CN111978362A

CN111978362A - Method for removing isocoryzanol in natural product hesperidin

Info

Publication number: CN111978362A
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: 2020-11-24
Anticipated expiration: 2040-09-07
Also published as: CN111978362B

Abstract

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

Description

Method for removing isocoryzanol in natural product hesperidin

Technical Field

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

Background

Hesperidin, also known as hesperidin, flavanonoside. Is an active flavonoid extracted from fruit or peel of Citrus plant of Rutaceae such as fructus Aurantii Immaturus, pericarpium Citri Tangerinae, pericarpium Citri Junoris, etc., and the pure product is pure white fine dendritic needle crystal. Has effects in reducing capillary fragility, protecting capillary, preventing capillary rupture hemorrhage (similar to vitamin P action), inhibiting blood cholesterol increase, and preventing arteriosclerosis, and can be used for treating hypertension and myocardial infarction; has good effects of resisting oxidation, reducing blood sugar, resisting inflammation, relieving pain, resisting tumor and resisting virus. Apart from being a pharmaceutical, food and cosmetic additive per se, hesperidin is mainly used as a raw material for the production of the drugs diosmin and methyl hesperidin. Diosmin which is prepared by taking hesperidin as a raw material and performing semi-chemical synthesis is used as a common medicament for treating various symptoms related to venous insufficiency and lymphatic insufficiency and acute hemorrhoids in Europe; through the reaction of hesperidin and a methylating agent, 3' -phenolic hydroxyl of hesperidin is methylated to generate methyl hesperidin, which has the vitamin P-like effect and can enhance the vitamin C effect when being used together with vitamin C; the methyl hesperidin has strong antiviral and antibacterial effects, and can inhibit the propagation of influenza virus when being applied in large dose; in addition, it can inhibit the activity of tyrosinase which causes skin blackening, can be used for treating skin diseases such as black speck and freckle, and has important application in the cosmetic and cosmetic industries.

The most extensive methods for extracting hesperidin from immature bitter orange, orange peel and the like at present are an aqueous solvent extraction method and an organic solvent extraction method, wherein the two methods are that a proper amount of alkali is added into a solvent, and after the hesperidin is dissolved in an alkaline solvent, the hesperidin is precipitated by using acid, which is called as an alkali-dissolving acid-precipitating method for short. Due to the complexity of the components of the pericarp, such as vegetable protein, pigment, vegetable tannin, pectin, starch, polysaccharide, various small molecular substances, inorganic salt and the like. The photo-flavonoids are of a few kinds, and have small structural difference with each other, and only have the difference of 1-2 methyl, hydroxyl or sugar molecules. Therefore, the hesperidin extracted from the plant is mixed with a plurality of natural impurity products which are similar or similar to the structure of the hesperidin, and mainly comprises the isocetyl glycoside and the like (the structure is shown below). The quality of raw materials for producing the hesperidin is evaluated, and besides the content index of the hesperidin, an important index, namely the content of the isocetylin, is also provided. The superior immature bitter orange product has high hesperidin content and low isonicotinyl; on the contrary, the inferior product of immature bitter orange has low hesperidin content and high content of isochinacoside. The purity of natural plant hesperidin extraction in the current market generally has three specifications, namely 85%, 90% and 95%, and only the best raw materials can produce products with 95% specifications. However, in any specification of hesperidin, a certain amount of the impurity of the isocetylin is contained in the hesperidin, the content is generally between 1 and 5 percent, and more than 5 percent is regarded as unqualified; european customer requirements are more strict, and are generally below 2.5%. Therefore, the raw material with high content of the isocoryzanol can not be used for the production of qualified hesperidin, and the waste of resources is caused. Whether hesperidin is used as a medicine per se or as a raw material for producing medicines such as diosmin, a high content (preferably more than 98%) of a main component and a low content of impurities are required, and particularly, a low content of isonicotinyl is required. Therefore, how to remove impurities such as the isocoryzanol and the like in the hesperidin extracted from natural plants becomes a difficult point in the hesperidin extraction industry. The problem is solved, and the method has important significance for improving income of fruit growers, expanding the application range of raw materials of hesperidin manufacturers, reasonably using natural resources and improving the yield and quality of the whole hesperidin industry.

