CN107158044B - Integrated extraction method of ganoderma lucidum fluid extract - Google Patents
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- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 3
- 210000003056 antler Anatomy 0.000 abstract description 14
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
- A61K36/074—Ganoderma
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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Abstract
The invention discloses an integrated extraction method of ganoderma lucidum fluid extract, which takes grass, antler and ganoderma lucidum as raw materials, adopts a complex enzymolysis method and an ultrasonic method to extract, and obtains the ganoderma lucidum fluid extract; wherein, the complex enzyme is 2wt% of cellulase, 0.8wt% of papain and 3wt% of pectinase, and the enzymolysis conditions are as follows: the enzymolysis temperature is 60 ℃, the pH value is 5.5, the enzymolysis time is 80min, and the ultrasonic treatment conditions are as follows: the ultrasonic power is 300W, the temperature is 35 ℃, and the ultrasonic time is 15 min. Compared with the traditional hydrothermal extraction method, the yield of the fluid extract extracted by adopting the compound enzymolysis synergistic ultrasonic method can be improved by 16.71 times, and the extraction time can be effectively shortened.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to an integrated extraction method of a Ganoderma lucidum fluid extract.
Background
The ganoderma lucidum is actually 'the medicine for the first time' recorded in the Shennong Ben Cao Jing, is regarded as a symbol of good luck and longevity and enjoys the reputation of 'immortal grass'. The Ganoderma lucidum with grass of Juncao antler has 3% of effective components higher than that of the common Ganoderma lucidum, has high safety, and has health promotion effects of improving immunity, improving sleep, resisting tumor, protecting liver, and adjuvant treating arthritis. It has been isolated from ganoderma lucidum: triterpenes, polysaccharides, nucleosides, sterols, alkaloids, furan derivatives, amino acids, polypeptides, inorganic elements, fatty acids, etc. The polysaccharide compound is an important chemical component contained in the ganoderma lucidum, is one of main effective components of the ganoderma lucidum, and is concerned by people due to the effects of resisting tumors, regulating immunity, resisting oxidation, resisting aging and the like. Modern scientific research shows that the triterpenes in the ganoderma lucidum is one of the main active ingredients, and has various pharmacological actions of resisting tumor, protecting liver, resisting oxidation, inhibiting platelet aggregation, resisting fungi and the like.
Cutting Ganoderma, extracting according to certain process parameters, concentrating, and evaporating to obtain dark brown paste called Ganoderma fluid extract. The glossy ganoderma extract is first prepared for producing glossy ganoderma tablet, capsule, granule or syrup, and the quality of glossy ganoderma extract plays a significant role in the forming process of glossy ganoderma preparation and the quality of finished product of preparation. By improving the fluid extract extraction method, the extraction rate of effective components in the fluid extract is improved, the production efficiency of the ganoderma lucidum fluid extract in the industry can be effectively improved, the production cost is reduced, and the quality and the stability of the medicament are improved, so that the modernization process of ganoderma lucidum products is accelerated.
Many researches on the field of cut-log lucid ganoderma exist, a large amount of documents can be found, and the technology and the method are relatively mature, but the researches on the grass-cultivated lucid ganoderma are not many, but the grass-cultivated lucid ganoderma is gradually popularized nationwide due to the advantages of low production cost, good finished product quality and the like, and the grass-cultivated lucid ganoderma is an important variety in the field of lucid ganoderma research. Particularly, the current research on the ganoderma lucidum fluid extract is blank, so the research on the ganoderma lucidum fluid extract is particularly significant.
The invention selects the grass-cultivated antler lucid ganoderma as the object of enzyme extraction, and the extraction efficiency of the effective components of the lucid ganoderma is improved by destroying the cell wall structure of the lucid ganoderma by complex enzyme; in the extraction process, the strong vibration, stirring action and strong cavitation effect generated by ultrasonic waves are utilized, so that the entry of active ingredients of substances into a solvent can be accelerated, the full contact between enzyme and an effective extract is improved, the extraction time is shortened, the extraction rate is improved, and a theoretical basis is provided for the improvement of a process for producing the ganoderma lucidum fluid extract. And provides basis for the application of the complex enzyme and ultrasonic wave synergistic technology in the aspect of traditional Chinese medicine preparation, and is favorable for better controlling the yield and quality of the ganoderma lucidum fluid extract.
