CN114213486A - Method for extracting and purifying nicotinamide mononucleotide from broccoli - Google Patents
Method for extracting and purifying nicotinamide mononucleotide from broccoli Download PDFInfo
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Abstract
The invention discloses a method for extracting and purifying nicotinamide mononucleotide from broccoli, which comprises the following steps: s1, obtaining broccoli powder; s2, freezing the broccoli powder by liquid nitrogen, adding an extractant to unfreeze, performing ultrasonic-assisted extraction, and filtering to obtain a broccoli extracting solution; s3, adjusting the pH value of the broccoli extracting solution to be neutral, then dropwise adding an organic solvent, and cooling and crystallizing to obtain the nicotinamide mononucleotide, wherein the extraction rate of the nicotinamide mononucleotide is high, the purity is high, and the efficiency is high.
Description
Technical Field
The invention relates to the field of nicotinamide mononucleotide, and in particular relates to a method for extracting and purifying nicotinamide mononucleotide from broccoli.
Background
Nicotinamide mononucleotide is an important precursor of the intracellular assembly of nicotinamide adenine dinucleotide (NAD +, also known as coenzyme I). NAD + is redox coenzyme of tricarboxylic acid cycle, NMN is used as intermediate in NAD + remediation pathway, has effects of resisting oxidation, reducing oxidative stress, enhancing energy metabolism, slowing down physiological deterioration of organism, and prolonging life. Mills et al found that nicotinamide mononucleotide can significantly improve age-related physiological decline of mice, such as inhibiting age-related weight gain, enhancing energy metabolism, improving insulin sensitivity and lipid distribution in plasma, and improving eye function; nicotinamide mononucleotide prevents age-related gene expression changes in a tissue-specific manner and enhances mitochondrial oxidative metabolism in skeletal muscle, fighting against senescence.
Currently, nicotinamide mononucleotide is mainly prepared by microbial fermentation, chemical synthesis or in vitro enzymatic catalysis. Nicotinamide mononucleotide is also rich in some fruits and vegetables, including broccoli, cabbage, cucumber, green soybean, avocado, etc. and needs to be extracted and purified to obtain the nicotinamide mononucleotide.
For example, CN202010472425.8 is a method for extracting nicotinamide mononucleotide from avocado, which adopts a mode of extracting solvent in cooperation with ultrasonic wave assisted extraction to extract nicotinamide mononucleotide from avocado, wherein the extraction rate is to be improved, and the product is not purified and has low purity;
PCT/CN2016/092455 discloses a method for extracting and purifying nicotinamide mononucleotide from broccoli, which comprises the steps of subjecting a crude product of nicotinamide mononucleotide prepared by a biocatalysis method to anion exchange column, nanofiltration, and adsorption and desorption by chelating resin to obtain a relatively pure finished product of nicotinamide mononucleotide.
Patent PCT/CN2015/096215 discloses a purification method of β -nicotinamide mononucleotide using reversed phase high performance liquid chromatography to prepare a column, wherein the stationary phase is octadecylsilane chemically bonded silica, the mobile phase a is hydrochloric acid solution with pH of 3-7, and the mobile phase B is ethanol, and gradient elution purification is performed. Obtaining purified nicotinamide mononucleotide.
The purification method and the preparation method of the patent CN 112159445A nicotinamide mononucleotide use silica gel column to carry out chromatographic separation on a beta-nicotinamide mononucleotide crude product, and alcohol is used as a mobile phase to carry out gradient elution.
The method is to purify by ion exchange resin or chromatographic silica gel column to obtain the finished product of nicotinamide mononucleotide, which is easy to mix with impurities such as solvent and the like, and has complex operation, low efficiency and expensive purification equipment.
Disclosure of Invention
In order to solve the problems, the invention provides a method for extracting and purifying nicotinamide mononucleotide from broccoli, which has high extraction rate, high purity and high efficiency of the nicotinamide mononucleotide.
