Two-time crystallization method for extracting hesperidin

Abstract

The invention discloses a twice crystallization method for extracting hesperidin, and belongs to the technical field of plant extraction. The method for extracting hesperidin comprises the following steps: cleaning and crushing a raw material rich in hesperidin; adding an impurity removal solvent, and carrying out impurity removal reaction; adding an extraction solvent into the raw materials subjected to impurity removal to carry out extraction reaction, wherein the extraction solvent is a mixed solution of low-level saturated monohydric alcohol with the volume percent of 40-80% and alkali with the mass percent of 0.3-1.0%; adding acid into the extracting solution to adjust the pH value to 7.5-8.5; carrying out a first crystallization reaction, adding acid into the residual mother liquor to adjust the pH value to 6.5-7.5, and carrying out a second crystallization reaction; the extraction, purification and recovery processes of the hesperidin are optimized through a two-time crystallization method of sequentially performing crystallization in an alcohol system and a water system. The method not only overcomes the defects of large wastewater discharge amount and complex process of the traditional extraction process, but also further improves the extraction yield of hesperidin, reduces the production cost, and makes the extraction process more efficient and environment-friendly.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant extraction, and specifically relates to a two-stage crystallization method for extracting hesperidin, and also relates to a hesperidin product prepared by this method. Background Art

[0002] Hesperidin is a flavonoid substance present in citrus plants of the Rutaceae family, mainly present in the mature and thinner peel system or pulp. Hesperidin has a dihydroflavonoid oxygen glycoside structure and is weakly acidic. The pure product is white needle-shaped crystals and is the main component of vitamin P. In recent years, research has shown that hesperidin has the effects of maintaining osmotic pressure, enhancing capillary toughness, shortening bleeding time, and reducing cholesterol, and is used clinically for the adjuvant treatment of cardiovascular system diseases. It can be formulated into various drugs for preventing arteriosclerosis and myocardial infarction. Or as an intermediate for synthesizing diosmin or other raw materials, and is one of the main components of the Chinese patent medicine Maitong; or as a natural food antioxidant, added to foods such as beverages, milk powder, potato powder, confectionery products, lard, and vegetable oils to prevent lipid oxidation and spoilage of foods; as a feed additive, it can promote the feeding and growth rate of young livestock and shorten the market time. As an important plant extract, hesperidin ranks among the top fifteen in the plant extract industry in terms of market share, and has good development prospects.

[0003] The current production process for extracting hesperidin is the alkali extraction and acid precipitation method, mainly through impurity removal with sodium carbonate solution and extraction with sodium hydroxide solution. The wastewater output per ton of raw materials processed is about 25 - 50 tons. The extraction agent in the alkali extraction contains a large amount of water-soluble impurities, such as pectin, cellulose, protein, etc., which are difficult to remove, reducing the purity of the product. The fundamental problem of the alkali extraction and acid precipitation process is the extremely large amount of wastewater generated, which no longer meets the needs of social development. The alcohol extraction process can effectively solve the problem of large wastewater volume, but the alcohol extraction process also has disadvantages in production applications, such as the yield of the first crystallization of the alcohol extract is lower than that of the water extraction process, large solvent consumption, and solvent consumption problems. Therefore, it is particularly important to develop a method for extracting hesperidin with small wastewater generation, simple operation, low cost, high extraction rate, and good product quality. Summary of the Invention

[0004] The purpose of the present invention is to overcome at least one deficiency of the prior art, and provide a two-stage crystallization method for extracting hesperidin and a hesperidin product prepared thereby.

[0005] The technical solution adopted by this application is:

[0006] A method for extracting hesperidin, comprising the following steps:

[0007] (1) Cleaning and pulverizing the raw material rich in hesperidin;

[0008] (2) Add a purification solvent to carry out a purification reaction, and perform solid-liquid separation to obtain the purified raw material and the purification liquid; the purification solvent is a mixed solution of a lower saturated monohydric alcohol with a volume percentage of 40%-80% and an alkali with a mass percentage of 0.1-0.8%;

[0009] (3) Add an extraction solvent to the purified raw material to carry out an extraction reaction, and perform solid-liquid separation to obtain the extract; the extraction solvent is a mixed solution of a lower saturated monohydric alcohol with a volume percentage of 40%-80% and an alkali with a mass percentage of 0.3-1.0%;

[0010] (4) Adjust the pH of the extract to 7.5-8.5 with acid; keep it at 40-60 °C for 1-2 h, and let it stand at 20-30 °C for 8-12 h to carry out the first crystallization reaction, and perform solid-liquid separation to obtain hesperidin crystals and the mother liquor;

[0011] (5) Adjust the pH of the mother liquor to 6.5-7.5 with acid, evaporate and concentrate to remove the lower saturated monohydric alcohol to obtain a concentrated solution, adjust the pH of the concentrated solution to 7.5-8.5, keep it at 60-80 °C for 1-2 h, and let it stand at 20-30 °C for 8-12 h to carry out the second crystallization reaction, and perform solid-liquid separation to obtain hesperidin crystals.

[0012] In some embodiments, the raw material in step (1) is the dried young fruit of oranges or tangerines.

[0013] In some embodiments, the crushing mesh number in step (1) is 2-40 meshes.

[0014] In some embodiments, the lower saturated monohydric alcohol is a C1-C4 saturated monohydric alcohol, including at least one of methanol and ethanol, preferably methanol; and / or, the alkali is at least one of sodium hydroxide, potassium hydroxide, and calcium hydroxide.

[0015] In some embodiments, the purification solvent in step (2) is a mixed solution of 40%-60% lower saturated monohydric alcohol and 0.4%-0.6% alkali.

[0016] In some embodiments, the extraction solvent in step (3) is a mixed solution of 40%-60% lower saturated monohydric alcohol and 0.4%-0.7% alkali.

[0017] In some embodiments, the purification solvent in step (2) is added in a ratio of 1:(2-20) kg:L of the material-liquid ratio, preferably the material-liquid ratio is 1:(2-10) kg / L, and more preferably the material-liquid ratio is 1:(6-10) kg / L;

[0018] And / or, the temperature of the purification reaction in step (2) is 10 °C-40 °C; the time of the purification reaction is 0.5 h-4 h.

[0019] In some of these embodiments, the extraction solvent in step (3) is added in a ratio of solid to liquid of 1:(2 - 20) kg:L, preferably 1:(2 - 10) kg / L, and more preferably 1:(6 - 10) kg / L;

[0020] and / or, the temperature of the extraction reaction in step (3) is 10°C - 40°C; the time of the extraction reaction is 0.5 h - 4 h.

[0021] In some of these embodiments, the raw materials after impurity removal in step (3) are divided into N groups. The first group of raw materials is extracted 4 times. The extract of the 3rd extraction is used as the 1st extraction solvent for the next group, and the extract of the 4th extraction is used as the 2nd extraction solvent for the next group, and so on. N is 3 - 6 groups, preferably 4 - 6 groups.

[0022] In some of these embodiments, the acid in step (5) is at least one of hydrochloric acid, phosphoric acid, and sulfuric acid.

[0023] An hesperidin product, which is prepared by the method described in any one of the above.

[0024] The beneficial effects of this application are:

[0025] This application optimizes the extraction, purification, and recovery processes of hesperidin through a two - crystallization method of sequential crystallization in an alcohol system and a water system. The alcohol solvent is recovered and recycled during the extraction process, which not only significantly improves the solvent utilization rate but also effectively reduces wastewater discharge, controlling the wastewater generation per ton of raw materials to less than 10 tons. This method not only overcomes the disadvantages of large wastewater discharge and complex processes in traditional extraction processes but also further improves the extraction yield of hesperidin, reduces production costs, and makes the extraction process more efficient and environmentally friendly. Description of the Drawings

[0026] Figure 1 The raw materials used in the examples.

