0052] (3) Determination of anthocyanin content in black wolfberry extract by pH differential method:

[0053] Take 1 mL of the obtained anthocyanin extract and centrifuge it at 4000 r / min for 10 min; take the supernatant after centrifugation and dilute it to 4 mL in pH 1.0 hydrochloric acid-potassium chloride and pH 4.5 hydrochloric acid-sodium acetate buffer, respectively; use a visible spectrophotometer (Shanghai Meixi Instrument Co., Ltd.) and distilled water as a reference to measure the absorbance values ​​at wavelengths of 530 nm and 700 nm, respectively, and calculate the anthocyanin content.

[0054] (4) Determination of anthocyanin components by high performance liquid chromatography:

[0055] The obtained anthocyanin extract was centrifuged at 4000 r / min for 10 min; the supernatant after centrifugation was hydrolyzed in a boiling water bath for 30 min, taken out and cooled on ice, and the volume was fixed again with the extract; centrifuged at 6000 r / min for 10 min, the supernatant was taken, filtered with a 0.22 μm aqueous filter membrane, and its components were determined by high performance liquid chromatography (Thermo Fisher U3000 high performance liquid chromatograph).

[0056] The chromatographic conditions are as follows: (a) chromatographic column: Cs column, 250 mm × 4.6 mm × 5 m; (b) mobile phase A is an aqueous solution containing 1% formic acid, and mobile phase B is an acetonitrile solution containing 1% formic acid; (c) detection wavelength: 530 nm; (d) column temperature: 35 °C; (e) injection volume: 20 μL.

[0057] The elution procedure is:

[0058] At 0 min, flow rate of 0.8 mL / min, 92% mobile phase A, 8% mobile phase B;

[0059] At 2 min, flow rate of 0.8 mL / min, 88% mobile phase A, 12% mobile phase B;

[0060] At 5 min, flow rate of 0.8 mL / min, 80% mobile phase A, 20% mobile phase B;

[0061] At 10 min, flow rate of 0.8 mL / min, 75% mobile phase A, 25% mobile phase B;

[0062] At 15 min, flow rate of 0.8 mL / min, 70% mobile phase A, 30% mobile phase B;

[0063] At 18 min, flow rate of 0.8 mL / min, 55% mobile phase A, 45% mobile phase B;

[0064] At 20 min, flow rate of 0.8 mL / min, 20% mobile phase A, 880% mobile phase B;

[0065] At 22 min, flow rate of 0.8 mL / min, 92% mobile phase A, 8% mobile phase B;

[0066] At 30 min, flow rate was 0.8 mL / min, 92% mobile phase A, 8% mobile phase B.

[0067] Example 3

[0068] The method of ultrasonic-assisted extraction of anthocyanins from black wolfberry of this embodiment comprises the following steps:

[0069] (1) Obtaining raw materials:

[0070] Take fresh black wolfberry, remove the stems, and grind into a homogenous slurry in a pre-cooled mortar.

[0071] (2) Sample preparation:

[0072] The black wolfberry homogenate obtained in step (1) was added with 75% ethyl extract containing 0.1% hydrochloric acid at a solid-liquid ratio of 1:70, and extracted for 10 minutes in a constant temperature ultrasonic extractor (Ningbo Licheng Instrument Co., Ltd.) at an ultrasonic temperature of 60° C. and an ultrasonic extraction power of 320 W to obtain anthocyanin extract.

[0073] (3) Determination of anthocyanin components by high performance liquid chromatography:

[0074] The obtained anthocyanin extract was centrifuged at 4000 r / min for 10 min; the supernatant after centrifugation was hydrolyzed in a boiling water bath for 30 min, taken out and cooled on ice, and the volume was fixed again with the extract; centrifuged at 6000 r / min for 10 min, the supernatant was taken, filtered with a 0.22 μm aqueous filter membrane, and its components were determined by high performance liquid chromatography (Thermo Fisher U3000 high performance liquid chromatograph).

[0075] The chromatographic conditions are as follows: (a) chromatographic column: Cs column, 250 mm × 4.6 mm × 5 m; (b) mobile phase A is an aqueous solution containing 1% formic acid, and mobile phase B is an acetonitrile solution containing 1% formic acid; (c) detection wavelength: 530 nm; (d) column temperature: 35 °C; (e) injection volume: 20 μL.

