Method for efficiently extracting naringin and hesperidin from fingered citron

Abstract

The invention provides a method for efficiently extracting naringin and hesperidin from fingered citron, and belongs to the technical field of plant active ingredient extraction. The extraction method comprises the following steps: degreasing and filtering fingered citron powder in advance, refluxing and extracting hesperidin in filter residues by using an alkaline ethanol solution, adsorbing filtrate by using macroporous resin, eluting by using ethanol, concentrating under reduced pressure, and recrystallizing to obtain the naringin. According to the method, the naringin and the hesperidin in the fingered citron are jointly extracted by adopting multiple extraction processes, so that the naringin and the hesperidin can be separated, the extraction rate and the purity of the naringin and the hesperidin can be improved, and meanwhile, the method also has the characteristics of small dosage of an organic solvent, high degreasing efficiency, less impurity dissolution and the like.

Description

Technical Field

[0001] The invention relates to the technical field of plant active ingredient extraction, and in particular to a method for efficiently extracting naringin and hesperidin from bergamot. Background Art

[0002] Buddha's hand, also known as "bergamot", has a unique palm-shaped appearance. The shell of Buddha's hand is yellow-orange, the surface is smooth, and the flesh is thick, hence the name "Buddha's hand". Buddha's hand is a common dietary and medicinal material. The fruit of Buddha's hand has a strong aroma and is often used to make food and beverages. In addition, Buddha's hand has obvious medicinal value. Buddha's hand contains a variety of chemical components with important pharmacological effects, mainly including polysaccharides, flavonoids and tannin compounds. The above ingredients can play an important pharmacological effect in various ways, such as anti-oxidation, anti-inflammatory, anti-tumor, hypoglycemic, hypolipidemic and so on. At present, the development and utilization of Buddha's hand is usually based on the extraction of flavonoids, and the residues after extraction are usually discarded, which wastes resources and pollutes the environment.

[0003] The active ingredients of bergamot include naringin and hesperidin. Among them, naringin is a dihydroflavonoid compound with multiple biological activities, such as preventing cancer, inhibiting microbial growth and the activity of many enzymes, anti-oxidation, reducing the levels of cholesterol and triglycerides in the blood and maintaining normal capillary permeability, etc., showing its potential medical application value. It is of great significance to find a simple and efficient method to extract naringin from bergamot plants. Hesperidin is a glycoside formed by hesperidin and rutin, namely hesperidin-7-O-rutinoside, which is a dihydroflavonoid derivative. It has a significant inhibitory effect on the scavenging effect of hydroxyl free radicals and the lipid peroxidation caused by hydroxyl free radicals through the cell membrane. Its anti-lipid oxidation effect can inhibit the formation of lipid peroxides in patients with myocardial infection and atherosclerosis. Hesperidin sodium sulfonate has different degrees of scavenging effects on 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radicals, hydroxyl free radicals, superoxide anion free radicals, and alkyl free radicals.

[0004] The existing extraction methods of naringin and hesperidin mainly include: alcohol extraction, hot water method, alkali extraction and acid precipitation method, ultrasound-assisted extraction method, enzyme hot water extraction method, etc. However, the above methods all have defects to varying degrees. For example, the invention patent with application number 201710128917.3 discloses a method for enzymatic extraction of naringin. This patent requires the use of an enzymatic system, which is costly, and has strict requirements on conditions such as temperature and pH, and is complicated to operate. The invention patent with publication number CN103833809A discloses a new extraction and separation process for naringin. This patent involves the evaporation of a large amount of water, the evaporation and recovery of a large amount of petroleum ether, and the evaporation and recovery of a large amount of n-hexane and n-butanol. There are problems such as complex procedures and high costs. In summary, it is necessary to develop a method for efficiently extracting naringin and hesperidin from Buddha's hand. Summary of the invention

[0005] In view of this, the present invention provides a method for efficiently extracting naringin and hesperidin from bergamot, comprising the following steps:

