CN111978362B

CN111978362B - Method for removing iso-naringin in natural product hesperidin

Info

Publication number: CN111978362B
Application number: CN202010925937.5A
Authority: CN
Inventors: 崔仁发
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-09-07
Filing date: 2020-09-07
Publication date: 2023-05-09
Anticipated expiration: 2040-09-07
Also published as: CN111978362A

Abstract

The invention provides a method for removing impurity iso-naringin in a plant extract hesperidin, which comprises the following steps: selectively reacting the 4' -phenolic hydroxyl of the naringin with a small amount of phenolic hydroxyl protecting groups to weaken the polarity of the naringin, and then adsorbing the naringin by nonpolar or low-polarity macroporous resin to remove the naringin; when the 4 '-phenolic hydroxyl of the naringin is selectively reacted, the 3' -phenolic hydroxyl of the hesperidin also participates in the reaction, but the polarity of the obtained product is weakened, and the product is adsorbed and removed by macroporous resin like the naringin. According to the invention, through the reactivity difference of the naringin and the active phenolic hydroxyl of the hesperidin, the chemical reaction is combined with resin adsorption to remove impurities, and a small amount of main components are sacrificed, so that a product with the naringin content as low as 0 and the hesperidin purity of more than 98% can be obtained, and the raw material requirement for producing high-purity diosmin is met.

Description

Method for removing iso-naringin in natural product hesperidin

Technical Field

The invention belongs to the technical field of plant extraction, and particularly relates to a purification method of natural extraction product hesperidin.

Background

Hesperidin, also known as hesperidin, and dihydroflavone glycoside. Is active flavonoid extracted from fruit or skin of Citrus plant of Rutaceae such as fructus Aurantii Immaturus, pericarpium Citri Tangerinae, and pericarpium Citri Junoris, and the pure product is pure white fine dendritic needle crystal. Has the functions of reducing the fragility of capillary blood vessels, protecting the capillary blood vessels, preventing capillary rupture bleeding (similar to the action of vitamin P), inhibiting the rise of blood cholesterol and preventing arteriosclerosis, and is a medicament for treating hypertension and myocardial infarction; has good effects of resisting oxidation, reducing blood sugar, relieving inflammation and pain, resisting tumor and resisting virus. In addition to itself as an additive for medicines, foods and cosmetics, hesperidin is mainly used as a raw material for producing the medicines diosmin and methyl hesperidin. Dioxomine which is prepared by semi-chemical synthesis by taking hesperidin as a raw material is used as a common medicament for treating various symptoms related to venous and lymphatic insufficiency and acute hemorrhoids in Europe; through the reaction of the hesperidin and a methylation reagent, the phenolic hydroxyl at the 3' -position of the hesperidin is methylated to generate methyl hesperidin, so that the effect of vitamin P is achieved, and the effect of vitamin C can be enhanced by combining the methyl hesperidin with the vitamin C; the methyl hesperidin has strong antiviral and antibacterial effects, and can inhibit the reproduction of influenza virus by large-dose application; in addition, it can inhibit the activity of tyrosinase, which causes skin to turn black, and can be used for treating skin diseases such as black spots, freckles, etc., and has important uses in the beauty and cosmetic industries.

The most widely used methods for extracting hesperidin from immature bitter orange, citrus peel and the like at present are a water solvent extraction method and an organic solvent extraction method, wherein proper alkali is added into a solvent, after the hesperidin is dissolved in an alkaline solvent, the hesperidin is precipitated by acid, and the alkali-acid precipitation method is used for short. Due to the complexity of the peel components, such as vegetable proteins, pigments, vegetable tannins, pectins, starches, polysaccharides, various small molecular substances, inorganic salts, and the like. Photoflavonoids are better species with very small structural differences from each other, with only 1-2 methyl, hydroxyl or sugar molecules. Thus, the hesperidin extracted from the plant is mixed with a plurality of natural impurity products similar or similar to the natural impurity products in structure, and mainly comprises naringin and the like (the structure is shown in the following figure). The quality of the raw materials for producing the hesperidin is evaluated, and besides the index of the hesperidin content, an important index, namely the content of the iso-naringin is also provided. The higher quality orange peel of the immature bitter orange has high hesperidin content and low iso-naringin content; on the contrary, the inferior orange peel glycoside of the immature bitter orange has low content and the iso-naringin has high content. At present, the purity of the hesperidin extracted from natural plants in the market is generally three specifications, namely 85%, 90% and 95%, and only the most excellent raw materials can produce products with the specification of 95%. However, no matter which specification of hesperidin, a certain amount of naringin impurities are contained in the hesperidin, the content is generally between 1 and 5 percent, and more than 5 percent is regarded as unqualified; the European client demands are more stringent, typically below 2.5%. Therefore, raw materials with high content of the naringin cannot be used for producing qualified hesperidin, and resource waste is caused. Hesperidin itself, whether used as a pharmaceutical product or as a raw material for the production of pharmaceutical products such as diosmin, is required to have a high content of the main component (preferably up to 98% or more) and a low content of impurities, in particular, a low content of iso-naringin. Therefore, how to remove impurities such as iso-naringin in natural plant extract hesperidin becomes a difficulty in the industry of hesperidin extraction. The solution of the problem has important significance for improving income of fruit growers, expanding raw material application range of hesperidin manufacturers, reasonably using natural resources and improving yield and quality of the whole hesperidin industry.