The inventor finds that the nicotinamide mononucleotide can be crystallized by a solution synthesis method, the nicotinamide mononucleotide is dissolved out of broccoli by a specific extracting agent and ultrasonic extraction, the extraction rate is high, and the nicotinamide mononucleotide is purified in a solid state by crystallization, and the purification process does not need to pass through a column, so that the purity is high, and the efficiency is high.
The technical scheme of the invention is to provide a method for extracting and purifying nicotinamide mononucleotide from broccoli, which comprises the following steps:
s1, obtaining broccoli powder;
s2, freezing the broccoli powder by liquid nitrogen, adding an extractant to unfreeze, performing ultrasonic-assisted extraction, and filtering to obtain a broccoli extracting solution;
s3, adjusting the pH value of the broccoli extracting solution to be neutral, then dropwise adding an organic solvent, and cooling and crystallizing to obtain the nicotinamide mononucleotide.
The extraction and purification process and mechanism of the scheme are as follows: the method comprises the steps of freezing broccoli cells by using liquid nitrogen, wherein in the quick freezing process, the outer surface of the cell wall shrinks along with the temperature reduction, but the inner part is still hot, so that the shrinkage is restrained to generate tensile stress, the inner surface is opposite to the inner surface and is under the action of compressive stress, when the cell wall is heated and thawed, the outer surface is restrained to generate compressive stress due to expansion, the inner surface generates tensile stress, as the thermal conductivity of a cellulose material forming the cell wall is small, the temperature change of the inner surface of the cell wall is much later than that of the outer surface, so that great temperature difference is generated on two sides of the wall, and impact stress is generated, the thermal stress is usually much larger and is applied to the cell wall at the highest speed, the cell wall loses ductility and becomes brittle, the broccoli cell wall generates fatigue cracks under the action of alternating stress, the cracks gradually expand in the fatigue process, and finally the cell wall is cracked, the broccoli extract is released, compared with the grinding extraction in the prior art, the cell wall is broken more thoroughly in the method, so that the broccoli extract is released more thoroughly, and the amide mononucleotide is released as much as possible;
after the extraction agent is added for thawing, substances in cell membranes can seep out of the membranes due to different concentrations of the substances in the membranes at the moment of cell wall rupture, and the substances in the cell membranes can be used as the thawing solution to accelerate the thawing speed on one hand, and can be used as the extraction agent to seep out contents on the other hand, so that the extraction efficiency is improved, the broccoli extracting solution with high extraction rate is obtained, and the loss of nicotinamide mononucleotide in the raw material extraction step is less;
the broccoli extract contains other substances except nicotinamide mononucleotide, and can be crystallized by a solution synthesis method for purification, nicotinamide mononucleotide crystals can be obtained by utilizing the property that the nicotinamide mononucleotide can be crystallized and precipitated in water and an organic solvent, and the purification step does not need to dilute and pass through a column, but directly purifies and crystallizes, so that the nicotinamide mononucleotide has high purity, less loss and simple operation.
Preferably, the extractant comprises a mixture of a surfactant and an aqueous acetic acid solution;
the mixture of the surfactant and the acetic acid aqueous solution is used as an extracting agent, the surfactant and the ultrasonic wave are combined to generate a cavitation effect, the extraction rate is improved, the acetic acid can stabilize nicotinamide mononucleotide and improve the solubility on one hand, and on the other hand, the acetic acid belongs to a weak electrolyte, plays a role of a surfactant promoter and further improves the extraction rate.
Preferably, the surfactant is a nonionic emulsifier, and does not ionize in aqueous solution, because it is not in an ionic state in solution, is more stable than an ionic surfactant, and has better synergistic effect with acetic acid;
more preferably, the nonionic emulsifier comprises one or more of alkylphenol ethoxylates and castor oil polyoxyethylene, and the nonionic emulsifier has excellent foaming performance, so that larger and more bubbles can be generated under the action of ultrasonic waves, and the bubbles are instantaneously shrunk and exploded under the continuous action of the ultrasonic waves to generate a higher-energy sonochemical effect, so that nicotinamide mononucleotide can be dissolved out to a greater extent.
Preferably, the organic solvent is one or more of tetrahydrofuran, acetonitrile and acetone, and more preferably, the volume ratio of the organic solvent to the extracting agent is 1: 5-6, under which nicotinamide mononucleotide crystals precipitate.