[0006] (1) drying, crushing and sieving fresh bergamot to obtain bergamot powder, defatting the bergamot powder with petroleum ether, and filtering to obtain filtrate a and filter residue a;

[0007] (2) drying the filter residue a in step (1), adding an alkaline ethanol solution, refluxing and extracting for 1-5 hours, filtering, collecting the filtrate b, adjusting the pH of the filtrate b to within the range of 4.5-6, and concentrating under reduced pressure until the ethanol in the filtrate b is completely removed to obtain a concentrated solution, cooling the concentrated solution for a period of time, precipitating crystals, collecting the precipitate, obtaining a crude hesperidin extract, filtering and collecting the filtrate c, drying the obtained crude hesperidin extract, and repeating the above extraction process to obtain high-purity hesperidin;

[0008] (3) The filtrate a and the filtrate c are mixed to obtain a crude extract mixture, and the concentration of the crude extract mixture is adjusted to 0.5-1 g / ml using pure water, followed by adsorption with a macroporous resin, elution with ethanol, concentration under reduced pressure, and recrystallization to obtain high-purity naringin.

[0009] Furthermore, the screening in step (1) is specifically through a sieve of 80-120 mesh.

[0010] Furthermore, the alkaline ethanol solution in step (2) is specifically a 50-80% ethanol solution containing 0.5-2.0% potassium hydroxide, wherein the ratio of potassium hydroxide to ethanol is a mass volume ratio.

[0011] Furthermore, in step (2), the mass volume ratio of the alkaline ethanol solution to the filter residue is 8-10:1.

[0012] Furthermore, the temperature of the reflux extraction in step (2) is 60-80° C., and the reflux extraction is performed under a nitrogen atmosphere.

[0013] Furthermore, the pH in step (2) is in the range of 4.5-5.5, and more preferably, the pH is 4.7.

[0014] Furthermore, the cooling temperature in step (2) is 0-4°C, and the cooling time is 12-18h.

[0015] Furthermore, in step (3), the mass volume ratio of the crude hesperidin extract to the alkaline ethanol solution is 1:-3:10-12.

[0016] Furthermore, in step (3), the ethanol concentration is 60-80%, the column flow rate is 1-3 mL / min, the elution rate is 0.1-1 mL / min, and the water wash volume is 5-8 BV.

[0017] Furthermore, in step (3), the column flow rate of ethanol is 2.5 mL / min, and the elution rate is 0.2 mL / min.

[0018] Compared with the prior art, the present invention has the following beneficial effects: compared with the traditional extraction method, the present invention reasonably designs the pH of the alkaline ethanol solution, the column flow rate and the elution rate parameters in the purification process of naringin, and simultaneously selects a specific type of alkaline reagent and a specific type of macroporous resin, so that each extraction step presents a synergistic effect, significantly improves the extraction rate and purity of naringin and hesperidin, has the characteristics of high process reliability, low organic solvent consumption, high degreasing efficiency, less impurity dissolution, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The invention is a process flow chart for extracting naringin and hesperidin from bergamot. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0021] The experimental methods or test methods described in the following examples are conventional methods unless otherwise specified; the raw materials and auxiliary agents are obtained from conventional commercial channels or prepared by conventional methods unless otherwise specified.

[0022] The bergamot selected in the following examples and comparative examples is from Fujian, and is called "Yongchun bergamot". The content of naringin and hesperidin in the bergamot powder obtained after drying, crushing and sieving is determined by high performance liquid chromatography, and it is known that the content of naringin is 4.5% and the content of hesperidin is 4.3%.

[0023] In the following examples and comparative examples, the test method of hesperidin is referred to (Master's thesis "Study on the extraction and purification of hesperidin from tangerine peel and comprehensive utilization of residues", Qi Bing, Jiangnan University); the test method of naringin is referred to (Master's thesis "Study on the extraction process of synephrine and naringin from citrus rot fruit", Long Jianyuan, Xiangtan University).

[0024] The macroporous resin is X-5 macroporous adsorption resin, purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S25964-500g; the glass chromatography column used is Φ25mm×300mm, purchased from Beijing Biolab Technology Co., Ltd., product number QN3314.

