CN105641001B - Ganoderma lucidum amino acid extract and preparation method and application thereof - Google Patents
Ganoderma lucidum amino acid extract and preparation method and application thereof Download PDFInfo
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- CN105641001B CN105641001B CN201610041216.1A CN201610041216A CN105641001B CN 105641001 B CN105641001 B CN 105641001B CN 201610041216 A CN201610041216 A CN 201610041216A CN 105641001 B CN105641001 B CN 105641001B
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- amino acid
- glossy ganoderma
- extract
- ganoderma
- ganoderma lucidum
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- 150000001413 amino acids Chemical class 0.000 title claims abstract description 156
- 239000000284 extract Substances 0.000 title claims abstract description 80
- 240000008397 Ganoderma lucidum Species 0.000 title claims abstract description 34
- 235000001637 Ganoderma lucidum Nutrition 0.000 title claims abstract description 34
- 238000000605 extraction Methods 0.000 title abstract description 33
- 238000002360 preparation method Methods 0.000 title abstract description 12
- 241000222336 Ganoderma Species 0.000 claims abstract description 131
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 74
- 239000007787 solid Substances 0.000 claims abstract description 30
- 239000000843 powder Substances 0.000 claims abstract description 20
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000000047 product Substances 0.000 claims abstract description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000000287 crude extract Substances 0.000 claims abstract description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000000706 filtrate Substances 0.000 claims abstract description 10
- 239000003814 drug Substances 0.000 claims abstract description 9
- 238000001035 drying Methods 0.000 claims abstract description 8
- 238000010992 reflux Methods 0.000 claims abstract description 8
- 229940079593 drug Drugs 0.000 claims abstract description 7
- 238000000967 suction filtration Methods 0.000 claims abstract 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 27
- 229910052799 carbon Inorganic materials 0.000 claims description 15
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- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 26
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical group OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 8
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 8
- IFBHRQDFSNCLOZ-IIRVCBMXSA-N 4-nitrophenyl-α-d-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC1=CC=C([N+]([O-])=O)C=C1 IFBHRQDFSNCLOZ-IIRVCBMXSA-N 0.000 description 7
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- 229920001282 polysaccharide Polymers 0.000 description 2
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- 235000019353 potassium silicate Nutrition 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000005211 surface analysis Methods 0.000 description 2
- 150000003648 triterpenes Chemical class 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
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- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
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- 244000131316 Panax pseudoginseng Species 0.000 description 1
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- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 241000222341 Polyporaceae Species 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
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- 230000001093 anti-cancer Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
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- 150000004665 fatty acids Chemical class 0.000 description 1
- 150000002240 furans Chemical class 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
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- 229940125396 insulin Drugs 0.000 description 1
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- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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- 201000003068 rheumatic fever Diseases 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
-
- 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
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
Landscapes
- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Mycology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Plant Substances (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention provides a ganoderma lucidum amino acid extract which is prepared by the following method: taking ganoderma lucidum powder, and mixing the ganoderma lucidum powder with the liquid material ratio of 10-30: adding 1 into a hydrochloric acid solution of 4-10 mol/L, heating and refluxing for 4-12 h, performing suction filtration to obtain a hydrolysate, evaporating the hydrolysate under reduced pressure, dissolving the residue in water, and adjusting the pH to 2-4 to obtain a crude extract; decolorizing the crude extract with activated carbon, filtering out the activated carbon, concentrating the filtrate under reduced pressure, adding absolute ethanol, adjusting the pH to 2-4, standing, performing centrifugal separation, collecting solid precipitate, and drying to constant weight to obtain the ganoderma amino acid extract; the invention discloses an extraction process of ganoderma lucidum amino acid for the first time, and the obtained ganoderma lucidum amino acid extract can be applied to the preparation of health-care products, medicines and the like for reducing blood sugar and resisting oxidation; the method has the advantages of short process flow, simple method, convenient operation and good economic and social benefits.
Description
(1) technical field
The present invention relates to a kind of Amino Acid of Glossy ganoderma extract and preparation method thereof, the Amino Acid of Glossy ganoderma extract can be applied
In preparing hypoglycemic and anti-oxidant relevant healthcare product and drug.
(2) background technique
Ganoderma lucidum (Ganoderma Lucidum Karst) is the drying fructification of On Polyporaceae red sesame or purple sesame, is
The traditional medicinal fungi in China, is recorded according to Shennong's Herbal, and ganoderma lucidum has tonifying middle-Jiao and Qi, strengthening by means of tonics, strengthens the body resistance to consolidate the constitution, macrobiosis
It lengthens one's life and other effects, and ganoderma lucidum is also listed in top grade in Compendium of Material Medica, recognizes ganoderma lucidum for the first time in 2000 version " Chinese Pharmacopoeias "
Medical value, as the legal Chinese medicine in China.Modern medicine proves that ganoderma lucidum contains several physiological active substances, including polysaccharide
Class, triterpenes, sterols, alkaloids, furan derivatives, amino polypeptide, inorganic elements, fatty acid etc..Ganoderma lucidum can be adjusted
It saves, enhance human immunity, have to neurasthenia, rheumatic arthritis, coronary heart disease, hypertension, hepatitis, diabetes, tumour etc.