[0027] Figure 2 The HPLC chromatogram of hesperidin in Example 1. Detailed Embodiments

[0028] The hesperidin extraction process provided by this application uses medium - to - high - concentration lower - order saturated monohydric alcohols + low - concentration alkaline solutions to extract hesperidin, and adopts a two - crystallization method. After alcohol extraction, hesperidin first crystallizes in alcohol to obtain a large amount of high - content products, and the lower - order saturated monohydric alcohol (such as methanol) is concentrated and recycled. Finally, the product is crystallized in a water system. This extraction process has the characteristics of high hesperidin content yield, high extraction efficiency, and low energy consumption, has high market value, and is worthy of popularization and application.

[0029] The present application provides a two - crystallization method for extracting hesperidin, comprising the following steps:

[0030] (1) Clean and crush the raw material rich in hesperidin;

[0031] (2) Add a impurity - removing solvent to carry out an impurity - removing reaction, and perform solid - liquid separation to obtain the impurity - removed raw material and the impurity - removing liquid; the impurity - removing solvent is a mixed solution of 40% - 80% lower - level saturated monohydric alcohol and 0.1% - 0.8% alkali;

[0032] (3) Add an extraction solvent to the impurity - removed raw material to carry out an extraction reaction, and perform solid - liquid separation to obtain the extraction liquid; the extraction solvent is a mixed solution of 40% - 80% lower - level saturated monohydric alcohol and 0.3% - 1.0% alkali;

[0033] (4) Adjust the pH of the extraction liquid to 7.5 - 8.5 with acid; keep it at 40 - 60 °C for 1 - 2 h, and let it stand at 20 - 30 °C for 8 - 12 h to carry out the first crystallization reaction, and perform solid - liquid separation to obtain hesperidin crystals and the mother liquor;

[0034] (5) Adjust the pH of the mother liquor to 6.5 - 7.5 with acid, evaporate and concentrate to remove the lower - level saturated monohydric alcohol to obtain a concentrated liquid, adjust the pH of the concentrated liquid to 7.5 - 8.5, keep it at 60 - 80 °C for 1 - 2 h, and let it stand at 20 - 30 °C for 8 - 12 h to carry out the second crystallization reaction, and perform solid - liquid separation to obtain hesperidin crystals.

[0035] In some of the embodiments, the raw material in step (1) is the dried young fruit of oranges or tangerines. The dried product is more conducive to long - term preservation and can be processed according to the production plan. The raw materials used in the embodiments are shown in Figure 1 .

[0036] The raw material can also be "Fructus Aurantii Immaturus" in traditional Chinese medicine. Fructus Aurantii Immaturus, a traditional Chinese medicine name, is the dried young fruit of Citrus aurantium L. and its cultivated varieties or Citrus sinensis Osbeck of the Rutaceae family. It is collected from the fallen fruits in May - June, the impurities are removed, cut transversely into two halves from the middle, and dried in the sun or by low - temperature drying. The smaller ones are directly dried in the sun or by low - temperature drying.

[0037] Among them, the young fruit of oranges or tangerines has a diameter not greater than 2 cm.

[0038] In some of the embodiments, the crushing mesh number in step (1) is 2 - 40 meshes, preferably 10 - 40 meshes, and more preferably 20 - 30 meshes.

[0039] In some of these embodiments, the lower saturated monohydric alcohol is a C1-C4 saturated monohydric alcohol, including at least one of methanol and ethanol, and / or, the base is at least one of sodium hydroxide, potassium hydroxide, and calcium hydroxide.

[0040] In some of these preferred embodiments, the impurity removal solvent in step (2) is a mixed solution of 40%-60% lower saturated monohydric alcohol and 0.4%-0.6% base.

[0041] In some of these embodiments, the impurity removal solvent in step (2) is added in a ratio of material to liquid (M:V) of 1:(2 - 20) kg / L, preferably the ratio of material to liquid (M:V) is 1:(2 - 10) kg / L, and more preferably the ratio of material to liquid (M:V) is 1:(6 - 10) kg / L.

[0042] In some of these embodiments, the temperature of the impurity removal reaction in step (2) is 10°C - 40°C; the time of the impurity removal reaction is 0.5 h - 4 h.

[0043] If the extraction process is directly carried out after the raw materials are cleaned and pulverized, due to the presence of impurities in the raw materials rich in hesperidin, it will affect the content / purity of hesperidin in the final product and the extraction yield. Therefore, in some embodiments of the present application, an impurity removal process is added before the extraction step to reduce impurity interference and lay a foundation for the efficient extraction of the target component hesperidin. By optimizing key parameters such as the ratio of the impurity removal liquid and the concentration of the base, the stability and extraction efficiency of the extraction process can be further improved, providing an important basis for determining the optimal extraction conditions.

[0044] In some of these preferred embodiments, the extraction solvent in step (3) is a mixed solution of 40%-60% lower saturated monohydric alcohol and 0.4%-0.7% base.

[0045] In some of these embodiments, the extraction solvent in step (3) is added in a ratio of material to liquid (M:V) of 1:(2 - 20) kg / L; preferably the ratio of material to liquid (M:V) is 1:(2 - 10) kg / L, and more preferably the ratio of material to liquid (M:V) is 1:(6 - 10) kg / L.

[0046] In some of these embodiments, the temperature of the extraction reaction in step (3) is 10°C - 40°C; the time of the extraction reaction is 0.5 h - 4 h.

[0047] In some of these embodiments, the raw materials after impurity removal in step (3) are divided into N groups. The first group of raw materials is extracted 4 times. The third extraction liquid is used as the first extraction solvent for the next group, and the fourth extraction liquid is used as the second extraction solvent for the next group, and so on. The N is 3 - 6, and more preferably 4 - 6. The third and fourth extraction liquids of the last group are not recycled.

[0048] The present application creatively adopts the method of cyclic extraction. Because the dissolving ability of the solvent may not be fully utilized in a single extraction process. Through extraction, the solvent can come into contact with new raw materials again, enabling its dissolving ability to be more fully exerted, ensuring the maximum utilization of the solvent, and thus better exerting the extraction effect of the solvent on the target component.

[0049] In industrial production or large-scale extraction processes, the cost of the solvent accounts for a relatively large proportion. By means of extraction, the usage amount of the new solvent is reduced. If extraction is not adopted, a large amount of solvent is required to extract each batch of raw materials, which not only increases the cost but also may cause waste of resources. Especially when a large amount of solvent is required in the extraction process and the solvent recovery cost is relatively high, this kind of waste is more obvious.

[0050] Although the extraction process may slightly increase the complexity of the operation, from the overall production process, it can obtain more target products without increasing too much time cost. Because during the extraction process, the equipment can run continuously without the need to frequently replace the new solvent for multiple independent extractions, thus improving the production efficiency to a certain extent, especially on a continuous extraction production line, this advantage is more obvious.

[0051] However, the number of extraction times is not the more the better. After exceeding a certain number of times, the extraction effect will be discounted and the cost will instead increase. In the present application, the preferred number of extraction times N is 3 - 6, more preferably 4 - 6, and the best is 5 times. The extraction strategy of about 5 times shows the best performance in terms of solvent saving, efficiency and cost balance. Exceeding 6 times will lead to diminishing marginal benefits and an increase in operation complexity. At the same time, the time, energy consumption, operation cost and solvent recovery cost may increase, offsetting the economic benefits brought by solvent saving.

