CN110144015B - Method for synchronously extracting and purifying ganoderan, ganoderma triterpenic acid and amino acid - Google Patents
Method for synchronously extracting and purifying ganoderan, ganoderma triterpenic acid and amino acid Download PDFInfo
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Abstract
The invention provides a method for synchronously extracting and purifying ganoderan, ganoderma triterpenic acid and amino acid, and belongs to the technical field of natural product extraction. The method takes ganoderma lucidum fruit bodies as starting raw materials, adopts alkali liquor for low-temperature ultrasonic extraction, simultaneously uses an antioxidant to protect polysaccharide and easily-oxidized components, improves the extraction rate of ganoderma lucidum polysaccharide and active components, adopts a ceramic membrane microfiltration technology, greatly increases the concentration efficiency and the rough separation effect, and simultaneously uses matched ion exchange resins for purification, alcohol precipitation and ultrafiltration, so that the contents of ganoderma lucidum polysaccharide, ganoderma lucidum triterpenic acid and amino acid are improved.
Description
Technical Field
The invention belongs to a method for extracting and separating natural products, and particularly relates to a method for synchronously extracting, separating and purifying active ingredients of ganoderma lucidum polysaccharide, ganoderma lucidum triterpenic acid and amino acid.
Background
Ganoderma (Ganoderma lucidum (Leys ex Fr.) Krast.) belongs to Basidiomycetes, Polyporaceae, and Ganoderma. Traditional Chinese medicine holds that ganoderma lucidum is sweet in taste, warm in nature and non-toxic, has the effects of nourishing, building body, strengthening body resistance and consolidating constitution, is a Chinese herbal medicine treasure, is entitled 'immortal grass', and has unique and excellent physiological efficiency. The 'Shennong Ben Cao Jing' in eastern Han and the 'Ben Cao gang mu' of Li Shizhen, a famous medical specialist in the Ming dynasty are the very definite records of the efficacy of Ganoderma lucidum. Modern researches have shown that the pharmacological components of Ganoderma are very rich, and the effective components of Ganoderma are in 10 kinds, including Ganoderma polysaccharides, Ganoderma polypeptides, triterpenes, amino acids, steroids, mannitol, coumarins, alkaloids, organic acids (mainly containing fumaric acid), and various trace elements such as Ge, P, Fe, Ca, Mn and Zn. The ganoderma lucidum can regulate and enhance human immunity, reduce blood fat and blood sugar, protect liver, inhibit obesity and arteriosclerosis and prevent cholesterol from being absorbed; has good prevention and treatment effects on neurasthenia, coronary heart disease, hypertension, chronic bronchitis, diabetes, tumor and the like. Recent studies have also shown that: ganoderma has antifatigue, skin caring, antiaging, and AIDS preventing and treating effects. At present, ganoderma lucidum is officially collected by national formulary as a medicine, is a new resource food approved by the state and can be used as both medicine and food.