Preferably, the temperature of the liquid nitrogen freezing is-180 ℃ to-200 ℃, the freezing time is 3-5min, and the purpose of the step is to provide a quick freezing environment for the broccoli molecules.
Preferably, the temperature for cooling crystallization is 4-8 ℃.
Preferably, the extraction temperature of the ultrasonic-assisted extraction is 55-60 ℃, and the extraction power is 500-600W.
Preferably, the pore size of the filtered filter paper is less than or equal to 5 um.
The invention has the beneficial effects that:
1. quick freezing and thawing are utilized to thoroughly destroy broccoli cells, the release rate of a dissolved substance is improved, an extractant is utilized to cooperate with ultrasonic assisted extraction, and the extraction rate of nicotinamide mononucleotide is improved;
2. the broccoli extracting solution is directly cooled and crystallized by using an organic solvent-water solution, and the broccoli extracting solution has high purity, less loss and high efficiency.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available by purchase.
The method for extracting and purifying nicotinamide mononucleotide from broccoli comprises the following steps:
s1, obtaining broccoli powder: taking 300g of broccoli stems and leaves, washing the broccoli stems and leaves with distilled water, cutting the broccoli stems and leaves into small blocks of 0.5cm multiplied by 0.5cm, drying the broccoli blocks in an oven at 45-50 ℃ to constant weight, sieving the broccoli blocks with a 80-mesh sieve to obtain broccoli powder with the particle size of less than or equal to 150 mu m, equally dividing the broccoli powder obtained by 300g of broccoli stems and leaves into 6 equal parts, and carrying out the following extraction and purification processes by taking 50g of the evenly divided broccoli powder as a research object.
S2, preparing an extracting agent: a. weighing 0.1-3g of surfactant, dispersing in 100ml of deionized water, and fully stirring until the surfactant is dissolved to obtain a surfactant solution, wherein the surfactant comprises one or more of alkylphenol polyoxyethylene ether and castor oil polyoxyethylene ether;
b. placing 0.5-2g of acetic acid in 100ml of deionized water, and fully stirring until the acetic acid is dissolved to obtain an acetic acid aqueous solution;
c. mixing a surfactant solution with an acetic acid aqueous solution to obtain the extractant;
extracting nicotinamide mononucleotide: putting 50g of broccoli powder into a liquid nitrogen tank at the temperature of-180-200 ℃, rapidly cooling and freezing for 3-5min, transferring to a beaker, adding 600ml of 500-one extractant, heating the beaker at the temperature of 30-40 ℃, and heating for 1-3min to unfreeze the broccoli powder; placing the unfrozen substances in an ultrasonic cleaner, adjusting the extraction temperature to 55-60 ℃, extracting at the power of 500-600W for 30-60min to obtain a solid-liquid mixture, filtering by using filter paper with the pore diameter less than or equal to 5 mu m, and performing solid-liquid separation to obtain a supernatant to obtain a broccoli extracting solution;
s3, crystallization and purification: putting the broccoli extracting solution into a crystallization kettle, adjusting the pH value of the broccoli extracting solution to 6-7 by using a phosphoric acid buffer solution, slowly dropwise adding 100ml of an organic solvent while stirring, wherein the organic solvent comprises one or more of tetrahydrofuran, acetonitrile and acetone, keeping the temperature of the solution at 30-40 ℃ in the dropwise adding process, reducing the temperature of the solution to 4-8 ℃ after the dropwise adding is finished, standing, waiting for crystal precipitation, and filtering the solution after the crystallization is finished to obtain the nicotinamide mononucleotide.
The present invention will be described in detail below by way of examples, but the scope of the present invention is not limited thereto.
