Natural neohesperidin and separation and purification method thereof

Abstract

The invention discloses natural neohesperidin and a separation and purification method thereof, and belongs to the technical field of natural product extraction.The high-purity neohesperidin is prepared from immature bitter oranges through smashing, extraction, coarse crystallization, organic solvent pulping, water pulping and recrystallization. The problems of complex process, long production period, high equipment requirement, high comprehensive production cost, high product ash content and the like in the existing neohesperidin separation and purification process are solved.

Description

Technical Field

[0001] The present invention discloses a natural neohesperidin and a separation and purification method thereof, and specifically relates to a natural neohesperidin extracted from Citrus aurantium immaturus and a separation and purification method for obtaining high-purity and low-ash neohesperidin, belonging to the technical field of natural product extraction. Background Art

[0002] Neohesperidin Neohesperidin is a natural dihydroflavonoid compound, which is widely found in the peels of citrus fruits, such as oranges, grapefruits, and tangerines. Neohesperidin has a variety of biological activities and pharmacological effects, mainly in terms of anti-oxidation, regulating blood lipids, and preventing cardiovascular diseases. Among them, anti-oxidation is one of its most important biological activities. Neohesperidin can exert its antioxidant effect by scavenging free radicals and preventing oxidative stress. In addition, neohesperidin can also prevent cardiovascular diseases by lowering blood lipids and regulating blood pressure. At the same time, neohesperidin is also an important intermediate, which is converted into neohesperidin dihydrochalcone with higher sweetness through hydrogenation. Its sweetness is 1500 to 1800 times that of sucrose, and it has low calories and will not affect the blood sugar and blood lipid levels of the human body. Therefore, neohesperidin dihydrochalcone is widely used as a food additive as a sweetener (flavor corrector) in fruit juice, fruit wine, beverages, cakes and pharmaceutical formulations, and is particularly suitable for diabetic patients.

[0003] Neohesperidin has a complex structure and is difficult to prepare by total synthesis. Therefore, it is generally converted into neohesperidin by chemical synthesis using naringin extracted from plants as the starting material, or extracted from Citrus aurantium, which is widely available and inexpensive. Compared with the synthesis of naringin, direct extraction from natural plants has its incomparable advantages. Its raw materials are widely available, and the products are pure and natural, which is a very promising way to obtain it.

[0004] At present, the separation and purification of neohesperidin can be mainly divided into two technical routes. One is the separation method using macroporous resin and (or) silica gel column, such as Chinese patents CN100335490C, CN1272339C, CN102477056A, CN105399787B, CN105481920B and CN109912666B, etc. The separation and purification of macroporous resin and silica gel column used in this technical route usually includes multiple steps, such as sample pretreatment, resin and silica gel loading, chromatographic separation, etc. Each step requires strict control of conditions and a large amount of organic solvents. Therefore, there are problems such as relatively complex operation, long time consumption and high production cost; The other type is to first use membrane filtration (ceramic membrane, microfiltration membrane, nanofiltration membrane, etc.), and then add flocculants for flocculation and inorganic salts to assist crystallization, such as Chinese patents CN106083940B, CN111777653B, CN112142807A, CN112125936A and CN1218953C. The membrane filtration equipment used in this type of technical route is expensive and has a huge amount of wastewater. At the same time, the introduction of flocculants and a large amount of inorganic salts to assist crystallization can easily lead to a large amount of solid waste and high-salt wastewater in the production process. Therefore, this technical route has high requirements on production equipment, complex procedures, and high comprehensive production costs.

[0005] In addition, Chinese patent CN101293906B also discloses that the crude extract concentrate is obtained by solvent reflux extraction using Citrus aurantium, Citrus aurantium immaturus, and orange as raw materials, and then the crude product of neohesperidin is obtained by salting out, and then the neohesperidin product is obtained by recrystallization using an ethanol-water system. This method adds a large amount of mineral salt for salting out, and a large amount of high-salt wastewater is generated during the production process; at the same time, the quality standard of "Plant Extract Neohesperidin" (standard number T / CCCMHPIE 1.53-2019) requires an ash content of ≤0.5%, and the patent method does not mention the ash content data. Repeating this method to prepare neohesperidin has an ash content of up to 12.5%, which does not meet the quality requirements.