Modern medicine proves that: the ganoderan can regulate and enhance human immunity, and can be used for preventing and treating serious diseases such as tumor, diabetes, AIDS, etc.; as reported by Bao-Mei Shao et al, the immunological activity of ganoderan (Immune receptors for polysaccharides from Garoderma lucidum, Biochemical and Biophysical Research Communication 323(2004)133-141), and Chen XP et al, the scavenging action of ganoderan on Free radicals and the influence on antioxidant enzymes and immunological activity in cervical cancer mice (Free radial scavenging of Garoderma lucidum polysaccharides and enzyme effects on antioxidant enzymes and immunological activity, Carbodreate Polymers,2009,77(2), 399). According to the literature, the high-purity ganoderma lucidum polysaccharide is basically used in the activity research; ganoderma triterpenic acid compounds are compounds with obvious pharmacological activity except ganoderma lucidum polysaccharide discovered in recent years, since Kubota et al isolated triterpenic compounds ganoderic acid A and B from Ganoderma lucidum fruiting body for the first time in 1982, research on ganoderic acid has attracted much attention, and more than 100 triterpenic acid compounds have been isolated from Ganoderma lucidum fruiting body successively. Ganoderic acid is generally distributed at the peripheral part of fruiting body, and increases with the increasing of the maturity of fruiting body, and the ganoderic acid has the functions of protecting liver, resisting tumor, reducing blood sugar, inhibiting histamine release, etc. (Enhancement of IL-2and IFN-g expression and NK cells activity induced in the anticancer or effect of organic acid Me in vivo. International immunological science, 2007, 7 (6): 864-870; Structure-activity relationship for inhibition of5 a-reduction of side tertiary amino isolated from door tissue. bioorganic & Medicinal Chemistry, 2006,14 (24): 8654-8660); in addition, ganoderma lucidum is also a 'storage library' of amino acids, 17 amino acids exist, 7 of the amino acids are necessary in human bodies, the amino acids account for more than 10% of ganoderma lucidum, free amino acids account for 30% -35% of total amino acids, most of the rest amino acids are combined with polysaccharide to form a sugar peptide chain, and the amino acids are easy to absorb by the human body, so that the ganoderma lucidum is often used as a health-care product and is favored by people. In conclusion, pharmacological research on active ingredients of ganoderma lucidum is clear at present, the clinical or health care requirement is large, the clarification requirement on the basis of medicinal substances is higher along with the improvement of the requirement of people on the medical care level, and therefore, the method has important significance on the extraction and purification of ganoderma lucidum polysaccharide, ganoderma lucidum triterpenic acid and amino acid and the application of the ganoderma lucidum polysaccharide, ganoderma lucidum triterpenic acid and amino acid as raw material medicines for medicines, health care products and the like.
At present, many reports are made on the separate purification of ganoderma lucidum polysaccharide, ganoderma lucidum triterpenic acid and amino acid, but the common problems of complex process, low yield and incapability of comprehensive utilization cause resource waste; in addition, because the difference of the physicochemical properties of the active components in the ganoderma is large, the general extraction method can fully extract the active components of the ganoderma only by double-solvent secondary extraction, the production cost is greatly increased, and some active components are distributed in cell walls, the active components are difficult to extract without breaking the walls by physical or chemical methods, which brings certain difficulty to the extraction process, the traditional physical treatment methods such as ultramicro crushing, high-pressure blasting and the like can achieve the effect of breaking the walls, but have high cost, the subsequent process treatment is troublesome, so that the method can not be widely applied, for example, the application No. 200810151025.6, namely the method for extracting the ganoderma lucidum fruiting body polysaccharide by using ultrahigh pressure is adopted, the extraction method has high cost, potential safety hazards exist, the production and popularization are not facilitated, and the structure of the active components can be changed under the ultrahigh pressure environment, only crude polysaccharide can be obtained due to no subsequent purification treatment process; for example, the publication number is CN1560072A entitled "a method for preparing ganoderma triterpenic acid and ganoderma polysaccharide from ganoderma", the method adopts supercritical extraction technology to extract active ingredients, the technology has high cost, is not easy to produce and popularize, and lacks subsequent purification technology, and the obtained ganoderma triterpenic acid content (60.2%) and ganoderma polysaccharide content (36%) are both low. In addition, at present, no research on simultaneous extraction of ganoderan, ganoderan triterpenic acid and amino acid is provided aiming at an extraction and purification method of ganoderma active ingredients, so that how to realize simultaneous extraction of the three active ingredients and save extraction and purification procedures is needed to be further researched and developed.
Therefore, it is urgently needed to provide a method for synchronously extracting and purifying the active ingredients of ganoderma lucidum polysaccharide, ganoderma lucidum triterpenic acid and amino acid, greatly improving the purity of the three active ingredients and increasing the yield of the three active ingredients.
Disclosure of Invention
The invention aims to solve the technical problems, and provides a method for synchronously extracting and purifying ganoderan, ganoderma triterpenic acid and amino acid, which greatly improves the purity of active ingredients, simplifies the purification process, reduces the cost and improves the benefit of ganoderma products.