[0076] The elution procedure is:

[0077] At 0 min, flow rate of 0.8 mL / min, 92% mobile phase A, 8% mobile phase B;

[0078] At 2 min, flow rate of 0.8 mL / min, 88% mobile phase A, 12% mobile phase B;

[0079] At 5 min, flow rate of 0.8 mL / min, 80% mobile phase A, 20% mobile phase B;

[0080] At 10 min, flow rate of 0.8 mL / min, 75% mobile phase A, 25% mobile phase B;

[0081] At 15 min, flow rate of 0.8 mL / min, 70% mobile phase A, 30% mobile phase B;

[0082] At 18 min, flow rate of 0.8 mL / min, 55% mobile phase A, 45% mobile phase B;

[0083] At 20 min, flow rate of 0.8 mL / min, 20% mobile phase A, 880% mobile phase B;

[0084] At 22 min, flow rate of 0.8 mL / min, 92% mobile phase A, 8% mobile phase B;

[0085] At 30 min, flow rate was 0.8 mL / min, 92% mobile phase A, 8% mobile phase B.

[0086] Experimental Example 1

[0087] This experimental example is used to illustrate the process and results of optimizing ultrasonic parameters using the response surface analysis method of the Box-Behnken Designs (BBD) central composite design model.

[0088] 1. Experimental Methods

[0089] First, the liquid-to-solid ratio, extraction time, extraction temperature, and extraction power were changed in turn to conduct a single factor test. Referring to Example 2, the anthocyanin content in the obtained black wolfberry anthocyanin extract was determined by the pH differential method, and the anthocyanin yield was calculated according to the following formula. Each treatment was repeated three times:

[0090] A=(A530-A700)pH1.0-(A530-A700)pH4.5;

[0091]

[0092] Where:

[0093] A——Absorbance difference;

[0094] (A 530nm -A 700nm)pH4.5——The absorbance difference of the sample solution with pH 4.5 at two wavelengths (530 and 700nm);

[0095] (A 530nm -A 700nm ) pH 1.0 - the absorbance difference of the sample solution at pH 1.0 at two wavelengths (530 and 700 nm);

[0096] C——anthocyanidin concentration (calculated as cyanidin-3-glucoside), mg / mL;

[0097] f——dilution multiple;

[0098] E——molar absorption constant of cyanidin-3-glucoside, 26900;

[0099] M – molecular weight of cyanidin-3-glucoside, 449.2;

[0100] L——Cuvette thickness, 1cm;

[0101] V——Volume of buffer after adjustment, mL.

[0102] The results are as follows Figures 1 to 4 As shown in the figure, the extraction time, liquid-to-solid ratio and extraction temperature have a significant impact on the extraction effect of anthocyanins from black wolfberry.

[0103] Next, the response surface methodology was used to optimize the design:

[0104] According to the results of the single factor test, the extraction power was fixed at 240 W, and the three factors of extraction time, extraction temperature, and solid-liquid ratio, which had a significant impact on the extraction effect of proanthocyanidins, were selected. The experimental design was carried out using Design Expert 10.0 software according to the Box-Behnken design principle, as shown in Tables 1 and 2.

[0105] Table 1 Experimental factors and level settings

[0106]

[0107] Table 2 Box-Behnken experimental design and results

[0108]

[0109] With extraction time A, liquid-to-solid ratio B, and extraction temperature C as independent variables and anthocyanin yield as response value y, a multivariate quadratic regression equation was established: Y = 9.32-0.17A + 0.14B-0.072C-0.16AB-

[0110] 0.067AC+0.13BC-0.27A2-0.39B2+0.031C2;

[0111] The significance of the linear relationship between each factor and the response value is determined by the F value test. The smaller the P value, the higher the significance of the variable. From the variance analysis table (Table 3), it can be seen that the linear relationship between the dependent variable and all independent variables is significant (R 2 =0.9837), the model significance level is less than 0.05, so the regression equation model is significant.

[0112] Table 3 Results of variance analysis of experimental data

[0113]

[0114] Note: ***The difference is extremely significant (p<0.0001); **The difference is significant (p<0.05).

[0115] 2. Experimental Results Analysis and Optimization

[0116] Design Expert 10.0 software was used to draw a picture based on the multivariate quadratic regression equation, and the response surface of the regression equation and its contour map were obtained ( Figures 5 to 10 ).

[0117] The suitable extraction conditions for ultrasonic-assisted extraction of anthocyanins from black wolfberry obtained by Design Expert 10.0 software are: extraction time 16.65 min, extraction temperature 38.3 °C, liquid-to-solid ratio 61.2:1 mL / g, and the maximum theoretical value of anthocyanin extraction rate is 9.389 mg / g.