Viewed from the molecular structure, the hesperidin, the isochinoside and the melissa glycoside are very close to each other. The difference between the isocarpin and the melittin is only the methylation of 4 '-phenolic hydroxyl, i.e. the isocarpin is phenolic hydroxyl, the melittin is the product of the methylation of 4' -phenolic hydroxyl of the isocarpin, i.e. the melittin is methylated isocarpin; and hesperidin has only one more phenolic hydroxyl group at the 3' position than melissa glucoside. Theoretically, the impurity of the isocoryzanol can be separated out through recrystallization, and the difficulty is that a proper solvent is difficult to find for dissolving the hesperidin. At present, solvents capable of dissolving hesperidin, such as pyridine, N-Dimethylformamide (DMF), morpholine, dimethyl sulfoxide (DMSO) and the like, are high-boiling-point solvents, and are not easy to separate from products completely after recrystallization, so that solvent residues are caused, which is disastrous to natural products of hesperidin as food or medicines. If the hesperidin is dissolved by an alkaline aqueous solvent such as a NaOH aqueous solvent and then acidified, impurities and the main components of the hesperidin are separated out together due to the similar pKa, so that the aim of removing the impurities cannot be fulfilled. Therefore, a new way must be found to find a reasonable route for purifying hesperidin.

Disclosure of Invention

On the basis of full research on documents and a large number of experiments, the invention creatively provides the purpose of removing impurities by combining chemical reaction with macroporous resin adsorption and sacrificing a small amount of main components according to the difference of chemical reactivity of active groups in molecular structures of impurity isonicotinyl and main component hesperidin and the small difference of molecular polarity after derivatization and conversion. The method is simple, low in cost, high in yield, high in product purity and good for purifying natural product hesperidin.

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

1. a dissolving step: slowly adding the crude hesperidin product into the dilute alkaline solution to dissolve the crude hesperidin product. And cooling after complete dissolution.

2. And (3) methylation: slowly dropping a small amount of methylating agent into the hesperidin solution, and keeping the temperature low in the dropping process. And after the dropwise addition is finished, heating for reaction.

3. A column passing step: and (3) passing the reacted solution through nonpolar or low-polarity macroporous adsorption resin to adsorb the impurities such as the methylated isoconoside, namely the melissoside, the methylated hesperidin, namely the methyl hesperidin, and the inherent component of the melissoside in the hesperidin.

4. Acidifying: acidifying the solution, standing for precipitation, and centrifuging to obtain high-purity hesperidin product.

5. An elution step: eluting macroporous adsorption resin with a large amount of pure water to obtain methyl hesperidin; eluting with a large amount of ethanol to obtain melissoside.

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: 3-1: 10; the amount of NaOH substance is 2-8 times, preferably 2-4 times of the amount of hesperidin substance.

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

Further, in the "methylation step", the methylating agent is dimethyl sulfate, methyl iodide, dimethyl carbonate and the like, and preferably dimethyl sulfate; the amount of dimethyl sulfate is 0.5-30 times, preferably 2-15 times of the amount of the isocetyl glycoside.

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

Further, the dropping time of the methylating agent in the "methylation step" is 0.5 to 6 hours, preferably 0.5 to 2.5 hours; the reaction time at elevated temperature is 2 to 10 hours, preferably 2 to 6 hours.

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

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

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, acidified to a pH of 6 to 8, preferably around 7. The acidification time is preferably 1 to 20 hours, preferably 4 to 10 hours. Slowly acidifying to facilitate the growth of hesperidin dendritic needle crystal.

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

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following description will be made more clearly and completely with reference to the specific embodiments. It is obvious that the embodiments described below are only partial implementations of the present application, and other similar or similar applications, which are proposed by those skilled in the art without making an inventive idea or effort, are considered infringements of the present invention.

The crude hesperidin used in the embodiment 1-3 is marked as "hesperidin 1"; the crude hesperidin used in examples 4-6 was labeled "hesperidin 2". The content, purity and impurities are summarized in Experimental data for removing the isocoryzanol by the methylation reaction of hesperidin.