In order to achieve the purpose, the invention adopts the technical scheme that the method for synchronously extracting and purifying the ganoderan, the ganoderma triterpenic acid and the amino acid comprises the following steps:
(1) pulverizing Ganoderma encarpium, sieving, adding alkali solution and reducing agent, ultrasonic extracting at 0-5 deg.C, centrifuging the extractive solution to obtain centrifugate and residue; the reducing agent comprises one of sodium borohydride, sodium bisulfite and sodium sulfite, the weight ratio of the reducing agent to the lucid ganoderma sporocarp is 1:20, and the concentration of the alkali liquor is 0.3-1 mol/L;
(2) adjusting the pH value of the centrifugate obtained in the step (1) to 2-4, standing for 8-16 hours, centrifuging, and respectively collecting the centrifugate and the precipitate;
(3) and (3) extracting and purifying amino acid and ganoderma lucidum polysaccharide:
(a) filtering the centrifugal clear liquid in the step (2) through a ceramic microfiltration membrane, and collecting filtrate and non-permeate;
(b) adsorbing the filtrate obtained in the step a by using cation exchange resin, eluting by using purified water and alkali liquor in sequence, detecting amino acid by ninhydrin color reaction, collecting eluent which shows positive reaction in the elution process of the alkali liquor, adjusting the pH value of the eluent to 4-7, heating and boiling for 2 hours, cooling, standing for 8-16 hours, filtering, concentrating the filtrate under reduced pressure, drying the concentrate in vacuum, pulverizing and mixing uniformly to obtain brown powdery amino acid;
(c) adding absolute ethyl alcohol into the non-permeate liquid in the step a until the alcohol content is 5-30%, carrying out alcohol precipitation, centrifuging, collecting centrifugate, adding absolute ethyl alcohol until the alcohol content is 50-95%, carrying out alcohol precipitation, centrifuging, and collecting precipitate;
(d) removing protein from the precipitate in the step c by using an organic reagent method, recovering residual organic reagent under reduced pressure, adsorbing the concentrated solution by using anion exchange resin, eluting by using purified water and a salt solution in sequence, detecting polysaccharide by using a sulfuric acid-anthrone method, and collecting the eluate with positive reaction;
(e) d, combining the eluates obtained in the step d, performing ultrafiltration purification, collecting the non-permeating liquid, and performing low-temperature vacuum drying on the non-permeating liquid to obtain white spongy dry ganoderma lucidum polysaccharide;
(4) dissolving the precipitate in the step (2) with alkali liquor, adjusting pH to 6-8, adding anion exchange resin, eluting with weak base ethanol solution, collecting the eluate with acetic anhydride-concentrated sulfuric acid developing to positive, adjusting pH of the eluate to 2-6, recovering solvent under reduced pressure, vacuum drying to obtain pale yellow solid, heating and dissolving with ethanol, cooling for recrystallization to obtain white needle-like crystal, and vacuum drying to obtain Ganoderma total triterpenic acid.
In the above method, the obtained brown powder is the raw material with amino acid content of above 60%, the white sponge-like dried substance is the raw material with ganoderan content of above 90%, and the white crystal granule is the raw material with ganoderan total triterpene acid content of above 92%.