[0052] In some of the preferred embodiments, in step (3), the raw materials after impurity removal are divided into 5 groups for extraction reaction, which can enable the solvent to be recycled. In addition, this design is also for better detecting the stability of the yield and hesperidin content. Once it is found that the extraction result fluctuates, the production plan can be adjusted in a timely manner to ensure the controllability of the process and the stability of the final product quality.

[0053] The crystallization processes in steps (4) and (5) are based on the principle that crystallization is the process of solute precipitation from a supersaturated solution to form a new phase. It utilizes the different solubilities of each component in the crude extract in a certain solvent. The same substance forms crystals to achieve the separation from other components. By taking advantage of the ring-opening and dissolution of hesperidin molecular structure under alkaline conditions and the ring-closing and precipitation under acidic conditions. The first crystallization is carried out in an alcohol system. Since hesperidin is more soluble in alcohol (the solubility of hesperidin in alcohol is 7-10 times greater than that in water), alcohol can not only be fully recovered and utilized, but also inhibit the precipitation of some water-soluble impurities. Therefore, a high-content product is obtained in the first crystallization. The second crystallization is carried out in an aqueous system to obtain the remaining product, and a high-yield product is obtained in total.

[0054] In some of these embodiments, in steps (4) and (5), during heat preservation, slow stirring can be carried out or no stirring is required. After the heat preservation treatment is completed, static treatment at 20-30 °C (room temperature) is carried out.

[0055] The "static" in this application is a verb, meaning to keep an object or liquid in a stationary and immobile state.

[0056] Obviously, the temperature of the heat preservation treatment in steps (4) and (5) is higher than that of the static treatment. And during static treatment, as the name implies, no stirring is required.

[0057] In some embodiments of this application, heat preservation is used to promote the formation of crystal nuclei. The principle is that during the crystallization process, heat preservation can provide a relatively stable temperature environment, enabling solute molecules to obtain sufficient energy and increasing the collision frequency between molecules. When the collision between solute molecules reaches a certain degree, it is more conducive to the formation of crystal nuclei. For example, during the crystallization of some organic compounds, heat preservation at a temperature slightly higher than room temperature (such as 30-40 °C) can maintain an appropriate movement rate of solute molecules and promote the formation of tiny crystallization centers. In the research of this application, it is found that in this technical solution, step (4) specifies heat preservation at 40-60 °C for 1-2 h, step (5) specifies heat preservation at 60-80 °C for 1-2 h, and combined with subsequent static treatment at 20-30 °C (room temperature), it is more conducive to the formation of hesperidin crystals.

[0058] And the static process provides a stable environment for the growth of crystals. After the formation of crystal nuclei, the further growth of crystals requires sufficient time and suitable space. During the static process, the disturbance of the solution can be avoided, enabling solute molecules to deposit orderly on the surface of crystal nuclei and gradually grow into larger crystals. If the solution is always in a stirred or shaken state, it is difficult for solute molecules to stack regularly on the surface of crystal nuclei, which may affect the growth morphology and size of crystals and thus the purity and yield of the final product.

[0059] In some of these embodiments, the acid in step (5) is at least one of hydrochloric acid, phosphoric acid, and sulfuric acid.

[0060] In some of these embodiments, in step (5), the mother liquor is adjusted to a pH of 6.5 - 7.5 with acid, evaporated and concentrated to remove the lower saturated monohydric alcohol, obtaining a concentrated solution, the pH of the concentrated solution is adjusted to 8, kept warm at 60 - 80 °C, and a second crystallization reaction is carried out. After solid - liquid separation, hesperidin crystals are obtained. The "mother liquor" refers to the saturated solution remaining after separating the precipitate or crystals during a chemical precipitation or crystallization process. Adjusting the pH of the mother liquor to 6.5 - 7.5 (neutral) with acid is to minimize the impact on hesperidin during the process of removing the lower saturated monohydric alcohol.

[0061] In some of these preferred embodiments, the holding temperature in step (4) is preferably 50 - 60 °C; and / or; the holding temperature in step (5) is preferably 70 - 80 °C.

[0062] In some of these preferred embodiments, the hesperidin crystals obtained in steps (4) and (5) are added to a 0.2% - 0.4% Na₂CO₃ aqueous solution according to a solid - to - liquid ratio of 1:(8 - 12) kg / L and stirred for 2 - 3 h for crystal washing, and then stirred with deionized water for 20 - 40 min for crystal washing.

[0063] In some of these preferred embodiments, the washed hesperidin crystals are filtered by a centrifuge and dried at 70 - 80 °C to obtain a hesperidin product.

[0064] A hesperidin product, which is prepared by the above - mentioned method. The hesperidin product can be used in foods, health products, drugs, cosmetics, and feeds.

[0065] The content of the hesperidin extracted in the examples was detected by a high - performance liquid chromatograph (HPLC). According to the Chinese Pharmacopoeia (2015) and relevant literature information, through the optimization of the mobile phase, the following liquid - phase parameter conditions of the method were determined:

[0066] Chromatographic column: Cortecs C18 column (4.6×100 mm, 3.5 μm);

[0067] Mobile phase: The volume ratio of acetonitrile to 0.1% acetic acid water is 17:83 (V:V), and an isocratic elution method is adopted;

[0068] Flow rate: 1 mL / min;

[0069] Column temperature: 35 °C;

[0070] Injection volume: 10 μL;

[0071] Detection wavelength: 283 nm;

[0072] Running time: 18 min.

[0073] The hesperidin content in the raw material Fructus Aurantii Immaturus of the example was obtained by heating and refluxing the raw material with dimethylformamide (i.e., N,N-dimethylformamide) and measuring it by high performance liquid chromatography. Since hesperidin is soluble in dimethylformamide, the obtained result can measure the hesperidin content in the raw material. The remainder is impurities relative to hesperidin, and there are many types of substances included in the impurities, such as other flavonoid substances, proteins, sugars, etc.

[0074] For the hesperidin product obtained by extracting with the technical solution of the present application, the content of hesperidin was measured by high performance liquid chromatography and compared with the result of the hesperidin content sampled and detected in the raw material to illustrate the beneficial effects of the technical solution of the present application.

[0075] In the example, the yield is (hesperidin content * weight of hesperidin) obtained by extraction / (hesperidin content * weight of the raw material used) in the raw material. That is, the total amount of hesperidin obtained by extraction / the total amount of hesperidin theoretically.

[0076] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. In the examples, the concentration of methanol is volume percentage concentration (V / V), and sodium hydroxide is mass percentage concentration.

[0077] For the experimental methods without specific conditions in the following examples of the present invention, they are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. All kinds of common chemical reagents used in the examples are commercially available products.

[0078] Example 1

[0079] The raw material is 100 kg of Fructus Aurantii Immaturus, and the hesperidin content in the sampled and detected raw material Fructus Aurantii Immaturus is 35.5%. Taking the raw material as an example, a two-crystallization method for extracting hesperidin is provided, including the following steps:

[0080] (1) 100 kg of Fructus Aurantii Immaturus raw material is soaked and washed with water, and the wet material is pulverized to 20 mesh, and divided into five groups for extraction, with about 20 kg of dry raw material in each group.