Example 1

A500 mL three-neck flask is taken, provided with a syringe with a needle (firstly sheathed in a rubber plug), an electric stirring device and a thermometer respectively, and placed in a fume hood. 150mL of purified water is measured and added into a three-neck flask. 3.0g of solid NaOH was added and dissolved by stirring. Then, 20.0g of hesperidin 1 was slowly added thereto, and stirred to be completely dissolved. Directly putting a large amount of solid ice blocks into the three-neck flask, and reducing the temperature to 8 ℃. 0.3mL of dimethyl sulfate was slowly pushed in from the syringe, and after completion of the dropwise addition within 5 minutes, the temperature of the solution in the flask was between 8 and 10 ℃ and the volume was about 300 mL. After the dropwise addition, the solution was allowed to stand overnight, the temperature of the solution was slowly returned to normal temperature, and the reaction was carried out for 10 hours with stirring.

The next morning, 9.5g of activated carbon was added, and the mixture was decolorized with stirring at 30 ℃ for 2 hours. Filtering, and cleaning the activated carbon. Acidifying with glacial acetic acid to neutrality, and a large amount of yellow precipitate is separated out within several minutes. Standing for 2 hours, and performing suction filtration. Oven drying at 60 deg.C to obtain 18.0g hesperidin. The yield of the isononaringin is reduced from 2.8 percent to 0.6 percent, and the yield is 92.5 percent. See the summary table below for specific data.

Example 2

150mL of pure water was poured into the three-necked flask, 3.5g of solid NaOH and 20.0g of hesperidin 1 were added, and the mixture was stirred until the components were completely dissolved. And (3) putting ice blocks into the flask, cooling to 0 ℃, slowly dropping 0.2mL of dimethyl sulfate, finishing the addition within 3 minutes, keeping the temperature at about 0 ℃ without great change, and keeping the volume of the solution at 300 mL. After the dropwise addition, the solution was slowly returned to normal temperature and heated to 30 ℃ to react for 4 hours.

Acidification to neutrality with dilute hydrochloric acid immediately results in a large amount of yellow precipitate in the solution. Standing for 2 hours, and performing suction filtration. Oven drying at 60 deg.C to obtain 18.6g hesperidin. The yield of the isononaringin is reduced from 2.8 percent to 0.8 percent and 93.0 percent.

Embodiment 3

150mL of pure water and 4.0g of solid NaOH are poured into the three-neck flask, 20.0g of hesperidin 1 is slowly added after dissolution, and stirring is carried out until complete dissolution. The temperature is reduced to-8 ℃ by ice cubes, 0.4mL of dimethyl sulfate is slowly pushed in by an injector, the temperature in the flask is slowly raised to about 0 ℃ after the addition is completed within 7 minutes, and the volume of the solution is 350 mL. The temperature of the solution is slowly restored to normal temperature, and the solution is heated and reacted for 3 hours at 40 ℃. Meanwhile, 200g of resin AB-8 is weighed and filled into a column, and is respectively treated with ethanol, dilute hydrochloric acid and dilute NaOH for later use.

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

Example 4

150mL of pure water, 3.5g of solid NaOH and 20.0g of hesperidin 2 were placed in a three-necked flask. Stirring until the mixture is completely dissolved, adding ice blocks into the solution, and cooling to-6 ℃. 0.7mL of dimethyl sulfate was slowly pushed in with a syringe and the addition was completed in 7 minutes. The temperature of the solution in the flask was gradually raised to about 0 ℃ and the volume was about 300 mL. The temperature of the solution was gradually raised to room temperature, and the solution was heated at 50 ℃ for 3 hours. Meanwhile, 500g of resin D-101 is weighed and filled into a column, and is respectively treated with ethanol, dilute hydrochloric acid and dilute NaOH for later use.

After the reaction is finished, 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, producing a large amount of pale yellow precipitate immediately. Standing for 3 hours, filtering, and drying at 60 ℃ to obtain 18.2g of hesperidin. The yield of the isononaringin is 91.0 percent, and 4.5 percent of the isononaringin is reduced to 0.4 percent.

Example 5

150mL of pure water, 4.0g of solid NaOH and 20.0g of hesperidin 2 were placed in a three-necked flask. Stirring until the mixture is completely dissolved, adding ice blocks into the solution, and cooling to-3 ℃. 1.0mL of dimethyl sulfate was slowly pushed in with a syringe and the addition was completed in 10 minutes. The temperature of the solution in the flask was gradually raised to around 2 ℃ and the volume was about 300 mL. The temperature of the solution is gradually raised to normal temperature, and the solution is heated and reacted for 3 hours at 30 ℃. Meanwhile, 500g of resin NKA-9 is weighed and loaded into a column, and is respectively treated by ethanol, dilute hydrochloric acid and dilute NaOH for standby.