Compared with the traditional process, the extraction and purification method disclosed by the invention has the advantages that the low-temperature alkali liquor ultrasonic extraction is adopted, the excellent extraction effect is achieved, meanwhile, the antioxidant is combined to protect the easily-oxidized active ingredients, the ganoderma lucidum polysaccharide, the ganoderma lucidum triterpenic acid and the amino acid peptide compounds are extracted simultaneously, then, the micro-filtration technology of the ceramic membrane is used for directly separating the micromolecule active ingredients from the macromolecule active ingredients, and the method is more suitable for production and application. On the other hand, the invention adopts ion exchange resin to purify the active ingredients, so that the ion exchange adsorption of the ganoderma triterpenic acid and amino acid compounds in an ionic state is directional adsorption, redundant ingredients are removed, the ganoderma polysaccharide is subjected to protein and pigment removal, crystallization and ultrafiltration in the application of the purification process of the ganoderma triterpenic acid and the ganoderma polysaccharide, the purity of the active ingredients is greatly improved, the purity of the prepared ganoderma polysaccharide is more than 90 percent, the purity of the total ganoderma triterpenic acid is more than 92 percent and the content of amino acid is more than 60 percent, the ganoderma resource is fully utilized, and all purification procedures in the invention are simple, easy to operate, low in cost and easy to widely use in production.
Further, the alkali liquor in the step (1) comprises sodium hydroxide solution or sodium carbonate solution or sodium bicarbonate solution.
Further, the ganoderma lucidum fruiting body in the step (1) is crushed and then screened by a 100-mesh screen.
Further, the pore size of the ceramic microfiltration membrane in the step a is 0.22um or 0.45um or 0.8 um.
Further, the cation exchange resin in the step b is 732 type cation exchange resin or D001 type cation exchange resin, the alkali liquor is 0-1mol/L ammonia water or 0-1mol/L sodium hydroxide aqueous solution, and the acid liquor used for adjusting the pH is 0-5mol/L hydrochloric acid aqueous solution or 0-5mol/L sulfuric acid aqueous solution.
Further, the organic reagent used for removing the protein in the step d is diethyl ether or petroleum ether.
Further, in step d, the anion exchange resin is 717 type anion exchange resin or DEAE cellulose anion exchange resin.
Further, the salt solution in the step d is 0-1mol/L sodium chloride solution.
Further, the anion exchange resin in the step (4) is a D311 type weak base anion exchange resin or a D941 type weak base anion exchange resin.
Further, the mass concentration of the weak base in the weak base ethanol solution in the step (4) is 1-10%, and the weak base is NaOH or Na2CO3Or NaHCO3The volume ratio of the weak base to the ethanol is 1: 1-5.
Compared with the prior art, the invention has the following beneficial effects:
the method adopted by the invention can simultaneously extract and purify the ganoderma lucidum sporocarp to obtain three active ingredients with the ganoderma lucidum polysaccharide content of more than 90 percent, the ganoderma lucidum triterpenic acid content of more than 92 percent and the amino acid content of more than 60 percent, the extraction and purification of the three substances can be completely carried out in one production process, and the production equipment is simple, the process route is easy to operate, the production cost is low, the production efficiency is high, and the industrial production can be carried out.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following embodiments, and it should be noted that the following embodiments are only for explaining and illustrating the present invention and are not intended to limit the present invention. The invention is not limited to the embodiments described above, but rather, may be modified within the scope of the invention.
