CN110144015A - A simultaneous extraction and purification method of Ganoderma lucidum polysaccharides, Ganoderma triterpene acids and amino acids - Google Patents

Abstract

The invention provides a synchronous extraction and purification method of ganoderma polysaccharides, ganoderma triterpene acids and amino acids, belonging to the technical field of natural product extraction. The method of the present invention uses the ganoderma lucidum fruiting body as the starting material, through the use of alkaline solution for low-temperature ultrasonic extraction, and simultaneously uses antioxidants to protect polysaccharides and easily oxidized components, thereby increasing the extraction rate of ganoderma polysaccharides and active components, and adopts ceramic membrane microfiltration technology, which greatly increases the concentration efficiency and rough separation effect. At the same time, the matching ion exchange resin is used for purification, alcohol precipitation and ultrafiltration technology, so that the contents of Ganoderma lucidum polysaccharide, Ganoderma triterpene acid and amino acid have all been improved. The method of the present invention starts from Extracting and purifying Ganoderma lucidum fruiting body to obtain a Ganoderma lucidum polysaccharide content of more than 90%, a Ganoderma triterpene acid content of more than 92%, and an amino acid content of more than 60%, which is more suitable for industrial production applications, and simplifies the extraction and purification steps and reduces costs.

Description

A simultaneous extraction and purification method of Ganoderma lucidum polysaccharides, Ganoderma triterpene acids and amino acids

technical field

The invention belongs to the extraction and separation method of natural products, in particular to the synchronous extraction, separation and purification method of Ganoderma lucidum polysaccharide, Ganoderma triterpene acid and amino acid active ingredients.

Background technique

Ganoderma lucidum (Leyss ex Fr.) Krast.) belongs to Basidiomycetes, Polyporaceae, Ganoderma fungus. Traditional Chinese medicine believes that Ganoderma lucidum is sweet, warm in nature, non-toxic, and has the effects of nourishing and strengthening the body, strengthening the body, and strengthening the body. As a treasure of Chinese herbal medicine in my country, it is known as the "immortal grass" and has unique and excellent physiological effects. The "Shen Nong's Materia Medica" in the Eastern Han Dynasty and the "Compendium of Materia Medica" by Li Shizhen, a famous medical scientist in the Ming Dynasty, have detailed and extremely positive records on the efficacy of Ganoderma lucidum. Modern research shows that the pharmacological components of Ganoderma lucidum are very rich, and the active ingredients reach 10 categories, including Ganoderma lucidum polysaccharides, Ganoderma lucidum polypeptides, triterpenoids, amino acids, steroids, mannitol, coumarin, alkaloids, organic acids (mainly containing fumaric acid), and various trace elements such as Ge, P, Fe, Ca, Mn and Zn, etc. Ganoderma lucidum can regulate and enhance human immunity, lower blood fat and blood sugar, protect liver, inhibit obesity and arteriosclerosis, prevent cholesterol absorption; it has good prevention and treatment for neurasthenia, coronary heart disease, high blood pressure, chronic bronchitis, diabetes and tumors effect. The latest research also shows that Ganoderma lucidum also has the functions of anti-fatigue, beauty and beauty, anti-aging, and prevention and treatment of AIDS. At present, Ganoderma lucidum has been officially recorded in the National Pharmacopoeia as a medicine, and at the same time it is a new resource food approved by the state, which can be used for both medicine and food.