From the molecular structure, the hesperidin, the naringin and the melissa glucoside are very close. The difference of whether the 4 '-phenolic hydroxyl group of the naringin and the melissa glucoside is methylated or not is that the naringin is phenolic hydroxyl group, and the melissa glucoside is a product of methylation of the 4' -phenolic hydroxyl group of the naringin, namely, the melissa glucoside is methylated naringin; whereas hesperidin has only one more phenolic hydroxyl group at the 3' position than melissa glucoside. Theoretically, the impurity naringin can be separated out by recrystallization, and it is difficult to find a suitable solvent to dissolve the hesperidin. Solvents such as pyridine, N-Dimethylformamide (DMF), morpholine, dimethyl sulfoxide (DMSO) and the like which can dissolve hesperidin are all high-boiling solvents, are not easy to separate from products after recrystallization, and cause solvent residues, which are disastrous for natural products of hesperidin as food or medicines. If the main components are dissolved in an alkaline aqueous solvent such as NaOH aqueous solvent and then acidified, impurities and hesperidin are separated out together due to the similar pKa, and the aim of removing impurities cannot be achieved. Therefore, a new way must be developed to find a reasonable way to purify hesperidin.

Disclosure of Invention

Based on the full investigation of documents and a large number of experiments, the invention creatively proposes the combination of chemical reaction and macroporous resin adsorption according to the difference of chemical reactivity of active groups in the molecular structure of the impurity naringin and the main ingredient hesperidin and the tiny difference of molecular polarity after derivative conversion, and the aim of removing impurities is achieved by sacrificing a small amount of main ingredient. The method is simple, the cost is low, the yield is high, and the purity of the obtained product is high, so that the method is a good method for purifying the natural product hesperidin.

The invention is realized by the following technical scheme, which comprises the following steps:

1. and (3) dissolving: in a dilute alkaline solution, the crude hesperidin is slowly added to dissolve the hesperidin. Cooling after complete dissolution.

2. Methylation: and slowly dropwise adding a small amount of methylation reagent into the hesperidin solution, and keeping a low temperature in the dropwise adding process. And (5) heating to react after the dripping is finished.

3. Column passing step: allowing the reacted solution to pass through nonpolar or low-polar macroporous adsorption resin, and adsorbing methylated naringin, such as melissa glucoside, methylated hesperidin, such as methyl hesperidin, and impurities such as essential component melissa glucoside in hesperidin.

4. And (3) an acidification step: acidifying the solution, standing for precipitation, and centrifuging to obtain high-purity hesperidin product.

5. Elution: eluting macroporous adsorption resin with a large amount of pure water to obtain methyl hesperidin; eluting with large amount of ethanol to obtain the final product.

Further, the alkali used in the "dissolving step" is NaOH, and the solvent is water. The mass ratio of the hesperidin to the solvent water is 1:1-1: 20, preferably 1:3 to 1:10; the amount of NaOH substance is 2-8 times, preferably 2-4 times the amount of hesperidin substance.

Further, after the dissolution in the "dissolution step", the temperature is lowered to-20 to 15℃and preferably-10 to 5 ℃.

Further, the methylating agent in the "methylating step" is dimethyl sulfate, methyl iodide, dimethyl carbonate, etc., preferably dimethyl sulfate; the amount of dimethyl sulfate is 0.5-30 times, preferably 2-15 times the amount of naringin.

Further, the temperature during the dropwise addition in the "methylation step" is-20 to 15 ℃, preferably-10 to 5 ℃; after the completion of the dropwise addition, the temperature is raised to 20-80 ℃, preferably 20-60 ℃.

Further, the dropwise addition time of the methylating agent in the "methylating step" is 0.5 to 6 hours, preferably 0.5 to 2.5 hours; the reaction time for heating is 2 to 10 hours, preferably 2 to 6 hours.

