A method for extracting high-purity naringin from young grapefruit fruit

Abstract

This invention discloses a method for extracting high-purity naringin from young grapefruit. The method includes: mechanically and chemically ball-milling pretreated young grapefruit with zirconium oxide beads to obtain ball-milled powder; adding an aqueous ethanol solution to the powder, heating and stirring to extract a crude extract; concentrating the crude extract and precipitating with alcohol to remove polysaccharides; filtering, concentrating the supernatant, adding deionized water, heating and stirring, and filtering while hot; repeating this operation 3-5 times for further purification; finally, concentrating, crystallizing, and drying to obtain the refined naringin product. This invention utilizes mechanical and chemical ball milling to efficiently destroy the cell structure of the raw material, significantly improving the extraction efficiency and yield of naringin (up to 76%), and replacing complex column chromatography and other processes with recrystallization purification. It has the advantages of simple operation, short time consumption, and low cost, which is conducive to industrial production.

Description

Technical Field

[0001] This invention belongs to the field of plant active ingredient extraction technology, specifically relating to a method for extracting high-purity naringin from young grapefruit fruit. Background Technology

[0002] Naringin is mainly found in the peel and young fruit of grapefruit and belongs to the flavonoid class of compounds. Naringin is a pale yellow or off-white powder with anti-inflammatory, antiviral, anticancer, analgesic, and blood pressure-lowering activities. It can lower blood cholesterol, reduce thrombus formation, improve local microcirculation and nutrient supply, and can be used to prevent and treat cardiovascular and cerebrovascular diseases. Furthermore, naringin can be further processed into high-end sweeteners, natural pigments, and other additives commonly used in food. Its market prospects and application potential are broad, attracting increasing attention.

[0003] Currently, mainstream extraction methods in the industry include ultrasonic-assisted extraction, microwave extraction, and supercritical CO2 extraction. Purification methods mostly employ macroporous resin adsorption, membrane separation, and column chromatography. Among these, supercritical CO2 extraction equipment is costly, and column chromatography is complex, time-consuming, and consumes a lot of solvent, making it unsuitable for industrial-scale mass production. Traditional grinding methods are incomplete, resulting in larger particle sizes of young grapefruit, leading to insufficient extraction of naringin, low extraction efficiency, low yield (usually 50%-60%), and difficulty in achieving a purity of over 90%. Furthermore, some processes suffer from solvent residue and damage to active ingredients. To achieve widespread application of naringin, a simple, efficient, and low-cost extraction technology is urgently needed. However, there is currently no mature technology or supporting equipment of this kind in this field. Therefore, how to provide a reasonable extraction solution to solve the problems of high cost, low efficiency, and complex processes in naringin extraction has become a pressing technical problem to be solved in this field.

[0004] Mechanochemical ball milling replaces traditional grinding. By using mechanical force to break down the tissue particle size and cell wall structure of young grapefruit, naringin is fully exposed. Compared with traditional grinding, it significantly improves extraction efficiency and solves the problems of insufficient extraction and low yield in existing technologies. Unlike existing technologies such as freeze grinding and ordinary pulverization, mechanochemical ball milling does not require the addition of additional reagents and achieves efficient extraction solely through mechanical action.

[0005] There are currently no reports on the use of mechanochemical ball milling technology for the extraction of naringin from young grapefruit fruits, combined with recrystallization to achieve high-purity purification. Summary of the Invention

[0006] To address the above problems, the present invention aims to provide a method for extracting high-purity naringin from young grapefruit.

[0007] The specific technical solution is as follows: A method for extracting high-purity naringin from immature grapefruit fruit includes the following steps: 1) Add the pretreated young grapefruit and zirconia beads together to a ball mill jar for ball milling to obtain ball milled powder; 2) Add an aqueous ethanol solution to the ball-milled powder, heat and stir to extract, and obtain crude naringin extract; 3) After concentrating the crude extract of naringin, add pure ethanol and precipitate overnight to remove polysaccharides; 4) After filtering to remove polysaccharides, concentrate the supernatant, add deionized water, heat and stir, filter while hot to remove impurities, continue to concentrate, add deionized water, heat and stir, filter while hot, and repeat this operation 3-5 times. 5) Concentrate, crystallize, collect the crystals, and dry them to obtain naringin refined product.

[0008] Furthermore, in step 1), the diameter of the zirconia beads is 10-15 mm, the ball milling speed is 300 rpm, the ball milling time is 10-60 min, and the ball-to-material ratio is 25-15:1.