Modern medicine has proved that Ganoderma lucidum polysaccharides can regulate and enhance human immunity, and thus be used for the prevention and treatment of major diseases such as tumors, diabetes, and AIDS; for example, Bao-Mei Shao reported the immune activity of Ganoderma lucidum polysaccharides (Immunereceptors for polysaccharides from Ganoderma lucidum, Biochemical and Biophysical Research Communication 323 (2004) 133-141), and Chen XP et al. revealed the scavenging effect of Ganoderma lucidum polysaccharides on free radicals and the effect on antioxidant enzymes and immune activity in mice with cervical cancer (Free radicalscavenging of Ganoderma lucidum polysaccharides and its effect on antioxidant antenzymes and immunity activities in cervical carcinoma rats, Carbohydrate Polymers, 2009, 77(2), 399). After reviewing the literature, high-purity Ganoderma lucidum polysaccharides were basically used for activity research; Ganoderma lucidum triterpenoids are another class of compounds with obvious pharmacological activity discovered in recent years besides Ganoderma lucidum polysaccharides. Since 1982, Kubota et al. Since the triterpenoids ganoderma acid A and B were isolated from Ganoderma lucidum, the research on ganoderma acid has attracted much attention. So far, more than 100 triterpene acid compounds have been isolated from the fruiting body of ganoderma lucidum. Ganoderma acid is generally distributed in the peripheral parts of the fruiting body, and increases with the maturity of the fruiting body. This kind of substance has the functions of protecting the liver, anti-tumor, lowering blood sugar, and inhibiting the release of histamine (Enhancement of IL-2and IFN-g expression and NKcells activity involved in the antitumor effect of ganoderic acid Me invivo. International Immunopharmacology, 2007, 7(6): 864-870; Structure-activity relationship for inhibition of 5a-reductase by triterpenoids isolated from Ganoderma lucidum. ): 8654-8660); In addition, Ganoderma lucidum is also a "reservoir" of amino acids. There are 17 amino acids, of which 7 are essential amino acids in the human body. Amino acids account for more than 10% of Ganoderma lucidum, and free amino acids account for 30% of the total amino acids. %-35%, most of the rest are combined with polysaccharides to form glycopeptide chains, and these amino acids are easily absorbed by the human body, so they are often used as health products and are favored by people. To sum up, the current pharmacological research on the active ingredients of Ganoderma lucidum has been very clear, and there is a large demand for clinical or health care. With the improvement of people's requirements for medical care, there is a higher requirement for the clarification of the medicinal material basis. It is of great significance to extract and purify Ganoderma lucidum polysaccharides, Ganoderma triterpene acids and amino acids and use them as raw materials for medicines and health products.

At present, there are many reports on the separate purification of Ganoderma lucidum polysaccharides, Ganoderma triterpene acids, and amino acids, but the process is generally complicated, the yield is not high, and they cannot be comprehensively utilized, resulting in waste of resources; Large, the general extraction method is double solvent extraction to fully extract the active ingredients of Ganoderma lucidum, which greatly increases the production cost, and some active ingredients are distributed in the cell wall, and it is difficult to extract the active ingredients without breaking the wall by physical or chemical methods. The extraction process has brought certain difficulties to the extraction process. Although traditional physical treatment methods such as superfine pulverization and high-pressure blasting can achieve the effect of breaking the wall, the cost is high and the follow-up process is troublesome, so it cannot be widely used. Application, such as application number 200810151025.6, titled "Using ultra-high pressure to extract polysaccharides from Ganoderma lucidum fruiting body", using ultra-high pressure extraction method to extract polysaccharides from Ganoderma lucidum, this extraction method has high cost and potential safety hazards, which is not conducive to production promotion, and Under ultra-high pressure environment, the structure of active ingredients may change, and because there is no subsequent purification process, only crude polysaccharides can be obtained; for example, the publication number is CN1560072A, and the name is "a method for preparing ganoderma triterpene acids and ganoderma polysaccharides from ganoderma lucidum." Method", this method adopts supercritical extraction technology to extract active ingredients, this technology is costly, not easy to produce and popularize, and lacks follow-up purification process, the obtained content of triterpene acid of Ganoderma lucidum (60.2%) and polysaccharide content of Ganoderma lucidum (36%) are lower. In addition, for the extraction and purification methods of active ingredients of Ganoderma lucidum, there is no research on the simultaneous extraction of Ganoderma lucidum polysaccharides, Ganoderma triterpene acids, and amino acids. How to achieve simultaneous extraction of these three active ingredients and save extraction and purification procedures requires further research. Research carried out.