[0081] (2) Impurity removal: The impurity removal solvent is a mixed solvent of 60% (V / V) methanol + 0.4% NaOH. The impurity removal solvent is added according to a material-liquid ratio of 1:6, and the reaction is carried out at 30 °C for 2 h to remove impurities, and the raw material after impurity removal and the impurity removal liquid are obtained by filtration. The solvent in the impurity removal liquid is recovered.

[0082] (3) Extraction: An extraction solvent is added to the raw material after impurity removal for extracting hesperidin. The extraction solvent is a mixed solvent of 60% methanol + 0.6% NaOH, the material-liquid ratio is 1:6 kg / L, the extraction temperature is 25 °C, and the extraction time for each pass is 2 h. The filter residue and the extraction liquid are obtained by filtration.

[0083] Five groups of raw materials are extracted by the method of cyclic extraction. Among them, the first group of raw materials is extracted 4 times. The extract of the 3rd extraction is used as the solvent for the 1st extraction of the next group (the second group), and the extract of the 4th extraction is used as the solvent for the 2nd extraction of the next group (the second group). The solvents for the 3rd and 4th extractions of the second group are new solutions. And so on. According to the above method, the extracts of the 3rd and 4th extractions of the fifth group are no longer recycled. Finally, all the extracts are combined.

[0084] (4) First crystallization: Add hydrochloric acid to the extract to adjust the pH to 8. Stir the solution evenly during pH adjustment. Keep it at 50 °C for 1 h, let it stand at 30 °C for 10 h, and then centrifuge and filter to obtain hesperidin crystals and the mother liquor after the first crystallization. Add the centrifuged crystals to 0.2% Na2CO3 aqueous solution at a solid-liquid ratio of 1:10 and stir for 2 h to wash the crystals. Then centrifuge and filter, and add deionized water at a solid-liquid ratio of 1:3 and stir for 30 min to wash the crystals.

[0085] (5) Second crystallization: Add hydrochloric acid to the mother liquor after the first crystallization to adjust the pH to 7. Rotavapor and concentrate to remove methanol. Adjust the pH of the concentrated solution to 8. Stir the solution evenly during pH adjustment. Keep it at 70 °C for 1 h, let it stand at 25 °C for 10 h, and then centrifuge and filter to obtain hesperidin crystals. Add the centrifuged crystals to 0.4% Na2CO3 aqueous solution at a solid-liquid ratio of 1:10 and stir for 2 h to wash the crystals. Then centrifuge and filter, and add deionized water at a solid-liquid ratio of 1:3 and stir for 30 min to wash the crystals.

[0086] (6) Filtration and drying: Filter using a centrifuge respectively and dry at 75 °C to obtain the hesperidin product.

[0087] Among them, for the hesperidin product obtained by the first crystallization, after detection, the methanol residue is 265 ppm. For the hesperidin product obtained by the second crystallization, after detection, the methanol residue is 97 ppm.

[0088] The total yield of hesperidin obtained by the method of this example is 92.32% (total yield of five groups of raw materials). The average content of hesperidin in the first crystallization is 95.57%, and the yield is 86.35%. The average content of hesperidin in the second crystallization is 91.29%, and the yield is 5.97%.

[0089] Example 2

[0090] The raw material is 100 kg of Fructus Aurantii Immaturus. After sampling and testing, the content of hesperidin in the raw material Fructus Aurantii Immaturus is 32.5%. Taking the above raw material as an example, a two-crystallization method for extracting hesperidin is provided, including the following steps:

[0091] (1) Soak and wash 100 kg of Fructus Aurantii Immaturus raw materials with water, crush the wet materials to 20 mesh, and divide them into five groups for extraction, with each group about 20 kg.

[0092] (2) Impurity removal: The impurity removal solvent is a mixed solvent of 60% methanol + 0.4% NaOH. The impurity removal solvent is added according to a material-liquid ratio of 1:6, and the reaction is carried out at 30 °C for 2 h to remove impurities. After filtration, the raw material after impurity removal and the impurity removal liquid are obtained.

[0093] (3) Extraction: A mixed solvent of 70% methanol + 0.6% NaOH is added to the raw material after impurity removal as the extraction solvent for extraction. The material-liquid ratio is 1:6, the extraction temperature is 25 °C, and the extraction time for each pass is 2 h to obtain the extraction liquid.

[0094] For the five groups of raw materials, a cyclic extraction method is adopted. Among them, the first group of raw materials is extracted 4 times. The extraction liquid of the 3rd extraction of the first group is used as the extraction solvent for the 1st extraction of the next group (the second group), and the extraction liquid of the 4th extraction of the first group is used as the extraction solvent for the 2nd extraction of the next group (the second group). The 3rd and 4th extraction solvents of the second group use fresh liquid. And so on, processed according to the above method, the 3rd extraction liquid and the 4th extraction liquid of the fifth group are no longer recycled. Finally, all the extraction liquids are combined.

[0095] (4) First crystallization: Hydrochloric acid is added to the extraction liquid to adjust the pH to 8. During pH adjustment, stirring is carried out to make the solution mix evenly. It is kept warm at 50 °C for 1 h, left to stand at 25 °C for 10 h, and then centrifuged and filtered to obtain hesperidin crystals and the mother liquor after the first crystallization; the centrifuged crystals are added to a 0.2% Na2CO3 aqueous solution according to a material-liquid ratio of 1:10 and stirred for 2 h for crystal washing; after centrifugation and filtration, deionized water is added according to a material-liquid ratio of 1:3 and stirred for 30 min for crystal washing.

[0096] (5) Second crystallization: Hydrochloric acid is added to the mother liquor after the first crystallization to adjust the pH to 7, and methanol is removed by rotary evaporation and concentration. The concentrated solution is adjusted to pH 8. During pH adjustment, stirring is carried out to make the solution mix evenly. It is kept warm at 70 °C for 1 h, left to stand at 25 °C for 10 h, and then centrifuged and filtered; the centrifuged crystals are added to a 0.4% Na2CO3 aqueous solution according to a material-liquid ratio of 1:10 and stirred for 2 h for crystal washing; after centrifugation and filtration, deionized water is added according to a material-liquid ratio of 1:3 and stirred for 30 min for crystal washing.

[0097] (6) Filtration and drying: Centrifugation filtration is used respectively, and drying is carried out at 75 °C to obtain the hesperidin product.

[0098] Among them, the methanol residue in the hesperidin product obtained by the first crystallization is 239 ppm, and the methanol residue in the hesperidin product obtained by the second crystallization is 103 ppm.

[0099] The total yield of hesperidin obtained by the method of this example is 92.78% (total yield of five groups of raw materials). The average content of hesperidin in the first crystallization is 93.92%, and the yield is 85.61%; the average content of hesperidin in the second crystallization is 90.37%.

[0100] Example 3

[0101] The raw material is 100 kg of Fructus Aurantii Immaturus. Through sampling and testing, the hesperidin content in the raw Fructus Aurantii Immaturus is 26.5%. Fructus Aurantii Immaturus from different origins and with different diameters has different contents, and raw materials from different batches are used for extraction.

[0102] Taking the above raw material as an example, a two - crystallization method for extracting hesperidin is provided, including the following steps:

[0103] (1) Soak and wash 100 kg of Fructus Aurantii Immaturus raw material with water, crush the wet material to 20 - mesh, and divide it into 5 groups for extraction, with each group being about 20 kg.

[0104] (2) Impurity removal: The impurity - removal solvent is a mixed solvent of 60% methanol + 0.4% NaOH. Add the impurity - removal solvent according to a solid - liquid ratio of 1:6, react at 30 °C for 2 h to remove impurities, and filter to obtain the raw material after impurity removal and the impurity - removal liquid.