[0009] Furthermore, in step 2), the mass concentration of the ethanol aqueous solution is 50-80%, and the ratio of grapefruit powder to ethanol aqueous solution is 0.025-0.1 g / mL.

[0010] Further, in step 2), heat to 50-80℃ and extract for 30-150 minutes.

[0011] Furthermore, in step 4), the volume ratio of the concentrated supernatant to deionized water is 1:1-5, and the heating temperature is 50-80℃.

[0012] Further, in step 5), the volume is concentrated to 1 / 5 to 1 / 2 of the original volume, and crystallization is carried out at 4-25℃ for 6-24 hours.

[0013] The beneficial effects of this invention are as follows: 1) This invention utilizes mechanochemical-assisted extraction of naringin by performing mechanochemical ball milling. The mechanical force of the ball milling breaks down the particle size of the young grapefruit tissue, allowing naringin to be fully exposed. Compared with traditional grinding, this significantly improves extraction efficiency and solves the problems of insufficient extraction and low yield in existing technologies, increasing the naringin yield to 76%. 2) No complex steps such as column chromatography and macroporous resin adsorption are required. Purification can be achieved through recrystallization, which is convenient, time-saving and effectively improves efficiency. Attached Figure Description

[0014] Figure 1 The effect of ball milling time on the extraction yield of naringin; Figure 2 The effect of the solid-liquid ratio on the extraction yield of naringin; Figure 3The effect of ethanol concentration on the extraction yield of naringin during crude extraction; Figure 4 The effect of temperature on the extraction yield of naringin during crude extraction; Figure 5 The effect of extraction time on the yield of naringin during crude extraction; Figure 6 The effect of concentration during crystallization on the purity and yield of recrystallized naringin; Figure 7 The effect of recrystallization time on the purity and yield of naringin during crystallization; Figure 8 The liquid chromatogram of naringin standard; Figure 9 This is a liquid chromatogram of naringin extract. Detailed Implementation

[0015] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto.

[0016] Example 1 Standard curve preparation: Accurately weigh 20 mg of naringin standard into a 100 mL volumetric flask, dissolve in methanol solution, and dilute to 100 mL to obtain a solution with a mass concentration of 2 mg / mL. Use this as the stock solution to dilute to mass concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 1.8 mg / mL, and 2 mg / mL, respectively. Plot the naringin standard curve using high-performance liquid chromatography (HPLC): Y = 8.2075x - 74.201 (R² + π / 4)². 2 =0.9994). See chromatogram. Figure 8 .

[0017] Sample determination method: The naringin extract was dissolved in methanol, filtered through a 0.45 μm filter membrane, and determined by HPLC.

[0018] Calculation formula: Naringin content m1 (mg / g) = C1 × V / M1 Naringin purity = m1 / M2 Naringin crystallization yield = M2 / m2; Where C1 is the concentration of the sample solution (mg / mL); V is the volume of the extract (mL); M1 is the mass of the raw material powder (g); m1 is the mass of naringin in the sample (g); m2 is the mass of the refined product before drying (g); and M2 is the mass of the refined product after drying (g).

[0019] Example 2 Method for extracting naringin from young grapefruit using hot reflux extraction To compare the extraction results, the hot reflux method was used to extract young grapefruit. The specific process is as follows: 60% ethanol at 20 times the amount of young grapefruit powder was used as the extraction solvent, and the extraction was carried out at 105℃ for 2 hours. After the extraction was completed, the extracts were combined, and polysaccharides were removed by alcohol precipitation. The content of the extracted liquid was then determined to be 112.31 mg / g.

[0020] Example 3 Optimal ball milling time 5.0 g of young grapefruit powder was added to a 25 mL polytetrafluoroethylene ball mill jar, followed by zirconia grinding beads as the grinding medium. The grinding beads had a diameter of 14 mm and a ball-to-powder ratio of 15:1. After mixing evenly, the ball mill jar was placed in a planetary ball mill, and the rotation speed was set to 300 rpm. After the reaction was completed, the product was separated from the zirconia beads, and the product was then heated and hydrolyzed with an ethanol solution (the mass concentration of ethanol during extraction was 70%, the material-to-liquid ratio was 0.05 g / mL, the extraction time was 30 min, and the extraction temperature was 70 °C) to obtain a crude extract of naringin.