Example 1
A method for synchronously extracting and purifying ganoderan, ganoderma triterpenic acid and amino acid comprises the following steps:
A. taking Ganoderma encarpium, pulverizing, sieving with 100 mesh sieve, weighing 1kg, adding 15 L0.3mol/L sodium hydroxide water solution, simultaneously adding 50g sodium borohydride, performing ultrasonic extraction at 0-5 deg.C for 3 hr, and centrifuging at 8000r/min to obtain centrifugate; adjusting pH to 3.5 with 1mol/L hydrochloric acid, standing overnight (12h), centrifuging at 8000r/min, and collecting the supernatant and precipitate respectively;
B. taking the centrifugal clear liquid, carrying out microfiltration by a 0.45um ceramic membrane, adding purified water with the same volume when the concentrated solution reaches 1L, continuing microfiltration, repeating the microfiltration once, and respectively collecting filtrate and unpermeated concentrated solution; adsorbing the filtrate by 732 type cation exchange resin, eluting with purified water and 0.5mol/L ammonia water in sequence, collecting the eluate with positive reaction in ninhydrin detection during ammonia water elution, adjusting pH to 4.8 with 1mol/L hydrochloric acid, heating and boiling for 2 hr, cooling, standing overnight (12 hr), filtering, concentrating the filtrate at 60 deg.C under reduced pressure to 150ml, evaporating to dryness in water bath, vacuum drying at 60 deg.C, taking out, pulverizing, and mixing to obtain brown powder with amino acid content of 63.5%; diluting the microfiltration non-permeate with equal volume of purified water, adding absolute ethyl alcohol under stirring until the alcohol content is 15%, standing overnight (12h), centrifuging at 8000r/min, collecting the supernatant, continuing to add absolute ethyl alcohol until the alcohol content is 85%, standing overnight, centrifuging at 8000r/min, collecting the precipitate, adding 500ml of purified water to dissolve, adding 200ml of diethyl ether to remove protein, separating the lower water layer and the organic phase by a separating funnel, recovering the solvent from the organic phase, repeatedly removing the protein from the water layer once, filtering, recovering the solvent to 100ml under reduced pressure, adsorbing by DEAE-cellulose anion exchange resin, washing the resin column with purified water, eluting with 0.1mol/L sodium chloride solution, collecting the eluate with positive reaction detected by sulfuric acid-anthrone reagent, combining the eluates, removing small molecular substances by a 10 KD ultrafiltration membrane, collecting the permeate, vacuum drying the non-permeated solution at low temperature to obtain ganoderan with polysaccharide content of 91.1%.
C. And (2) centrifuging the precipitate in the step A, dissolving the precipitate by using a 1% sodium hydroxide solution, adding the alkali liquor until the pH value is 7.4, adsorbing the obtained solution by using D311 type anion exchange resin, washing a resin column by using purified water, eluting by using a 3% sodium hydroxide ethanol solution (volume ratio is 1: 1), collecting an eluent with positive coloration of an acetic anhydride-concentrated sulfuric acid reagent, adjusting the pH value to 4.2 by using 1mol/L hydrochloric acid, recovering the solvent under reduced pressure, evaporating to dryness in a water bath, drying in vacuum to obtain a light yellow solid, adding 15 times of absolute ethyl alcohol, heating to dissolve the light yellow solid completely, cooling, crystallizing, filtering to obtain a white needle-shaped crystal, drying in vacuum to obtain a white powdery solid, and detecting that the total triterpenic acid content of the lucid ganoderma is 93.6%.
The detection method involved in the embodiment of the invention is as follows:
the content of amino acid is determined by ultraviolet-visible spectrophotometry, U-2000 type ultraviolet-visible spectrophotometer has ninhydrin reagent as developer, arginine as reference substance, and detection wavelength of 576 nm; the content of ganoderan is determined by ultraviolet-visible spectrophotometry, U-2000 ultraviolet-visible spectrophotometer, anthrone sulfate solution as developer, detection wavelength of 625nm, and glucose as reference; an ultraviolet spectrophotometry for measuring total triterpenic acid content of Ganoderma, U-2000 type ultraviolet visible spectrophotometer, and ganoderic acid B as reference substance, with detection wavelength of 526 nm.
