CN108642117B - Method for extracting limonin and nomilin - Google Patents
Method for extracting limonin and nomilin Download PDFInfo
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Abstract
The invention relates to a method for extracting limonin and nomilin, which comprises the following steps: degreasing the raw materials, and performing enzymolysis by using wheat starter compound enzyme liquid; removing the supernatant to obtain an enzymolysis substrate; adding sodium salicylate solution for extraction, and separating the extract; adjusting the pH value to 4.6-5.0, taking supernatant liquid, putting the supernatant liquid on macroporous resin, eluting with 20-25% ethanol solution to remove impurities, eluting with 85-90% ethanol solution, collecting eluent, recovering ethanol, and drying to obtain an extract A; heating and dissolving the obtained extract A with a mixed solution of dichloromethane and isopropanol, filtering to remove insoluble substances, cooling, and crystallizing to obtain limonin crystals; evaporating the mother liquor under reduced pressure to obtain extract B, heating and dissolving with mixed solution of ethyl acetate and ethanol, filtering to remove insoluble substances, cooling, and crystallizing to obtain nomilin crystal. The extraction method has low cost, high yield and product purity, and reduced environmental pollution.
Description
Technical Field
The invention belongs to the technical field of extraction, separation and purification of effective components in natural plants, and particularly relates to a method for extracting limonin and nomilin.
Background
Limonin (Limonin) and Nomilin (Nomilin) are the major limonoids in citrus, which are secondary metabolites of triterpenes, found primarily in rutaceae and meliaceae plants, and are particularly high in citrus seeds. The limonin compounds in the tangerine seed mainly comprise limonin compounds aglycone and glucoside, wherein the aglycone substances mainly comprise limonin, nomilin, obacunone, iso-obacunone and the like, and the glucoside substances are generally D-ring opening and a glycosyl is added. Limonin, alternative name: phellodendron lactone and evodia fruit lactone, molecular formula C26H30O8470.53, pure white, bitter and crystalline; nomilin, formula C28H34O9Molecular weight 514.57, white powder, bitter taste.
With the determination of the structure of the limonin compounds and the clarification of the biological function of the limonin compounds, the development and the utilization of the limonin compounds in citrus fruits have wide market prospects. The prior knowledge of limonoids has been to remove the limonoids from citrus fruit to enhance the commercial value of citrus juice. Later researches find that the limonin compounds have important biological activity in the aspects of promoting cancer cell apoptosis, resisting bacteria, viruses, malaria, inflammation, insect antifeedant and the like. At present, the demand of people for limonin compounds is increasing day by day, and large-scale extraction and separation of limonin analogues and application research as functional food additives at home and abroad are started; and the limonin compounds can be predicted to have wider application prospects in the near future.
At present, the extraction and separation process of limonin compounds has the problems of large organic solvent consumption, low yield, low purity and high extraction cost. Chinese patent CN101531666A discloses a process for extracting limonin from pomelo kernel, which describes that an alcohol organic solvent is used as an extraction solvent, and limonin with purity of 92% and yield of 0.25% is obtained by chromatography and crystallization, and the yield and purity of limonin are both low, and the dosage of the organic solvent is large. Chinese patent CN101367823A discloses a method for separating limonin, evodiamine and rutaecarpine from fructus evodiae, which obtains limonin with a purity of 99% by organic solvent extraction, column chromatography and high-efficiency preparative liquid phase separation, and the extraction process has the problem of low yield, only 0.1%, and high cost, and is only suitable for experimental preparation at present. Chinese patent CN102153615A discloses a method for extracting active ingredients from orange seeds, which adopts ultrafiltration treatment, and carries out affinity chromatography on filtrate, and beta-glucosaccharase, glucoamylase and alpha-amylase are subjected to enzymolysis, so as to obtain limonin with the yield of 0.62-0.65%, the purity of 98-100%, the yield of nomilin of 0.085-0.11% and the purity of 94-97%. The extraction method still has the defects of incomplete enzymolysis, high cost and difficult large-scale production of membrane treatment. Chinese patent CN103421077A discloses a method for separating and purifying limonin compounds from pomelo fruits, which adopts three steps of resin primary purification, high-speed countercurrent chromatography purification and semi-preparative liquid chromatography separation, adopts experimental-grade instruments, and is difficult to expand production.
Disclosure of Invention
In order to overcome the problems in the extraction process of limonin and nomilin, the invention provides a method for extracting limonin and nomilin, which can promote the dissolution of limonin and nomilin in tangerine seeds and solve the problems of large organic solvent dosage, low extraction yield and high extraction cost in the existing process for extracting limonin. The process of the invention is applicable to limonin and nomilin-containing feedstocks such as seeds of rutaceae and meliaceae plants, especially citrus plants (e.g., citrus kernels, citrus cores, etc.).
