CN113121633A - Method for separating various mogrosic acid monomers from momordica grosvenori roots by continuous method - Google Patents
Method for separating various mogrosic acid monomers from momordica grosvenori roots by continuous method Download PDFInfo
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Abstract
The invention relates to a method for separating various mogrosic acid monomers from momordica grosvenori roots by a continuous method, which comprises the following steps: after enzymolysis of momordica grosvenori roots under the weak acidic condition, percolating and extracting the momordica grosvenori roots by using an alcohol-water solution under the alkaline condition, filtering, decoloring and concentrating, extracting the obtained concentrated solution under the acidic condition to obtain momordica grosvenori total acid, carrying out gradient elution separation on the momordica grosvenori total acid by using a chromatograph, and sequentially carrying out momordica grosvenori acid B, momordica grosvenori acid A, momordica grosvenori acid D and momordica grosvenori acid C according to the polar outflow order. The invention adopts the percolation extraction after the enzymolysis under the alkaline condition, and simultaneously hydrolyzes the mogroside to lead the mogroside to become free mogroside, salify and leach the free mogroside, thereby improving the yield of the mogroside. The method for extracting and separating the total acid of the momordica grosvenori from the momordica grosvenori roots has the advantages of coherent and simple process, strong operability, low production cost, no use of toxic and harmful chemical solvents, safety, environmental protection, high product content and high yield, and is suitable for industrial production.
Description
Technical Field
The invention relates to a separation method of natural triterpenic acid monomers, in particular to a method for separating various momordica acid monomers from momordica grosvenori roots by a continuous method.
Background
The momordica grosvenori is a famous and precious local specialty of Guilin, is mainly produced in Yongfu county and Longsheng county, and is also one of the first approved medicinal and edible materials in China. Mogroside is a specific natural high-power sweetener in fructus momordicae, has the sweetness of 300 times that of cane sugar and zero heat, has the effects of clearing heat, moistening lung, relieving cough, moistening intestines and relaxing bowels, and has prevention and treatment effects on obesity, constipation, diabetes and the like. Mogroside as food is safe and nontoxic, and can be used for various foods in an unlimited way according to the national mandatory standard GB2760 food additive use standard.
In the current production practice, the industrialized production and utilization of the momordica grosvenori are mainly focused on the extraction and separation of the mogrosides in the momordica grosvenori fruits, and natural resources such as roots, stems, leaves and the like of the momordica grosvenori fruits are not utilized and are treated as wastes. The momordica grosvenori roots are bitter in taste and slightly cold in nature, have the effects of clearing heat, eliminating dampness, dredging collaterals and relieving pain, are special medicinal materials commonly used by Guangxi folks, and are commonly used for treating cough, pharyngitis, rheumatoid arthritis, tumor resistance and the like. Because the scale of the planting and production processing of the momordica grosvenori is increased day by day, a large amount of momordica grosvenori roots are abandoned every year. Therefore, various natural components with physiological activity are extracted and separated from the momordica grosvenori roots, so that the waste is changed into valuable, and the method has important practical significance for protecting the environment, promoting the development of the whole industry of the momordica grosvenori and promoting the development of agricultural economy in planting areas.
Consistent with mogroside, mogroside is also a specific cucurbitane-type compound in momordica grosvenori plants; in contrast, mogroside belongs to the triterpene saponin class, and mogroside belongs to the triterpene acid class.
The existence form of the momordica grosvenori acid in the momordica grosvenori root is divided into two types: the first is in the free state, such as: mogroside A, mogroside B, mogroside C, mogroside D, mogroside E, etc.; the other is combined with glycosyl, and exists in the form of acid glycoside (glycoside), such as mogroside II and mogroside II.
The chemical structures of the momordica grosvenori acid A and the momordica grosvenori acid B are shown as follows:
the chemical structures of the mogroside C and the mogroside D are shown as follows:
the existing extraction method can only extract free total mogrosides or the mixture of the two, and has no method for simultaneously extracting and converting the mogrosides into free mogrosides, so that the yield of extracting the mogrosides from the roots of the momordica grosvenori in the prior art is not high.
The national scholars Wang Xuefen et al have isolated a variety of free mogrosides from the ethyl acetate fraction of the ethanol extract of Momordica grosvenori roots, including first, second, third, fourth, and fifth (Wang Xuefen et al, 1996, 1998; Sijian Yong et al, 1999).
Two triterpenic acid glycosides with good water solubility, i.e., mogroside A II and mogroside B II (Lufenglai, etc., 2010), were qualitatively and quantitatively analyzed from Momordica grosvenori roots by scholars in China.
CN201910046314.8 discloses a triterpene compound, a preparation method and application thereof, and after verification, mogroside II and mogroside II are obtained.