Example 2
A method for synchronously extracting and purifying ganoderan, ganoderma triterpenic acid and amino acid comprises the following steps:
A. taking lucid ganoderma sporocarp, crushing and sieving the lucid ganoderma sporocarp with a 100-mesh sieve, weighing 1kg, adding 15 L0.5mol/L sodium carbonate aqueous solution, simultaneously adding 50g sodium borohydride, carrying out ultrasonic extraction at 0-5 ℃ for 3 hours, and centrifuging at 8000r/min to obtain a centrifugal liquid; adjusting pH to 3.1 with 1mol/L hydrochloric acid, standing overnight (8h), centrifuging at 8000r/min, and collecting the supernatant and precipitate respectively;
B. taking the centrifugal clear liquid, carrying out microfiltration by a 0.22um ceramic membrane, adding purified water with the same volume when the concentrated solution reaches 1L, continuing microfiltration, repeating the microfiltration once, and respectively collecting filtrate and unpermeated concentrated solution; adsorbing the filtrate by using D001 type cation exchange resin, eluting by using purified water and 0.5mol/L sodium hydroxide solution in sequence, collecting eluent with positive reaction in ninhydrin detection in an alkali liquor elution process, adjusting pH to 4.8 by using 1mol/L hydrochloric acid, filtering, concentrating the filtrate at 60 ℃ under reduced pressure to 150ml, evaporating in a water bath, drying at 60 ℃ in vacuum, taking out, powdering and mixing uniformly to obtain brown powder, and detecting that the content of amino acid is 61.2%; diluting the microfiltration non-permeate with equal volume of purified water, adding absolute ethyl alcohol under stirring until the alcohol content is 20%, standing overnight, centrifuging at 8000r/min, collecting the supernatant, continuing to add absolute ethyl alcohol until the alcohol content is 90%, standing overnight (8h), centrifuging at 8000r/min, collecting the precipitate, adding 500ml of purified water to dissolve, adding 200ml of diethyl ether to remove protein, separating the lower water layer and the organic phase by a separating funnel, recovering the solvent from the organic phase, repeatedly removing the protein from the water layer once, filtering, recovering the solvent to 100ml under reduced pressure, adsorbing by DEAE-cellulose anion exchange resin, washing the resin column with purified water, eluting with 0.3mol/L sodium chloride solution, collecting the eluate with positive reaction detected by sulfuric acid-anthrone reagent, combining the eluates, removing small molecular substances by a 10 KD ultrafiltration membrane, collecting the permeate, vacuum drying the non-permeated solution at low temperature to obtain ganoderan with polysaccharide content of 90.3%.
C. And (2) centrifuging the precipitate in the step A, dissolving the precipitate by using a 1% sodium hydroxide solution, adding the alkali liquor until the pH value is 6.2, adsorbing the obtained solution by using D941 type anion exchange resin, washing a resin column by using purified water, eluting by using an 8% sodium carbonate ethanol solution (volume ratio of 1: 1), collecting an eluent with positive coloration of an acetic anhydride-concentrated sulfuric acid reagent, adjusting the pH value to 4.2 by using 1mol/L hydrochloric acid, recovering the solvent under reduced pressure, evaporating to dryness in a water bath, performing vacuum drying to obtain a light yellow solid, adding 15 times of absolute ethyl alcohol, heating to dissolve the light yellow solid completely, cooling, crystallizing, filtering to obtain white needle-shaped crystals, and performing vacuum drying to obtain a white powdery solid, wherein the content of total triterpene acid of the lucid ganoderma is detected to be 94.3%.
The method of detection of the active ingredient was the same as in example 1.
Example 3
A method for synchronously extracting and purifying ganoderan, ganoderma triterpenic acid and amino acid comprises the following steps:
A. pulverizing Ganoderma fruiting body, sieving with 150 mesh sieve, weighing 1kg, adding 15L 1mol/L sodium bicarbonate water solution, simultaneously adding 50g sodium sulfite, ultrasonic extracting at 0-5 deg.C for 3 hr, and centrifuging at 8000r/min to obtain centrifugate; adjusting pH to 3.8 with 1mol/L hydrochloric acid, standing overnight (10h), centrifuging at 8000r/min, and collecting the supernatant and precipitate respectively;