Further, the amount of the methylation reagent required in the "methylation step" is based on monitoring the content of the naringin in real time by liquid chromatography, and when the peak of the naringin is reduced to a low level (the peak height or the peak area is reduced to 10% or less of the initial value or even lower), the dropwise addition of the methylation reagent is stopped.

Further, the time required for the temperature-rising reaction in the methylation step is also based on monitoring the peak height or peak area of the naringin in real time by liquid chromatography, and the reaction is stopped when the peak height or peak area is reduced to below 10% of the initial value or even lower.

Further, in the "column passing step", the resin used is a nonpolar or weakly polar macroporous adsorbent resin such as AB-8, SPD-300, LSA-40, D101, CAD-40, NKA-9, polyamide column, etc.

Further, in the "acidification step", the acid used is preferably hydrochloric acid, and is acidified to a pH of about 6 to 8, preferably about 7. The acidification time is preferably 1 to 20 hours, preferably 4 to 10 hours. Slowly acidizing to facilitate the growth of the hesperidin dendritic needle-like crystal.

Further, in the eluting step, pure water and ethanol are respectively adopted for eluting, so that methyl hesperidin and melissa glucoside can be respectively obtained. The volume of the pure water and the ethanol is 1-10BV, preferably 2-6BV.

Disclosure of Invention

In order to better understand the technical solution of the present invention, the present invention will be more clearly and completely described in the following in connection with specific embodiments. It is apparent that the embodiments described below are only some, but not all, implementations of the present application and that other similar or similar applications of the invention, as proposed by those skilled in the art without making the inventive concepts and the efforts, are deemed to be infringement of this invention.

The crude hesperidin used in examples 1-3 is labeled "hesperidin 1"; the crude hesperidin used in examples 4-6 was labeled "hesperidin 2". The content, purity and impurity are shown in the summary of experimental data of the removal of the iso-naringin by the methylation reaction of the hesperidin.

Example 1

One 500mL three-neck flask is taken, and a syringe with a needle (sleeved in a rubber plug first), an electric stirring thermometer and a thermometer are respectively arranged in a fume hood. 150mL of purified water was measured and added to the three-necked flask. 3.0g of solid NaOH was added and dissolved with stirring. Then 20.0g of hesperidin 1 is slowly added, stirred and completely dissolved. A large amount of solid ice cubes are directly put into the three-neck flask, and the temperature is reduced to 8 ℃. Slowly pushing dimethyl sulfate into the syringe to obtain 0.3mL of dimethyl sulfate, and after the dropwise addition in 5 minutes, the temperature of the solution in the flask is 8-10 ℃ and the volume is about 300mL. After the completion of the dropwise addition, overnight, the temperature of the solution was slowly returned to normal temperature, and the reaction was stirred for 10 hours.

The next morning, 9.5g of activated carbon was added and decolorized with stirring at 30℃for 2 hours. Filtering to clean the active carbon. Acidizing with glacial acetic acid to neutrality, and precipitating a large amount of yellow precipitate within several minutes. Standing for 2 hours, and filtering. Oven-drying at 60deg.C to obtain 18.0g hesperidin. The naringin content is reduced from 2.8% to 0.6%, and the yield is 92.5%. Specific data are presented in the summary table below.

Example 2

Into a three-necked flask, 150mL of pure water was poured, 3.5g of solid NaOH and 20.0g of hesperidin 1 were added, and the mixture was stirred until complete dissolution. Ice blocks are put into the flask to be cooled to 0 ℃, dimethyl sulfate is slowly dripped into the flask for 0.2mL, the temperature change is small after the addition is completed within 3 minutes, the temperature is kept at about 0 ℃, and the volume of the solution is 300mL. After the completion of the dropwise addition, the solution was slowly returned to normal temperature, heated to 30℃and reacted for 4 hours.

Acidifying with diluted hydrochloric acid until it is neutral, and precipitating a great deal of yellow precipitate in solution. Standing for 2 hours, and filtering. Oven-drying at 60deg.C to obtain 18.6g hesperidin. The naringin content is reduced from 2.8% to 0.8%, and the yield is 93.0%.

Example 3

150mL of pure water, 4.0g of solid NaOH was poured into the three-necked flask, and after dissolution, 20.0g of hesperidin 1 was slowly added and stirred until complete dissolution. The temperature is reduced to-8 ℃ by ice cubes, 0.4mL of dimethyl sulfate is slowly pushed in by a syringe, the addition is completed within 7 minutes, the temperature in the flask is slowly increased to about 0 ℃, and the volume of the solution is 350mL. The temperature of the solution is slowly restored to normal temperature, and the reaction is heated at 40 ℃ for 3 hours. Simultaneously, 200g of resin AB-8 is weighed and packed, and is treated with ethanol, dilute hydrochloric acid and dilute NaOH respectively for later use.