Therefore, there is an urgent need to provide a method that can simultaneously extract and purify Ganoderma lucidum polysaccharides, Ganoderma triterpene acids and amino acid active ingredients, and greatly improve the purity and yield of the above three active ingredients.

Contents of the invention

The purpose of the present invention is to solve the above-mentioned technical problems, and provide a synchronous extraction and purification method of Ganoderma lucidum polysaccharides, Ganoderma triterpene acids and amino acids, which greatly improves the purity of active ingredients, simplifies the purification process, reduces costs, and improves the benefits of Ganoderma lucidum products .

In order to achieve the above object, the technical solution adopted in the present invention is a synchronous extraction and purification method of Ganoderma lucidum polysaccharides, Ganoderma triterpene acids and amino acids, comprising the following steps:

(1) Ganoderma lucidum fruiting bodies are crushed and sieved, adding lye and reducing agent for ultrasonic extraction at 0-5°C, centrifuging the extract to obtain centrifugate and medicinal residues; the reducing agent includes sodium borohydride, hydrogen sulfite One of sodium or sodium sulfite, the weight ratio of the reducing agent to the ganoderma lucidum fruiting body is 1:20, and the concentration of the lye is 0.3-1mol/L;

(2) adjust the pH value of the centrifugate obtained in step (1) to 2-4, place it for 8-16 hours, centrifuge, and collect the centrifuge liquid and precipitate respectively;

(3) Extraction and purification of amino acids and ganoderma polysaccharides:

(a) filtering the centrifugal supernatant in step (2) through a ceramic microfiltration membrane, collecting the filtrate and the non-permeated liquid;

(b) Adsorb the filtrate in step a by cation exchange resin, elute with purified water and lye successively, detect amino acids with ninhydrin chromogenic reaction, and collect eluate showing positive reaction during elution with lye , adjust the pH of the eluent to 4-7, heat and boil for 2 hours, cool, stand for 8-16 hours, filter, concentrate the filtrate under reduced pressure, dry the concentrate in vacuum, powder and mix to obtain brown powder amino acid;

(c) Add absolute ethanol to the non-permeated liquid in step a until the alcohol content is 5-30%, ethanol precipitation, centrifuge, collect the centrifugate, then add absolute ethanol until the alcohol content is 50-95%, alcohol sink, centrifuge, and collect the precipitate;

(d) remove the protein from the precipitate in step c using the organic reagent method, recover the residual organic reagent under reduced pressure, absorb the concentrated solution through an anion exchange resin, and sequentially elute with purified water and salt solution, and detect the polysaccharide with the sulfuric acid-anthrone method, Collect the eluate with positive reaction;

(e) combining the eluents in step d, purifying by ultrafiltration, collecting the non-permeated liquid, and vacuum-drying the non-permeated liquid at low temperature to obtain a white spongy dried Ganoderma lucidum polysaccharide;

(4) Dissolve the precipitate in step (2) with lye, adjust the pH to 6-8, apply anion exchange resin, elute with weak base ethanol, and collect the acetic anhydride-concentrated sulfuric acid to develop a positive eluate , the pH of the eluent was adjusted to 2-6, the solvent was recovered under reduced pressure, vacuum-dried, it was light yellow solid, dissolved by heating with ethanol, cooled and recrystallized to obtain white needle-shaped crystals, dried in vacuum to obtain the total triterpene acids of Ganoderma lucidum.

In the above method, the obtained brown powder is a raw material with an amino acid content of more than 60%, the off-white spongy dry matter is a raw material with a Ganoderma polysaccharide content of more than 90%, and the white crystal particles are a raw material with a total triterpene acid content of Ganoderma lucidum with a content of more than 92%.