B. taking the centrifugal clear liquid, carrying out microfiltration by a 0.8um ceramic membrane, adding purified water with the same volume when the concentrated solution reaches 1L, continuing microfiltration, repeating the microfiltration once, and respectively collecting filtrate and unpermeated concentrated solution; adsorbing the filtrate by using D001 type cation exchange resin, eluting by using purified water and 0.8mol/L sodium hydroxide solution in sequence, collecting eluent with positive reaction in ninhydrin detection in an alkali liquor elution process, adjusting pH to 5.7 by using 1mol/L hydrochloric acid, filtering, concentrating the filtrate at 60 ℃ under reduced pressure to 150ml, evaporating in a water bath, drying at 60 ℃ in vacuum, taking out, powdering and mixing uniformly to obtain brown powder, and detecting that the content of amino acid is 67.2%; diluting the microfiltration non-permeate with purified water with the same volume, adding absolute ethyl alcohol under stirring until the alcohol content is 10%, standing overnight (10h), centrifuging at 8000r/min, collecting the centrifugal clear liquid, continuously adding absolute ethyl alcohol until the alcohol content is 90%, standing overnight, centrifuging at 8000r/min, collecting the precipitate, then adding 500ml of purified water to dissolve, adding 200ml of petroleum ether to remove protein, separating the lower water layer and the organic phase by a separating funnel, recovering the solvent from the organic phase, repeatedly removing the protein from the water layer once, filtering, recovering the solvent to 100ml under reduced pressure, adsorbing by 711 type anion exchange resin, washing the resin column with purified water, eluting with 0.8mol/L sodium chloride solution, collecting the eluate of which the sulfuric acid-anthrone reagent shows a positive reaction, combining the eluates, removing small molecular substances through a 10 KD ultrafiltration membrane, and collecting the permeate, vacuum drying the non-permeated solution at low temperature to obtain ganoderan with polysaccharide content of 93.7%.
C. And (2) centrifuging the precipitate in the step A, dissolving the precipitate by using a 1% sodium hydroxide solution, adding the alkali liquor until the pH value is 7.8, adsorbing the obtained solution by using D941 type anion exchange resin, washing a resin column by using purified water, eluting by using a 10% sodium bicarbonate ethanol solution (volume ratio of 1: 2), collecting an eluent with positive color development of an acetic anhydride-concentrated sulfuric acid reagent, adjusting the pH value to 4.2 by using 1mol/L hydrochloric acid, recovering the solvent under reduced pressure, evaporating by using a water bath, drying in vacuum to obtain a light yellow solid, adding 15 times of anhydrous ethanol, heating to completely dissolve, cooling, crystallizing, filtering to obtain white needle-shaped crystals, drying in vacuum to obtain a white powdery solid, and detecting the total triterpenic acid content of the lucid ganoderma to be 92.9%.
The method of detection of the active ingredient was the same as in example 1.
Comparative example 1
According to the method of example 1, alkali solution and reducing agent are not added in the step A, ultrasonic extraction is directly carried out at low temperature, and finally the obtained amino acid content is 52.6%, the ganoderma lucidum polysaccharide content is 83.5%, and the ganoderma lucidum total triterpene acid content is 84.3%.
Comparative example 2
According to the method of the embodiment 2, alkali liquor is not added in the step A, reducing agent is directly added at normal temperature for ultrasonic extraction, and the final obtained amino acid content is 55.8%, the ganoderma lucidum polysaccharide content is 84.3%, and the ganoderma lucidum total triterpene acid content is 86.1%.
Comparative example 3
According to the method of the embodiment 3, alkali liquor and reducing agent are added in the step A to carry out ultrasonic extraction at normal temperature, the filtered liquid is absorbed by adopting 001X 7 type cation exchange resin in the step B, and the ganoderma lucidum total triterpenic acid is absorbed and extracted by adopting AB-8 macroporous absorption resin in the step C, so that the content of the obtained amino acid is 57.4%, the content of ganoderma lucidum polysaccharide is 85.1%, and the content of the ganoderma lucidum total triterpenic acid is 88.3%.