The above prior art does not disclose a method for simultaneously and continuously obtaining a plurality of high-content mogrosic acid monomers from the roots of momordica grosvenori.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for separating various mogrosic acid monomers from momordica grosvenori roots, which has the advantages of coherent and simple process, strong operability, low production cost, no use of toxic and harmful chemical solvents, safety, environmental protection, high product content and high yield, and is suitable for industrial production.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for separating various mogrosic acid monomers from momordica grosvenori roots by a continuous method comprises the following steps:
after enzymolysis of momordica grosvenori roots under the weak acidic condition, percolating and extracting the momordica grosvenori roots by using an alcohol-water solution under the alkaline condition, filtering, decoloring and concentrating, extracting the obtained concentrated solution under the acidic condition to obtain momordica grosvenori total acid, carrying out gradient elution separation on the momordica grosvenori total acid by using a chromatograph, and sequentially carrying out momordica grosvenori acid B, momordica grosvenori acid A, momordica grosvenori acid D and momordica grosvenori acid C according to the polar outflow order.
Further, the weak acidic condition is pH5-6, the acidic condition is pH 2-5, the alkaline condition is pH 13.0-13.7; such acid-base modifying substances are well known in the art, such as hydrochloric acid, sulfuric acid, acetic acid, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, and the like.
Preferably, 0.2-0.5 wt% of borate of Momordica grosvenori root is also added for enzymolysis. The borate is used for protecting the molecular structures of the first and second mogrosides from being damaged by beta-glucosidase. Because the mogroside A and the mogroside B belong to carbon-loss tetracyclic triterpenic acid, the oxygen bridge structure in the molecular structure is unstable, and certain damage can occur in the enzymolysis process. The root of the momordica grosvenori is also the reason that the momordica grosvenori acid A and the momordica grosvenori acid B are distributed in the momordica grosvenori plants only in a small amount, the types and the quantities of the glucohydrolase are small, and the starch storage is rich. The inventor finds that the borate can effectively protect the oxygen bridge structure and prevent the oxygen bridge structure from being degraded and destroyed by beta-glucosidase. The borate is at least one of sodium borate and potassium borate.
The enzymolysis is that the crushed momordica grosvenori roots are put into an enzyme solution with the pH value of 5-6, the enzyme is beta-glucosidase, and the dosage of the enzyme is 0.5-2 wt% of the momordica grosvenori roots. The conditions of the enzymatic hydrolysis are not particularly limited, and are well known in the art, and specifically, the enzymatic hydrolysis temperature is 20 to 30 ℃ and the enzymatic hydrolysis time is 12 to 24 hours.
The purpose of adding beta-glucosidase for enzymolysis is firstly to destroy the cell wall in the cell tissue of the momordica grosvenori root so as to be convenient for the rapid leaching of the momordica grosvenori acid and the momordica grosvenori acid glycoside in the percolation process, and secondly to carry out enzymolysis on part of the momordica grosvenori acid glycoside into the momordica grosvenori acid. If the dosage of the enzyme is too small or the enzymolysis time is too short, the purpose cannot be achieved; if the dosage of the enzyme is too much or the enzymolysis time is too long, the waste of materials, energy and time can be caused, and the starch in the momordica grosvenori roots is over gelatinized, so that the flow rate of the subsequent percolation process is seriously slow.
In the alcohol water solution obtained by percolation extraction, the volume concentration of alcohol is 60-95%, and the alcohol is at least one of methanol, ethanol and isopropanol. The volume consumption of the alcohol-water solution is 8-10 times of the mass of the momordica grosvenori roots, and the unit is L/kg. The flow rate of the percolation extraction is 0.5-2.0 BV/h, preferably 0.8-1.5 BV/h. The purpose of using alkaline alcohol water solution for percolation is that firstly, the mogrosides in the raw materials after enzymolysis and the mogrosides which are not subjected to enzymolysis are fully leached (the mogrosides reacts with alkali to form salt which can be dissolved in alcohol, and the mogrosides can be easily dissolved in alcohol), and secondly, the mogrosides which are not subjected to enzymolysis are hydrolyzed into the mogrosides so as to improve the yield of the mogrosides.
The filtration is not particularly limited, and examples thereof include microfiltration, ultrafiltration, and ceramic membrane filtration. Preferably ceramic membrane filtration, the aperture of the ceramic membrane is 10-100nm, and the filtration pressure of the ceramic membrane is 0.2-0.5 Mpa. The purpose of the ceramic membrane filtration is to remove suspended substances (organic insoluble substances, fine silt, raw material powder and the like) in the percolate, ensure the clarification of the solution and prevent the emulsification in the subsequent extraction process.