[0006] In summary, based on the deficiencies of the prior art, the present invention provides a method for preparing neohesperidin with low requirements on production equipment and simple and efficient separation and purification process. The prepared neohesperidin meets the quality standards of plant extract neohesperidin. Summary of the invention

[0007] The present invention aims to provide a method for separating and purifying natural neohesperidin. The method uses Citrus aurantium as a raw material and obtains high-purity neohesperidin through crushing, extraction, coarse crystallization, organic solvent beating, water beating and recrystallization. The method solves the problems of complex procedures, long production cycle, high equipment requirements, high comprehensive production cost, high product ash content, etc. in the existing process for separating and purifying neohesperidin. The method is a new process for extracting high-purity neohesperidin from Citrus aurantium. To this end, the present invention also provides natural neohesperidin obtained by the separation and purification method.

[0008] The present invention is achieved through the following technical scheme: A method for separating and purifying natural neohesperidin comprises the following steps: (1) Crushing: crushing the immature bitter orange into coarse powder; (2) Extraction: The crude powder of Citrus aurantium is extracted by heating with an organic solvent, the filtrate is filtered to obtain a filtrate, and the extract is concentrated by vacuum distillation; (3) Coarse crystallization: add pure water or low concentration sulfate solution to the extract, cool the system to 0-10°C, keep warm for crystallization or add neohesperidin seed crystals and keep warm for crystallization, filter and collect the filter cake; (4) Organic solvent beating: the filter cake is beating with an organic solvent, filtered, and the filter cake is collected; (5) Water beating: The filter cake is beating with water, filtered, and the filter cake is collected to obtain a crude product; (6) Recrystallization: The crude product is dissolved by heating in an organic solvent, and then cooled and crystallized to obtain high-purity neohesperidin.

[0009] The organic solvent in step (2) is 40-95% ethanol or 40-95% methanol; the amount of the organic solvent used is 3-4 times the weight of the Fructus Aurantii Immaturus; the heating extraction temperature is 50-75° C.; the extraction time is 2-4 hours; and the number of extractions is 2 times.

[0010] The amount of pure water used in step (3) is 1.5 to 2.5 times the volume of the extract weight; the heat preservation crystallization time is 6 to 24 hours.

[0011] The low-concentration sulfate aqueous solution described in step (3) is selected from one of sodium sulfate, potassium sulfate or magnesium sulfate, and its salt concentration ranges from 1 to 5%.

[0012] The organic solvent in step (4) is selected from one of ethyl acetate, methyl formate, isopropyl formate, ethyl formate, methyl acetate, tetrahydrofuran, methyl tert-butyl ether, acetonitrile, acetone, toluene or dichloromethane; the amount of organic solvent used for pulping is 3 to 5 times the volume of the filter cake weight; the pulping time is 0.5 to 1 hour, and the pulping temperature is 20 to 60°C.

[0013] The amount of water used for pulping in step (5) is 2 to 3 times the volume of the filter cake weight; the pulping time is 0.5 to 1 hour; and the pulping temperature is 20 to 40°C.

[0014] The organic solvent for recrystallization in step (6) is 50-90% ethanol or 50-90% methanol; the amount of the organic solvent is 2-4 times the volume of the crude product; the crystallization temperature is 5-15°C, and the crystallization time is 10-24h.

[0015] A natural neohesperidin obtained by the above separation and purification method, wherein the natural neohesperidin contains ≥95% neohesperidin and ≤0.5% ash, and meets the quality standard of "Neohesperidin from Plant Extract" (standard number T / CCCMHPIE 1.53-2019).