[0025] Example 1

[0026] A method for efficiently extracting naringin and hesperidin from bergamot, the specific steps are as follows:

[0027] (1) drying, crushing and passing fresh bergamot through an 80-mesh sieve to obtain a bergamot powder, refluxing and degreasing the bergamot powder with petroleum ether, and filtering to obtain a filtrate a and a filter residue a;

[0028] (2) taking 100 kg of the filter residue a described in step (1), drying it at 50° C., adding it to an extraction tank containing 1000 kg of 0.5% potassium hydroxide in 80% ethanol solution, extracting it under reflux at 80° C. in a nitrogen atmosphere for 1 h, filtering it with suction, collecting the filtrate b, adjusting the pH of the filtrate b to 4.7 with a hydrochloric acid solution, placing it in a concentration tank and concentrating it under reduced pressure until the ethanol in the filtrate b is completely removed, and when about 150 kg of concentrated solution is obtained, transferring the concentrated solution to a crystallization tank, cooling it at 4° C. for 12 h, crystallizing it, collecting the precipitate, obtaining a crude hesperidin extract, filtering and collecting the filtrate c, drying the obtained crude hesperidin extract, and repeating the above extraction process once to obtain high-purity hesperidin;

[0029] (3) The filtrate a and the filtrate c are mixed to obtain a crude extract mixture, the concentration of the crude extract mixture is adjusted to 0.5 g / ml with pure water, the pH of the mixture is adjusted to 5.5 with hydrochloric acid, the sample is loaded at a rate of 2.5 mL / min, loaded on a macroporous resin adsorption column, eluted with 80% ethanol as an eluent, the elution rate is 0.2 mL / min, the water washing volume is 5 BV, the eluate is concentrated under reduced pressure, and recrystallized to obtain high-purity naringin.

[0030] The results show that the extraction rates of naringin and hesperidin are 95.3% and 96.7% respectively. The purity of naringin and hesperidin is 97.1% and 98.2% respectively as determined by high performance liquid chromatography.

[0031] Example 2

[0032] A method for efficiently extracting naringin and hesperidin from bergamot, the specific steps are as follows:

[0033] (1) drying, crushing and passing fresh bergamot through a 120-mesh sieve to obtain a bergamot powder, refluxing and degreasing the bergamot powder with petroleum ether, and filtering to obtain a filtrate a and a filter residue a;

[0034] (2) taking 100 kg of the filter residue a described in step (1), drying it at 50° C., adding it to an extraction tank containing 1000 kg of 2.0% potassium hydroxide in 50% ethanol solution, extracting it under reflux at 60° C. in a nitrogen atmosphere for 2 h, filtering it with suction, collecting the filtrate b, adjusting the pH of the filtrate b to 4.7 with a hydrochloric acid solution, placing it in a concentration tank and concentrating it under reduced pressure until the ethanol in the filtrate b is completely removed, and when about 150 kg of concentrated solution is obtained, transferring the concentrated solution to a crystallization tank, cooling it at 2° C. for 18 h, crystallizing it, collecting the precipitate, obtaining a crude hesperidin extract, filtering and collecting the filtrate c, drying the obtained crude hesperidin extract, and repeating the above extraction process once to obtain high-purity hesperidin;

[0035] (3) The filtrate a and the filtrate c are mixed to obtain a crude extract mixture, the concentration of the crude extract mixture is adjusted to 1 g / ml with pure water, the pH of the mixture is adjusted to 5.5 with hydrochloric acid, the sample is loaded at a rate of 3 mL / min, loaded on a macroporous resin adsorption column, eluted with 80% ethanol as an eluent, the elution rate is 1 mL / min, the water washing volume is 8 BV, the eluate is concentrated under reduced pressure, and recrystallized to obtain high-purity naringin.

[0036] The results show that the extraction rates of naringin and hesperidin are 94.1% and 95.3% respectively. The purity of naringin and hesperidin is 96.7% and 95.9% respectively as determined by high performance liquid chromatography.