The decolorization is carried out by using an alumina or magnesia chromatographic column, the dosage of the alumina or magnesia is 0.1-0.3 time (kg/kg) of the weight of the raw material of the momordica grosvenori roots, and the height-diameter ratio of the chromatographic column is 5-8: 1, the flow rate of the ceramic membrane filtrate passing through an alumina or magnesia chromatographic column is 0.5-2.0 BV/h. The purpose of the decolorization using an alumina or magnesia column is to remove fat-soluble impurities such as pigments in the ceramic membrane filtrate.
The concentration is carried out under reduced pressure until no alcohol smell is generated.
The extraction is carried out by using a low-polarity organic solvent, and the low-polarity organic solvent is selected from at least one of petroleum ether, diethyl ether, No. 6 solvent oil, No. 120 solvent oil, ethyl acetate, cyclohexane, n-hexane and heptane. The volume consumption of the low-polarity organic solvent is 5-10 times of the weight of the raw material of the momordica grosvenori roots, and the unit is L/kg. The purpose of extracting by using the low-polarity organic solvent is to extract the free momordica grosvenori acid in the acidic concentrated solution, and the water-soluble impurities are remained in the acid water, thereby realizing the separation of the momordica grosvenori acid and the water-soluble impurities.
Preferably, the extraction is performed 2-4 times, each time in amounts not exceeding 15% of each other, more preferably, the amount of the low-polarity organic solvent used for the extraction is decreased progressively. For example, for 2 extractions, the solvent used for the first extraction is 55-65% and the solvent used for the second extraction is 35-45%.
In the separation by chromatography, a high-pressure liquid chromatographic column is adopted, and the column pressure is 5-15 Mpa; the grain size of the chromatographic packing is 5-20 μm; the volume consumption of the chromatographic packing is 20-40 times of the weight of the total acid sample of the momordica grosvenori, and the unit is L/kg; the height-diameter ratio of the chromatographic packing column is 2-5: 1; the flow rate of the mobile phase elution is 20-40 BV/h, preferably, the column pressure of the high-pressure preparation liquid chromatographic column is 10-12Mpa, the particle size of the chromatographic packing is 10-15 μm, and the height-diameter ratio of the chromatographic packing to the column is 2.5-3: 1 the flow rate of the elution of the mobile phase is 20-25 BV/h,
the filler in the chromatographic column is a polymer, and is specifically polystyrene, polymethacrylate, polyhydroxymethacrylate or polyvinyl alcohol.
The gradient elution is to obtain momordica grosvenori acid B and momordica grosvenori acid A in sequence by adopting mobile phase I elution; eluting with mobile phase II to obtain fructus Siraitiae Grosvenorii acid D and fructus Siraitiae Grosvenorii acid C. The mobile phase I is an alcohol-water mixed solution, wherein the volume fraction of alcohol is 15-20%; the mobile phase II is an alcohol-water-acetic acid mixed solution, the volume fraction of the alcohol is 25-35%, the volume fraction of the acetic acid is 0.2-1%, the alcohol is methanol and/or ethanol, and a small amount of acetic acid is added into the mobile phase II for preventing target components from trailing and crossing in the elution process, so that the full and rapid separation of various target components is facilitated.
In a preferred technical scheme of the invention, under the condition that the column pressure is 10-12Mpa, the particle size of the chromatographic packing is 10-15 mu m, and the height-diameter ratio of the chromatographic packing filled with the column is 2.5-3: 1, under the condition that the flow rate of elution of a mobile phase is 20-25 BV/h, timing from the beginning of elution, and collecting a fraction B of mogroside in 16-22 min; collecting fraction A of mogroside at 26-34 min; and after the first fraction of the mogroside is collected, the mobile phase II is used, the second fraction of the mogroside is collected for 39-47min at the same flow rate, and the third fraction of the mogroside is collected for 52-59 min.
In the invention, 1BV is equal to 1 percolator raw material packed column volume or 1 filler (alumina, magnesia, chromatographic polymer filler) packed column volume.
In a preferred technical scheme of the invention, the method for separating a plurality of mogrosic acid monomers from momordica grosvenori roots by the continuous method comprises the following steps:
(1) enzymolysis: taking dried momordica grosvenori roots, crushing, mixing with beta-glucosidase, adjusting pH to be faintly acid, stirring uniformly, and performing enzymolysis at room temperature;
(2) alkaline alcohol percolation: putting the raw materials after enzymolysis into a percolator, and percolating and extracting by using alkaline methanol or ethanol to obtain percolate;
(3) ceramic membrane filtration: filtering the percolate with ceramic membrane to obtain ceramic membrane filtrate;
(4) and (3) decoloring: passing the ceramic membrane filtrate through an alumina or magnesia chromatographic column to obtain a decolorized solution;
(5) and (3) concentrating under reduced pressure: concentrating the decolorized solution under reduced pressure until no alcohol exists to obtain a concentrated solution;
(6) acid adjustment: adjusting the pH value of the concentrated solution to acidity by using acid to obtain acidic concentrated solution;
(7) extraction and concentration: extracting the acidic concentrated solution with low-polarity organic solvent for 2-3 times, standing for layering, separating, collecting and combining organic solvent layers, concentrating the organic layers under reduced pressure, and vacuum drying to obtain fructus Siraitiae Grosvenorii total acid product;
(8) preparing chromatographic separation of the mogroside monomer: firstly, dissolving and diluting a momordica grosvenori total acid sample by using a mobile phase, injecting the sample into a high-pressure preparation liquid chromatographic column, and finally eluting the sample by using the mobile phase; collecting eluate fractions of different time periods, concentrating and drying respectively to obtain four mogroside monomers, namely mogroside B, mogroside A, mogroside D and mogroside C in sequence.