[0118] Experimental Example 2

[0119] This experimental example is used to verify the effectiveness and feasibility of the method of the present invention.

[0120] (1) Obtaining raw materials:

[0121] Take fresh black wolfberry, remove the stems, and grind into a homogenous slurry in a pre-cooled mortar.

[0122] (2) Sample preparation:

[0123] The black wolfberry homogenate obtained in step (1) was added with 75% ethyl extract containing 0.1% hydrochloric acid at a solid-liquid ratio of 1:61.2, and extracted for 16.65 minutes in a constant temperature ultrasonic extractor (Ningbo Licheng Instrument Co., Ltd.) at an ultrasonic temperature of 38.3°C and an ultrasonic extraction power of 240W to obtain anthocyanin extract.

[0124] (3) Determination of anthocyanin content in black wolfberry extract by pH differential method:

[0125] Take 1 mL of the obtained anthocyanin extract and centrifuge it at 4000 r / min for 10 min; take the supernatant after centrifugation and dilute it to 4 mL in pH 1.0 hydrochloric acid-potassium chloride and pH 4.5 hydrochloric acid-sodium acetate buffer, respectively; use a visible spectrophotometer (Shanghai Meixi Instrument Co., Ltd.) and distilled water as a reference to measure the absorbance values ​​at wavelengths of 530 nm and 700 nm, respectively.

[0126] Under these conditions, the anthocyanin extraction amount was 9.377 mg / g, which was close to the theoretical yield, verifying the effectiveness of this model.

[0127] (4) Determination of anthocyanin components by high performance liquid chromatography:

[0128] The obtained anthocyanin extract was centrifuged at 4000 r / min for 10 min; the supernatant after centrifugation was hydrolyzed in a boiling water bath for 30 min, taken out and cooled on ice, and the volume was fixed again with the extract; centrifuged at 6000 r / min for 10 min, the supernatant was taken, filtered with a 0.22 μm aqueous filter membrane, and its components were determined by high performance liquid chromatography (Thermo Fisher U3000 high performance liquid chromatograph).

[0129] The chromatographic conditions are as follows: (a) chromatographic column: Cs column, 250 mm × 4.6 mm × 5 m; (b) mobile phase A is an aqueous solution containing 1% formic acid, and mobile phase B is an acetonitrile solution containing 1% formic acid; (c) detection wavelength: 530 nm; (d) column temperature: 35 °C; (e) injection volume: 20 μL.

[0130] The elution procedure is:

[0131] At 0 min, flow rate of 0.8 mL / min, 92% mobile phase A, 8% mobile phase B;

[0132] At 2 min, flow rate of 0.8 mL / min, 88% mobile phase A, 12% mobile phase B;

[0133] At 5 min, flow rate of 0.8 mL / min, 80% mobile phase A, 20% mobile phase B;

[0134] At 10 min, flow rate of 0.8 mL / min, 75% mobile phase A, 25% mobile phase B;

[0135] At 15 min, flow rate of 0.8 mL / min, 70% mobile phase A, 30% mobile phase B;

[0136] At 18 min, flow rate of 0.8 mL / min, 55% mobile phase A, 45% mobile phase B;

[0137] At 20 min, flow rate of 0.8 mL / min, 20% mobile phase A, 880% mobile phase B;

[0138] At 22 min, flow rate of 0.8 mL / min, 92% mobile phase A, 8% mobile phase B;

[0139] At 30 min, flow rate was 0.8 mL / min, 92% mobile phase A, 8% mobile phase B.

[0140] Under the above HPLC conditions, delphinium pigment, cyanidin and geranium pigment were determined. Fig.11 This is the chromatogram of anthocyanin mixed standard (in the figure, peaks 1 to 4 correspond to delphinium pigment, cyanidin, petunia pigment, geranium pigment, peony pigment, and malva pigment, respectively). Fig.12 The chromatogram of anthocyanins of black wolfberry was measured for this experimental example (in the figure, peaks 1, 2, and 4 correspond to delphinium pigment, cyanidin pigment, and geranium pigment, respectively).

[0141] In summary, the present invention uses the response surface analysis method of the Box-Behnken Designs central composite design model to optimize the four factors affecting anthocyanin extraction, namely, the solid-liquid ratio, ultrasonic extraction temperature, extraction time, and extraction power, to obtain the optimal ultrasonic extraction parameters; based on the above optimal ultrasonic extraction parameters, the present invention obtains a method for ultrasonic-assisted extraction of anthocyanins from black wolfberry with simple operation, short extraction time, high extraction efficiency, and low cost.