The technical scheme of the invention is as follows:
a method for extracting limonin and nomilin, comprising: degreasing a raw material containing limonin and nomilin to obtain a pretreatment material; carrying out enzymolysis by adopting wheat starter compound enzyme liquid; removing supernatant after enzymolysis to obtain an enzymolysis substrate; adding sodium salicylate solution for extraction, and separating the extract; adjusting the pH value to 4.6-5.0, taking the supernatant, loading the supernatant on macroporous resin, eluting with 25-30% (volume fraction, the same below) of ethanol solution to remove impurities, eluting with 85-90% of ethanol solution, collecting the eluate, recovering ethanol, and drying to obtain an extract A; heating and dissolving the obtained extract A with a mixed solution of dichloromethane and isopropanol, filtering to remove insoluble substances, cooling, and crystallizing to obtain limonin crystals; and (3) evaporating the filtrate from which the limonin crystals are removed to dryness under reduced pressure to obtain an extract B, heating and dissolving the extract B by using a mixed solution of ethyl acetate and ethanol, filtering to remove insoluble substances, cooling, and crystallizing to obtain the nomilin crystals.
Further, degreasing treatment may be performed by a method conventional in the art, for example, degreasing with petroleum ether. To further enhance the degreasing effect, the raw material may be pulverized to 80-100 mesh.
Further, the wheat koji compound enzyme liquid is obtained by leaching brewing wheat koji, and preferably leaching by using physiological saline or Phosphate Buffer Solution (PBS). Raw wheat koji and cooked wheat koji commonly used in the brewing field can be adopted.
Further, the preparation method of the wheat koji compound enzyme liquid comprises the following steps: crushing the wheat starter to 80-100 meshes, adding physiological saline or phosphate buffer (pH value is 6.0-6.5) with the weight 2-3 times of that of the wheat starter, leaching for 8-12 h at the temperature of 4-6 ℃, and filtering to obtain the wheat starter complex enzyme solution.
The wheat starter complex enzyme liquid contains complex enzyme systems such as amylase, cellulase and protease, and is used for enzymolysis of starch, cellulose, protein and the like in the pretreated material, so that the dissolution of limonin compounds is promoted, and the structural damage to the limonin substances is less.
Further, the enzymolysis conditions of the wheat koji compound enzyme liquid are as follows: the pH value is 6.0-6.5; the temperature is 40-45 ℃. The enzymolysis time is generally 6-8 h. Proper amount of physiological saline or phosphate buffer solution can be added in the enzymolysis process.
The enzymolysis process is to carry out enzymolysis on corresponding substances such as cellulose, protease, starch and the like in the processed tangerine seed material by using complex enzyme systems such as amylase, cellulase, protease and the like contained in the wheat starter complex liquid under certain conditions, so that the dissolution of limonin compound glucoside is promoted. In addition, enzymes such as amylase, glycosidase and the like in the enzyme system can also hydrolyze limonin compound glucoside, promote the dropping of glycosyl in the structure and form the limonin compound.
Further, the concentration of the sodium salicylate solution is 1.5-1.7 mol/L, and preferably 1.6 mol/L.
Further, the weight ratio of the sodium salicylate solution to the dry weight of the enzymolysis backing material is 6-7: 1, and preferably 6.5: 1.
Further, the extraction temperature of the sodium salicylate solution is 52-55 ℃, and the extraction time is 6-8 hours; preferably, the extraction temperature is 53 ℃, and the extraction time is 6-8 h.
In general, each batch can be extracted three times using the method described above.
The sodium salicylate can combine with limonin substance to form soluble salt, thereby promoting the dissolution of limonin substance in the zymolysis substrate.
Further, adjusting the pH of the extract of the sodium salicylate solution to 4.6-5.0. Conventional pH regulators may be used, hydrochloric acid being preferred. After the pH is adjusted, the sodium salicylate is gradually changed into salicylic acid under the acidic condition, the solubility of the sodium salicylate is reduced, the sodium salicylate is separated out from the extraction solution and is removed by centrifugation, so that the macroporous resin adsorption in the subsequent steps is facilitated. The invention also researches and discovers that if the pH value is too low, the yield of the limonin substances is reduced.
Further, XAD-6 type macroporous resins are preferably used.
Further, eluting with 25-30% ethanol solution to remove impurities.
Further, eluting with 85-90% ethanol solution, and collecting eluate.
The low-concentration ethanol (with the concentration of 25-30%) is used for eluting, so that impurities adsorbed by the resin are washed away, and the subsequent crystallization and purification operations are facilitated. The research of the invention finds that the low-concentration ethanol eluent has higher impurity content and almost has no limonin compounds; the high concentration eluent has high limonin substance content.
Further, the dichloromethane and isopropanol mixed solution is composed of dichloromethane and isopropanol according to a volume ratio of 1: 1.8. Further, the weight ratio of the dichloromethane and isopropanol mixed solution to the dry weight of the extract A is 11-12: 1.
Further, the mixed solution of ethyl acetate and ethanol is composed of ethyl acetate and isopropanol according to a volume ratio of 5: 1. Further, the weight ratio of the ethyl acetate and ethanol mixed solution to the dry weight of the extract B is 12-14: 1.
The starting materials used in the present invention are commercially available or may be prepared by methods conventional in the art.
On the basis of the common knowledge in the field, the above preferred conditions can be combined with each other to obtain the preferred embodiments of the invention.