Example 1
A method for extracting and purifying nicotinamide mononucleotide from broccoli comprises the following steps:
s1, obtaining broccoli powder: taking 300g of broccoli stems and leaves, washing the broccoli stems and leaves with distilled water, cutting the broccoli stems and leaves into small blocks of 0.5cm multiplied by 0.5cm, drying the broccoli blocks in an oven at 45-50 ℃ to constant weight, sieving the broccoli blocks with a 80-mesh sieve to obtain broccoli powder with the particle size of less than or equal to 150 mu m, equally dividing the broccoli powder obtained by the 300g of broccoli stems and leaves into 6 equal parts, and carrying out the following extraction and purification processes by taking 50g of the evenly divided broccoli powder as a research object;
s2, preparing an extracting agent: a. weighing 0.1g of alkylphenol polyoxyethylene ether, dispersing in 100ml of deionized water, and fully stirring until the alkylphenol polyoxyethylene ether is dissolved;
b. placing 0.5g of acetic acid in 100ml of deionized water, and fully stirring until the acetic acid is dissolved to obtain an acetic acid aqueous solution;
c. mixing the surfactant solution with acetic acid water solution to obtain an extractant;
extracting nicotinamide mononucleotide: putting 50g of broccoli powder into a liquid nitrogen tank at the temperature of-180 ℃, rapidly cooling and freezing for 3min, transferring the broccoli powder into a beaker, adding 500ml of an extracting agent, heating the beaker at the temperature of 30 ℃ for 3min, and unfreezing the broccoli powder; placing the thawed substance in an ultrasonic cleaner, adjusting extraction temperature to 55 deg.C, extracting at 500W for 30min to obtain solid-liquid mixture, filtering with filter paper with pore diameter equal to 5 μm, and separating solid and liquid to obtain supernatant to obtain broccoli extractive solution;
s3, crystallization and purification: putting the broccoli extracting solution into a crystallization kettle, adjusting the pH value of the broccoli extracting solution to 7 by using a phosphoric acid buffer solution, slowly dropwise adding 100ml of tetrahydrofuran while stirring, keeping the temperature of the solution at 30 ℃ in the dropwise adding process, reducing the temperature of the solution to 4 ℃ after the dropwise adding is finished, standing, waiting for crystal precipitation, and filtering the solution after the crystallization is finished to obtain the nicotinamide mononucleotide.
Example 2
A method for extracting and purifying nicotinamide mononucleotide from broccoli comprises the following steps:
s1, obtaining broccoli powder: taking 50g of broccoli powder obtained by evenly dividing in the example 1 as a research object, and carrying out the following extraction and purification processes;
s2, preparing an extracting agent: a. weighing 3g of castor oil polyoxyethylene ether, dispersing in 100ml of deionized water, and fully stirring until the castor oil polyoxyethylene ether is dissolved to obtain a surfactant solution;
b. placing 2g of acetic acid in 100ml of deionized water, and fully stirring until the acetic acid is dissolved to obtain an acetic acid aqueous solution;
c. mixing a surfactant solution with an acetic acid aqueous solution to obtain the extractant;
extracting nicotinamide mononucleotide: putting 50g of broccoli powder into a liquid nitrogen tank at the temperature of-200 ℃, rapidly cooling and freezing for 5min, transferring the broccoli powder into a beaker, adding 600ml of an extracting agent, heating the beaker at the temperature of 40 ℃ for 1min, and unfreezing the broccoli powder; placing the thawed substance in an ultrasonic cleaner, adjusting extraction temperature to 60 deg.C, extracting at 600W for 60min to obtain solid-liquid mixture, filtering with filter paper with pore diameter equal to 5 μm, and separating solid and liquid to obtain supernatant to obtain broccoli extractive solution;
s3, crystallization and purification: and (2) putting the broccoli extracting solution into a crystallization kettle, adjusting the pH value of the broccoli extracting solution to 6 by using a phosphoric acid buffer solution, slowly dropwise adding 100ml of acetonitrile into the broccoli extracting solution while stirring, keeping the temperature of the solution at 40 ℃ in the dropwise adding process, reducing the temperature of the solution to 8 ℃ after the dropwise adding is finished, standing the solution for the precipitation of crystals, and filtering the solution after the crystallization is finished to obtain the nicotinamide mononucleotide.