The above-mentioned extraction and purification method of the present invention, compared with the traditional process, the present invention adopts low-temperature lye ultrasonic extraction to achieve excellent extraction effect, combined with antioxidants to protect the easily oxidized active ingredients, the Ganoderma lucidum polysaccharide, Ganoderma lucidum Triterpene acids and amino acid peptide compounds are extracted at the same time, and then use ceramic membrane microfiltration technology to directly separate small molecule active ingredients from macromolecular active ingredients, which is more suitable for production applications. On the other hand, the present invention uses ion exchange resins to purify the active components, so that the ion exchange adsorption of ganoderma triterpene acids and amino acid compounds in the ionic state is directional adsorption, and redundant components are removed, so that ganoderma lucidum polysaccharides are deproteinized. And the application of pigment, crystallization and ultrafiltration in the purification process of Ganoderma lucidum triterpene acid and Ganoderma polysaccharide greatly improves the purity of active ingredients. The content is more than 60%, fully utilizes the ganoderma lucidum resources, and all the purification procedures in the present invention are simple, easy to operate, low in cost, and easy to be widely used in production.

Further, the lye described in step (1) includes sodium hydroxide solution or sodium carbonate solution or sodium bicarbonate solution.

Further, the ganoderma lucidum fruiting body described in step (1) is crushed and passed through a 100-mesh sieve.

Further, the pore size of the ceramic microfiltration membrane in step a is 0.22um or 0.45um or 0.8um.

Further, the cation exchange resin described in step b is 732-type cation-exchange resin or D001-type cation-exchange resin, the lye is 0-1mol/L ammonia water or 0-1mol/L sodium hydroxide aqueous solution, and the pH adjustment adopts The acid liquid is 0-5mol/L hydrochloric acid aqueous solution or 0-5mol/L sulfuric acid aqueous solution.

Further, the organic reagent used to remove protein in step d is diethyl ether or petroleum ether.

Further, the anion exchange resin described in step d is 717 type anion exchange resin or DEAE cellulose anion exchange resin.

Further, the salt solution in step d is 0-1mol/L sodium chloride solution.

Further, the anion exchange resin described in step (4) is D311 type weakly basic anion exchange resin or D941 type weakly basic anion exchange resin.

Further, the mass concentration of the weak base in the weak base ethanol liquid described in step (4) is 1-10%, and the weak base is NaOH or Na ₂ CO ₃ or NaHCO ₃ , the volume of the weak base and ethanol The ratio is 1:1-5.

Compared with the prior art, the beneficial effects of the present invention are as follows:

The method adopted in the present invention can simultaneously extract and purify from the fruit body of Ganoderma lucidum to obtain three active ingredients with a content of Ganoderma lucidum polysaccharides of more than 90%, a content of Ganoderma triterpene acids of more than 92%, and an amino acid content of more than 60%. The extraction of the three substances Purification 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 industrial production can be carried out.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in detail below in conjunction with the examples. It must be pointed out that the following examples are only used to explain and illustrate the present invention, and are not intended to limit the present invention . Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the invention still belong to the protection scope of the present invention.

Example 1

A simultaneous extraction and purification method of Ganoderma lucidum polysaccharides, Ganoderma triterpene acids and amino acids is carried out according to the following steps:

A. Take Ganoderma lucidum fruiting bodies, crush them through a 100-mesh sieve, weigh 1 kg, add 15 L of 0.3 mol/L sodium hydroxide aqueous solution, and add 50 g of sodium borohydride at the same time, ultrasonically extract at 0-5 °C for 3 hours, and then use 8000 r/L Centrifuge for 1 min to obtain the centrifugate; use 1mol/L hydrochloric acid to adjust the pH to 3.5, leave it overnight (12h), centrifuge at 8000r/min, collect the centrifuge supernatant and precipitate respectively, and set aside;