Specifically, the method for extracting limonin and nomilin comprises the following steps:
a) pretreating tangerine seeds, selecting the tangerine seeds which are not mildewed and dried, crushing by adopting a crusher, and sieving by using a sieve of 80-100 meshes; adding petroleum ether for degreasing, separating liquid phase, distilling under reduced pressure, and recovering solvent to obtain byproduct semen Citri Reticulatae extract oil; decompressing and evaporating the degreased bottom material to dryness to obtain a degreased tangerine seed pretreatment material;
b) preparing wheat starter complex enzyme liquid, namely taking brewing wheat starter, crushing and sieving, adding physiological saline or phosphate buffer solution for leaching, filtering and centrifuging to obtain wheat starter complex enzyme liquid;
c) performing enzymolysis treatment, namely adding a buffer solution into the degreased tangerine seed pretreatment material, adding wheat koji compound enzyme solution into the degreased tangerine seed pretreatment material for heat preservation enzymolysis, and removing supernatant after the enzymolysis is finished to obtain an enzymolysis bottom material;
d) extracting with water cosolvent, adding the zymolysis substrate into the sodium salicylate solution, extracting under certain conditions, and separating to obtain extractive solution;
e) adsorbing with macroporous resin, eluting, adjusting pH of the extractive solution, centrifuging, adsorbing supernatant with XAD-6 type macroporous resin, eluting with low concentration ethanol to remove impurities, eluting with high concentration ethanol, collecting eluate, distilling under reduced pressure, recovering ethanol, and drying to obtain extract A;
f) crystallizing, drying extract A at 40 deg.C, adding the dried extract into the mixed solution of dichloromethane and isopropanol, heating for dissolving, filtering to remove insoluble substances, cooling, and crystallizing to obtain limonin crystal; evaporating the mother liquor under reduced pressure to obtain an extract B, adding a mixed solution of ethyl acetate and ethanol into the extract B, heating and dissolving, filtering to remove insoluble substances, cooling, and crystallizing to obtain a crystal which is a nomilin crystal.
The wheat koji in the step b) is raw wheat koji and/or cooked wheat koji adopted in the brewing process. The preparation conditions of the compound enzyme liquid are as follows: crushing the wheat starter to 80-100 meshes, wherein the liquid-material ratio is 2-3L/kg, the leaching temperature is 4-8 ℃, and the leaching time is 8-16 h. Preferably, the liquid-material ratio is 2L/kg, the leaching temperature is 4-6 ℃, and the leaching time is 8-12 h.
Unless otherwise specified, the liquid-to-material ratio in the present invention refers to liquid in volume L and solid in weight kg.
The enzymolysis treatment conditions in the step c) are as follows: the liquid-material ratio is 4-8L/kg, the addition amount of the wheat koji compound enzyme liquid is 1-5% (volume fraction), the pH of the solution is 6.0-6.5, the temperature is 40-45 ℃, and the time is 6-8 h. Preferably, the liquid-material ratio is 6-8L/kg, the addition amount of the wheat koji compound enzyme liquid is 2-3% (volume fraction), the pH value of the solution is 6.5, the enzymolysis temperature is 45 ℃, and the time is 7 hours.
The buffer solution in the steps b) and c) is phosphate buffer solution or normal saline with the pH value of 6.0-6.5.
The extraction conditions of the water cosolvent in the step d are as follows: the extraction solvent is 1.5-1.7 mol/L sodium salicylate solution, the liquid-material ratio is 6-7L/kg, the extraction temperature is 52-55 ℃, the extraction time is 6-8 h, and each batch of materials is extracted for three times. Preferably, the concentration of the sodium salicylate is 1.6mol/L, the liquid-material ratio is 6.5L/kg, the extraction temperature is 53 ℃, and the extraction time is 7 h.
The pH value of the extract in the step e) is adjusted to 4.6-5.0 by using hydrochloric acid, and the pH value is preferably 4.8.
The low-concentration ethanol solution is 25-30% ethanol solution. The high-concentration ethanol solution is 85-90% ethanol solution.
The dichloromethane and isopropanol mixed solution in the step f) consists of dichloromethane and isopropanol according to the volume ratio of 1: 1.8; the liquid-material ratio of the extract A to the extract A is 11-12L/kg.
The mixed solution of ethyl acetate and ethanol in the step f) consists of ethyl acetate and isopropanol in a volume ratio of 5: 1; the liquid-material ratio of the extract B to the extract B is 12-14L/kg.