[0037] Example 3

[0038] A method for efficiently extracting naringin and hesperidin from bergamot, the specific steps are as follows:

[0039] (1) drying, crushing and passing fresh bergamot through a 100-mesh sieve to obtain a bergamot powder, refluxing and degreasing the bergamot powder with petroleum ether, and filtering to obtain a filtrate a and a filter residue a;

[0040] (2) taking 100 kg of the filter residue a described in step (1), drying it at 50° C., adding it to an extraction tank containing 1000 kg of 1.0% potassium hydroxide in 60% ethanol solution, extracting it under reflux at 80° C. in a nitrogen atmosphere for 5 h, filtering it with suction, collecting the filtrate b, adjusting the pH of the filtrate b to 5.5 with a hydrochloric acid solution, placing it in a concentration tank and concentrating it under reduced pressure until the ethanol in the filtrate b is completely removed, and obtaining about 150 kg of concentrated solution, transferring the concentrated solution to a crystallization tank, cooling it at 4° C. for 18 h, precipitating crystals, collecting the precipitate, obtaining a crude hesperidin extract, filtering and collecting the filtrate c, drying the obtained crude hesperidin extract, and repeating the above extraction process to obtain high-purity hesperidin;

[0041] (3) The filtrate a and the filtrate c are mixed to obtain a crude extract mixture, the concentration of the crude extract mixture is adjusted to 1 g / ml with pure water, the pH of the mixture is adjusted to 5.5 with hydrochloric acid, the sample is loaded at a rate of 2 mL / min, loaded on a macroporous resin adsorption column, eluted with 80% ethanol as an eluent, the elution rate is 0.5 mL / min, the water washing volume is 8 BV, the eluate is concentrated under reduced pressure, and recrystallized to obtain high-purity naringin.

[0042] The extraction rates of naringin and hesperidin were calculated to be 95.8% and 96.2% respectively. The purity of naringin and hesperidin was determined to be 97.4% and 98.1% respectively by high performance liquid chromatography.

[0043] Comparative Example 1

[0044] The difference from Example 1 is that in step (2), the pH of the filtrate b is adjusted to 4.0, and the remaining steps are consistent with Example 1.

[0045] The results show that the extraction rates of naringin and hesperidin are 90.7% and 90.5% respectively. The purity of naringin and hesperidin is 91.0% and 91.2% respectively as determined by high performance liquid chromatography.

[0046] Comparative Example 2

[0047] The difference from Example 1 is that in step (2), the pH of the filtrate b is adjusted to 6.5, and the remaining steps are consistent with Example 1.

[0048] The results show that the extraction rates of naringin and hesperidin are 85.1% and 83.3% respectively. The purity of naringin and hesperidin is 86.4% and 84.2% respectively as determined by high performance liquid chromatography.

[0049] Comparative Example 3

[0050] The difference from Example 1 is that the concentration of potassium hydroxide in step (2) is 0.1%, and the other steps are consistent with Example 1.

[0051] The results show that the extraction rates of naringin and hesperidin are 85.3% and 84.2% respectively. The purity of naringin and hesperidin is 86.9% and 84.8% respectively as determined by high performance liquid chromatography.

[0052] Comparative Example 4

[0053] The difference from Example 1 is that the concentration of potassium hydroxide in step (2) is 2.5%, and the remaining steps are consistent with Example 1.

[0054] The results show that the extraction rates of naringin and hesperidin are 76.2% and 72.9% respectively. The purity of naringin and hesperidin is 78.3% and 73.5% respectively as determined by high performance liquid chromatography.

[0055] Comparative Example 5

[0056] The difference from Example 1 is that step (2) uses 80% ethanol solution containing 0.5% sodium hydroxide, and the other steps are consistent with Example 1.

[0057] The results show that the extraction rates of naringin and hesperidin are 89.0% and 85.6% respectively. The purity of naringin and hesperidin is 89.9% and 86.3% respectively as determined by high performance liquid chromatography.