[0021] A single-factor experiment was conducted by changing the ball milling time to 10 min, 30 min, 45 min, and 60 min, and the results are as follows: Figure 1 As shown, the extraction yield of naringin was 102.68 mg / g after grinding for 10 min, 115.25 mg / g after grinding for 30 min, 115.88 mg / g after grinding for 45 min, and 113.85 mg / g after grinding for 60 min. Figure 1 It can be observed that the extraction rate of naringin increases with the increase of grinding time, but when the grinding time exceeds 30 minutes, the extraction rate of naringin does not increase significantly.

[0022] Example 4 Optimal material-liquid ratio Add 5.0 g of young grapefruit powder to a 25 mL polytetrafluoroethylene ball milling jar, then add zirconia grinding beads as the grinding medium. The grinding beads have a diameter of 14 mm and the ball-to-powder ratio is 15:1. After mixing evenly, place the ball milling jar into a planetary ball mill, set the speed to 300 rpm, and the grinding time to 30 min. After the reaction is complete, separate the product from the zirconia beads, and then heat the product with an ethanol solution for hydrolysis (the mass concentration of ethanol during extraction is 70%, the extraction time is 30 min, and the extraction temperature is 70℃) to obtain a crude extract of naringin.

[0023] The amount of ethanol solution added was varied, with solid-liquid ratios of 0.025 g / mL, 0.05 g / mL, 0.067 g / mL, and 0.1 g / mL. The results are shown below. Figure 2When the material-to-liquid ratio was 0.025 g / mL, the naringin content was 106.31 mg / g; when the material-to-liquid ratio was 0.05 mg / mL, the naringin content was 115.74 mg / g; when the material-to-liquid ratio was 0.067 g / mL, the naringin content was 115.26 mg / g; and when the material-to-liquid ratio was 0.1 g / mL, the naringin content was 102.41 mg / g.

[0024] It can be observed that within the solid-liquid ratio range of 0.025-0.05 g / mL, as the amount of ethanol solution increases, the dissolution environment of naringin becomes more sufficient, the extraction is more complete, and the yield increases significantly. However, when the solid-liquid ratio exceeds 0.05 g / mL, excessive ethanol consumption will lead to an increase in the impurity content in the crude extract. Therefore, the solid-liquid ratio is determined to be 0.05 g / mL.

[0025] Example 5

[0026] Optimal Ethanol Concentration 5.0 g of young grapefruit powder was added to a 25 mL polytetrafluoroethylene ball milling jar, followed by zirconia grinding beads as the grinding medium. The grinding beads had a diameter of 14 mm and a ball-to-powder ratio of 15:1. After mixing evenly, the ball milling jar was placed in a planetary ball mill. The speed was set to 300 rpm and the grinding time was 30 min. After the reaction was completed, the product was separated from the zirconia beads, and the product was then heated and hydrolyzed with an ethanol solution (extraction material-to-liquid ratio 0.05 g / mL, extraction time 30 min, extraction temperature 70 °C) to obtain a crude extract of naringin.

[0027] The ethanol concentration during extraction was changed to 50%, 60%, 70%, and 80%, and the results are shown in the figure. Figure 3 When the ethanol concentration is 50%, the extraction yield of naringin is 112.15 mg / g; when the ethanol concentration is 60%, the extraction yield of naringin is 121.31 mg / g; when the ethanol concentration is 70%, the extraction yield of naringin is 115.74 mg / g; and when the ethanol concentration is 80%, the extraction yield of naringin is 108.8 mg / g.

[0028] Figure 3 It is known that high-concentration ethanol is volatile, and the concentration of the system fluctuates greatly during the extraction process, which disrupts the dissolution-precipitation balance of naringin. Some naringin fails to dissolve completely or precipitates prematurely, thus reducing the extraction yield. Therefore, an ethanol concentration of 60% is preferred.

[0029] Example 6 Optimal extraction temperature 5.0 g of young grapefruit powder was added to a 25 mL polytetrafluoroethylene ball milling jar, followed by zirconia grinding beads as the grinding medium. The grinding beads had a diameter of 14 mm and a ball-to-powder ratio of 15:1. After mixing evenly, the ball milling jar was placed in a planetary ball mill. The rotation speed was set to 300 rpm and the grinding time was 30 min. After the reaction was completed, the product was separated from the zirconia beads, and the product was then heated and hydrolyzed with an ethanol solution (the mass concentration of ethanol during extraction was 60%, the material-to-liquid ratio was 0.05 g / mL, and the extraction time was 30 min) to obtain a crude extract of naringin.