Disclosure of Invention
The invention aims to provide an integrated extraction method of ganoderma lucidum fluid extract, which can obviously improve the yield of the ganoderma lucidum fluid extract and effectively shorten the extraction time.
In order to achieve the purpose, the invention adopts the following technical scheme:
an integrated extraction method of ganoderma lucidum fluid extract is characterized in that grass, antler and ganoderma lucidum are used as raw materials, and a complex enzymolysis method and an ultrasonic method are adopted for extraction to obtain the ganoderma lucidum fluid extract. The method specifically comprises the following steps:
1) cutting Ganoderma into pieces, oven drying at 65 deg.C, pulverizing, and sieving to obtain Ganoderma powder with fineness of 80 mesh;
2) adding acetic acid-sodium acetate buffer solution and complex enzyme into the obtained Ganoderma powder for enzymolysis;
3) after enzymolysis, placing the mixture in a water bath at 100 ℃ for 10min to inactivate the enzyme, cooling to room temperature, and performing ultrasonic extraction;
4) filtering the obtained extract, concentrating under reduced pressure, and freeze drying to obtain the Ganoderma fluid extract.
The adding amount of the complex enzyme in the step 2) is 2wt% of cellulase, 0.8wt% of papain and 3wt% of pectinase; the conditions of the compound enzymolysis are as follows: the enzymolysis temperature is 60 deg.C, pH is 5.5, and the enzymolysis time is 80 min.
The ultrasonic extraction conditions in the step 3) are as follows: the ultrasonic power is 300W, the temperature is 35 ℃, and the ultrasonic time is 15 min.
The invention has the following remarkable advantages: the yield of the fluid extract of the grass-cultivated antler ganoderma lucidum of the traditional hydrothermal method is 2.71 percent, while the yield of the fluid extract of the grass-cultivated antler ganoderma lucidum of the invention of the compound enzymolysis synergistic ultrasonic method is 45.29 percent, namely, the fluid extract yield of the grass-cultivated antler ganoderma lucidum of the invention is improved by 16.71 times compared with the conventional method, and the extraction time can be effectively shortened. And the extraction process does not need high temperature, the damage of the high temperature to the chemical molecular structure of ganoderma lucidum polysaccharide and the like is avoided, and the removal capacity, the reducing power and the ORAC value of polysaccharide content and DPPH free radical are obviously higher than those of the fluid extract obtained by the conventional method.
Detailed Description
1. Materials and methods
1.1 materials
1.1.1 subjects
Grass of fungus, antler and glossy ganoderma, purchased from Fujian agriculture and forestry university.
1.1.2 Main Instrument
Ultracentrifugal grinders (RetschZM 200, germany); a numerical control ultrasonic cleaner (KQ-500 DE type, ultrasonic instruments ltd of Kunshan city), an ultraviolet visible spectrophotometer (Beijing general analytical instrument ltd), a pH meter (Reye PHS-3C, made by Xiamen Jing technology ltd, Fuzhou), a magnetic stirrer (IKA-Werke GmbH and Co. KG), a freeze dryer (Beijing Bo Yi kang experimental instrument ltd), a constant temperature water bath (Shanghai Yangrong biochemical instrument factory), a rotary evaporator RE52CS (Shanghai Yangrong biochemical instrument factory), a SHZ-D (III) circulating water type vacuum pump (KQ-500 DE type, digital display constant temperature water bath HH-6 (national Hua electrical instruments ltd).
1.1.3 Primary reagents
Cellulase 400 (mu/mg), papain (1000 mu/mg), pectinase (500 mu/mg), acetic acid, sodium acetate, disodium hydrogen phosphate, and sodium dihydrogen phosphate.