[0105] (3) Extraction: Add a mixed solvent of 70% methanol + 0.6% NaOH to the raw material after impurity removal as the extraction solvent for extraction. The solid - liquid ratio is 1:6, the extraction temperature is 25 °C, and the extraction time for each pass is 2 h to obtain the extraction liquid.

[0106] For the five groups of raw materials, a circulating - extraction method is adopted. Among them, the first group of raw materials is extracted 4 times. The third extraction liquid of the first group is used as the first - time extraction solvent for the next group (the second group), and the fourth extraction liquid of the first group is used as the second - time extraction solvent for the next group (the second group). The third and fourth extraction solvents for the second group use fresh liquid. And so on, according to the above method for treatment, the third and fourth extraction liquids of the fifth group are not recycled. Finally, all the extraction liquids are combined.

[0107] (4) First crystallization: Adjust the pH of the extraction liquid to 8 with hydrochloric acid. Stir during pH adjustment to make the solution mix evenly, keep it warm at 50 °C for 1 h, stand at 25 °C for 10 h, and centrifuge and filter to obtain hesperidin crystals and the mother liquor after the first crystallization; Add the centrifuged crystals to 0.2% Na2CO3 aqueous solution according to a solid - liquid ratio of 1:10 and stir for 2 h to wash the crystals; Centrifuge and filter, and then add deionized water according to a solid - liquid ratio of 1:3 and stir for 30 min to wash the crystals.

[0108] (5) Second crystallization: Adjust the pH of the mother liquor after the first crystallization to 7 with hydrochloric acid, rotary evaporate and concentrate to remove methanol, adjust the pH of the concentrated solution to 8. Stir during pH adjustment to make the solution mix evenly, keep it warm at 70 °C for 1 h, stand at 25 °C for 10 h, and centrifuge and filter; Add the centrifuged crystals to 0.4% Na2CO3 aqueous solution according to a solid - liquid ratio of 1:10 and stir for 2 h to wash the crystals; Centrifuge and filter, and then add deionized water according to a solid - liquid ratio of 1:3 and stir for 30 min to wash the crystals.

[0109] (6) Filtration and drying: Filter with a centrifuge respectively and dry at 75 °C to obtain the hesperidin product.

[0110] Among them, the methanol residue in the hesperidin product obtained by the first crystallization is 233 ppm, and the methanol residue in the hesperidin product obtained by the second crystallization is 89 ppm.

[0111] The total yield of hesperidin obtained by the method of this example is 92.72% (total yield of five groups of raw materials). The average content of hesperidin in the first crystallization is 91.81%, and the yield is 83.98%; the average content of hesperidin in the second crystallization is 88.66%.

[0112] Example 4

[0113] Example 4 is a small-scale test. The raw material used is 10 kg of Fructus Aurantii Immaturus. The content of hesperidin in the raw material Fructus Aurantii Immaturus is detected by sampling to be 35.5%. Taking the said raw material as an example, a two-stage crystallization method for extracting hesperidin is provided, including the following steps:

[0114] (1) 10 kg of Fructus Aurantii Immaturus raw materials are soaked in water for cleaning, and the wet materials are pulverized to 20 meshes, and divided into five groups for extraction, 2 kg for each group.

[0115] (2) Impurity removal: The impurity removal solvents are respectively selected as mixed solutions with concentrations of 60% methanol + 0.1% NaOH, 60% methanol + 0.2% NaOH, 60% methanol + 0.4% NaOH, 60% methanol + 0.6% NaOH, and 60% methanol + 0.8% NaOH (see Table 1 for details). According to the material-liquid ratio of 1:6, the impurity removal solvent is added, and the reaction is carried out at 30 °C for 2 h to remove impurities, and the raw materials and impurity removal liquid after impurity removal are obtained by filtration.

[0116] Other steps are the same as those in Example 1, that is, each group is continued to be divided into five groups of raw materials, and the method of cyclic nested extraction is adopted. For example, when in step (3) for group A, it is continued to be divided into five groups of raw materials, and the method of cyclic nested extraction is used to extract hesperidin.

[0117] The results are as follows:

[0118] Table 1

[0119]

[0120] Note: The average content % of hesperidin in the said product is the average hesperidin content calculated from the hesperidin products obtained by the first crystallization and the hesperidin products obtained by the second crystallization of five groups of raw materials.

[0121] The impurity removal solvent uses an aqueous solution of methanol added with a weakly basic substance. Among them, the weakly basic substance reacts with the strong acidic substance in the impurity, and the dosage of sodium hydroxide base is the most critical. Therefore, through research in this application, the impurity removal solvent is selected as a mixed solution of 40%-80% lower saturated monohydric alcohol and 0.1%-0.8% base; more preferably a mixed solution of 40%-60% lower saturated monohydric alcohol and 0.4%-0.6% base; the optimal base dosage is 0.4% sodium hydroxide solution.

[0122] The impurity-removing solvent of 60% methanol + sodium hydroxide, with the sodium hydroxide concentration below 0.4%. Compared with the impurity-removing effect of 0.4% sodium hydroxide, the hesperidin content in the hesperidin product after extraction is relatively lower, and the average yield has little difference; when the sodium hydroxide concentration is above 0.4%, compared with the impurity-removing effect of 0.4% sodium hydroxide, the hesperidin content after extraction slightly increases, while the yield significantly decreases. Therefore, the optimal impurity-removing solvent is the mixed solution of 60% methanol + 0.4% sodium hydroxide.

[0123] Example 5

[0124] Example 5 is a small-scale test. The raw material used is 10 kg of Fructus Aurantii Immaturus. The hesperidin content in the raw material Fructus Aurantii Immaturus is detected by sampling to be 35.5%. Taking the said raw material as an example, a two-crystallization method for extracting hesperidin is provided, including the following steps:

[0125] (1) 10 kg of Fructus Aurantii Immaturus raw material is soaked in water for cleaning, and the wet material is crushed to 20 mesh, and then divided into 5 groups for extraction, with 2 kg in each group.

[0126] (2) Impurity removal: The impurity-removing solvents are respectively selected as the mixed solvents of 40% methanol + 0.4% NaOH, 50% methanol + 0.4% NaOH, 60% methanol + 0.4% NaOH, 70% methanol + 0.4% NaOH, and 80% methanol + 0.4% NaOH (see Table 2 for details). According to the material-liquid ratio of 1:6, the impurity-removing solvent is added, and the mixture is stirred and extracted at 30 °C for 2 h to remove impurities. The hesperidin content in each pass of the liquid and the residue is measured.

[0127] Other steps are the same as those in Example 1, that is, each group is further divided into five groups of raw materials, and the cyclic extraction method is adopted. For example, when in step (3) for group F, it is further divided into five groups of raw materials, and the cyclic extraction method is used to extract hesperidin.

[0128] The results are as follows:

[0129] Table 2

[0130]

[0131] Note: The average content % of hesperidin in the said product is the average hesperidin content calculated from the hesperidin products obtained by the first crystallization and the second crystallization of the five groups of raw materials.

[0132] The solubility of sodium hydroxide in methanol is relatively small, and water is needed to increase the solubility of sodium hydroxide in methanol and to balance the removal of alcohol-soluble and water-soluble impurities in the raw materials. If the proportion of methanol in the impurity removal solvent is too high, the solubility of sodium hydroxide is small, and there are few water-soluble impurities in the impurity removal liquid, resulting in a low product content and yield. If the proportion of methanol in the impurity removal solvent is too low, a large amount of hesperidin is lost during the impurity removal process, the product yield is low, and the energy consumption for recycling low-concentration methanol is high. Based on the above reasons and test results, the best impurity removal solvent is a mixed solution of 60% methanol + 0.4% sodium hydroxide.