[0030] The extraction temperature was changed to 50℃, 60℃, 70℃, and 80℃ respectively, and the results are shown in [the table below]. Figure 4 The extract yield of naringin was 128.66 mg / g at 50℃, 134.81 mg / g at 60℃, 121.31 mg / g at 70℃, and 118.59 mg / g at 80℃.

[0031] from Figure 4 It was found that increasing the temperature can improve the yield of naringin, but when the temperature exceeds 60℃, the ethanol evaporation rate accelerates, leading to unstable concentration in the extraction system. Furthermore, high temperatures can damage the flavonoid core structure of naringin, resulting in a decrease in yield. Therefore, the optimal extraction temperature is 60℃.

[0032] Example 7 Optimal extraction time Add 5.0 g of young grapefruit powder to a 25 mL polytetrafluoroethylene ball mill jar, then add zirconia grinding beads as the grinding medium. The grinding beads have a diameter of 14 mm and the ball-to-powder ratio is 15:1. After mixing evenly, place the ball mill jar into a planetary ball mill, set the speed to 300 rpm, and the grinding time to 30 min. After the reaction is complete, separate the product from the zirconia beads, and then heat the product with an ethanol solution for hydrolysis (the mass concentration of ethanol during extraction is 60%, the material-to-liquid ratio is 0.05 g / mL, and the extraction temperature is 60 °C) to obtain a crude extract of naringin.

[0033] The extraction time was changed to 30 min, 60 min, 90 min, 120 min, and 150 min respectively. The results are shown in [the table below]. Figure 5 The extraction yield of naringin was 134.81 mg / g when the extraction time was 30 min; 149.73 mg / g when the extraction time was 60 min; 147.89 mg / g when the extraction time was 90 min; 143.42 mg / g when the extraction time was 120 min; and 137.62 mg / g when the extraction time was 150 min.

[0034] from Figure 5 It was observed that the extraction time increased with the increase of naringin, but after 1 hour, naringin had basically reached dissolution equilibrium. Further extending the extraction time would increase the amount of impurities dissolved, leading to a decrease in the purity of the crude extract, as well as increasing energy consumption and production time. Therefore, an extraction time of 60 minutes is preferred.

[0035] Example 8 Effect of concentration factor on the purity and yield of naringin recrystallization Extraction: 5.0 g of young grapefruit powder was added to a 25 mL polytetrafluoroethylene ball mill jar, followed by zirconia grinding beads as the grinding medium. The grinding beads had a diameter of 14 mm and a ball-to-powder ratio of 15:1. After mixing evenly, the ball mill jar was placed in a planetary ball mill. The rotation speed was set to 300 rpm and the grinding time was 30 min. After the reaction was completed, the product was separated from the zirconia beads. The product was then heated and hydrolyzed with an ethanol solution (the ethanol concentration during extraction was 60%, the material-to-liquid ratio was 0.05 g / mL, the extraction temperature was 60℃, and the extraction time was 60 min) to obtain a crude extract of naringin. The crude extract contained a total mass of 0.749 g of naringin.

[0036] Crystallization: The crude extract was concentrated to a certain multiple of its original volume, and pure ethanol was added for alcohol precipitation at 4°C overnight to remove polysaccharides. The polysaccharides were removed by filtration, and the supernatant was concentrated. Deionized water was added at a volume ratio of 1 / 2.5, and the mixture was heated and stirred at 70°C. The mixture was filtered while hot, and the supernatant was concentrated, heated and stirred, and filtered while hot again. This process was repeated three times until the mixture was supersaturated. Crystals were allowed to precipitate at 4°C for 24 hours. The crystals were collected, dried, and the purity and yield of the naringin were calculated.

[0037] The concentration volume ratios were set to 1 / 2, 1 / 3, 1 / 4, and 1 / 5, respectively. The purity results of the refined product are shown below. Figure 6 When the concentration volume is half the original volume, the purity of naringin is 76.2% and the yield is 67.3%; when the concentration volume is one-third the original volume, the purity of naringin is 85.7% and the yield is 70.1%; when the concentration volume is one-quarter the original volume, the purity of naringin is 88% and the yield is 76.3%; when the concentration volume is one-fifth the original volume, the purity of naringin is 90.5% and the yield is 75%. It can be observed that as the concentration increases, both purity and yield gradually improve. In particular, when concentrated to 1 / 4 of the original volume, the purity can reach as high as 88%. However, excessively high concentrations can easily lead to crystal bursting, resulting in a large number of impurities encapsulated in the crystals, significantly reducing purity. Furthermore, the crystals tend to clump together, making them difficult to separate. Therefore, it is preferable to concentrate to 1 / 4 of the original volume.