[0058] Comparative Example 6

[0059] The difference from Example 1 is that the macroporous resin column in step (3) is D101 macroporous resin, and the other steps are consistent with Example 1.

[0060] The results show that the extraction rate of naringin is 88.3%. The purity of naringin is 89.0% as determined by high performance liquid chromatography.

[0061] Comparative Example 7

[0062] The difference from Example 1 is that the column flow rate in step (3) is 5 mL / min, and the remaining steps are consistent with Example 1.

[0063] The results show that the extraction rate of naringin is 75.8%, and the purity of naringin is 78.2% as determined by high performance liquid chromatography.

[0064] Comparative Example 8

[0065] The difference from Example 1 is that the elution rate in step (3) is 1.2 mL / min, and the remaining steps are consistent with Example 1.

[0066] The results show that the extraction rate of naringin is 73.1%. The purity of naringin measured by high performance liquid chromatography is 74.0%.

[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for efficiently extracting naringin and hesperidin from bergamot, characterized in that: The following steps are involved: (1) drying, crushing and sieving fresh bergamot to obtain bergamot powder, defatting the bergamot powder with petroleum ether, and filtering to obtain filtrate a and filter residue a; (2) drying the filter residue a in step (1), adding an alkaline ethanol solution, refluxing and extracting for 1-5 hours, filtering, collecting the filtrate b, adjusting the pH of the filtrate b to within the range of 4.5-6, and concentrating under reduced pressure until the ethanol in the filtrate b is completely removed to obtain a concentrated solution, cooling the concentrated solution for a period of time, precipitating crystals, collecting the precipitate, obtaining a crude hesperidin extract, filtering and collecting the filtrate c, drying the obtained crude hesperidin extract, and repeating the above extraction process to obtain high-purity hesperidin; (3) The filtrate a and the filtrate c are mixed to obtain a crude extract mixture, and the concentration of the crude extract mixture is adjusted to 0.5-1 g / ml using pure water, followed by adsorption with a macroporous resin, elution with ethanol, concentration under reduced pressure, and recrystallization to obtain high-purity naringin.

2. The method for efficiently extracting naringin and hesperidin from bergamot according to claim 1, characterized in that: The sieving in step (1) is specifically sieving through a sieve with a mesh size of 80-120.

3. The method for efficiently extracting naringin and hesperidin from bergamot according to claim 1, characterized in that: The alkaline ethanol solution in step (2) is specifically a 50-80% ethanol solution containing 0.5-2.0% potassium hydroxide, wherein the ratio of potassium hydroxide to ethanol is a mass volume ratio.

4. The method for efficiently extracting naringin and hesperidin from bergamot according to claim 1, characterized in that: The mass volume ratio of the alkaline ethanol solution and the filter residue in step (2) is 8-10:

1.

5. The method for efficiently extracting naringin and hesperidin from bergamot according to claim 1, characterized in that: The temperature of the reflux extraction in step (2) is 60-80° C., and the reflux extraction is carried out under a nitrogen atmosphere.

6. The method for efficiently extracting naringin and hesperidin from bergamot according to claim 1, characterized in that: The pH in step (2) is in the range of 4.5-5.

5.

7. The method for efficiently extracting naringin and hesperidin from bergamot according to claim 1, characterized in that: The cooling temperature in step (2) is 0-4°C and the cooling time is 12-18h.

8. The method for efficiently extracting naringin and hesperidin from bergamot according to claim 1, characterized in that: The concentration of ethanol in step (3) is 60-80%, and the column flow rate is 1-3 mL / min.

9. The method for efficiently extracting naringin and hesperidin from bergamot according to claim 1, characterized in that: The elution rate in step (3) is 0.1-1 mL / min, and the water washing volume is 5-8 BV.

10. The method for efficiently extracting naringin and hesperidin from bergamot according to claim 1, characterized in that: The column flow rate of ethanol in step (3) is 2.5 mL / min, and the elution rate is 0.2 mL / min.

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