[0133] In summary, through research in this application, the impurity removal solvent is a mixed solution of 40%-80% lower saturated monohydric alcohol and 0.1-0.8% alkali; more preferably, it is a mixed solution of 40%-60% lower saturated monohydric alcohol and 0.4%-0.6% alkali; the best is a mixed solution of 60% methanol + 0.4% sodium hydroxide.

[0134] Example 6

[0135] Example 6 is a small-scale test. The raw material used is 10 kg of Fructus Aurantii Immaturus. The content of hesperidin in the raw material Fructus Aurantii Immaturus is detected by sampling to be 35.5%. Taking the above raw material as an example, a two-stage crystallization method for extracting hesperidin is provided, including the following steps:

[0136] (1) Soak and wash 10 kg of Fructus Aurantii Immaturus raw material with water, crush the wet material to 10 mesh, and divide it into five groups for extraction, with 2 kg in each group.

[0137] (2) Impurity removal: The impurity removal solvent is a mixed solution of 60% methanol + 0.4% NaOH. Add the impurity removal solvent according to a material-liquid ratio of 1:5, react at 30 °C for 2 h to remove impurities, and filter to obtain the raw material after impurity removal and the impurity removal liquid. Recover the solvent from this impurity removal liquid.

[0138] (3) Extraction: Add an extraction solvent to the raw material after impurity removal for the extraction of hesperidin. The extraction solvent is a mixed solution of 60% methanol + 0.8% NaOH, the material-liquid ratio is 1:5 kg / L, the extraction temperature is 25 °C, and the extraction time for each pass is 2 h. Filter to obtain the filter residue and the extraction liquid.

[0139] The five groups of raw materials adopt a method of cyclic countercurrent extraction. Among them, the first group of raw materials is extracted 4 times. The third extraction liquid is used as the first extraction solvent for the next group (the second group), and the fourth extraction liquid is used as the second extraction solvent for the next group (the second group). The third and fourth extraction solvents for the second group use fresh liquid. And so on, and processed according to the above method, the third and fourth extraction liquids of the fifth group are no longer recycled. Finally, all the extraction liquids are combined.

[0140] (4) First crystallization: Adjust the pH of the extract to 8 with hydrochloric acid. Stir the solution evenly during pH adjustment, keep it at 40 °C for 2 h, let it stand at 25 °C for 10 h, and then centrifuge and filter to obtain hesperidin crystals and the mother liquor after the first crystallization. Add the centrifuged crystals to 0.2% Na2CO3 aqueous solution at a solid-liquid ratio of 1:10, stir for 2 h for crystal washing, centrifuge and filter, and then add deionized water at a solid-liquid ratio of 1:3, stir for 30 min for crystal washing.

[0141] (5) Second crystallization: Adjust the pH of the mother liquor after the first crystallization to 7 with hydrochloric acid, remove methanol by rotary evaporation and concentration. Adjust the pH of the concentrated solution to 8, stir the solution evenly during pH adjustment, keep it at 60 °C for 1 h, let it stand at 25 °C for 10 h, and then centrifuge and filter to obtain hesperidin crystals. Add the centrifuged crystals to 0.4% Na2CO3 aqueous solution at a solid-liquid ratio of 1:10, stir for 2 h for crystal washing, centrifuge and filter, and then add deionized water at a solid-liquid ratio of 1:3, stir for 30 min for crystal washing.

[0142] (6) Filtration and drying: Filter with a centrifuge respectively and dry at 75 °C to obtain hesperidin products. Among them, for the hesperidin products obtained by the first crystallization, after detection, the residual methanol is 291 ppm, and for the hesperidin products obtained by the second crystallization, after detection, the residual methanol is 109 ppm.

[0143] The total yield of hesperidin obtained by the method of this example is 89.73% (total yield of five groups of raw materials). The average content of hesperidin in the first crystallization is 92.69%, and the yield is 82.35%; the average content of hesperidin in the second crystallization is 88.78%.

[0144] Test Example 1: Methanol detection and HPLC analysis of hesperidin

[0145] In the general rules of the Chinese Pharmacopoeia 2020 edition, it is stipulated that the raw materials of drugs need to meet the requirements of residual solvents. According to the ICH Q3C classification, methanol belongs to Class 3 solvents (low toxicity), and the corresponding concentration limit is usually 5000 ppm (0.5%).

[0146] The detection of methanol in the crystals is to detect methanol in the dried hesperidin products, and then calculate the content of methanol in the detected samples, with the unit of ppm, where ppm means "parts per million".

[0147] The methanol residue standard of hesperidin needs to be combined with the following basis:

[0148] Drug grade: Referring to the general rules of the pharmacopoeia, the limit is generally ≤5000 ppm (0.5%).

[0149] Food and plant extracts: It can be based on specific industry standards or the general requirements of GB 2760. Usually, the limit is similar to that of the drug grade.

[0150] After analysis, the methanol residues in the hesperidin products obtained in the above examples all meet the standards.

[0151] Figure 2 It is the liquid chromatogram of hesperidin in Example 1. Figure 2 The results are shown in Table 3.

[0152] Table 3

[0153] Sequence Name Retention time, min 1 Narirutin 6.009 2 Hesperidin 8.903 3 Neohesperidin 19.951 4 Hesperetin 24.716

[0154] Comparative Example 1

[0155] Comparative Example 1 is a small-scale test, using a conventional crystallization method (such as single crystallization method or one-step crystallization method).

[0156] The specific steps are as follows:

[0157] Steps (1)-(3) are the same as those in Example 1, and the hesperidin content in the raw materials is also the same. It is the same batch of Fructus Aurantii Immaturus raw materials, and the raw material dosage is adjusted to 10 kg.

[0158] After completing steps (1)-(3) and obtaining the extract, continue with the following experimental steps.

[0159] (4) Crystallization: Adjust the pH of the extract to 7.0, rotary evaporate and concentrate to remove methanol. Add hydrochloric acid to the concentrated solution to adjust the pH to 8. Stir the solution evenly during pH adjustment, keep it at 70 °C for 1 h, let it stand at 25 °C for 10 h, and centrifuge and filter; Add the centrifuged crystals to 0.2% Na2CO3 aqueous solution at a solid-liquid ratio of 1:10 and stir for 2 h to wash the crystals; Centrifuge and filter, and then add deionized water at a solid-liquid ratio of 1:3 and stir for 30 min to wash the crystals.

[0160] (5) Filtration and drying: Filter with a centrifuge and dry at 75 °C to obtain the hesperidin product. The total yield of the hesperidin product obtained by single crystallization is 90.17%, and the hesperidin content in the crystalline product is 92.23%.

[0161] Compared with the results of Example 1, the total yield of hesperidin in Example 1 is 92.32%, the hesperidin content in single crystallization is 95.57%, and the yield is 86.35%; the hesperidin content in secondary crystallization is 91.29%, and the yield is 5.97%. (The yield of secondary crystallization is the total amount of hesperidin obtained in the second crystallization / the total amount of theoretical hesperidin, so it will be lower than the first yield).