Preferably, 0.1-0.5 wt% of borate of the roots of momordica grosvenori is also added in the enzymolysis of the step (1).
The principle of the method of the invention is as follows:
the raw material of the momordica grosvenori roots after enzymolysis is percolated and extracted by adopting alkaline methanol or ethanol, so that various free momordica grosvenori acids in the raw material can be salified and leached, and momordica grosvenori acid glycosides (the skeleton structure is glucoside of the momordica grosvenori acids) which are not fully enzymolyzed in the raw material can be hydrolyzed to be free momordica grosvenori acids, salified and leached, so that the momordica grosvenori roots can be obtained by killing two birds with one stone, and the yield of the momordica grosvenori acids can be improved; removing fat-soluble impurities such as pigments and the like by utilizing the Momordica grosvenori acid salt without being adsorbed by aluminum oxide or magnesium oxide; and finally, reacting the mogrosite with acid to restore the free state of the mogrosite, and performing phase inversion on the mogrosite into an organic solvent through extraction to realize separation of the mogrosite from water-soluble impurities, thereby obtaining a total acid product of the mogrosite. Because the polarity difference of various mogrosides is very small, the mogrosides cannot be effectively separated by adopting a conventional elution method, and various high-content mogroside monomers can be accurately collected and obtained by performing high-pressure preparative chromatography on a total mogroside product and adopting a mode of gradient elution and segmented collection.
The method has the following beneficial effects:
(1) the invention adopts the method of enzymolysis and alkali alcohol percolation, which can ensure the full enzymolysis or hydrolysis of the mogroside, can also form salt of the mogroside and completely leach the mogroside, and improves the yield of the total acid of the grosvenor momordica.
(2) The method for extracting and separating the total acid of the momordica grosvenori from the momordica grosvenori roots is coherent and simple in process, strong in operability, low in production cost, safe and environment-friendly, high in product content and high in yield, does not use toxic and harmful chemical solvents, and is suitable for industrial production; in addition, the invention also provides a method for continuously obtaining a plurality of high-content mogroside monomers.
(3) In the invention, a certain amount of borate is also added in the enzymolysis process, so that the mogroside A and the mogroside B with 'oxygen bridge structures' can be protected, and the yield of the mogroside A and the mogroside B is improved.
Detailed Description
The present invention will be further described with reference to the following examples.
The momordica grosvenori roots (dried) used in the embodiment of the invention are purchased from momordica grosvenori planting bases of the limited biological resource of the southern Hunan Huacheng, wherein the content of total momordica grosvenori acid is 0.76 wt% (free total acid content plus momordica grosvenori acid glycoside converted into the content of momordica grosvenori acid, HPLC detection shows that the content of total momordica grosvenori acid is 0.49 wt%, the total content of momordica grosvenori acid glycoside is 0.46 wt%, and the converted content of momordica grosvenori acid is 0.27 wt%, so that the content of total momordica grosvenori acid is 0.49 wt% +0.27 wt% > -0.76 wt%); the high-pressure preparative liquid chromatography used in the embodiment of the invention is purchased from Jiangsu Hanbang science and technology Limited; the adjuvants or chemicals used in the examples of the present invention are commercially available in the usual manner unless otherwise specified. In the embodiment of the invention, the content of mogroside and mogroside is detected by adopting a high performance liquid chromatography (external standard).
The detection method of the invention for the momordica grosvenori acid uses High Performance Liquid Chromatography (HPLC), and the instrument comprises the following steps: waters1525 high performance liquid phase; a chromatographic column: elette C18(ODS), 4.6 × 250 mm; mobile phase: acetonitrile-water (40: 60); flow rate: 0.8 ml/min; detection wavelength: 230 nm; column temperature: 35 ℃; the sample injection amount is 10 uL.