Preferably, the method for extracting limonin and nomilin comprises the following steps:
1) pretreating tangerine seeds: selecting mildew-free dried tangerine seeds, crushing by using a crusher, and sieving by using a sieve of 80-100 meshes for later use;
2) degreasing treatment: taking the crushed tangerine seeds for degreasing treatment, wherein the degreasing conditions are as follows: the degreasing solvent is petroleum ether with a boiling range of 60-90 ℃, the liquid-material ratio is 4-6L/kg, the temperature is 60 ℃ and the water bath lasts for 5-8 h, and the degreased tangerine seed pretreatment material is obtained after degreasing;
3) preparing wheat starter complex enzyme liquid: crushing the brewing wheat starter by a crusher, and sieving by a 100-mesh sieve; adding a phosphate buffer solution with the pH value of 6.0-6.5 for leaching, wherein the leaching conditions are as follows: the liquid-material ratio is 2-3L/kg, the temperature is 4-8 ℃, and the time is 8-16 h; then filtering the leaching solution with 4 layers of gauze and filter paper in sequence, centrifuging the filtrate for 30min at 10000rpm and 4 ℃, taking the supernatant of centrifugation, passing through 0.22 mu m under the aseptic condition to obtain wheat starter complex enzyme solution, and storing at 4 ℃ for later use;
4) and (3) enzymolysis treatment: adding a wheat starter compound enzyme solution into the degreased tangerine seed pretreatment material for enzymolysis, wherein the conditions include adding a phosphate buffer solution with pH of 6.0-6.5 (or physiological saline with pH of 6.0-6.5), the liquid-material ratio is 4-8L/kg, adding the wheat starter compound enzyme solution, the adding amount of the wheat starter compound enzyme solution is 1-5% of the total volume, the pH value of the solution is 6.0-6.5, the enzymolysis temperature is 40-45 ℃, stirring and extracting for 6-8 h, centrifuging and removing supernatant to obtain an enzymolysis substrate;
5) extracting a water cosolvent: adding 1.5-1.7 mol/L sodium salicylate solution into the enzymolysis bottom material for extraction, wherein the liquid-material ratio is 6-7L/kg, the temperature is 52-55 ℃, and the time is 6-8 hours; the extraction process adopts a three-stage four-pot type countercurrent extraction mode, and after extraction is finished, an extracting solution is separated;
6) macroporous resin adsorption and desorption: adjusting the pH of the extracting solution to 4.6-5.0 by using 1mol/L hydrochloric acid, and centrifugally separating and precipitating salicylic acid (which can be recycled); collecting supernatant, performing resin adsorption, and performing column packing (10.8 × 200cm) on pretreated XAD-6 type macroporous adsorbent resin with resin content of 50L and flow rate of 50ml/min for adsorption; after adsorption is finished, firstly washing with 25-30% ethanol with 2-4 times of column volume to remove impurities; and eluting the macroporous resin by using 85-90% ethanol with the volume of 3-6 times of that of the column, collecting the eluent, distilling under reduced pressure, and recovering the ethanol to obtain an extract A.
7) Crystallizing limonin: drying the extract A at 40 ℃, adding a mixed solution of dichloromethane and isopropanol (volume ratio is 1:1.8) according to a liquid-material ratio of 11-12L/kg, heating to 40 ℃, filtering to remove insoluble substances while the mixture is hot, cooling, and crystallizing at 4 ℃ to obtain limonin crystals; and (4) evaporating the mother liquor under reduced pressure to obtain an extract B.
8) And (3) performing Nomlein crystallization: adding ethyl acetate and ethanol (volume ratio is 5:1) solution into the extract B according to the liquid-material ratio of 12-14L/kg, heating to 40 ℃ for dissolving, filtering to remove insoluble substances while the extract is hot, cooling, and crystallizing at 4 ℃ to obtain white crystals, wherein the obtained crystals are nomilin crystals.
The yield of the semen citri reticulatae limonin prepared by the method is 0.75-0.80%, and the purity is more than 98%; the yield of the nomilin is 0.12-0.14%, and the purity is more than 96%.
In conclusion, compared with the prior art, the extraction method of limonin and nomilin has the following advantages:
1. the extraction method of limonin and nomilin adopts wheat koji compound enzyme liquid to act on the orange kernel degreasing treatment substrate, wherein cellulase, protease and amylase contained in the substrate can promote the dissolution of limonin substances in the substrate, and the amylase can also promote the conversion of the glycoside of the amylase into the limonin substances, so that the extraction rate of a target product is increased.
2. The extraction method of limonin and nomilin adopts a water cosolvent extraction method, reduces the use amount of organic solvents in the whole extraction process, and reduces the environmental pollution.
3. The wheat koji leaching liquor is used as the complex enzyme for hydrolyzing the tangerine seed bottom material, and the wheat koji is used as the enzyme source because the market price of the wheat koji is 4-10 yuan/kg, so that the wheat koji leaching liquor has the advantage of low cost compared with the complex enzyme liquid prepared by mixing single enzyme or single enzyme, and has the purpose of reducing the production cost.
Drawings
FIG. 1 is a chromatogram of a solution of a limonin and nomilin mixed control;
FIG. 2 is a standard curve for limonin content measurement;
FIG. 3 is a standard curve for measuring the content of nomilin.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.
Example 1
1) Pretreating tangerine seeds: selecting mildew-free dried tangerine seeds, crushing by a crusher, and sieving by a sieve of 80-100 meshes for later use.
2) Degreasing treatment: taking the crushed tangerine seeds for degreasing treatment, wherein the degreasing conditions are as follows: the degreasing solvent is petroleum ether with a boiling range of 60-90 ℃, the liquid-material ratio is 5L/kg, the temperature is 60 ℃ water bath, the time is 6h, and the tangerine seed pretreatment material is obtained after degreasing.