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The present invention has low requirements on production equipment. Compared with the prior art, it does not use precision filtration equipment such as ceramic membranes, microfiltration membranes, and nanofiltration membranes.

[0017] (2) The process of the present invention is simple and efficient. High-purity neohesperidin can be obtained by only using pulping and recrystallization operations. Compared with the prior art, no macroporous resin and silica gel column separation and purification are used, which greatly simplifies the purification process of neohesperidin, significantly shortens the product production cycle, and has strong practicality.

[0018] (3) The process of the method of the present invention is highly operable, the product purity is high, it is environmentally friendly, and it is suitable for industrial production.

[0019] (4) In the method of the present invention, no or only a small amount of inorganic salt is used to assist crystallization, and the crude crystals are first slurried with an organic solvent to remove organic impurities to improve purity, and then slurried with water to remove salt, thereby obtaining high-purity, low-ash neohesperidin. The continuous coordination of these steps is also the key to obtaining high-purity, low-ash neohesperidin in the present invention.

[0020] (5) The neohesperidin obtained by the method of the present invention has high purity and an ash content of less than 0.5%, meeting the quality requirements of "Neohesperidin from Plant Extracts". BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the HPLC spectrum of the neohesperidin product obtained in Example 4.

[0022] Figure 2 This is the HPLC spectrum of the neohesperidin product obtained in Example 7.

[0023] Figure 3 This is the HPLC spectrum of the neohesperidin product obtained in Example 9. DETAILED DESCRIPTION

[0024] The invention objectives, technical solutions and beneficial effects of the present invention are further described in detail below.

[0025] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the claimed invention, and unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0026] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0027] Embodiment 1: Take 2kg of Citrus aurantium and crush it to obtain Citrus aurantium powder. Add 6L 95% ethanol to the Citrus aurantium powder and reflux extract for 3h, filter while hot, and reflux extract the filter cake with 6L 95% ethanol for the second time; combine the extracts and concentrate under reduced pressure to obtain 560g of extract. Add 850g of water to the extract, stir at 0-10℃ for 10h, and filter to obtain 70g of filter cake, which is the crude crystal product. The crude crystal product was slurried with 230mL of ethyl acetate at 50℃ for 30min, filtered while hot to obtain 58g of filter cake, and the filter cake was slurried with 120mL of water at 25-30℃ for 30min and filtered to obtain 50g of crude neohesperidin. The crude product was heated with 200mL of 70% ethanol to reflux and dissolve, then cooled to 5-10℃, and kept warm for crystallization for 16h, filtered and dried to obtain 42g of neohesperidin finished product with a purity of 97.88% and an ash content of 0.15%.

[0028] Embodiment 2: Take 2kg of Citrus aurantium and crush it to obtain Citrus aurantium powder. Add 6L 70% methanol to the Citrus aurantium powder and reflux extract for 2h, filter while hot, and reflux extract the filter cake with 6L 70% methanol for the second time; combine the extracts and concentrate under reduced pressure to obtain 720g of extract. Add 1100g of water to the extract, stir at 0-10℃ for 18h, and filter to obtain 90g of filter cake, which is a crude crystal product. The crude crystal product was slurried with 300mL of tetrahydrofuran at 30℃ for 30min, and 75g of filter cake was filtered. The filter cake was slurried with 150mL of water at 25-30℃ for 30min and filtered to obtain 62g of crude neohesperidin. The crude product was heated with 200mL of 60% methanol to reflux and dissolve, then cooled to 5-10℃, and kept warm for crystallization for 12h, filtered and dried to obtain 54g of neohesperidin finished product, with a purity of 96.80% and an ash content of 0.21%.