After the reaction is finished, the solution is passed through a pretreated AB-8 macroporous adsorption column. The solution is acidified to be neutral by dilute hydrochloric acid, a large amount of sediment is generated in the solution immediately, and the solution is stood for 3 hours and filtered by suction. Oven-drying at 60deg.C to obtain 18.0g hesperidin. The content of the naringin is reduced to 0.4% by 2.8%, and the yield is 90.0%.

Example 4

150mL of pure water, 3.5g of solid NaOH, and 20.0g of hesperidin 2 were placed in a three-necked flask. After stirring to complete dissolution, ice was added to the solution and cooled to-6 ℃. 0.7mL of dimethyl sulfate was slowly pushed in with a syringe and the addition was completed within 7 minutes. The temperature of the solution in the flask was gradually increased to around 0℃and the volume was about 300mL. The temperature of the solution is slowly increased to normal temperature, and the solution is heated to 50 ℃ for 3 hours. Simultaneously, 500g of resin D-101 is weighed and filled into a column, and is treated with ethanol, dilute hydrochloric acid and dilute NaOH respectively for later use.

After the reaction is completed, the solution is cooled to normal temperature and then passes through the pretreated D-101 resin. The effluent was acidified to neutrality with dilute hydrochloric acid, yielding a large amount of pale yellow precipitate immediately. Standing for 3 hours, filtering, and drying at 60 ℃ to obtain 18.2g of hesperidin. The naringin content is reduced to 0.4% by 4.5%, and the yield is 91.0%.

Example 5

150mL of pure water, 4.0g of solid NaOH, and 20.0g of hesperidin 2 were placed in a three-necked flask. After stirring to complete dissolution, ice was added to the solution and cooled to-3 ℃. 1.0mL of dimethyl sulfate was slowly pushed in with a syringe and the addition was completed within 10 minutes. The temperature of the solution in the flask was gradually increased to around 2℃and the volume was about 300mL. The temperature of the solution is slowly increased to normal temperature, and the reaction is heated at 30 ℃ for 3 hours. Simultaneously, 500g of resin NKA-9 is weighed and packed, and is treated with ethanol, dilute hydrochloric acid and dilute NaOH respectively for later use.

After the reaction is completed, the solution is cooled to normal temperature and passes through the pretreated NKA-9 resin. The effluent was acidified to neutrality with dilute hydrochloric acid, yielding a large amount of pale yellow precipitate immediately. Standing for 3 hours, suction filtering, and drying at 60 ℃ to obtain 17.6g of hesperidin. The content of the naringin is reduced to 0.2% by 4.5%, and the yield is 88.2%.

The adsorption column is eluted with a small amount of pure water and discarded. Eluting with a large amount of pure water, spotting the eluent on a thin layer chromatography plate, and stopping eluting when no obvious fluorescence exists; the adsorption column is eluted by ethanol until the thin layer chromatography plate has no obvious fluorescence. The water eluent and the ethanol eluent are respectively distilled to 1/20-1/10 of the original volume. Adding 2 times of absolute ethyl alcohol into the water concentrated solution, uniformly stirring, and standing in a refrigerator for crystallization; the ethanol concentrated solution is directly put into a refrigerator for standing and crystallization. After overnight filtration, drying at 60℃gave 0.1g of methyl hesperidin and 0.4g of melissa glucoside, respectively.

Example 6

150mL of pure water, 4.2g of solid NaOH, and 20.0g of hesperidin 2 were placed in a three-necked flask. After stirring to complete dissolution, ice was added to the solution and cooled to-10 ℃. 1.3mL of dimethyl sulfate was slowly pushed in with a syringe and the addition was completed within 12 minutes. The temperature of the solution in the flask was gradually increased to around-5℃and the volume was about 300mL. The temperature of the solution is slowly increased to normal temperature, and the reaction is heated at 30 ℃ for 3 hours. Meanwhile, 500g of resin SPD-30 is weighed and packed, and is treated with ethanol, dilute hydrochloric acid and dilute NaOH respectively for later use.

After the reaction is completed, the solution is cooled to normal temperature and then passes through the pretreated SPD-30 resin. The effluent was acidified to neutrality with dilute hydrochloric acid, yielding a large amount of pale yellow precipitate immediately. Standing for 3 hours, suction filtering, and drying at 60 ℃ to obtain 17.1g of hesperidin. The content of the naringin is reduced to 0.1% by 4.5%, and the yield is 85.6%.