Example 1
(1) Enzymolysis: taking 10kg of dried momordica grosvenori roots, crushing the momordica grosvenori roots into particles with the particle size of 2-3mm, adding 10kg of hydrochloric acid aqueous solution (pH is 5), 0.02kg of sodium borate and 0.1kg of beta-glucosidase, uniformly stirring, and carrying out enzymolysis for 24 hours at room temperature;
(2) alkaline alcohol percolation: putting the raw materials after enzymolysis into a percolator, percolating and extracting with 80L alkaline methanol (the volume percentage concentration of the methanol is 70%, wherein the volume percentage concentration of the methanol contains sodium hydroxide is 1%), wherein the percolation flow rate is 1.0 BV/h, and obtaining a percolation solution;
(3) ceramic membrane filtration: filtering the percolate with ceramic membrane with pore diameter of 50nm (filtering pressure 0.4Mpa) to obtain ceramic membrane filtrate;
(4) and (3) decoloring: passing the ceramic membrane filtrate through an alumina chromatographic column (the dosage of alumina is 3kg, the height-diameter ratio of the chromatographic column is 6: 1, the column flow rate is 1.5 BV/h) to obtain a decolorized solution;
(5) and (3) concentrating under reduced pressure: concentrating the decolorized solution under reduced pressure until no alcohol exists to obtain a concentrated solution;
(6) acid adjustment: regulating the pH value of the concentrated solution to 3 by hydrochloric acid to obtain an acidic concentrated solution;
(7) extraction and concentration: extracting the acidic concentrated solution with 80L petroleum ether for 3 times, standing for layering, separating, collecting and combining organic solvent layers, concentrating the organic layer under reduced pressure, and vacuum drying to obtain fructus Siraitiae Grosvenorii total acid 0.080 kg;
(8) preparing chromatographic separation of the mogroside monomer: firstly, 0.050kg of a momordica grosvenori total acid sample is dissolved and diluted by a mobile phase I (a methanol-water mixed solvent, wherein the volume ratio of methanol is 15%), a solution with the solid concentration of 15% is injected into a high-pressure preparation liquid chromatography column (the column pressure is 10Mpa, the type of a filler of the chromatography column is polyhydroxymethacrylate, the particle size of the chromatography filler is 10 mu m, the using amount of the chromatography filler is 2L, and the height-diameter ratio of the chromatography filler filled in the column is 3: 1), and the momordica grosvenori total acid sample is eluted by the mobile phase I at the flow rate of 20 BV/h. Timing from the beginning of elution, collecting eluent fraction A at 16-20min, collecting eluent fraction B at 26-31min, collecting fraction B, and after fraction B is collected, using mobile phase II (methanol-water-acetic acid mixed solvent, methanol volume ratio is 25%, acetic acid volume ratio is 0.5%), collecting eluent fraction C at 39-45min and collecting eluent fraction D at 52-56min under the same flow rate. Concentrating and drying the fraction A of the eluent to obtain 11.15g of a high-content mogroside B product; concentrating and drying the fraction B of the eluent to obtain 19.84g of a high-content mogroside A product; concentrating and drying the fraction C of the eluent to obtain 5.23g of a high-content diced momordica grosvenori acid product; and concentrating and drying the eluent fraction D to obtain 3.82g of a high-content propyl mogroside product.
Through high performance liquid chromatography (external standard) determination, the content of the obtained fructus momordicae total acid product is 85.25% (wherein, the content of the momordica grosvenori acid B is 22.65%, the content of the momordica grosvenori acid A is 41.32%, the content of the momordica grosvenori acid D is 10.86%, the content of the momordica grosvenori acid C is 7.93%), and the yield of the fructus momordicae total acid is 89.74%. In the step of preparative chromatographic separation, the content of the high-content mogroside B product obtained in the embodiment of the invention is 98.23%, the content of the mogroside A product is 99.17%, the content of the mogroside D product is 98.52%, and the content of the mogroside C product is 97.78%.