[0029] Embodiment 3: Take 2kg of Citrus aurantium and crush it to obtain Citrus aurantium powder. Add 6L 40% ethanol to the Citrus aurantium powder and reflux extract for 3h, filter while hot, and reflux extract the filter cake with 6L 70% ethanol for the second time; combine the extracts and concentrate under reduced pressure to obtain 770g of extract. Add 1200g of 5% magnesium sulfate aqueous solution to the extract, stir at 0-10℃ for 10h, and filter to obtain 98g of filter cake, which is a crude crystal product. The crude crystal product was slurried with 250mL acetonitrile at 60℃ for 30min, and 85g of filter cake was filtered. The filter cake was slurried with 250mL water at 25-30℃ for 30min and filtered to obtain 74g of crude neohesperidin. The crude product was heated with 300mL 90% ethanol to reflux and dissolve, then cooled to 5-10℃, and kept warm for crystallization for 10h, filtered and dried to obtain 61g of neohesperidin finished product with a purity of 98.90% and an ash content of 0.1%.

[0030] Embodiment 4: Take 2kg of Citrus aurantium and crush it to obtain Citrus aurantium powder. Add 6L 50% ethanol to the Citrus aurantium powder and reflux extract for 4h, then filter it while hot. Use 6L 50% ethanol to reflux extract for the second time; combine the extracts and concentrate under reduced pressure to obtain 810g of extract. Add 1200g of 1% potassium sulfate aqueous solution and a small amount of neohesperidin seed to the extract, stir at 0-10℃ for 6h and filter to obtain 130g of filter cake, which is a crude crystal product. The crude crystal product was slurried with 500mL ethyl acetate at 60℃ for 30min, filtered to obtain 117g of filter cake, and the filter cake was slurried with 350mL water at 35-40℃ for 30min and filtered to obtain 102g of crude neohesperidin. The crude product was heated to reflux with 200 mL of 70% ethanol to dissolve, then cooled to 5-10°C and kept warm for 24 hours to crystallize. After filtering and drying, 89 g of the finished product of neohesperidin was obtained with a purity of 98.10% and an ash content of 0.08%. The HPLC spectrum of the finished product of neohesperidin is shown in Figure 1 .

[0031] Embodiment 5: Take 2kg of Citrus aurantium and crush it to obtain Citrus aurantium powder. Add 6L of 50% methanol to the Citrus aurantium powder and reflux extract for 3h, then filter it while hot. Use 6L of 50% methanol to reflux extract for the second time; combine the extracts and concentrate under reduced pressure to obtain 800g of extract. Add 1200g of water and a small amount of neohesperidin seed to the extract, stir at 0-10℃ for 24h, and then filter to obtain 127g of filter cake, which is a crude crystal product. The crude crystal product is slurried with 500mL of methyl formate at 60℃ for 30min, and filtered to obtain 115g of filter cake. The filter cake is slurried with 350mL of water at 20-25℃ for 30min and filtered to obtain 103g of crude neohesperidin. The crude product was heated to reflux with 300 mL of 70% methanol to dissolve, then cooled to 5-10°C and kept warm for 18 hours for crystallization. After filtration and drying, 92 g of neohesperidin was obtained with a purity of 97.58% and an ash content of 0.1%.

[0032] Embodiment 6: Take 2kg of Citrus aurantium and crush it to obtain Citrus aurantium powder. Add 6L 50% methanol to the Citrus aurantium powder and reflux extract for 3h, then filter it while hot. Use 6L 50% methanol to reflux extract for the second time; combine the extracts and concentrate under reduced pressure to obtain 800g of extract. Add 1200g of water and a small amount of neohesperidin seeds to the extract, stir at 0-10℃ for 24h, and filter to obtain 127g of filter cake, which is a crude crystal product. The crude crystal product was slurried with 500mL of ethyl acetate at 60℃ for 30min, and 115g of filter cake was filtered. The filter cake was slurried with 350mL of water at 20-25℃ for 30min and filtered to obtain 103g of crude neohesperidin. The crude product was heated with 300mL of 70% methanol to reflux and dissolve, then cooled to 5-10℃, and kept warm for crystallization for 18h, filtered and dried to obtain 92g of neohesperidin finished product, with a purity of 96.90% and an ash content of 0.13%.