Example 3
A method for extracting and purifying nicotinamide mononucleotide from broccoli comprises the following steps:
s1, obtaining broccoli powder: taking 50g of broccoli powder obtained by evenly dividing in the example 1 as a research object, and carrying out the following extraction and purification processes;
s2, preparing an extracting agent: a. weighing 2g of alkylphenol polyoxyethylene ether, dispersing the alkylphenol polyoxyethylene ether in 100ml of deionized water, and fully stirring until the alkylphenol polyoxyethylene ether is dissolved to obtain a surfactant solution;
b. putting 1g of acetic acid into 100ml of deionized water, and fully stirring until the acetic acid is dissolved to obtain an acetic acid aqueous solution;
c. mixing a surfactant solution with an acetic acid aqueous solution to obtain the extractant;
extracting nicotinamide mononucleotide: putting 50g of broccoli powder into a liquid nitrogen tank at the temperature of-190 ℃, rapidly cooling and freezing for 4min, transferring the broccoli powder into a beaker, adding 550ml of an extracting agent, heating the beaker at the temperature of 35 ℃ for 2min, and unfreezing the broccoli powder; placing the thawed substance in an ultrasonic cleaner, adjusting extraction temperature to 58 deg.C, extracting at 550W for 50min to obtain solid-liquid mixture, filtering with filter paper with pore diameter equal to 5 μm, and separating solid and liquid to obtain supernatant to obtain broccoli extractive solution;
s3, crystallization and purification: and (2) putting the broccoli extracting solution into a crystallization kettle, adjusting the pH value of the broccoli extracting solution to 7 by using a phosphoric acid buffer solution, slowly dropwise adding 100ml of acetone into the broccoli extracting solution while stirring, keeping the temperature of the solution at 35 ℃ in the dropwise adding process, reducing the temperature of the solution to 6 ℃ after the dropwise adding is finished, standing the solution for the precipitation of crystals, and filtering the solution after the crystallization is finished to obtain the nicotinamide mononucleotide.
Example 4
A method for extracting and purifying nicotinamide mononucleotide from broccoli comprises the following steps:
s1, obtaining broccoli powder: taking 50g of broccoli powder obtained by evenly dividing in the example 1 as a research object, and carrying out the following extraction and purification processes;
s2, preparing an extracting agent: a. weighing 1.2g of castor oil polyoxyethylene ether, dispersing in 100ml of deionized water, and fully stirring until the castor oil polyoxyethylene ether is dissolved to obtain a surfactant solution;
b. putting 1.5g of acetic acid into 100ml of deionized water, and fully stirring until the acetic acid is dissolved to obtain an acetic acid aqueous solution;
c. mixing a surfactant solution with an acetic acid aqueous solution to obtain the extractant;
extracting nicotinamide mononucleotide: putting 50g of broccoli powder into a liquid nitrogen tank at the temperature of-200 ℃, rapidly cooling and freezing for 3min, transferring the broccoli powder into a beaker, adding 500ml of an extracting agent, heating the beaker at the temperature of 40 ℃ for 3min, and unfreezing the broccoli powder; placing the thawed substance in an ultrasonic cleaner, adjusting extraction temperature to 60 deg.C, extracting at 600W for 60min to obtain solid-liquid mixture, filtering with filter paper with pore diameter equal to 5 μm, and separating solid and liquid to obtain supernatant to obtain broccoli extractive solution;
s3, crystallization and purification: putting the broccoli extracting solution into a crystallization kettle, adjusting the pH value of the broccoli extracting solution to 6.5 by using a phosphoric acid buffer solution, slowly dropwise adding 100ml of acetonitrile into the broccoli extracting solution while stirring, keeping the temperature of the solution at 40 ℃ in the dropwise adding process, reducing the temperature of the solution to 4 ℃ after the dropwise adding is finished, standing the solution for the precipitation of crystals, and filtering the solution after the crystallization is finished to obtain the nicotinamide mononucleotide.