1.2 methods
1.2.1 extraction of Ganoderma lucidum fluid extract
Cutting the fruiting body of Ganoderma lucidum into pieces, oven drying at 65 deg.C, pulverizing, sieving with 80 mesh sieve, weighing 5.0 g of the obtained Ganoderma powder, adding 100.0 mL of buffer solution with certain pH (pH of 4.6-5.5 in acetic acid-sodium acetate buffer solution), adding appropriate amount of enzyme, mixing, and performing enzymolysis at constant temperature. After the reaction is finished, placing the reaction product in a water bath at 100 ℃ for 10min to inactivate the enzyme, then cooling to room temperature, filtering the extracting solution, concentrating the extracting solution to about 10mL by a rotary evaporator under reduced pressure, freeze-drying the extracting solution to constant weight, and calculating the extraction rate of the obtained ganoderma lucidum fluid extract. The calculation method is as follows:
extraction rate/% = of ganoderma lucidum fluid extract
×100%。
1.2.2 Complex enzyme dosage ratio orthogonal test design
Under the enzymolysis conditions of 60min, 50 deg.C and pH5, the dosage of cellulase, papain and pectinase are used as orthogonal test3 factors of experiment. Each factor was set to 4 levels, and L was performed16(43) Orthogonal test (table 1).
TABLE 1 Complex enzyme dosage ratio orthogonal test factor horizon
1.2.3 orthogonal experimental design of enzymolysis condition of complex enzyme
According to the test result of the optimal compound enzyme dosage ratio, the temperature, the pH value and the enzyme extraction time are taken as investigation factors, each factor is set to 3 levels, and L9 (3) is carried out3) Orthogonal test (table 2).
TABLE 2 Complex enzyme enzymolysis condition orthogonal test factor horizon
1.2.4 ultrasonic condition orthogonal experimental design
According to the results of the optimal compound enzyme dosage ratio test and the optimal enzymolysis condition test, taking time, temperature and power as investigation factors, setting 3 levels for each factor, and carrying out L9（33) Orthogonal test (table 3).
TABLE 3 ultrasonic condition orthogonal experiment factor horizon
1.2.5 comparison of Complex enzyme-ultrasonic synergistic extraction method with conventional Water bath extraction method
Weighing 8 parts of 5.0 g of ganoderma lucidum powder, wherein 4 parts of ganoderma lucidum powder are extracted according to the optimal process conditions of complex enzyme synergistic ultrasonic extraction, and adding 100mL of 40% ethanol solution into the other 4 parts of ganoderma lucidum powder respectively, and leaching in a water bath at 60 ℃ for 80 min.
2 results and analysis
2.1.1 Complex enzyme dosage ratio orthogonal test
TABLE 4 Quadrature test results for complex enzyme dosage ratio
It can be seen from the results of the orthogonal tests obtained in table 4 that K4 of the factor a is higher than K1, K2 and K3, it can be seen that the level of 4 of the factor a is better than other levels, K4 of the factor B is higher than K1, K2 and K3, it can be seen that the level of 4 of the factor B is better than other levels, K3 of the factor C is higher than K1, K2 and K4, and it can be seen that the level of 3 of the factor C is better than other levels. Therefore, the compound enzyme dosage ratio of the fungus grass antler ganoderma lucidum fluid extract is determined to be A4B4C3, namely 2.0 percent of cellulase (A), 0.8 percent of papain (B) and 3.0 percent of pectinase (C), and the average extraction rate of the ganoderma lucidum fluid extract is 56.81 percent under the enzymolysis conditions of 60min, 50 ℃ and pH value of 4.6.