Example 2
(1) Enzymolysis: taking 10kg of dried momordica grosvenori roots, crushing the momordica grosvenori roots into particles with the particle size of 2-3mm, adding 15kg of dilute hydrochloric acid aqueous solution (pH is 6), 0.05kg of sodium borate and 0.2kg of beta-glucosidase, uniformly stirring, and carrying out enzymolysis at room temperature for 18 hours;
(2) alkaline alcohol percolation: putting the raw materials after enzymolysis into a percolator, percolating and extracting with 100L alkaline ethanol (the volume percentage concentration of the ethanol is 80%, wherein the volume percentage concentration of the ethanol contains 0.8% of potassium hydroxide) at the percolation flow rate of 1.5 BV/h to obtain percolate;
(3) ceramic membrane filtration: filtering the percolate with ceramic membrane with pore diameter of 50nm (filtering pressure 0.4Mpa) to obtain ceramic membrane filtrate;
(4) and (3) decoloring: passing the ceramic membrane filtrate through an alumina chromatographic column (the use amount of alumina is 2kg, the height-diameter ratio of the chromatographic column is 7: 1, and the column flow rate is 2.0 BV/h) to obtain a decolorized solution;
(5) and (3) concentrating under reduced pressure: concentrating the decolorized solution under reduced pressure until no alcohol exists to obtain a concentrated solution;
(6) acid adjustment: regulating the pH value of the concentrated solution to 3 by using sulfuric acid to obtain an acidic concentrated solution;
(7) extraction and concentration: extracting the acidic concentrated solution with 60L n-hexane for 2 times, standing for layering, separating, collecting and combining organic solvent layers, concentrating the organic layer under reduced pressure, and vacuum drying to obtain 0.081kg of fructus Siraitiae Grosvenorii total acid product;
(8) preparing chromatographic separation of the mogroside monomer: firstly, 0.05kg of a momordica grosvenori total acid sample is dissolved and diluted by a solution with the solid concentration of 15% by using a mobile phase I (ethanol-water mixed solvent, wherein the volume ratio of ethanol is 18%), a high-pressure preparation liquid chromatographic column is injected (the column pressure is 12Mpa, the type of a filler of the chromatographic column is polymethacrylate, the particle size of the chromatographic filler is 15 mu m, the using amount of the chromatographic filler is 1.8L, and the height-diameter ratio of the chromatographic filler filled in the column is 2.8: 1), and the momordica grosvenori total acid sample is eluted by using the mobile phase I at the flow rate of 25 BV/h. Timing from the beginning of elution, collecting fraction A at 18-22min, collecting fraction B at 29-34min, collecting fraction B, and collecting mobile phase II (ethanol-water-acetic acid mixed solvent, ethanol volume ratio of 29% and acetic acid volume ratio of 0.4%) at the same flow rate, collecting fraction C at 43-47min, and collecting fraction D at 55-59 min. Concentrating and drying the fraction A of the eluent to obtain 11.26g of a high-content mogroside B product; concentrating and drying the fraction B of the eluent to obtain 19.85g of a high-content mogroside A product; concentrating and drying the fraction C of the eluent to obtain 5.32g of a high-content diced momordica grosvenori acid product; and concentrating and drying the eluent fraction D to obtain 3.87g of a high-content propyl mogroside product.
Through high performance liquid chromatography (external standard) determination, the content of the obtained momordica grosvenori total acid product is 86.31% (wherein, the content of momordica grosvenori acid B is 23.77%, the content of momordica grosvenori acid A is 41.64%, the content of momordica grosvenori acid D is 11.21%, the content of momordica grosvenori acid C is 8.05%), and the yield of the momordica grosvenori total acid is 91.99%. In the step of preparative chromatographic separation, the content of the high-content mogroside B product obtained in the embodiment of the invention is 98.65%, the content of the mogroside A product is 98.59%, the content of the mogroside D product is 97.81%, and the content of the mogroside C product is 96.43%.
Example 3
(1) Enzymolysis: taking 10kg of dried momordica grosvenori roots, crushing the momordica grosvenori roots into particles with the particle size of 2-3mm, adding 15kg of dilute sulfuric acid aqueous solution (pH is 5), 0.03kg of sodium borate and 0.15kg of beta-glucosidase, uniformly stirring, and carrying out enzymolysis for 20 hours at room temperature;
(2) alkaline alcohol percolation: putting the raw materials after enzymolysis into a percolator, percolating and extracting with 90L alkaline methanol (the volume percentage concentration of the methanol is 85%, wherein the volume percentage concentration of the methanol contains sodium hydroxide 1.5%), wherein the percolation flow rate is 0.8 BV/h, and obtaining a percolation solution;
(3) ceramic membrane filtration: filtering the percolate with ceramic membrane with aperture of 100nm (filtration pressure 0.2Mpa) to obtain ceramic membrane filtrate;
(4) and (3) decoloring: passing the ceramic membrane filtrate through a magnesia chromatographic column (the use amount of magnesia is 2.6kg, the height-diameter ratio of the chromatographic column is 8: 1, the flow rate of the chromatographic column is 1.0 BV/h) to obtain a decolorized solution;
(5) and (3) concentrating under reduced pressure: concentrating the decolorized solution under reduced pressure until no alcohol exists to obtain a concentrated solution;
(6) acid adjustment: regulating the pH value of the concentrated solution to 4 by hydrochloric acid to obtain an acidic concentrated solution;
(7) extraction and concentration: extracting the acidic concentrated solution with 100L 6# solvent oil for 3 times, standing for layering, separating, collecting and combining organic solvent layers, concentrating the organic layers under reduced pressure, and vacuum drying to obtain fructus Siraitiae Grosvenorii total acid 0.076 kg;
(8) preparing chromatographic separation of the mogroside monomer: firstly, 0.05kg of a momordica grosvenori total acid sample is dissolved and diluted by a solution with the solid concentration of 10% by using a mobile phase I (a methanol-water mixed solvent, wherein the volume ratio of methanol is 20%), a high-pressure preparation liquid phase chromatographic column is injected (the column pressure is 10Mpa, the filler type of the chromatographic column is polyhydroxymethacrylate, the particle size of the chromatographic filler is 10 mu m, the using amount of the chromatographic filler is 2L, and the height-diameter ratio of the chromatographic filler filled in the column is 3: 1), and the momordica grosvenori total acid sample is eluted by using the mobile phase I at the flow rate of 22 BV/h. Timing from the beginning of elution, collecting eluent fraction A at 17-21min, collecting eluent fraction B at 27-32min, collecting fraction B, and after fraction B is collected, using mobile phase II (methanol-water-acetic acid mixed solvent, methanol volume ratio is 27%, acetic acid volume ratio is 0.7%), collecting eluent fraction C at 41-46min and eluent fraction D at 53-58min at the same flow rate. Concentrating and drying the fraction A of the eluent to obtain 11.50g of a high-content mogroside B product; concentrating and drying the fraction B of the eluent to obtain 20.54g of a high-content mogroside A product; concentrating and drying the fraction C of the eluent to obtain 6.16g of a high-content mogroside butyl product; and concentrating and drying the eluent fraction D to obtain 3.86g of a high-content propyl mogroside product.