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

The adsorption column is eluted with a small amount of pure water and discarded. Eluting with a large amount of pure water, and stopping eluting when the eluate does not have obvious fluorescence; eluting the adsorption column with ethanol until no fluorescence is evident on the TLC plate. The water eluent and the ethanol eluent are respectively distilled in a rotary manner to 1/20-1/10 of the original volume. Adding 2 times volume of absolute ethyl alcohol into the water concentrated solution, uniformly stirring, and then placing in a refrigerator for standing and crystallizing; directly placing the ethanol concentrated solution into a refrigerator, standing and crystallizing. After the night, the mixture is filtered by suction and dried at 60 ℃ to respectively obtain 0.1g of methyl hesperidin and 0.4g of melissoside.

Example 6

150mL of pure water, 4.2g of solid NaOH and 20.0g of hesperidin 2 were placed in a three-necked flask. Stirring until the mixture is completely dissolved, adding ice blocks into the solution, and cooling to-10 ℃. 1.3mL of dimethyl sulfate was slowly pushed in with a syringe and the addition was completed over 12 minutes. The temperature of the solution in the flask was gradually raised to about-5 ℃ and the volume was about 300 mL. The temperature of the solution is gradually raised to normal temperature, and the solution is heated and reacted for 3 hours at 30 ℃. Meanwhile, 500g of resin SPD-30 is weighed and filled into a column, and is respectively treated by ethanol, dilute hydrochloric acid and dilute NaOH for standby.

After the reaction is finished, 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, producing a large amount of pale yellow precipitate immediately. Standing for 3 hours, filtering, and drying at 60 ℃ to obtain 17.1g of hesperidin. The yield of the isononaringin is 85.6 percent, and 4.5 percent of the isononaringin is reduced to 0.1 percent.

The adsorption column is eluted with a small amount of pure water and discarded. Then eluting with a large amount of pure water and ethanol until no fluorescence is evident. The water eluent and the ethanol eluent are respectively distilled in a rotary manner to 1/20-1/10 of the original volume. Adding 2 times volume of absolute ethyl alcohol into the water concentrated solution, uniformly stirring, and then placing in a refrigerator for standing and crystallizing; directly placing the ethanol concentrated solution into a refrigerator, standing and crystallizing. After the night, the mixture is filtered by suction and dried at 60 ℃ to respectively obtain 0.2g of methyl hesperidin and 0.6g of melissoside.

Summary of Experimental data

Data summarization of experiment for removing isocoryzanol by methylation of hesperidin

Sample (I)	Content (wt.)	Purity of	Isonaringin	Melissside	Hesperetin (Hesperetin)	Adsorbent and process for producing the same	Dimethyl sulfate	Yield of
									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%	Is free of	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 of isononaringin in plant extract hesperidin is characterized by comprising the following steps:

1.1, dissolving a crude product of hesperidin by using a proper amount of alkaline solution, and cooling;

1.2, dropwise adding a small amount of phenolic hydroxyl protective agent into the solution at low temperature, and then heating for reaction;

1.3, passing the reaction solution through nonpolar or low-polarity macroporous adsorption resin, and adsorbing the isononaringin, hesperidin and the like with protected phenolic hydroxyl groups and the melissa glycoside on a column for removing;

1.4, acidifying the passing liquid, and standing to obtain a high-purity hesperidin product;

and 1.5, eluting the adsorption column with pure water and then with alcohol to respectively obtain hesperidin and melissoside with protected phenolic hydroxyl groups.

2. The method for removing the impurity of the isochinacoside in the hesperidin according to claim 1, wherein in step 1.1, the alkaline solution is an aqueous solution or an organic solution of hydroxide of alkali metal and alkaline earth metal, carbonate (hydrogen) salt, and the temperature is-20-30 ℃. The alkali is LiOH, NaOH, KOH, Ca (OH)₂、CaO、Mg(OH)₂、Na₂CO₃、NaHCO₃、K₂CO₃、KHCO₃And the like, organic solvents mainly include short-chain alcohols such as methanol, ethanol, (iso) propanol, (tert) butanol, and other non-alcohols such as acetone, ethyl acetate, pyridine, N-Dimethylformamide (DMF), morpholine, dimethyl sulfoxide (DMSO), dioxane, and the like.