Comparative example 1
A method for extracting and purifying nicotinamide mononucleotide from broccoli directly uses ethanol as an extracting agent and then utilizes crystallization purification to obtain nicotinamide mononucleotide in the embodiment, and comprises the following steps:
s1, obtaining broccoli powder: taking 50g of broccoli powder obtained by evenly dividing in the example 1 as a research object, and carrying out the following extraction and purification processes;
s2, extracting nicotinamide mononucleotide: transferring 50g of broccoli powder to a beaker, adding 500ml of ethanol as an extractant, placing the broccoli powder in an ultrasonic cleaner, adjusting the extraction temperature to 60 ℃, extracting for 60min at the extraction power of 600W to obtain a solid-liquid mixture, filtering the solid-liquid mixture by using filter paper with the aperture equal to 5um, and carrying out solid-liquid separation to obtain a supernatant, thus obtaining a broccoli extracting solution;
s3, crystallization and purification: putting the broccoli extracting solution into a crystallization kettle, adjusting the pH value of the broccoli extracting solution to 7 by using a phosphoric acid buffer solution, slowly dropwise adding 100ml of an organic solvent while stirring, wherein the organic solvent comprises one or more of tetrahydrofuran, acetonitrile and acetone, keeping the temperature of the solution at 40 ℃ in the dropwise adding process, reducing the temperature of the solution to 4 ℃ after the dropwise adding is finished, standing, waiting for crystal precipitation, and filtering the solution after the crystallization is finished to obtain the nicotinamide mononucleotide solid.
Comparative example 2
The extraction and purification method of nicotinamide mononucleotide in broccoli comprises the following steps:
s1, obtaining broccoli powder: taking 50g of broccoli powder obtained by evenly dividing in the example 1 as a research object, and carrying out the following extraction and purification processes;
s2, preparing an extracting agent: a. weighing 0.1-3g of surfactant, dispersing in 100ml of deionized water, and fully stirring until the surfactant is dissolved to obtain a surfactant solution, wherein the surfactant comprises one or more of alkylphenol polyoxyethylene ether and castor oil polyoxyethylene ether;
b. placing 0.5-2g of acetic acid in 100ml of deionized water, and fully stirring until the acetic acid is dissolved to obtain an acetic acid aqueous solution;
c. mixing a surfactant solution with an acetic acid aqueous solution to obtain the extractant;
extracting nicotinamide mononucleotide: putting 50g of broccoli powder into a liquid nitrogen tank at the temperature of-180-200 ℃, rapidly cooling and freezing for 3-5min, transferring to a beaker, adding 600ml of 500-one extractant, heating the beaker at the temperature of 30-40 ℃, and heating for 1-3min to unfreeze the broccoli powder; placing the unfrozen substances in an ultrasonic cleaner, adjusting the extraction temperature to 55-60 ℃, extracting at the power of 500-600W for 30-60min to obtain a solid-liquid mixture, filtering by using filter paper with the pore diameter less than or equal to 5 mu m, and performing solid-liquid separation to obtain a supernatant to obtain a broccoli extracting solution;
s3, purification: sampling broccoli extract, and then loading the broccoli extract on a reversed-phase high performance liquid chromatography preparation column (the diameter and the length of the column are 5cm x 30cm), wherein the stationary phase is forty-eight alkyl silane bonded silica gel, the mobile phase A is hydrochloric acid solution, the mobile phase B is 40 wt% ethanol, the flow rate is 2500ml/min, the detection wavelength is 260nm, and the gradient is B%: 4 percent and the loading amount is 8g, gradient elution purification is carried out for 40min, and the solvent is evaporated after the target peak is collected to obtain the purified beta-nicotinamide mononucleotide.
Test method
The total nicotinamide mononucleotide products obtained in examples 1-4 and comparative examples 1-2 are weighed, the nicotinamide mononucleotide content is determined by liquid phase mass spectrometry, and the nicotinamide mononucleotide purity is calculated according to the ratio of the nicotinamide mononucleotide content to the nicotinamide mononucleotide content, wherein the conditions for performing the nicotinamide mononucleotide content by the liquid phase mass spectrometry are as follows: a chromatographic column: syncronis C18 (100X 2.1mm, 1.7 μm); sample introduction amount: 10 mu L of the solution; column temperature: 35 ℃; flow rate: 0.2 min/mL; mobile phase: the phase A is an organic phase containing 100% acetonitrile; phase D was an aqueous phase containing 0.1% formic acid. The mass spectrometry conditions were as follows: an ion source: electrospray ionization ESI; the scanning mode is as follows: positive ion scan ESI (+); the detection mode is as follows: multiple Reaction Monitoring (MRM).