[0142] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for ultrasonic-assisted extraction of anthocyanins from black wolfberry, characterized in that: The steps include: (1) Remove the stems of fresh black wolfberries and grind them into a homogenous slurry in a pre-cooled mortar; (2) adding 75% ethyl extract containing 0.1% hydrochloric acid to the black wolfberry homogenate obtained in step (1) at a solid-liquid ratio of 1: (30-70), extracting in a constant temperature ultrasonic extractor for 10-60 minutes at an ultrasonic temperature of 20-60° C. and an ultrasonic extraction power of 160 W-320 W to obtain an anthocyanin extract.

2. The method for ultrasonic-assisted extraction of anthocyanins from black wolfberry according to claim 1, characterized in that: The material-liquid ratio, ultrasonic temperature, ultrasonic extraction power and ultrasonic extraction time in step (2) are all optimized and determined by response surface methodology.

3. The method for ultrasonic-assisted extraction of anthocyanins from black wolfberry according to claim 1, characterized in that: The material-liquid ratio is specifically 1:61.2, the ultrasonic temperature is 38.3°C, the ultrasonic extraction power is 240W, and the ultrasonic extraction time is 16.65min.

4. The method for ultrasonic-assisted extraction of anthocyanins from black wolfberry according to claim 1, characterized in that: The method also includes the step of determining the anthocyanin content in the black wolfberry extract by using a pH differential method, and / or the step of determining the anthocyanin component of the black wolfberry by using a high performance liquid chromatography method.

5. The method for ultrasonic-assisted extraction of anthocyanins from black wolfberry according to claim 4, characterized in that: The specific steps of the pH differential method for determining the anthocyanin content in the black wolfberry extract are: taking the obtained anthocyanin extract and centrifuging it at 4000r / min for 10min; taking the supernatant after centrifugation, diluting it in pH1.0 and pH4.5 buffer solutions respectively, and determining the absorbance value using a visible spectrophotometer.

6. The method for ultrasonic-assisted extraction of anthocyanins from black wolfberry according to claim 4, characterized in that: The specific steps of the high performance liquid chromatography method for determining the anthocyanin components of black wolfberry are: taking the obtained anthocyanin extract and centrifuging it at 4000r / min for 10min; taking the supernatant after centrifugation, hydrolyzing it in a boiling water bath for 30min, taking it out and cooling it on ice, and making up the volume again with the extract; centrifuging it at 6000r / min for 10min, taking the supernatant, filtering it with a 0.22μm aqueous phase filter membrane, and determining its components through a high performance liquid chromatograph.

7. The method for ultrasonic-assisted extraction of anthocyanins from black wolfberry according to claim 6, characterized in that: Thermo Fisher U3000 high performance liquid chromatograph was used, and the chromatographic conditions were as follows: (a) chromatographic column: Cs column, 250 mm×4.6 mm×5 m; (b) mobile phase A was an aqueous solution containing 1% formic acid, and mobile phase B was an acetonitrile solution containing 1% formic acid; (c) detection wavelength: 530 nm; (d) column temperature: 35°C; (e) injection volume: 20 μL; The elution program was as follows: at 0 min, flow rate of 0.8 mL / min, 92% mobile phase A, 8% mobile phase B; at 2 min, flow rate of 0.8 mL / min, 88% mobile phase A, 12% mobile phase B; at 5 min, flow rate of 0.8 mL / min, 80% mobile phase A, 20% mobile phase B; at 10 min, flow rate of 0.8 mL / min, 75% mobile phase A, 25% mobile phase B; at 15 min, flow rate of 0.8 mL / min Flow rate, 70% mobile phase A, 30% mobile phase B; at 18 min, flow rate of 0.8 mL / min, 55% mobile phase A, 45% mobile phase B; at 20 min, flow rate of 0.8 mL / min, 20% mobile phase A, 880% mobile phase B; at 22 min, flow rate of 0.8 mL / min, 92% mobile phase A, 8% mobile phase B; at 30 min, flow rate of 0.8 mL / min, 92% mobile phase A, 8% mobile phase B.

8. The method for ultrasonic-assisted extraction of anthocyanins from black wolfberry according to claim 6, characterized in that: High performance liquid chromatography determined that the anthocyanin components of black wolfberry are delphinium pigment, cornflower pigment and geranium pigment.

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