2.1, according to claim 2, characterized in that in step 1.1, the base is preferably NaOH and the solvent is preferably water. On one hand, the effect of NaOH dissolves hesperidin, and on the other hand, neutralizes acid released in the process of protecting phenolic hydroxyl. The addition amount of NaOH is 2-8 times of the amount of hesperidin.

2.2, according to claim 2, characterized in that in step 1.1, NaOH is preferably added in an amount 2-4 times the amount of naringin substance, and the temperature is preferably-10 ℃.

3. The method for removing the impurity of the isocetyl glycoside in the hesperidin according to claim 1, wherein in step 1.2, the phenolic hydroxyl group protecting agent is used for converting the phenolic hydroxyl group at the 4' -position of the isocetyl glycoside into ether or ester, so that the ether formation protection is mainly performed. The ethers mainly comprise methyl ether, ethyl ether, silicon ether, tert-butyl ether, benzyl ether, tetrahydropyrane ether, allyl ether and the like; the esters are mainly formate, acetate, benzoate and their derivatives. The phenolic hydroxyl protective agent is dripped at low temperature (-20-30 ℃), and then the temperature is raised for reaction (20-80 ℃).

3.1, according to claim 3, characterized in that, in step 1.2, the methyl ether protective agent of the phenolic hydroxyl group is mainly dimethyl sulfate, methyl iodide, dimethyl carbonate, dimethyl phosphite, methyl trifluoromethanesulfonate, etc.; the ethyl ether protective agent of the phenolic hydroxyl is mainly diethyl sulfate; the phenolic hydroxyl silyl ether protective agent mainly comprises Trimethylsilane (TMS), Triethylsilane (TES), tri-tert-butylsilane (TBS), tert-butyldimethylsilane (TBDMS), tert-butyldiphenylsilane (TBDPS) and the like; the tert-butyl ether protective agent of the phenolic hydroxyl group is mainly isobutylene, and the benzyl ether protective agent is mainly benzyl chloride, benzyl bromide and the like. The method of claim 3, step 1.2, wherein the methyl ester protecting agent of the phenolic hydroxyl group is mainly an adduct of formic acid, methyl acetic anhydride, DMF and benzoyl chloride; the ethyl ester protective agent of the phenolic hydroxyl is mainly acetic anhydride, acetyl chloride, ethyl acetate, pentafluorophenyl acetate and the like; the benzyl ester protective agent of the phenolic hydroxyl group is mainly benzoate, p-phenylbenzoate, 2,4, 6-trimethylbenzoate and the like; the allyl ether protecting agent of the phenolic hydroxyl is mainly allyl chloride, allyl bromide and the like.

3.2, according to claims 3 and 3.1, characterized in that in step 1.2, the protection of the phenolic hydroxyl group is preceded by preferably a methyl ether; the protecting agent is preferably dimethyl sulfate.

3.3, according to claims 2.2 and 3.2, characterized in that in step 1.2, the crude hesperidin is dissolved with aqueous NaOH, the temperature is reduced to-10 to 5 ℃, and then the phenolic hydroxyl group protecting agent dimethyl sulfate is slowly dropped. The dropping time is 0.5-2.5 hours. After the dropwise addition, the temperature is raised to 20-60 ℃ for reaction. Monitoring the content of the isocetyl glycoside by liquid chromatography, and determining whether to supplement a methylation reagent according to the chromatographic peak height or peak area of the isocetyl glycoside. When the isocononalin chromatographic peak was reduced to a low level or even 0, the reaction was stopped. The reaction time is 2-10 hours.

3.4, according to claim 3.3, characterized in that in step 1.2, the methylating agent is 0.5 to 30 times, preferably 2 to 15 times the amount of the isocetyl glycoside substance.