3) Preparing wheat starter complex enzyme liquid: taking the brewing wheat starter, crushing by a crusher, and sieving by a 100-mesh sieve. Adding phosphate buffer solution with pH of 6.0 for leaching, wherein leaching conditions are as follows: the liquid-material ratio is 2.5L/kg, the temperature is 6 ℃, and the time is 12 h. Then filtering the leaching solution with 4 layers of gauze and filter paper in sequence, centrifuging the filtrate at 10000rpm and 4 ℃ for 30min, taking the supernatant of centrifugation, passing through 0.22 mu m under aseptic condition to obtain wheat koji compound enzyme solution, and storing at 4 ℃ for later use.
4) And (3) enzymolysis treatment: adding wheat koji compound enzyme solution into the pre-treated material of the tangerine seeds for enzymolysis, wherein the conditions comprise adding phosphate buffer solution with pH of 6.0, the liquid-material ratio is 6L/kg, the adding amount of the wheat koji compound enzyme solution is 3 percent of the total volume, the pH value of the solution is 6.0, the enzymolysis temperature is 40 ℃, stirring and extracting for 8 hours, and centrifuging to remove supernatant to obtain an enzymolysis bottom material.
5) Extracting a water cosolvent: adding solvent into the zymolysis substrate for extraction, wherein the extraction condition is that the extraction solvent is 1.6mol/L sodium salicylate solution, the liquid-material ratio is 6L/kg, the temperature is 52 ℃, and the time is 8 h. The extraction process adopts a three-stage four-pot type countercurrent extraction mode, and the extract is separated after the extraction is finished.
6) Macroporous resin adsorption and desorption: adjusting pH of the extractive solution to 4.8 with 1mol/L hydrochloric acid, and centrifuging at 4000rpm for 10min to separate precipitate. Collecting supernatant, performing resin adsorption, and performing column packing (10.8 × 200cm) on pretreated XAD-6 type macroporous adsorbent resin with resin content of 50L and flow rate of 50ml/min for adsorption; after adsorption, washing with ethanol with the volume fraction of 28 percent which is 3 times of the column volume to remove impurities; eluting macroporous resin with 88% ethanol with 4 times column volume, collecting eluate, distilling under reduced pressure, and recovering ethanol to obtain extract A.
7) Crystallizing limonin: drying the extract A at 40 deg.C, adding mixed solution of dichloromethane and isopropanol (volume ratio of 1:1.8) at liquid-to-material ratio of 11L/kg, heating to 40 deg.C, filtering to remove insoluble substances, cooling, and crystallizing at 4 deg.C to obtain limonin crystal. And (4) evaporating the mother liquor under reduced pressure to obtain an extract B.
8) And (3) performing Nomlein crystallization: adding ethyl acetate and ethanol (volume ratio of 5:1) solution into the extract B according to the liquid-material ratio of 12L/kg, heating to 40 deg.C for dissolving, filtering to remove insoluble substances, cooling, and crystallizing at 4 deg.C to obtain white crystal, wherein the obtained crystal is Nomlein crystal.
With the method, the yield of the obtained active ingredient limonin is 0.79%, and the purity is 98.6%; the yield of nomilin was 0.13% with a purity of 96.8%.
Example 2
Steps 1) -3) are the same as in example 1.
4) And (3) enzymolysis treatment: adding wheat koji compound enzyme solution into the pre-treated material of the tangerine seeds for enzymolysis, wherein the conditions comprise adding phosphate buffer solution with pH of 6.0, the liquid-material ratio is 8L/kg, the adding amount of the wheat koji compound enzyme solution is 5 percent of the total volume, the pH value of the solution is 6.2, the enzymolysis temperature is 42 ℃, stirring and extracting for 8h, and centrifuging to remove supernatant to obtain an enzymolysis bottom material.
5) Extracting a water cosolvent: adding solvent into the zymolysis substrate for extraction, wherein the extraction condition is that the extraction solvent is 1.7mol/L sodium salicylate solution, the liquid-material ratio is 7L/kg, the temperature is 53 ℃, and the time is 8 h. The extraction process adopts a three-stage four-pot type countercurrent extraction mode, and the extract is separated after the extraction is finished.
Steps 6) -8) are the same as in example 1.
With the method, the yield of the obtained active ingredient limonin is 0.77%, and the purity is 99.1%; the yield of nomilin was 0.12% with a purity of 96.2%.
Example 3
Steps 1) to 4) were the same as in example 1
5) Extracting a water cosolvent: adding solvent into the zymolysis substrate for extraction, wherein the extraction condition is that the extraction solvent is 1.5mol/L sodium salicylate solution, the liquid-material ratio is 6L/kg, the temperature is 52 ℃, and the time is 8 h. The extraction process adopts a three-stage four-pot type countercurrent extraction mode, and the extract is separated after the extraction is finished.
6) Macroporous resin adsorption and desorption: adjusting pH of the extractive solution to 5.0 with 1mol/L hydrochloric acid, and centrifuging at 4000rpm for 10min to separate precipitate. Collecting supernatant, performing resin adsorption, and performing column packing (10.8 × 200cm) on pretreated XAD-6 type macroporous adsorbent resin with resin content of 50L and flow rate of 50ml/min for adsorption; after adsorption, washing with ethanol with the volume fraction of 25 percent which is 3 times of the column volume to remove impurities; eluting macroporous resin with 85% ethanol of 5 times column volume, collecting eluate, distilling under reduced pressure, and recovering ethanol to obtain extract A.