[0038] Example 9 Effect of crystallization time on the purity and yield of naringin crystals Extraction: 5.0 g of young grapefruit fruit powder was added to a 25 mL polytetrafluoroethylene ball mill jar. Zirconia grinding beads (14 mm diameter, ball-to-powder ratio 15:1) were then added as the grinding medium. After thorough mixing, the jar was placed in a planetary ball mill at 300 rpm for 30 min. After the reaction, the product was separated from the zirconium beads. The product was then hydrolyzed with an ethanol solution under heating (ethanol concentration 60%, material-to-liquid ratio 0.05 g / mL, extraction temperature 60℃, extraction time 60 min) to obtain a crude extract of naringin. The total mass of naringin in the crude extract was 0.749 g.

[0039] Crystallization: The crude extract was concentrated to 1 / 4 of its original volume, and pure ethanol was added for alcohol precipitation at 4°C overnight to remove polysaccharides. The polysaccharides were removed by filtration, and the supernatant was concentrated. Deionized water was added at a volume ratio of 1 / 2.5, and the mixture was heated and stirred at 70°C. The mixture was then filtered while hot. This process of concentrating the supernatant, heating and stirring, and filtering while hot was repeated three times until supersaturation. Crystals were collected at 4°C for a period of time and dried to obtain the refined naringin product. The purity and yield of the crystals were calculated. The liquid phase diagram of the refined product is shown below. Figure 9 .

[0040] The crystallization time was set to 6h, 12h, 18h, and 24h, and the results are shown below. Figure 7 When the crystallization time was 6 hours, the purity of the naringin refined product was 90% and the yield was 45%; when the crystallization time was 12 hours, the purity of the naringin refined product was 93% and the yield was 76%; when the crystallization time was 18 hours, the purity of the naringin refined product was 91% and the yield was 76.2%; when the crystallization time was 24 hours, the purity of the naringin refined product was 88% and the yield was 76.3%.

[0041] A crystallization time of 6 hours is insufficient, resulting in incomplete crystallization. Crystallization times of 12 hours or more do not significantly improve the yield, but slightly decrease the purity (trace impurities slowly precipitate and adsorb onto the crystal surface) and increase the industrial production cycle. Therefore, crystallization at 4°C for 12 hours is the optimal crystallization time.

Claims

1. A method for extracting high-purity naringin from young grapefruit, characterized in that, Includes the following steps: 1) Add the pretreated young grapefruit and zirconia beads together to a ball mill jar for ball milling to obtain ball milled powder; 2) Add an aqueous ethanol solution to the ball-milled powder, heat and stir to extract, and obtain crude naringin extract; 3) After concentrating the crude extract of naringin, add pure ethanol and precipitate overnight to remove polysaccharides; 4) After filtering to remove polysaccharides, concentrate the supernatant, add deionized water, heat and stir, filter while hot to remove impurities, continue to concentrate, add deionized water, heat and stir, filter while hot, and repeat this operation 3-5 times. 5) Concentrate, crystallize, collect the crystals, and dry them to obtain naringin refined product.

2. The method for extracting high-purity naringin from young grapefruit as described in claim 1, characterized in that, In step 1), the diameter of the zirconia beads is 10-15 mm, the ball milling speed is 300 rpm, the ball milling time is 10-60 min, and the ball-to-material ratio is 25-15:

1.

3. The method for extracting high-purity naringin from young grapefruit as described in claim 1, characterized in that, In step 2), the mass concentration of the ethanol aqueous solution is 50-80%, and the ratio of grapefruit powder to ethanol aqueous solution is 0.025-0.1 g / mL.

4. The method for extracting high-purity naringin from young grapefruit as described in claim 1, characterized in that, In step 2), heat to 50-80℃ and extract for 30-150 minutes.

5. The method for extracting high-purity naringin from young grapefruit as described in claim 1, characterized in that, In step 4), the volume ratio of the concentrated supernatant to deionized water is 1:1-5, and the heating temperature is 50-80℃.

6. The method for extracting high-purity naringin from immature grapefruit as described in claim 1, characterized in that, In step 5), the volume is concentrated to 1 / 5 to 1 / 2 of the original volume, and crystallization is carried out at 4-25℃ for 6-24 hours.

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