B. Take the supernatant and microfilter it through a 0.45um ceramic membrane. When the concentrated solution reaches 1 L, add an equal volume of purified water to continue the microfiltration, repeat the microfiltration once, and collect the filtrate and the non-permeated concentrated solution respectively; The filtrate is adsorbed by 732-type cation exchange resin, eluted with purified water and 0.5mol/L ammonia water in sequence, and the ninhydrin test positive reaction eluate is collected during the elution process of ammonia water, and then the pH is adjusted with 1mol/L hydrochloric acid To 4.8, heat to boil for 2 hours, cool, stand overnight (12h), filter, concentrate the filtrate to 150ml under reduced pressure at 60°C, evaporate to dryness in a water bath, dry in a vacuum at 60°C, take out the powder and mix well to obtain a brown powder for detection of amino acids The content is 63.5%; add the same volume of purified water to dilute the non-permeate liquid from the microfiltration, stir and add absolute ethanol to 15% alcohol content, let it stand overnight (12h), centrifuge at 8000r/min, collect the supernatant, and continue to add Absolute ethanol until the alcohol content is 85%, leave it overnight, centrifuge at 8000r/min, collect the precipitate, then add 500ml of purified water to dissolve, add 200ml of ether to remove protein, separate the lower aqueous layer and organic phase with a separatory funnel, and recover the organic phase Solvent, the water layer was repeatedly deproteinized once, filtered, and the solvent was recovered under reduced pressure to 100ml, adsorbed on DEAE-cellulose anion exchange resin, first washed the resin column with purified water, then eluted with 0.1mol/L sodium chloride solution, and collected Sulfuric acid-anthrone reagent detected the eluate with a positive reaction, combined the eluate, passed through a 10 KD ultrafiltration membrane to remove small molecular substances, collected as the permeate, and dried the non-permeate in vacuum at low temperature to obtain Ganoderma lucidum polysaccharide. The detected polysaccharide content was 91.1%.

C. Dissolve the precipitate in A after centrifugation with 1% sodium hydroxide solution, and add the lye until the pH is 7.4, and the resulting solution is adsorbed by D311 anion exchange resin, first wash the resin column with purified water, and then use 3% hydrogen Sodium oxide ethanol solution (volume ratio 1:1) was used to elute, and the acetic anhydride-conc. Dry in vacuo to obtain a light yellow solid, add 15 times the amount of absolute ethanol to heat and dissolve completely, cool, crystallize, filter to obtain white needle-like crystals, dry in vacuo to obtain a white powdery solid, the total triterpene acid content of Ganoderma lucidum is 93.6 %.

The detection method involved in the embodiment of the present invention is as follows:

The content of amino acid is measured by UV-visible spectrophotometry, U-2000 type UV-visible spectrophotometer, the chromogenic agent is ninhydrin reagent, the reference substance is arginine, and the detection wavelength is 576nm; the content of Ganoderma lucidum polysaccharide is measured by UV-visible spectrophotometer Method, U-2000 UV-visible spectrophotometer, anthrone sulfate solution as chromogenic agent, detection wavelength is 625nm, reference substance is glucose; Determination of total triterpene acid content of Ganoderma lucidum by UV spectrophotometry, U-2000 UV-visible spectrophotometry Photometer, the reference substance is ganoderma acid B, and the detection wavelength is 526nm.

Example 2

A simultaneous extraction and purification method of Ganoderma lucidum polysaccharides, Ganoderma triterpene acids and amino acids is carried out according to the following steps:

A. Take Ganoderma lucidum fruiting bodies, crush through a 100-mesh sieve, weigh 1 kg, add 15 L of 0.5 mol/L sodium carbonate aqueous solution, and add 50 g of sodium borohydride at the same time, ultrasonically extract at 0-5 ° C for 3 hours, and centrifuge at 8000 r/min to obtain Centrifugate: use 1mol/L hydrochloric acid to adjust the pH to 3.1, let it stand overnight (8h), centrifuge at 8000r/min, collect the centrifuge supernatant and precipitate respectively, and set aside;