3.5, according to claim 3.3, characterized in that in step 1.2, the 4 'phenolic hydroxyl group of the isononaringin and the 3' phenolic hydroxyl group of the hesperidin are competitively methylated to be converted into the melissa glycoside and the methyl hesperidin respectively, and the polarity of the products is weakened; but the activity of the isocetyl glycoside and the hesperidin phenolic hydroxyl is different, namely the activity of the 3 ' -position phenolic hydroxyl of the hesperidin is smaller due to the large steric hindrance effect of the adjacent 4 ' -position methoxyl group, and the activity of the 4 ' -position phenolic hydroxyl of the isocetyl glycoside is relatively larger due to the small steric hindrance effect of the adjacent hydrogen atom. Therefore, the conversion rate of the isocoryzanol is higher under the condition; more importantly, the product of the conversion of the isocoryzanol is an impurity component in the natural product of hesperidin. By methylation, interconversion between impurity components is achieved without the generation of new harmful impurity components. Meanwhile, the hesperidin conversion product methyl hesperidin has the vitamin P-like effect as the hesperidin, is widely applied to the fields of food, cosmetics and medicines, and is a safe component.

3.6, according to claims 3 and 3.1, characterized in that in step 1.2, the phenol hydroxyl protecting agent at the 4 'position of the isononaringin can also be trimethylchlorosilane, benzyl chloride, benzyl bromide and the like, which can also easily react with the phenol hydroxyl at the 4' position of the isononaringin, the polarity of the product is weakened, and the later stage can also be removed by passing through a nonpolar or low-polarity macroporous adsorption column. However, the transformation product is a brand new species which is not contained in the natural product hesperidin and has certain potential safety hazard. The protecting agent is preferably a methylating agent.

4. The method for removing impurity of the isononaringin in the hesperidin according to claim 1, wherein in step 1.3, the nonpolar or weakly polar macroporous adsorption resin is AB-8, SPD-300, LSA-40, D-101, CAD-40, NKA-9, polyamide column, etc., or adsorption with activated carbon can be used instead of resin, which is preferred because the resin can be used more efficiently and repeatedly.

The method for removing the impurity of the isochinoside in the hesperidin according to claim 1, wherein in the step 1.3, substances adsorbed by the nonpolar or low-polarity resin are mainly products with reduced polarity after the derivation and conversion of the active phenolic hydroxyl groups of the isochinoside and the hesperidin, and the natural extracted product hesperidin which is originally mixed with the melissa officinalis glycoside.

4.2, according to claims 3.2-3.5, characterized in that in step 1.3, the adsorbed substances of the apolar or weakly polar resin are mainly melissoside methylated from isonicotinyl, methyl hesperidin methylated from hesperidin, and the natural extraction product hesperidin, which is in itself mixed with melissoside.

5. The method for removing the impurity of the isochinoside from the hesperidin according to claim 1, wherein in step 1.5, the hesperidin phenolic hydroxyl group is protected, so that the water solubility is increased, and the hesperidin phenolic hydroxyl group can be eluted by pure water; whereas the melissoside needs to be eluted with alcohol.

5.1, according to claim 4.2, in step 1.5, the product methyl hesperidin, after the phenolic hydroxyl group of hesperidin is protected, is easily soluble in water and can be eluted from the resin by pure water; the product obtained after the phenol hydroxyl group of the isocoryzanol is protected is melissoside which can be eluted by alcohol, and the alcohol is preferably ethanol.

6. The method for purifying the natural extraction product hesperidin is considered to fall into the protection scope of the patent on the premise of not proposing creative ideas by modifying the difference of the reaction activities of chemical reagents or biological enzymes by utilizing the 4 '-phenolic hydroxyl of the isocarringoside and the 3' -phenolic hydroxyl of the hesperidin to weaken the polarity of the chemical reagents and then carrying out separation such as macroporous resin, crystallization, recrystallization and the like. On the contrary, the purification method of the natural extraction product hesperidin is considered to fall into the protection scope of the patent on the premise of not proposing creative ideas by modifying the difference of the reaction activity of chemical reagents or biological enzymes by utilizing the 4 '-phenolic hydroxyl of the isocarringoside and the 3' -phenolic hydroxyl of the hesperidin to enhance the polarity of the chemical reagents or the biological enzymes, such as methyl esterification, ethylene oxide addition, acrylonitrile addition, chloroacetic acid reaction and the like, and then separating the chemical reagents or the biological enzymes, such as macroporous resin treatment, crystallization, recrystallization and the like.