The range R reflects the change amplitude of the K value when the same factor is at different levels, and is the difference between the maximum value and the minimum value corresponding to different levels under the same factor. The larger the variation range is, the more important the factor has influence on the test result. The worst R values of cellulase (a), papain (B), and pectinase (C) were 17.64, 15.84, and 13.45, respectively, from which it was deduced that cellulase (a) had the greatest effect on the results and pectinase (C) had the least effect on the results. The three have the effects of cellulase (A), papain (B) and pectinase (C) from large to small, wherein the cellulase (A) is the most important and the pectinase (C) is the least important. According to visual analysis data, the G value of the factor A, B, C is larger than 1.5, namely all the factors are main factors, different levels are selected to have larger influence on the extraction of the grass-cultivated ganoderma lucidum fluid extract, and the influence degree is A, B, C in sequence.
In order to further judge whether the test error and the test condition influence the test result, the orthogonal test data is subjected to variance analysis and significance test to find out the main factors playing a leading role in the factors. From the results of the analysis of the variance in the orthogonal experiments in table 5, it can be seen that the cellulase (a), the papain (B) and the pectinase (C) have significant effects on the extraction rate of the Ganoderma lucidum karst extract, i.e., the cellulase (a), the papain (B) and the pectinase (C) are all main effective factors.
TABLE 5 analysis of variance in the complex enzyme dosage ratio test
2.1.2 Experimental validation of extraction Process
The optimal combination A4B4C3, derived from the orthogonal experiments, was repeated three times. The result shows that the average value of the extraction rate of the ganoderma lucidum fluid extract is 56.81%, and RSD =6.65%, which is the maximum value.
2.2 Complex enzyme enzymolysis Condition orthogonal test
TABLE 6 orthogonal test results of complex enzyme enzymolysis conditions
It can be seen from the results of the orthogonal test in table 6 that k3 of the factor a is higher than k1 and k2, it is known that the level of 3 of the factor a is better than other levels, k2 of the factor B is higher than k1 and k3, it is known that the level of 2 of the factor B is better than other levels, k2 of the factor C is higher than k1 and k3, and it is deduced that the level of 2 of the factor C is better than other levels. Therefore, the enzymolysis condition of the grass-cultivated antler ganoderma lucidum fluid extract is determined to be A3B2C2, namely the enzymolysis temperature (A) is 60 ℃, the pH value (B) is 5.5, and the time (C) is 80 minutes. The R values at temperature (a), ph (b) and time (C) were 3.01, 3.30 and 11.83, respectively, and it was found that the effect of time (C) on the results was the largest and the effect of temperature (a) on the results was the smallest. The factor primary and secondary arrangement sequence is C > B > C, namely the most important factor time is 80min (C), pH5.5 (B), and temperature is 40 ℃ (A). From visual analysis data, the G value of the factor A, B is less than 1.5, namely the temperature (A) and pH (B) factors are secondary factors, and the G value of the time (C) is more than 1.5, namely the time (C) factor is an important factor.
In order to further judge whether the test error and the test condition influence the test result, the orthogonal test data is subjected to variance analysis and significance test to find out the main factors playing a leading role in the factors. From the results of the analysis of variance in the orthogonal experiment in table 7, it can be seen that the temperature (a), the ph (b) and the time (C) have no significant influence on the extraction rate of the Ganoderma lucidum karst extract, and the temperature (a), the ph (b) and the time (C) are not main effect factors.
TABLE 7 results of analysis of variance under the Complex enzyme enzymolysis conditions
2.3 ultrasonic Condition orthogonal experiment
TABLE 8 results of ultrasonic condition orthogonal experiments
It can be intuitively seen from the orthogonal experimental results in table 8 that k2 of the factor a is higher than k1 and k3, it can be known that the level of 2 of the factor a is better than other levels, k3 of the factor B is higher than k1 and k2, the level of 3 of the factor B is better than other levels, k1 of the factor C is higher than k2 and k3, and the level of 1 of the derivative factor C is better than other levels. Therefore, the ultrasonic condition of the fluid extract of the grass, antler and ganoderma lucidum is determined to be A2B3C1, namely the ultrasonic time (A) is 15min, the power (B) is 500W, and the temperature is 35 ℃. From the range analysis, the factor primary and secondary arrangement order is C > A > B, namely the most important factor is temperature (C), and the second is time (A) and power (B). From visual analysis data, the G value of the factor A, B, C is less than 1.5, namely, the time (A), power (B) and temperature (C) factors are all secondary factors.