B. Take the supernatant and microfilter it through a 0.22um ceramic membrane. When the concentrated solution reaches 1 L, add an equal volume of purified water to continue the microfiltration, repeat the microfiltration once, and collect the filtrate and the unpermeated concentrated solution respectively; The filtrate is adsorbed by D001 type cation exchange resin, eluted with purified water and 0.5mol/L sodium hydroxide solution in sequence, and the eluate that is positive for ninhydrin in the elution process of alkaline solution is collected, and then used 1mol/L Adjust the pH to 4.8 with L hydrochloric acid, filter, concentrate the filtrate under reduced pressure at 60°C to 150ml, evaporate to dryness in a water bath, dry in a vacuum at 60°C, take it out and mix it to obtain a brown powder, the amino acid content of which is 61.2%. Dilute with an equal volume of purified water, stir and add absolute ethanol until the alcohol content is 20%, let stand overnight, centrifuge at 8000r/min, collect the supernatant, continue to add absolute ethanol until the alcohol content is 90%, let stand overnight (8h), centrifuge at 8000r/min, collect the precipitate, then add 500ml of purified water to dissolve, add 200ml of ether to remove protein, separate the lower water layer and the organic phase with a separatory funnel, recover the solvent from the organic phase, repeat the protein removal once in the water layer, and filter , the solvent was recovered under reduced pressure to 100ml, adsorbed on DEAE-cellulose anion exchange resin, the resin column was washed with purified water first, and then eluted with 0.3mol/L sodium chloride solution, and the sulfuric acid-anthrone reagent was collected for positive reaction. The eluents were combined, passed through a 10 KD ultrafiltration membrane to remove small molecular substances, collected as the permeate, and the non-permeate was vacuum-dried at low temperature to obtain Ganoderma lucidum polysaccharide. The detected polysaccharide content was 90.3%.

C. Dissolve the precipitate in A after centrifugation with 1% sodium hydroxide solution, and add the lye until the pH is 6.2, and the resulting solution is adsorbed by D941 anion exchange resin, first wash the resin column with purified water, and then use 8% carbonic acid Sodium ethanol solution (volume ratio 1:1) was used for elution, and the acetic anhydride-concentrated sulfuric acid reagent was collected for positive color development, and then the pH was adjusted to 4.2 with 1mol/L hydrochloric acid, and the solvent was recovered under reduced pressure, evaporated to dryness in a water bath, and vacuum Dry to obtain a light yellow solid, add 15 times the amount of absolute ethanol to heat and dissolve completely, cool, crystallize, filter to obtain white needle-shaped crystals, vacuum dry to obtain a white powdery solid, and the total triterpene acid content of Ganoderma lucidum is 94.3%. .

The detection method of active ingredient is the same as embodiment 1.

Example 3

A simultaneous extraction and purification method of Ganoderma lucidum polysaccharides, Ganoderma triterpene acids and amino acids is carried out according to the following steps:

A. Take Ganoderma lucidum fruiting bodies, crush them through a 150-mesh sieve, weigh 1 kg, add 15 L of 1mol/L sodium bicarbonate aqueous solution, and add 50 g of sodium sulfite at the same time, perform ultrasonic extraction at 0-5°C for 3 hours, and centrifuge at 8000r/min to obtain a centrate; Use 1mol/L hydrochloric acid to adjust the pH to 3.8, leave it overnight (10h), centrifuge at 8000r/min, collect the supernatant and precipitate respectively, and set aside;

B. Take the supernatant and microfilter it through a 0.8um ceramic membrane. When the concentrated solution reaches 1 L, add an equal volume of purified water to continue the microfiltration, repeat the microfiltration once, and collect the filtrate and the unpermeated concentrated solution respectively; The filtrate is adsorbed by D001 type cation exchange resin, eluted with purified water and 0.8mol/L sodium hydroxide solution in sequence, and the eluate that is positive for ninhydrin in the elution process of alkaline solution is collected, and then used 1mol/L Adjust the pH to 5.7 with L hydrochloric acid, filter, concentrate the filtrate under reduced pressure at 60°C to 150ml, evaporate to dryness in a water bath, dry in a vacuum at 60°C, take out the powder and mix well to obtain a brown powder, the amino acid content of which is 67.2%; Add an equal volume of purified water to dilute the liquid, stir and add absolute ethanol until the alcohol content is 10%, leave it overnight (10h), centrifuge at 8000r/min, collect the supernatant, continue to add absolute ethanol until the alcohol content is 90% , put it overnight, centrifuge at 8000r/min, collect the precipitate, then add 500ml of purified water to dissolve, add 200ml of petroleum ether to remove protein, separate the lower aqueous layer and organic phase with a separatory funnel, recover the solvent from the organic phase, repeat the protein removal once in the aqueous layer, Filtrate, recover the solvent to 100ml under reduced pressure, put it on 711 type anion exchange resin for adsorption, first wash the resin column with purified water, then use 0.8mol/L sodium chloride solution to elute, and collect the washed column with positive reaction detected by the sulfuric acid-anthrone reagent. Remove the liquid, combine the eluents, remove small molecular substances through a 10 KD ultrafiltration membrane, collect the permeate, and dry the non-permeate in a low-temperature vacuum to obtain Ganoderma lucidum polysaccharides. The detected polysaccharide content is 93.7%.