Steps 7) -8) are the same as in example 1.
With the method, the yield of the obtained active ingredient limonin is 0.78%, and the purity is 98.2%; the yield of nomilin was 0.13% with a purity of 97.1%.
Comparative example 1
Steps 1) -2) are the same as in example 1.
3) And (3) enzymolysis treatment: taking an orange core pretreatment material, adding a compound enzyme solution (consisting of 100U/mg beta-glucanase, 20U/mg glucoamylase and 20U/mg alpha-amylase in a mass ratio of 1: 1) for enzymolysis, wherein the conditions are that a phosphate buffer solution with the pH of 6.0 is added, the liquid-material ratio is 6L/kg, the adding amount of the compound enzyme solution is 7 percent of the total volume, the pH value of the solution is 6.0, the enzymolysis temperature is 45 ℃, stirring and extracting are carried out for 10 hours, and a supernatant is removed by centrifugation, so as to obtain an enzymolysis bottom material.
Steps 4) -7) are the same as steps 5-8 in example 1
By using the method, the yield of the obtained active ingredient limonin is 0.65 percent, and the purity is 98.1 percent; the yield of nomilin was 0.09% with a purity of 94.2%.
Comparative example 2
Steps 1) to 4) were the same as in example 1
5) And (3) organic solvent extraction, namely adding an organic solvent into the enzymolysis bottom material for extraction, wherein the extraction conditions are as follows: extracting with 85% acetone at a ratio of 20L/kg for 30min, tank-extracting for three times, mixing extractive solutions, distilling under reduced pressure, recovering acetone, and drying to obtain extract.
Steps 6) -7) are the same as steps 7) -8) of example 1.
By using the method, the yield of the obtained active ingredient limonin is 0.72 percent, and the purity is 93.2 percent; the yield of nomilin was 0.11% with a purity of 89.1%.
Comparative example 3
Steps 1) to 4) were the same as in example 1
5) And (3) organic solvent extraction, namely adding an organic solvent into the enzymolysis bottom material for extraction, wherein the extraction conditions are as follows: extracting with 95% ethanol at a liquid-to-material ratio of 20L/kg for 30min for three times, mixing the extractive solutions, distilling under reduced pressure, recovering ethanol, and drying to obtain extract.
Step 6) -)7 is identical to step 7) -8) of example 1.
By using the method, the yield of the obtained active ingredient limonin is 0.73 percent, and the purity is 92.2 percent; the yield of nomilin was 0.10% with a purity of 87.6%.
The quality indexes of the limonin and nomilin extract products of examples 1-3 and comparative examples 1-3 are shown in table 1.
TABLE 1 data table of yield and purity of extraction process for each example and comparative example
| Item | Limonin yield% | Purity of limonin% | Nomlein yield% | Nomlein purity% |
| Example 1 | 0.79 | 98.6 | 0.13 | 96.8 |
| Example 2 | 0.77 | 99.1 | 0.12 | 96.2 |
| Example 3 | 0.78 | 98.2 | 0.13 | 97.1 |
| Comparative example 1 | 0.65 | 98.1 | 0.09 | 94.2 |
| Comparative example 2 | 0.72 | 93.2 | 0.11 | 89.1 |
| Comparative example 3 | 0.73 | 92.2 | 0.10 | 87.6 |
As can be seen from the results in Table 1, examples 1-3 have higher yields and purities of limonin and nomilin than comparative examples 1-3. The extraction yields of limonin and nomilin in comparative examples 2-3 are very different from those of examples 1-3, but the purity of the extracted products is low.
Wherein, the determination method of each parameter in table 1 is as follows:
method for detecting content of limonin and nomilin
The instrument comprises the following steps: agilent model 200 hplc; a DAD diode array detector; digital display constant temperature water bath; an ultraviolet-visible spectrophotometer; chromatographic column of Dikmac18Column (250 mm. times.4.6 mm, 5 μm).
Materials: the limonin and the nomilin reference substances are purchased from Sichuan Vickers Biotechnology Co., Ltd (the purity is more than or equal to 98%), the used reagent methanol is chromatographic pure, petroleum ether and dichloromethane are analytically pure, and ultrapure water is used;
the method comprises the following steps of carrying out chromatographic conditions on a chromatographic column DikmAC18Columns (250 mm. times.4.6 mm, 5 μm); mobile phase: methanol-water (65:35), flow rate 1.0mL/min, column temperature: the detection wavelength is 210nm at 30 ℃; the loading amount is 10 muL.
Preparing standard solution, precisely weighing appropriate amount of limonin and nomilin reference substances, respectively placing in a volumetric flask, diluting with chromatographic methanol to scale, shaking up, and making the concentrations of the limonin and nomilin respectively 0.372mg/ml and 0.242 mg/ml. Taking the solution as mother liquor to prepare a series of standard solutions with concentration gradient, wherein the concentrations of the limonin are 0.0372mg/ml, 0.0744mg/ml, 0.1488mg/ml, 0.2232mg/ml, 0.2976mg/ml and 0.3720mg/ml respectively; the concentration of nomilin is 0.0484mg/ml, 0.0968mg/ml, 0.1452mg/ml, 0.1936mg/ml and 0.2420mg/ml respectively.