The test results are shown in table 1:
| total mass of product (mg) | Nicotinamide mononucleotide mass (mg) | Nicotinamide mononucleotide purity (%) | |
| Example 1 | 0.61 | 0.60 | 98 |
| Example 2 | 0.56 | 0.55 | 99 |
| Example 3 | 0.56 | 0.54 | 98 |
| Example 4 | 0.58 | 0.56 | 97 |
| Comparative example 1 | 0.36 | 0.34 | 96 |
| Comparative example 2 | 0.51 | 0.49 | 97 |
Furthermore, those skilled in the art will appreciate that while some embodiments herein include some features included in other embodiments, rather than other features, combinations of features of different embodiments are meant to be within the scope of the application and form different embodiments. For example, in the claims, any of the claimed embodiments may be used in any combination.
Those of ordinary skill in the art will understand that: the above embodiments are only used for illustrating the technical solutions of the present application, and not for limiting the same, although the present application is described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it is possible to modify the solutions described in the previous embodiments or to substitute some or all of the technical features thereof without departing from the scope of the claims.
Claims (10)
1. A method for extracting and purifying nicotinamide mononucleotide from broccoli is characterized by comprising the following steps:
s1, obtaining broccoli powder;
s2, freezing the broccoli powder by liquid nitrogen, adding an extractant to unfreeze, performing ultrasonic-assisted extraction, and filtering to obtain a broccoli extracting solution;
s3, adjusting the pH value of the broccoli extracting solution to be neutral, then dropwise adding an organic solvent, and cooling and crystallizing to obtain the nicotinamide mononucleotide.
2. The method for extracting and purifying nicotinamide mononucleotide from broccoli as claimed in claim 1, wherein said extracting agent comprises a mixture of a surfactant and an aqueous solution of acetic acid.
3. The method for extracting and purifying nicotinamide mononucleotide from broccoli as claimed in claim 2, wherein said surfactant is a nonionic emulsifier.
4. The method for extracting and purifying nicotinamide mononucleotide from broccoli according to claim 3, wherein the nonionic emulsifier comprises one or more of alkylphenol ethoxylate and castor oil ethoxylate.
5. The method for extracting and purifying nicotinamide mononucleotide from broccoli as claimed in claim 1, wherein said organic solvent is one or more of tetrahydrofuran, acetonitrile and acetone.
6. The method for extracting and purifying nicotinamide mononucleotide from broccoli according to claim 1, wherein the volume ratio of the organic solvent to the extracting agent is 1: 5-6.
7. The method for extracting and purifying nicotinamide mononucleotide from broccoli according to claim 1, wherein the temperature of freezing with liquid nitrogen is-180 ℃ to-200 ℃, and the freezing time is 3-5 min.
8. The method for extracting and purifying nicotinamide mononucleotide from broccoli according to claim 1, wherein the temperature for cooling and crystallization is 4-8 ℃.
9. The method as claimed in claim 1, wherein the extraction temperature of the ultrasonic-assisted extraction is 55-60 ℃, and the extraction power is 500-600W.
10. The method for extracting and purifying nicotinamide mononucleotide from broccoli according to claim 1, wherein the pore size of the filtered filter paper is less than or equal to 5 um.
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| CN115010772A (en) * | 2022-06-16 | 2022-09-06 | 国珍健康科技（北京）有限公司 | Nicotinamide mononucleotide-containing pine pollen extract, extract and preparation method thereof |
| CN115769894A (en) * | 2022-12-13 | 2023-03-10 | 福迈香港基因工程有限公司 | Preparation method of extract containing high-activity NMN |
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| CN115769894A (en) * | 2022-12-13 | 2023-03-10 | 福迈香港基因工程有限公司 | Preparation method of extract containing high-activity NMN |
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