C. Dissolve the precipitate in A after centrifugation with 1% sodium hydroxide solution, and add the lye until the pH is 7.8, the resulting solution is absorbed by D941 anion exchange resin, first wash the resin column with purified water, and then use 10% carbonic acid Elute with sodium hydrogen ethanol solution (volume ratio 1:2), collect the acetic anhydride-conc. Vacuum-dried to obtain a light yellow solid, add 15 times the amount of absolute ethanol to heat and dissolve completely, cool, crystallize, filter to obtain white needle-shaped crystals, vacuum-dry to obtain a white powdery solid, and the total triterpene acid content of Ganoderma lucidum is 92.9% %.

The detection method of active ingredient is the same as embodiment 1.

Comparative example 1

According to the method of Example 1, without adding lye and reducing agent in step A, ultrasonic extraction is directly carried out at low temperature, the final amino acid content is 52.6%, the polysaccharide content of Ganoderma lucidum is 83.5%, and the total triterpene acid content of Ganoderma lucidum is 84.3% %.

Comparative example 2

According to the method of Example 2, no lye is added in step A, and a reducing agent is directly added at room temperature for ultrasonic extraction. The final amino acid content is 55.8%, the polysaccharide content of Ganoderma lucidum is 84.3%, and the total triterpene acid content of Ganoderma lucidum is 86.1% %.

Comparative example 3

According to the method of Example 3, add lye and reducing agent in step A to carry out ultrasonic extraction at room temperature, use 001×7 type cation exchange resin to adsorb the filtrate in step B, and use AB-8 macroporous in step C The total triterpene acids of the ganoderma lucidum are adsorbed and extracted by the adsorption resin, and the final amino acid content is 57.4%, the polysaccharide content of the ganoderma lucidum is 85.1%, and the total triterpene acid content of the ganoderma lucidum is 88.3%.

Claims (10)