[0033] Embodiment 7: Take 2kg of Citrus aurantium and crush it to obtain Citrus aurantium powder. Add 6L 60% ethanol to the Citrus aurantium powder and reflux extract for 3h, filter while hot, and reflux extract the filter cake with 6L 60% ethanol for the second time; combine the extracts and concentrate under reduced pressure to obtain 780g of extract. Add 1150g of 2% sodium sulfate aqueous solution to the extract, stir at 0-10℃ for 24h, and filter to obtain 116g of filter cake, which is a crude crystal product. The crude crystal product was slurried with 580mL of dichloromethane at 20℃ for 60min, and 102g of filter cake was filtered. The filter cake was slurried with 305mL of water at 30-35℃ for 60min and filtered to obtain 93g of crude neohesperidin. The crude product was heated with 370mL of 90% ethanol to reflux and dissolve, then cooled to 5-10℃, and kept warm for crystallization for 18h, filtered and dried to obtain 85g of neohesperidin finished product, with a purity of 97.93% and an ash content of 0.1%. The HPLC spectrum of the finished product of neohesperidin is shown in Figure 2 .

[0034] Embodiment 8: Take 2kg of Citrus aurantium and crush it to obtain Citrus aurantium powder. Add 8L 90% ethanol to the Citrus aurantium powder and reflux extract for 2h, filter while hot, and reflux extract the filter cake with 8L 60% ethanol for the second time; combine the extracts and concentrate under reduced pressure to obtain 890g of extract. Add 1800g of water and a small amount of neohesperidin seeds to the extract, stir at 0-10℃ for 24h, and filter to obtain 140g of filter cake, which is a crude crystal product. The crude crystal product was slurried with 420mL of toluene at 60℃ for 60min, and 125g of filter cake was filtered. The filter cake was slurried with 250mL of water at 30-35℃ for 60min and filtered to obtain 110g of crude neohesperidin. The crude product was heated with 220mL of 85% ethanol to reflux and dissolve, then cooled to 5-10℃, and kept warm for 12h for crystallization, filtered and dried to obtain 95g of neohesperidin finished product with a purity of 98.27% and an ash content of 0.05%.

[0035] Embodiment 9: Take 2kg of Citrus aurantium and crush it to obtain Citrus aurantium powder. Add 6L 80% ethanol to the Citrus aurantium powder and reflux extract for 4h, then filter it while hot. Use 6L 80% ethanol to reflux extract for the second time; combine the extracts and concentrate under reduced pressure to obtain 870g of extract. Add 1280g of 2% sodium sulfate aqueous solution and a small amount of neohesperidin seed to the extract, stir at 0-10℃ for 12h, and then filter to obtain 170g of filter cake, which is a crude crystal product. The crude crystal product was slurried with 510mL of ethyl acetate at 50℃ for 60min, and filtered to obtain 155g of filter cake. The filter cake was slurried with 310mL of water at 25-30℃ for 60min and filtered to obtain 138g of crude neohesperidin. The crude product was heated to reflux with 280 mL of 75% ethanol to dissolve, then cooled to 5-10°C and kept warm for 12 hours to crystallize. The product was filtered and dried to obtain 105 g of neohesperidin with a purity of 99.55% and an ash content of 0.05%. The HPLC spectrum of the neohesperidin product is shown in Figure 3 .

[0036] Comparative Example 1: Neohesperidin was prepared by referring to the method of patent CN101293906B.

[0037] Take 2kg of Citrus aurantium and crush it to obtain Citrus aurantium powder. Add 10L of 70% ethanol to the Citrus aurantium powder and reflux extract 3 times, each time for 1.5h, combine the three extracts and concentrate to 10L. Add 400g of sodium sulfate and 4L of ethyl acetate, stir at 40℃ for 1h, filter, reflux and dissolve the filter cake with 2.2L of 85% ethanol, filter while hot at 50℃, and let the filtrate crystallize at room temperature. Filter to obtain white needle-shaped crystals, with a product purity of 95.1% and an ash content of 12.5%.