[0162] Through comparison, it is found that the primary crystallization of Comparative Example 1 is slightly lower than the secondary crystallization of Example 1 in terms of the total yield. After the first crystallization in the secondary crystallization of Example 1, a product with a high content of 95.57% and a yield accounting for 86.35% can be obtained. Although the content of the product in the second crystallization decreases to 91.29%, which is only 0.94% lower than the content of the product in the primary crystallization of D1, it still reaches the content of the salable product.

[0163] The level of hesperidin content is the grading standard for hesperidin products. Generally speaking, products with a hesperidin content of more than 90% are first-class products, and products with a hesperidin content of 70%-90% are second-class products. The price of hesperidin products is also related to the hesperidin content. Therefore, the present application can obtain hesperidin products with high yields and high contents.

[0164] The total yield of the secondary crystallization in Example 1 is 2.15% higher than that of the primary crystallization in Comparative Example 1, which means more output and higher product profit. Moreover, 86.35% of the products obtained by the two crystallizations are high-content hesperidin with a content of over 95%, which cannot be achieved by the primary crystallization. Compared in terms of both yield and content, the technical solution of the example in the present application has significant advantages.

[0165] Comparative Example 2

[0166] Comparative Example 2 is a small-scale test, and the pH adjustment of acid addition in the crystallization step is adjusted to 6.5.

[0167] The specific steps are as follows:

[0168] Steps (1)-(3) are the same as those in Example 1, and the hesperidin content in the raw materials is also the same. It is the same batch of immature bitter orange raw materials, and the raw material dosage is adjusted to 10 kg.

[0169] After completing steps (1)-(3) and obtaining the extract, continue with the following experimental steps.

[0170] (4) Primary crystallization: Add hydrochloric acid to the extract to adjust the pH to 6.5. Stir the solution evenly during pH adjustment, keep it at 50°C for 1 h, stand still at 25°C for 10 h, and then perform centrifugal filtration to obtain hesperidin crystals and the mother liquor after the first crystallization; Add the centrifuged crystals to the 0.2% Na2CO3 aqueous solution at a solid-liquid ratio of 1:10 and stir for 2 h for crystal washing; Perform centrifugal filtration, and then add deionized water at a solid-liquid ratio of 1:3 and stir for 30 min for crystal washing.

[0171] (5) Second crystallization: The mother liquor after the first crystallization is adjusted to pH 7 with hydrochloric acid, and the methanol is removed by rotary evaporation and concentration. The concentrated solution is adjusted to pH 6.5. During the pH adjustment, the solution is stirred to make it uniformly mixed. It is kept at 70 °C for 1 h, left standing at 25 °C for 10 h, and then centrifuged and filtered; The centrifuged crystals are added to a 0.4% aqueous solution of Na2CO3 at a solid-liquid ratio of 1:10 and stirred for 2 h to wash the crystals; Centrifuged and filtered, and then deionized water is added at a solid-liquid ratio of 1:3 and stirred for 30 min to wash the crystals.

[0172] (6) Filtration and drying: Centrifuged and filtered respectively, and dried at 75 °C to obtain the hesperidin product.

[0173] For this comparative example method, the total yield of hesperidin is 88.35%, the average content of hesperidin in the first crystallization is 93.24%, and the yield is 83.81%; The average content of hesperidin in the second crystallization is 85.38%. Compared with the results of Example 1, in Example 1, the total yield of hesperidin is 92.32%, the average content of hesperidin in the first crystallization is 95.57%, and the yield is 86.35%; The average content of hesperidin in the second crystallization is 91.29%.

[0174] It is found by comparison that when the pH is adjusted to 6.5 in the crystallization step, both the hesperidin content and the yield of the product decrease.

[0175] Comparative Example 3

[0176] Comparative Example 3 is a small-scale test, and the temperature-raising and heat-preserving steps are not carried out in the first crystallization and the second crystallization of the crystallization step.

[0177] The specific steps are as follows:

[0178] Steps (1)-(3) are the same as those in Example 1, and the hesperidin content in the raw materials is also the same. It is the same batch of Fructus Aurantii Immaturus raw materials, and the raw material dosage is adjusted to 10 kg.

[0179] After completing steps (1)-(3) and obtaining the extract, the following experimental steps are continued.

[0180] (4) First crystallization: The extract is adjusted to pH 8 with hydrochloric acid. During the pH adjustment, the solution is stirred to make it uniformly mixed. It is left standing at 25 °C for 10 h, and then centrifuged and filtered to obtain hesperidin crystals and the mother liquor after the first crystallization; The centrifuged crystals are added to a 0.2% aqueous solution of Na2CO3 at a solid-liquid ratio of 1:10 and stirred for 2 h to wash the crystals, centrifuged and filtered, and then deionized water is added at a solid-liquid ratio of 1:3 and stirred for 30 min to wash the crystals.

[0181] (5) Second crystallization: Adjust the pH of the mother liquor after the first crystallization to 7 with hydrochloric acid, rotary evaporate and concentrate to remove methanol, adjust the pH of the concentrated solution to 8, stir the solution evenly during pH adjustment, let it stand at 25 °C for 10 h, and centrifuge and filter to obtain hesperidin crystals; Add the centrifuged crystals to 0.4% Na2CO3 aqueous solution at a solid-liquid ratio of 1:10 and stir for 2 h to wash the crystals, centrifuge and filter, and then add deionized water at a solid-liquid ratio of 1:3 and stir for 30 min to wash the crystals.

[0182] (6) Filtration and drying: Filter with a centrifuge respectively and dry at 75 °C to obtain the hesperidin product.

[0183] For the method of this comparative example, the total yield of hesperidin was 89.25% (total yield of five groups of raw materials), the average content of hesperidin in the first crystallization was 95.29%, and the yield was 82.91%; the average content of hesperidin in the second crystallization was 90.60%. Compared with the results of Example 1, the total yield of hesperidin in Example 1 was 92.32%, the average content of hesperidin in the first crystallization was 95.57%, and the yield was 86.35%; the average content of hesperidin in the second crystallization was 91.29%.

[0184] Because the heating and heat preservation process was not carried out, the content of the hesperidin product decreased slightly, but not significantly. However, the lack of the heat preservation process affected the yields of the two crystallizations (compared with Example 1, the total yield decreased by 3.07%). The absence of the heating and heat preservation process led to the hindrance of hesperidin crystal nucleation formation, the decrease of growth rate and the increase of impurity entrainment through the triple mechanisms of reducing the supersaturation of the extraction solution or mother liquor, slowing down molecular diffusion, and increasing structural frustration, ultimately reducing the crystallization yield of hesperidin.

[0185] This comparative example fully illustrates the importance of the heat preservation treatment, and this step will affect the product yield. Under the condition of high product yield, the profit will increase accordingly.

[0186] Comparative Example 4

[0187] Comparative Example 4 was a small-scale test, and ethanol was used to replace methanol in the solvents for the extraction and impurity removal steps.

[0188] The specific steps are as follows:

[0189] Step (1) was the same as that in Example 1, and the content of hesperidin in the raw materials was also the same. It was the same batch of Fructus Aurantii Immaturus raw materials, and the raw material dosage was adjusted to 10 kg. After completing step (1), continue with the following experimental steps.

[0190] (2) Impurity removal: The impurity removal solvent was a mixed solution of 60% ethanol + 0.4% NaOH. Add the impurity removal solvent according to a solid-liquid ratio of 1:6, react at 30 °C for 2 h to remove impurities, and filter to obtain the raw materials after impurity removal and the impurity removal solution. Recover the solvent from the impurity removal solution.