The adsorption column is eluted with a small amount of pure water and discarded. Eluting with large amounts of pure water and ethanol until no significant fluorescence is observed. The water eluent and the ethanol eluent are respectively distilled to 1/20-1/10 of the original volume. Adding 2 times of absolute ethyl alcohol into the water concentrated solution, uniformly stirring, and standing in a refrigerator for crystallization; the ethanol concentrated solution is directly put into a refrigerator for standing and crystallization. After overnight filtration, drying at 60℃gave 0.2g of methyl hesperidin and 0.6g of melissa glucoside, respectively.

Summary of experimental data

Experimental data summarizing for removing naringin by hesperidin methylation reaction

Sample of	Content of	Purity of	Naringin	Melissa officinalis glycoside	Hesperetin	Adsorbent and process for producing the same	Dimethyl sulfate	Yield is good
									Hesperidin 1	81.8%	88.2%	2.8%	2.6%	5.5%
Case 1	83.5%	86.9%	0.6%	3.7%	2.4%	Activated carbon	0.3mL	92.5%
									Case 2	83.1%	86.5%	0.8%	4.5%	2.9%	Without any means for	0.2mL	93.0%
Case 3	95.4%	98.0%	0.4%	0.2%	1.0%	AB-8	0.4mL	90.0%
									Hesperidin 2	87.1%	92.0%	4.5%	1.8%	1.0%
Case 4	95.5%	98.3%	0.4%	0.3%	0.5%	D-101	0.7mL	91.0%
									Case 5	96.1%	98.0%	0.2%	0.2%	0.4%	NKA-9	1.0mL	88.2%
Case 6	95.2%	97.5%	0.1%	0.1%	0.4%	SPD-30	1.3mL	85.6%

Claims

1. A method for removing impurity naringin in plant extract hesperidin, which is characterized by comprising the following steps:

step one: dissolving the crude hesperidin with a proper amount of alkaline solution, and cooling;

step two: slowly dripping a small amount of phenolic hydroxyl protective agent into the solution at low temperature, and then heating to 20-50 ℃ for reaction;

step three: allowing the reaction solution to pass through nonpolar or low-polar macroporous adsorption resin, and adsorbing the naringin and hesperidin with the phenolic hydroxyl groups protected together with the melissa glucoside on a column for removal;

step four: acidifying the liquid, and standing to obtain a high-purity hesperidin product;

step five: eluting the adsorption column with pure water and then alcohol to obtain hesperidin and melissa glucoside with phenolic hydroxyl groups protected respectively;

the phenolic hydroxyl protecting agent is selected from dimethyl sulfate, methyl iodide and methyl triflate.

2. The method according to claim 1, wherein in the first step, the alkaline solution is an aqueous solution of an alkali metal or alkaline earth metal hydroxide, carbonate, bicarbonate; the base is selected from LiOH, naOH, KOH, ca (OH) ₂ 、Mg(OH) ₂ 、Na ₂ CO ₃ 、NaHCO ₃ 、K ₂ CO ₃ 、KHCO ₃ 。

3. The method according to claim 1, wherein in the second step, the phenolic hydroxyl protecting agent is added dropwise at-20 to 30 ℃ for 0.5 to 2.5 hours; after the dripping is finished, the temperature is raised to 20-50 ℃ for reaction, and the reaction time is 2-10 hours.

4. The method according to claim 1, wherein in the second step, the amount of the substance of the phenolic hydroxyl protecting agent is 0.5 to 30 times the amount of the naringin substance; judgment basis of drop-adding is that drop-adding is stopped when the peak height or area of the naringin on the chromatographic monitoring chart is reduced to 10% or even lower of the initial value.

5. The method of claim 1, wherein in step three, the nonpolar or weakly polar macroporous adsorbent resin is one of AB-8, SPD-30, LSA-40, D-101, CAD-40, NKA-9, polyamide column.

6. The method according to claim 1, wherein in step five, the polarity of the product obtained after derivatization of the phenolic hydroxyl groups of the iso-naringin and hesperidin is reduced and the product is adsorbed when passing through a macroporous resin; both are separated by eluting with alcohol and water, respectively, according to their solubility.

7. The method according to claim 1, wherein in step five, the hesperidin derivative, methyl hesperidin, is readily soluble in water and eluted from the resin with pure water; the naringin derivative product, such as melissa glucoside, is eluted with alcohol.