Through high performance liquid chromatography (external standard) determination, the content of the obtained momordica grosvenori total acid product is 89.53% (wherein, the content of the momordica grosvenori acid B is 24.23%, the content of the momordica grosvenori acid A is 42.71%, the content of the momordica grosvenori acid D is 12.59%, the content of the momordica grosvenori acid C is 8.19%), and the yield of the momordica grosvenori total acid is 89.53%. In the step of preparative chromatographic separation, the content of the mogroside B product obtained in the embodiment of the invention is 97.31%, the content of the mogroside A product is 96.89%, the content of the mogroside D product is 96.92%, and the content of the mogroside C product is 95.73%.
Example 4
The other conditions and procedure were the same as in example 1 except that sodium borate was not added in step (1). Finally, 0.076kg of grosvenor momordica fruit total acid product is obtained, and the content of grosvenor momordica fruit total acid is 83.57 percent (wherein, the content of the momordica grosvenori acid B is 21.36 percent, the content of the momordica grosvenori acid A is 38.87 percent, the content of the momordica grosvenori acid D is 11.40 percent, and the content of the momordica grosvenori acid C is 8.36 percent) through the determination of a high performance liquid chromatography (external standard).
Carrying out high-pressure preparation of a liquid chromatographic column on 0.050kg of a grosvenor momordica fruit total acid product under the same condition to finally obtain 10.42g of a high-content grosvenor momordica acid B product with the content of 98.17%; 18.57g of high-content mogroside A product, with the content of 98.85%; 5.48g of high-content diced momordica grosvenori acid product, and the content is 98.54%; 4.01g of high-content propyl mogroside product, with the content of 97.81%.
Claims (10)
1. A method for separating various mogrosic acid monomers from momordica grosvenori roots by a continuous method comprises the following steps: after enzymolysis of momordica grosvenori roots under the weak acidic condition, percolating and extracting the momordica grosvenori roots by using an alcohol-water solution under the alkaline condition, filtering, decoloring and concentrating, extracting the obtained concentrated solution under the acidic condition to obtain momordica grosvenori total acid, carrying out gradient elution separation on the momordica grosvenori total acid by using a chromatograph, and sequentially carrying out momordica grosvenori acid B, momordica grosvenori acid A, momordica grosvenori acid D and momordica grosvenori acid C according to the polar outflow order.
2. The method of claim 1, wherein the weakly acidic condition is a pH of 5 to 6, the acidic condition is a pH of 2 to 5, and the basic condition is a pH of 13.0 to 13.7.
3. The method of claim 1, wherein the enzymatic hydrolysis is further added with 0.2-0.5 wt% of borate of mogrosia grosvenori roots; preferably, the borate is at least one selected from sodium borate and potassium borate.
4. The method of claim 1, wherein the enzymatic hydrolysis is performed by adding the pulverized luo han guo root into an enzyme solution having a pH of 5-6, wherein the enzyme is β -glucosidase and the amount of the enzyme is 0.5-2 wt% of the luo han guo root.
5. The method of claim 1, wherein the alcohol concentration of the aqueous percolation extract is 60-95% by volume; the volume consumption of the alcohol-water solution is 8-10 times of the mass of the momordica grosvenori roots, and the unit is L/kg. The flow rate of the percolation extraction is 0.5-2.0 BV/h, preferably 0.8-1.5 BV/h.