[0038] It can be seen from the above embodiments and comparative examples that the purity of the neohesperidin products prepared above all reaches more than 95%. However, since comparative example 1 adopts a salting-out method when extracting the crude neohesperidin, a large amount of high-salt wastewater will be generated in the process, which is not conducive to the control of the ash content in the product (ash is generated when the raw material extracts neohesperidin, generally referring to the various inorganic salts extracted), and its ash content is 12.5%, which does not meet the quality requirements of "Plant Extract Neohesperidin". In addition, for comparative example 1, even if an ethanol-water system is used for recrystallization, the purity of the product of comparative example 1 can only reach 95.1%, which cannot reach the same purity of the method of the present invention. Therefore, for the method of the present invention, through the reasonable coordination of the steps of crude crystallization, organic solvent beating and recrystallization, not only can the purity of the neohesperidin finished product be improved, up to 99.55%, but also the ash content of the product can be reduced to less than 0.5%, meeting the quality requirements of "Plant Extract Neohesperidin".

[0039] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for separating and purifying natural neohesperidin, characterized in that: The following steps are involved: (1) Crushing: crushing the immature bitter orange into coarse powder; (2) Extraction: The crude powder of Citrus aurantium is extracted by heating with an organic solvent, the filtrate is filtered to obtain a filtrate, and the extract is concentrated by vacuum distillation; (3) Coarse crystallization: add pure water or low concentration sulfate solution to the extract, cool the system to 0-10°C, keep warm for crystallization or add neohesperidin seed crystals and keep warm for crystallization, filter and collect the filter cake; (4) Organic solvent beating: the filter cake is beating with an organic solvent, filtered, and the filter cake is collected; (5) Water beating: The filter cake is beating with water, filtered, and the filter cake is collected to obtain a crude product; (6) Recrystallization: The crude product is dissolved by heating in an organic solvent, and then cooled and crystallized to obtain high-purity neohesperidin.

2. The separation and purification method according to claim 1, characterized in that: The organic solvent in step (2) is 40-95% ethanol or 40-95% methanol; the amount of the organic solvent used is 3-4 times the weight of the Fructus Aurantii Immaturus; the heating extraction temperature is 50-75° C.; the extraction time is 2-4 hours; and the number of extractions is 2 times.

3. The separation and purification method according to claim 1, characterized in that: The amount of pure water used in step (3) is 1.5 to 2.5 times the volume of the extract weight; the heat preservation crystallization time is 6 to 24 hours.

4. The separation and purification method according to claim 1, characterized in that: The low-concentration sulfate aqueous solution described in step (3) is selected from one of sodium sulfate, potassium sulfate or magnesium sulfate, and its salt concentration ranges from 1 to 5%.

5. The separation and purification method according to claim 1, characterized in that: The organic solvent in step (4) is selected from one of ethyl acetate, methyl formate, isopropyl formate, ethyl formate, methyl acetate, tetrahydrofuran, methyl tert-butyl ether, acetonitrile, acetone, toluene or dichloromethane; the amount of organic solvent used for pulping is 3 to 5 times the volume of the filter cake weight; the pulping time is 0.5 to 1 hour, and the pulping temperature is 20 to 60°C.

6. The separation and purification method according to claim 1, characterized in that: The amount of water used for pulping in step (5) is 2 to 3 times the volume of the filter cake weight; the pulping time is 0.5 to 1 hour; and the pulping temperature is 20 to 40°C.

7. The separation and purification method according to claim 1, characterized in that: The organic solvent for recrystallization in step (6) is 50-90% ethanol or 50-90% methanol; the amount of the organic solvent is 2-4 times the volume of the crude product; the crystallization temperature is 5-15°C, and the crystallization time is 10-24h.

8. A natural neohesperidin obtained by the separation and purification method according to any one of claims 1 to 7, characterized in that: The natural neohesperidin contains neohesperidin ≥ 95% and ash ≤ 0.5%.

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