[0191] (3) Extraction: Add an extraction solvent to the raw materials that have been purified to extract hesperidin. The extraction solvent is a mixed solution of 60% ethanol + 0.6% NaOH, the material-liquid ratio is 1:6 kg / L, the extraction temperature is 25°C, the extraction time for each pass is 2 h, and the filter residue and extraction solution are obtained by filtration. Five groups of raw materials are subjected to cyclic extraction in the same method as before.

[0192] Steps (4), (5), and (6) are the same as those in Example 1.

[0193] For this comparative example method, the total yield of hesperidin is 87.23%, the average content of hesperidin in the first crystallization is 91.71%, and the yield is 82.15%; the average content of hesperidin in the second crystallization is 89.33%. Compared with the results of Example 1, in Example 1, the total yield of hesperidin is 92.32%, the content in the first crystallization is 95.57%, and the yield is 86.35%; the content in the second crystallization is 91.29%.

[0194] It is found by comparison that when the methanol in the extraction and purification steps is replaced with ethanol, both the content and yield of hesperidin in the product decrease.

[0195] Methanol is CH3OH and ethanol is C2H5OH. Their structures are similar, both containing hydroxyl groups, but the carbon chain lengths are different. This may affect their physical and chemical properties such as polarity, boiling point, and solubility. Their extraction efficiencies may vary due to different molecular characteristics. Methanol is more likely to extract low-fat-soluble substances, has a low solubility in lipid and wax components, and is commonly used in the extraction of plant active ingredients such as flavonoids and saponins. Ethanol has better lipid solubility and can dissolve some lipid-soluble components such as vitamin E and plant essential oils.

[0196] In actual production, it is necessary to balance the content and yield of hesperidin. A high content without a high yield cannot ensure the maximization of benefits in profit calculation. Therefore, the technical solution of this application has better profits for the obtained hesperidin products on the premise of maintaining a high yield and a certain standard of hesperidin content, and is suitable for large-scale industrial promotion.

[0197] Test Example 2: Wastewater calculation

[0198] Calculate the wastewater in the production process and compare it with the technology mentioned in the literature of the prior art

[0199] Taking Example 1 as an example, the amount of wastewater discharged per ton of raw material extraction is calculated. By extracting 5 groups of raw materials, the first group of raw materials is extracted 4 times, the third extraction liquid is used as the first extraction solvent for the next group (i.e., the second group), the fourth extraction liquid is used as the second extraction solvent for the next group (i.e., the second group), and the third and fourth extraction solvents of the second group are new liquid, and so on. Five groups of raw materials are extracted, the impurity removal and extraction material-liquid ratio is 1:6, the extraction solvent is applied for cyclic extraction, 60% of which is methanol. Recycled water (i.e., water used for refined products (i.e., water used in the crystal washing process) generates about 5 tons of wastewater per ton of material.

[0200] The alkaline water extraction process, even if the same parameters as the alkaline extraction process are used, will produce about 12.5 tons of wastewater per ton of material. Generally speaking, the water extraction method has weak impurity removal and extraction effects on hesperidin, and it is necessary to increase the material-liquid ratio, so that the amount of wastewater produced per ton of raw material will be greater. For example: Li Hong's paper "Research on Process Conditions for Extraction of Hesperidin" uses saturated lime water to extract hesperidin, and the extraction efficiency is highest when the material-liquid ratio is 1:10. Zhang Juhua's paper "Research on Industrial Co-production Process of Hesperidin and Synephrine in Citrus Aurantium" uses saturated lime water and 0.1% NaOH aqueous solution with a material-liquid ratio of 1:40 to extract hesperidin, and the extraction rate is equivalent. When hesperidin is extracted with a material-liquid ratio of 1:40, it is preliminarily estimated that the wastewater produced per ton of raw material is about 30 tons.

[0201] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A method for extracting hesperidin, characterized in that, It includes the following steps: (1) Clean and crush the raw materials rich in hesperidin; (2) Add an impurity-removing solvent to carry out an impurity-removing reaction, and perform solid-liquid separation to obtain the impurity-removed raw materials and the impurity-removing liquid; the impurity-removing solvent is a mixed solution of a lower saturated monohydric alcohol with a volume percentage of 40%-80% and an alkali with a mass percentage of 0.1%-0.8%; (3) Add an extraction solvent to the impurity-removed raw materials to carry out an extraction reaction, and perform solid-liquid separation to obtain the extraction liquid; the extraction solvent is a mixed solution of a lower saturated monohydric alcohol with a volume percentage of 40%-80% and an alkali with a mass percentage of 0.3%-1.0%; (4) Adjust the pH of the extraction liquid to 7.5-8.5 with acid; keep it warm at 40-60°C for 1-2 h, let it stand at 20-30°C for 8-12 h, carry out the first crystallization reaction, and perform solid-liquid separation to obtain hesperidin crystals and the mother liquor; (5) Adjust the pH of the mother liquor to 6.5-7.5 with acid, evaporate and concentrate to remove the lower saturated monohydric alcohol to obtain a concentrated liquid, adjust the pH of the concentrated liquid to 7.5-8.5, keep it warm at 60-80°C for 1-2 h, let it stand at 20-30°C for 8-12 h, carry out the second crystallization reaction, and perform solid-liquid separation to obtain hesperidin crystals.

2. The method according to claim 1, characterized in that In the step (1), the raw materials are the dried young fruits of oranges or tangerines.

3. The method according to claim 1, characterized in that In the step (1), the crushing mesh number is 2-40 meshes, preferably 10-40 meshes, and more preferably 20-30 meshes.

4. The method according to claim 1, characterized in that, The lower saturated monohydric alcohol is a C1-C4 saturated monohydric alcohol, including at least one of methanol and ethanol, preferably methanol; and / or, the alkali includes at least one of sodium hydroxide, potassium hydroxide, and calcium hydroxide.

5. The method according to any one of claims 1-4, characterized in that, In the step (2), the impurity-removing solvent is a mixed solution of 40%-60% lower saturated monohydric alcohol and 0.4%-0.6% alkali.

6. The method according to any one of claims 1-4, characterized in that, In the step (3), the extraction solvent is a mixed solution of 40%-60% lower saturated monohydric alcohol and 0.4%-0.7% alkali.

7. The method according to any one of claims 1-4, characterized in that In the step (2), the impurity-removing solvent is added according to a material-liquid ratio of 1:(2-20) kg:L, preferably a material-liquid ratio of 1:(2-10) kg / L, and more preferably a material-liquid ratio of 1:(6-10) kg / L; and / or, in the step (2), the temperature of the impurity-removing reaction is 10°C-40°C; the time of the impurity-removing reaction is 0.5 h-4 h.

8. The method according to any one of claims 1 to 4, characterized in that, In the step (3), the extraction solvent is added according to a material-liquid ratio of 1:(2-20) kg:L, preferably a material-liquid ratio of 1:(2-10) kg / L, and more preferably a material-liquid ratio of 1:(6-10) kg / L; and / or, in the step (3), the temperature of the extraction reaction is 10°C-40°C; the time of the extraction reaction is 0.5 h-4 h.

9. The method according to any one of claims 1 to 4, characterized in that In the step (3), the impurity-removed raw materials are divided into N groups, the first group of raw materials is extracted 4 times, the third extraction liquid is used as the first extraction solvent for the next group, the fourth extraction liquid is used as the second extraction solvent for the next group, and so on, where N is 3-6 groups, preferably 4-6 groups.

10. A hesperidin product, characterized in that, The hesperidin product is prepared by the method according to any one of claims 1-9.

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