Preparation of test sample solution: the limonin and nomilin sample are precisely weighed, the limonin sample is dissolved by adopting chromatographic grade methanol to prepare a solution with the content of 0.2500mg/ml, and the nomilin sample is prepared into a solution with the content of 0.2000 mg/ml.
As a result: the mixed control (limonin and nomilin) was tested under the chromatographic conditions described above, indicating that good separation of the two compounds from the citrus seed was achieved (fig. 1). And measuring the areas of the peaks of the limonin and the nomilin according to the same chromatographic conditions to obtain a linear regression equation of the concentrations y (mg/ml) of the limonin and the nomilin by taking the areas of the peaks as horizontal coordinates x. The results show that limonin is in the range of 0.0372-0.3720mg/mL (figure 2), and nomilin is in good linear relation in the range of 0.0484-0.2420 mg/mL (figure 3).
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A method for extracting limonin and nomilin, comprising: defatting seeds of citrus plants to obtain a pretreatment material; carrying out enzymolysis by adopting wheat starter compound enzyme liquid; removing supernatant after enzymolysis to obtain an enzymolysis substrate; adding sodium salicylate solution for extraction, and separating the extract; adjusting the pH value to 4.6-5.0, taking supernatant liquid, putting the supernatant liquid on macroporous resin, eluting with 25-30% ethanol solution to remove impurities, eluting with 85-90% ethanol solution, collecting eluent, recovering ethanol, and drying to obtain an extract A; heating and dissolving the obtained extract A with a mixed solution of dichloromethane and isopropanol, filtering to remove insoluble substances, cooling, and crystallizing to obtain limonin crystals; evaporating the filtrate without limonin under reduced pressure to obtain extract B, heating and dissolving with mixed solution of ethyl acetate and ethanol, filtering to remove insoluble substances, cooling, and crystallizing to obtain nomilin crystal;
the preparation method of the wheat koji compound enzyme liquid comprises the following steps: crushing the wheat starter to 80-100 meshes, adding physiological saline or phosphate buffer solution with the weight 2-3 times of that of the wheat starter, leaching for 8-12 hours at the temperature of 4-6 ℃, and filtering to obtain the wheat starter complex enzyme solution.
2. The method of claim 1, wherein the enzymatic conditions are: the pH is 6.0-6.5, and the temperature is 40-45 ℃.
3. The method according to claim 1, wherein the concentration of the sodium salicylate solution is 1.5-1.7 mol/L;
the weight ratio of the sodium salicylate solution to the dry weight of the enzymolysis backing material is 6-7: 1;
the extraction temperature of the sodium salicylate solution is 52-55 ℃.
4. The method of claim 3, wherein the concentration of the sodium salicylate solution is 1.6 mol/L; the weight ratio of the sodium salicylate solution to the dry weight of the enzymolysis backing material is 6.5: 1; the extraction temperature of the sodium salicylate solution was 53 ℃.
5. The process of claim 1, wherein the macroporous resin is of the XAD-6 type.
6. The method according to claim 1, wherein the dichloromethane and isopropanol mixed solution is prepared by mixing dichloromethane and isopropanol in a volume ratio of 1: 1.8; the weight ratio of the dichloromethane and isopropanol mixed solution to the dry weight of the extract A is 11-12: 1; and/or the presence of a gas in the gas,
the mixed solution of the ethyl acetate and the ethanol consists of the ethyl acetate and the ethanol according to the volume ratio of 5: 1; the weight ratio of the ethyl acetate and ethanol mixed solution to the dry weight of the extract B is 12-14: 1.