Claims (5)
1. A method for synchronously extracting and purifying ganoderan, ganoderma triterpenic acid and amino acid is characterized by comprising the following steps: (1) crushing ganoderma lucidum sporocarp, sieving with a 100-mesh sieve, adding alkali liquor and a reducing agent, carrying out ultrasonic extraction at 0-5 ℃, and centrifuging an extracting solution to obtain a centrifugal liquid and dregs; the alkali liquor comprises a sodium hydroxide solution, a sodium carbonate solution or a sodium bicarbonate solution, the reducing agent comprises one of sodium borohydride, sodium bisulfite or sodium sulfite, the weight ratio of the reducing agent to the lucid ganoderma sporocarp is 1:20, and the concentration of the alkali liquor is 0.3-1 mol/L; (2) adjusting the pH value of the centrifugate obtained in the step (1) to 2-4, standing for 8-16 hours, centrifuging, and respectively collecting the centrifugate and the precipitate; and (3) extracting and purifying amino acid and ganoderma lucidum polysaccharide: filtering the centrifugal clear liquid in the step (2) through a ceramic microfiltration membrane, and collecting filtrate and non-permeate; the aperture of the ceramic microfiltration membrane is 0.22um or 0.45um or 0.8 um; (b) passing the filtrate obtained in the step a through 732 type cation exchange resin or D001 type cation exchange resin, sequentially eluting with purified water and alkali liquor, wherein the alkali liquor is 0-1mol/L ammonia water or 0-1mol/L sodium hydroxide aqueous solution, detecting amino acid by ninhydrin color reaction, collecting eluent which shows positive reaction in the alkali liquor elution process, adjusting the pH of the eluent to 4-7 by using 0-5mol/L hydrochloric acid aqueous solution or 0-5mol/L sulfuric acid aqueous solution, heating, boiling for 2 hours, cooling, standing for 8-16 hours, filtering, concentrating the filtrate under reduced pressure, drying the concentrate in vacuum, powdering and mixing uniformly to obtain brown powdery amino acid; (c) adding absolute ethyl alcohol into the non-permeate liquid in the step a until the alcohol content is 5-30%, carrying out alcohol precipitation, centrifuging, collecting centrifugate, adding absolute ethyl alcohol until the alcohol content is 50-95%, carrying out alcohol precipitation, centrifuging, and collecting precipitate; (d) deproteinizing the precipitate in the step c by using an organic reagent method, recovering residual organic reagent under reduced pressure, adsorbing the concentrated solution by using anion exchange resin, eluting by using purified water and a salt solution in sequence, detecting polysaccharide by using a sulfuric acid-anthrone method, and collecting the eluate showing a positive reaction; (e) combining the eluents obtained in the step d, performing ultrafiltration purification, collecting the non-permeating liquid, and performing low-temperature vacuum drying on the non-permeating liquid to obtain white spongy dry ganoderma lucidum polysaccharide;
(4) dissolving the precipitate in the step (2) with alkaline solution, adjusting pH to 6-8, adding anion exchange resin, wherein the anion exchange resin is D311 type weak base anion exchange resin or D941 type weak base anion exchange resin, eluting with weak base ethanol solution, collecting acetic anhydride-concentrated sulfuric acid to develop positive eluent, adjusting pH of the eluent to 2-6, recovering solvent under reduced pressure, vacuum drying to obtain pale yellow solid, heating and dissolving with ethanol, cooling for recrystallization to obtain white needle-shaped crystal, and vacuum drying to obtain total triterpene acid of Ganoderma.
2. The method for synchronously extracting and purifying ganoderan, ganoderic acid and amino acids according to claim 1, wherein the organic reagent used for removing proteins in step d is diethyl ether or petroleum ether.
3. The method for synchronously extracting and purifying ganoderan, ganoderic acid and amino acids according to claim 1, wherein the anion exchange resin in step d is 717 type anion exchange resin or DEAE cellulose anion exchange resin.
4. The method for synchronously extracting and purifying ganoderan, ganoderic triterpenic acid and amino acid according to claim 1, wherein the salt solution in step d is 0-1mol/L sodium chloride solution.
5. The method for synchronously extracting and purifying ganoderan, ganoderic triterpenic acid and amino acid according to claim 1, wherein the mass concentration of weak base in the weak base ethanol solution in the step (4) is 1-10%, and the weak base is Na2CO3Or NaHCO3The volume ratio of the weak base to the ethanol is 1: 1-5.
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