1. a synchronous extraction and purification method of ganoderma polysaccharide, ganoderma triterpene acid and amino acid, is characterized in that, described method comprises the following steps: (1) Ganoderma lucidum fruiting bodies are crushed and sieved, adding lye and reducing agent for ultrasonic extraction at 0-5°C, centrifuging the extract to obtain centrifugate and medicinal residues; the reducing agent includes sodium borohydride, hydrogen sulfite One of sodium or sodium sulfite, the weight ratio of the reducing agent to the ganoderma lucidum fruiting body is 1:20, and the concentration of the lye is 0.3-1mol/L; (2) adjust the pH value of the centrifugate obtained in step (1) to 2-4, place it for 8-16 hours, centrifuge, and collect the centrifuge liquid and precipitate respectively; (3) Extraction and purification of amino acids and ganoderma polysaccharides: (a) filtering the centrifugal supernatant in step (2) through a ceramic microfiltration membrane, collecting the filtrate and the non-permeated liquid; (b) Adsorb the filtrate in step a by cation exchange resin, elute with purified water and lye successively, detect amino acids with ninhydrin chromogenic reaction, and collect eluate showing positive reaction during elution with lye , adjust the pH of the eluent to 4-7, heat and boil for 2 hours, cool, stand for 8-16 hours, filter, concentrate the filtrate under reduced pressure, dry the concentrate in vacuum, powder and mix to obtain brown powder amino acid; (c) Add absolute ethanol to the non-permeated liquid in step a until the alcohol content is 5-30%, ethanol precipitation, centrifuge, collect the centrifugate, then add absolute ethanol until the alcohol content is 50-95%, alcohol sink, centrifuge, and collect the precipitate; (d) remove the protein from the precipitate in step c using the organic reagent method, recover the residual organic reagent under reduced pressure, absorb the concentrated solution through an anion exchange resin, and sequentially elute with purified water and salt solution, and detect the polysaccharide with the sulfuric acid-anthrone method, Collect the eluate with positive reaction; (e) Combine the eluents in step d, purify by ultrafiltration, collect the non-permeate liquid, and dry the non-permeate liquid under low-temperature vacuum to obtain the off-white spongy dried Ganoderma lucidum polysaccharide; (4) Step ( 2) Dissolve the precipitate in lye, adjust the pH to 6-8, apply anion exchange resin, elute with weak base ethanol, collect the eluate that is positive in color by acetic anhydride-concentrated sulfuric acid, and adjust the pH of the eluate To 2-6, the solvent was recovered under reduced pressure, dried under vacuum, and turned into a light yellow solid, which was dissolved by heating with ethanol, cooled and recrystallized to obtain white needle crystals, dried under vacuum, and the total triterpene acids of Ganoderma lucidum were obtained. 2. the synchronous extraction and purification method of Ganoderma lucidum polysaccharide, Ganoderma triterpene acid and amino acid according to claim 1, is characterized in that, lye described in step (1) comprises sodium hydroxide solution or sodium carbonate solution or sodium bicarbonate solution. 3. The synchronous extraction and purification method of Ganoderma lucidum polysaccharide, Ganoderma triterpene acid and amino acid according to claim 1, characterized in that, the Ganoderma lucidum fruiting body described in step (1) is crushed and passed through a 100-mesh sieve. 4. The synchronous extraction and purification method of Ganoderma lucidum polysaccharide, Ganoderma triterpene acid and amino acid according to claim 1, characterized in that the pore size of the ceramic microfiltration membrane in step a is 0.22um or 0.45um or 0.8um. 5. the synchronous extraction and purification method of Ganoderma lucidum polysaccharide, Ganoderma triterpene acid and amino acid according to claim 1, is characterized in that, the cation exchange resin described in the step b is 732 type cation exchange resin or D001 type cation exchange resin, the The lye is 0-1mol/L ammonia water or 0-1mol/L sodium hydroxide aqueous solution, and the acid solution used for pH adjustment is 0-5mol/L hydrochloric acid aqueous solution or 0-5mol/L sulfuric acid aqueous solution. 6. The synchronous extraction and purification method of Ganoderma lucidum polysaccharide, Ganoderma triterpene acid and amino acid according to claim 1, characterized in that, the organic reagent used in step d to remove protein is ether or petroleum ether. 7. The synchronous extraction and purification method of Ganoderma lucidum polysaccharide, Ganoderma triterpene acid and amino acid according to claim 1, characterized in that the anion exchange resin described in step d is 717 type anion exchange resin or DEAE cellulose anion exchange resin. 8. The synchronous extraction and purification method of Ganoderma lucidum polysaccharide, Ganoderma triterpene acid and amino acid according to claim 1, characterized in that the salt solution in step d is 0-1mol/L sodium chloride solution. 9. the synchronous extraction and purification method of Ganoderma lucidum polysaccharide, Ganoderma triterpene acid and amino acid according to claim 1, is characterized in that, the anion exchange resin described in step (4) is D311 type weakly basic anion exchange resin or D941 type Weakly basic anion exchange resin. 10. the synchronous extraction and purification method of Ganoderma lucidum polysaccharide, Ganoderma triterpene acid and amino acid according to claim 1, is characterized in that, the mass concentration of weak base in the weak base ethanol liquid described in step (4) is 1-10% , the weak base is NaOH or Na ₂ CO ₃ or NaHCO ₃ , and the volume ratio of the weak base to ethanol is 1:1-5.