7. The method of claim 1, wherein the citrus seed is citrus or grapefruit seed.
8. The method of claim 1, comprising the steps of:
a) pretreating tangerine seeds, selecting the tangerine seeds which are not mildewed and dried, crushing, and sieving by a sieve of 80-100 meshes; adding petroleum ether for degreasing, separating liquid phase, distilling under reduced pressure, and recovering solvent to obtain byproduct semen Citri Reticulatae extract oil; decompressing and evaporating the degreased bottom material to dryness to obtain a degreased tangerine seed pretreatment material;
b) preparing wheat starter complex enzyme liquid, namely taking brewing wheat starter, crushing and sieving, adding physiological saline or phosphate buffer solution for leaching, filtering and centrifuging to obtain wheat starter complex enzyme liquid; wherein the wheat starter is crushed to 80-100 meshes, the liquid-material ratio is 2-3L/kg, the leaching temperature is 4-6 ℃, and the leaching time is 8-12 hours;
c) performing enzymolysis treatment, namely adding a buffer solution into the degreased tangerine seed pretreatment material, adding wheat koji compound enzyme solution into the degreased tangerine seed pretreatment material for heat preservation enzymolysis, and removing supernatant after the enzymolysis is finished to obtain an enzymolysis bottom material; wherein the liquid-material ratio is 4-8L/kg, the addition amount of the wheat starter complex enzyme liquid is 1-5%, the pH of the solution is 6.0-6.5, the temperature is 40-45 ℃, and the time is 6-8 h;
d) extracting with water cosolvent, adding the zymolysis substrate into the sodium salicylate solution, extracting under certain conditions, and separating to obtain extractive solution;
e) adsorbing with macroporous resin, eluting, adjusting pH of the extractive solution, centrifuging, adsorbing supernatant with XAD-6 type macroporous resin, eluting with low concentration ethanol to remove impurities, eluting with high concentration ethanol, collecting eluate, distilling under reduced pressure, recovering ethanol, and drying to obtain extract A;
f) crystallizing, drying extract A at 40 deg.C, adding the dried extract into the mixed solution of dichloromethane and isopropanol, heating for dissolving, filtering to remove insoluble substances, cooling, and crystallizing to obtain limonin crystal; and (3) evaporating the filtrate from which the limonin crystals are removed to dryness under reduced pressure to obtain an extract B, adding a mixed solution of ethyl acetate and ethanol into the extract B, heating and dissolving, filtering to remove insoluble substances, cooling, and crystallizing to obtain the nomilin crystals.
9. The method according to claim 8, wherein the enzymolysis conditions in step c) are as follows: the liquid-material ratio is 6-8L/kg, the addition amount of the wheat starter complex enzyme liquid is 2-3%, the pH value of the solution is 6.0-6.5, the enzymolysis temperature is 42 ℃, and the time is 7 hours.
10. The method of claim 1, comprising the steps of:
1) pretreating tangerine seeds: selecting mildew-free dried tangerine seeds, crushing, and sieving with a sieve of 80-100 meshes for later use;
2) degreasing treatment: taking the crushed tangerine seeds for degreasing treatment, wherein the degreasing conditions are as follows: the degreasing solvent is petroleum ether with a boiling range of 60-90 ℃, the liquid-material ratio is 4-6L/kg, the temperature is 60 ℃ and the water bath lasts for 5-8 h, and the degreased tangerine seed pretreatment material is obtained after degreasing;
3) preparing wheat starter complex enzyme liquid: taking brewing wheat koji, crushing, and sieving with a 100-mesh sieve; adding a phosphate buffer solution with the pH value of 6.0-6.5 for leaching, wherein the leaching conditions are as follows: the liquid-material ratio is 2-3L/kg, the temperature is 4-6 ℃, and the time is 8-12 h; then filtering the leaching solution with 4 layers of gauze and filter paper in sequence, centrifuging the filtrate for 30min at 10000rpm and 4 ℃, taking the centrifuged supernatant, and filtering the supernatant through a filter membrane with the aperture of 0.22 mu m under the aseptic condition to obtain a wheat starter complex enzyme solution;
4) and (3) enzymolysis treatment: adding a phosphate buffer solution with the pH of 6.0-6.5 or physiological saline with the pH of 6.0-6.5 into the degreased tangerine seed pretreatment material for enzymolysis, wherein the liquid-material ratio is 4-8L/kg, adding the wheat starter complex enzyme solution, wherein the adding amount of the wheat starter complex enzyme solution is 1-5% of the total volume, the pH value of the solution is 6.0-6.5, the enzymolysis temperature is 40-45 ℃, stirring and extracting for 6-8 h, centrifuging and removing supernatant to obtain an enzymolysis bottom material;
5) extracting a water cosolvent: adding 1.5-1.7 mol/L sodium salicylate solution into the enzymolysis bottom material for extraction, wherein the liquid-material ratio is 6-7L/kg, the temperature is 52-55 ℃, and the time is 6-8 hours; the extraction process adopts a three-stage four-pot type countercurrent extraction mode, and after extraction is finished, an extracting solution is separated;
6) macroporous resin adsorption and desorption: adjusting the pH of the extracting solution to 4.6-5.0 by using 1mol/L hydrochloric acid, and centrifugally separating and precipitating salicylic acid; taking supernatant, and adsorbing with XAD-6 type macroporous adsorbent resin; firstly, washing with 25-30% ethanol with 2-4 times of column volume to remove impurities; eluting the macroporous resin by using 85-90% ethanol with 3-6 times of column volume, collecting eluent, distilling under reduced pressure, and recovering ethanol to obtain an extract A;
7) crystallizing limonin: drying the extract A at 40 ℃, adding a mixed solution of dichloromethane and isopropanol according to a liquid-material ratio of 11-12L/kg, heating to 40 ℃, filtering to remove insoluble substances while the extract is hot, cooling, and crystallizing at 4 ℃ to obtain limonin crystals; evaporating the filtrate without limonin crystal under reduced pressure to obtain extract B;
8) and (3) performing Nomlein crystallization: adding ethyl acetate and an ethanol solution into the extract B according to the liquid-material ratio of 12-14L/kg, heating to 40 ℃ for dissolving, filtering to remove insoluble substances while the extract is hot, cooling, and crystallizing at 4 ℃ to obtain a white crystal, wherein the obtained crystal is a nomilin crystal.
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