In order to further judge whether the test error and the test condition influence the test result, the orthogonal test is subjected to variance analysis to find out the main factors playing a leading role in all the factors. As can be seen from the results of the ANOVA in Table 9, the significant level of A, B, C is greater than 0.05, which indicates that the time (A), the power (B) and the temperature (C) have no significant effect on the extraction rate of the Ganoderma lucidum karst fluid extract, i.e. the time (A), the power (B) and the temperature (C) are not the main effect factors (P is greater than 0.05) affecting the extraction rate of the Ganoderma lucidum karst fluid extract.
TABLE 9 experimental results of variance of ultrasonic testing
2.4 Complex enzyme-ultrasonic synergic extraction method compared with traditional water bath extraction method
In order to investigate the influence of the complex enzyme-ultrasonic synergistic extraction method and the traditional hydrothermal extraction method on the extraction rate of the ganoderma lucidum fluid extract, 8 parts of 5.0 g of ganoderma lucidum powder are weighed, wherein 4 parts of ganoderma lucidum powder are extracted according to the optimal process conditions of complex enzyme-ultrasonic synergistic extraction, and 100mL of water is respectively added into the other 4 parts of ganoderma lucidum powder for hydrothermal extraction at 92 ℃ for 11 hours.
The result shows that the average yield of the fluid extract of the grass antler ganoderma lucidum extracted by the traditional hydrothermal method is 2.71 percent, wherein the average content of polysaccharide is 0.23g, while the average yield of the fluid extract of the grass antler ganoderma lucidum extracted by the complex enzyme synergistic ultrasonic method is 45.29 percent, wherein the average content of polysaccharide is 2.95g, namely the yield of the fluid extract of the grass antler ganoderma lucidum extracted by the complex enzyme synergistic ultrasonic method is 16.71 times that extracted by the traditional hydrothermal method, and the polysaccharide content in the fluid extract is 12.83 times that extracted by the traditional hydrothermal method. This is probably because the ultrasonic-assisted extraction technology, as a novel separation technology, has a stirring effect, a strong resonance effect, a cavitation effect due to high acceleration, and the like, and enhances the mass transfer process, so that the effective components enter the solvent more quickly, the extraction efficiency is improved, and the activity of the organic components is not weakened due to the generation of high temperature. Meanwhile, the ganoderma lucidum has hard cell texture, thicker cell wall and larger mass transfer resistance in the extraction process, and the mass transfer process of the effective components is enhanced by destroying the cell structure through enzymolysis.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (1)
1. An integrated extraction method of ganoderma lucidum fluid extract is characterized by comprising the following steps: extracting with Ganoderma lucidum karst and Ganoderma amboinense as raw materials by complex enzymolysis in combination with ultrasonic method to obtain Ganoderma fluid extract; the method specifically comprises the following steps:
1) cutting Ganoderma into pieces, oven drying at 65 deg.C, pulverizing, and sieving to obtain Ganoderma powder with fineness of 80 mesh;
2) adding acetic acid-sodium acetate buffer solution and complex enzyme into the obtained Ganoderma powder for enzymolysis;
3) after enzymolysis, placing the mixture in a water bath at 100 ℃ for 10min to inactivate the enzyme, cooling to room temperature, and performing ultrasonic extraction;
4) filtering the obtained extract, concentrating under reduced pressure, and freeze drying to obtain Ganoderma fluid extract;
the composition and the dosage of the complex enzyme are 2wt% of cellulase, 0.8wt% of papain and 3wt% of pectinase;
the enzymolysis temperature is 60 ℃, the pH value is 5.5, and the enzymolysis time is 80 min;
the ultrasonic method comprises the following treatment conditions: the ultrasonic power is 300W, the temperature is 35 ℃, and the ultrasonic time is 15 min.
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