6. The method according to claim 1, wherein the filtration is ceramic membrane filtration, the pore size of the ceramic membrane is 10-100nm, and the pressure of the ceramic membrane filtration is 0.2-0.5 Mpa; the decolorization is carried out by using an alumina or magnesia chromatographic column, the dosage of the alumina or magnesia is 0.1-0.3 time (kg/kg) of the weight of the raw material of the momordica grosvenori roots, and the height-diameter ratio of the chromatographic column is 5-8: 1, the flow rate of the ceramic membrane filtrate passing through an alumina or magnesia chromatographic column is 0.5-2.0 BV/h.
7. The method according to claim 1, wherein the extraction is an extraction with a low polar organic solvent selected from at least one of petroleum ether, diethyl ether, # 6 mineral spirits, # 120 mineral spirits, ethyl acetate, cyclohexane, n-hexane, heptane; the volume consumption of the low-polarity organic solvent is 5-10 times of the weight of the raw material of the momordica grosvenori roots, and the unit is L/kg.
8. The method according to claim 1, wherein the separation is carried out by chromatography using a high pressure preparative liquid chromatography column at a column pressure of 5-15 Mpa; the grain size of the chromatographic packing is 5-20 μm; the volume consumption of the chromatographic packing is 20-40 times of the weight of the total acid sample of the momordica grosvenori, and the unit is L/kg; the height-diameter ratio of the chromatographic packing column is 2-5: 1; the flow rate of the mobile phase elution is 20-40 BV/h, preferably, the column pressure of the high-pressure preparation liquid chromatographic column is 10-12Mpa, the particle size of the chromatographic packing is 10-15 μm, and the height-diameter ratio of the chromatographic packing to the column is 2.5-3: 1 the flow rate of elution of the mobile phase is 20-25 BV/h; further preferably, the filler is polystyrene, polymethacrylate, polyhydroxymethacrylate or polyvinyl alcohol.
9. The method according to claim 1, wherein the gradient elution is performed by eluting with a mobile phase I to obtain mogroside B and mogroside A in sequence; eluting with mobile phase II to obtain fructus Siraitiae Grosvenorii acid D and fructus Siraitiae Grosvenorii acid C. The mobile phase I is an alcohol-water mixed solution, wherein the volume fraction of alcohol is 15-20%; the mobile phase II is an alcohol-water-acetic acid mixed solution, the volume fraction of the alcohol is 25-35%, and the volume fraction of the acetic acid is 0.2-1%;
preferably, the column pressure is 10-12Mpa, the particle size of the chromatographic packing is 10-15 μm, and the ratio of height to diameter of the chromatographic packing in the column is 2.5-3: 1, collecting the fraction B of the mogroside at 16-22min from the beginning of elution under the condition that the flow rate of the mobile phase elution is 20-25 BV/h; collecting fraction A of mogroside at 26-34 min; and after the first fraction of the mogroside is collected, the mobile phase II is used, the second fraction of the mogroside is collected for 39-47min at the same flow rate, and the third fraction of the mogroside is collected for 52-59 min.
10. The method of any one of claims 1 to 9, comprising the steps of:
(1) enzymolysis: taking dried momordica grosvenori roots, crushing, adding beta-glucosidase, adjusting the pH to be weakly acidic, uniformly stirring, and performing enzymolysis at room temperature;
(2) alkaline alcohol percolation: putting the raw materials after enzymolysis into a percolator, and percolating and extracting by using alkaline methanol or ethanol to obtain percolate;
(3) ceramic membrane filtration: filtering the percolate with ceramic membrane to obtain ceramic membrane filtrate;
(4) and (3) decoloring: passing the ceramic membrane filtrate through an alumina or magnesia chromatographic column to obtain a decolorized solution;
(5) and (3) concentrating under reduced pressure: concentrating the decolorized solution under reduced pressure until no alcohol exists to obtain a concentrated solution;
(6) acid adjustment: adjusting the pH value of the concentrated solution to acidity by using acid to obtain acidic concentrated solution;
(7) extraction and concentration: extracting the acidic concentrated solution with low-polarity organic solvent for 2-3 times, standing for layering, separating, collecting and combining organic solvent layers, concentrating the organic layers under reduced pressure, and vacuum drying to obtain fructus Siraitiae Grosvenorii total acid product;
(8) preparing chromatographic separation of the mogroside monomer: firstly, dissolving and diluting a momordica grosvenori total acid sample by using a mobile phase, injecting the sample into a high-pressure preparation liquid chromatographic column, and finally eluting the sample by using the mobile phase; collecting eluate fractions of different time periods, concentrating and drying respectively to obtain four mogroside monomers, namely mogroside B, mogroside A, mogroside D and mogroside C in sequence;
preferably, 0.1-0.5 wt% of borate of the roots of momordica grosvenori is also added in the enzymolysis of the step (1).
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