Good synergistic therapeutic action.China consumer there's a widespread conviction that ganoderma lucidum class health care product has anticancer, improves the function such as immunity at hypoglycemic
Effect, national about 2,000,000,000 yuans of total sales volume of ganoderma lucidum class health product in 2014, growth momentum is preferable.
Contain 18 kinds of amino acid in ganoderma lucidum, wherein the relative amount of essential amino acid is higher than 50%, more edible than generally
Bacterium is high by 40%.Pertinent literature reports the amino acid content of five kinds of ganoderma lucidum finished products of In The Taishan Area, destroys except tryptophan is hydrolyzed
And outside being not detected, other 17 kinds of amino acid contents are abundant, and the total content of amino acid is up to 12.87%, and contain 7 kinds of human bodies
Essential amino acid is worth deep development to utilize.Amino acid is the basic unit that protein molecule is constituted in organism, with biology
Vital movement has close relationship, is one of indispensable nutritional ingredient in organism.Human body lacks any required
Amino acid, so that it may cause physiological function abnormal, influence being normally carried out for antibody metabolism, finally result in disease.Equally, if human body
It is interior to lack certain nonessential amino acid, antibody dysbolism can be generated.Such as arginine and citrulling are very heavy to urea is formed
It wants；Cystine insufficiency of intake can cause insulin to reduce, blood glucose rise.It is reported at present about the research of ganoderma lucidum chemical composition analysis
Polysaccharide and triterpenes components are biased toward, the research report isolated to Amino Acid of Glossy ganoderma and its extraction is less.
It is relatively more to extract the condition that Amino Acid of Glossy ganoderma needs to optimize for ganoderma lucidum complex chemical composition, so the present invention selects to ring
Answer face method.Response phase method utilizes reasonable test design method, and obtains certain data by test, using polynary secondary returning
Return the functional relation between equation model factor and response, optimal procedure parameters are sought by the analysis to regression equation, are
Solve a kind of statistical method of Multivariable.The main advantage of response surface is that test number (TN) can be reduced, and evaluates multiple ginsengs
Several interaction relationships.
The present invention extracts Amino Acid of Glossy ganoderma using ganoderma lucidum as raw material, with Hydrochloric Acid Hydrolysis Method, and is optimized by response phase method and extracted
The technique of the liquid-to-solid ratio of Amino Acid of Glossy ganoderma, extraction time and concentration of hydrochloric acid, and pass through active carbon decoloring, ethanol precipitation, centrifugation
The Amino Acid of Glossy ganoderma extract purified is dried, provides value guidance for the industrialized production of Amino Acid of Glossy ganoderma.The present invention is same
When also found the Amino Acid of Glossy ganoderma extract have hypoglycemic and oxidation resistance, be Amino Acid of Glossy ganoderma in health care product and pharmaceutical preparations
Application provide scientific basis.
(3) summary of the invention
The present invention provides a kind of Amino Acid of Glossy ganoderma extract and preparation method thereof, the Amino Acid of Glossy ganoderma extract tool
There is hypoglycemic and oxidation resistance, can be applied to prepare hypoglycemic and anti-oxidant relevant healthcare product and drug.
The present invention optimizes the method for extracting Amino Acid of Glossy ganoderma by response phase method, using design expert software, according to
On liquid-to-solid ratio, extraction time and concentration of hydrochloric acid, these influence the factor that Amino Acid of Glossy ganoderma extracts to Box-Behnken design principle
It optimizes, to improve the recovery rate of Amino Acid of Glossy ganoderma, shortens extraction time, reduce energy consumption and product cost.
The present invention adopts the following technical scheme:
A kind of Amino Acid of Glossy ganoderma extract, the Amino Acid of Glossy ganoderma extract are prepared as follows to obtain:
(1) sour water solution: taking ganoderma powder, is that 4~10mol/L hydrochloric acid solution is added in 10~30:1 (mL/g) by liquid-to-solid ratio
In, be heated to reflux 4~12h, filter to obtain hydrolyzate, by gained hydrolyzate evaporated under reduced pressure, residue is dissolved in water, and adjust pH to
2~4, obtain crude extract；
(2) it purifies: active carbon is added into crude extract obtained by step (1), decolourize 15~55min at 50~90 DEG C, takes out
Deactivation charcoal is filtered out, is added dehydrated alcohol after filtrate decompression is concentrated, adjusts pH to 2~4, stand 20 in 2~6 DEG C~for 24 hours, it
10~15min is centrifugated under the revolving speed of 2000~3000r/min afterwards, removes supernatant liquor, collects solid precipitating, in 50~
Freeze-day with constant temperature obtains the Amino Acid of Glossy ganoderma extract to constant weight in 70 DEG C of baking ovens.
The present invention optimizes the method for extracting Amino Acid of Glossy ganoderma by response phase method, using design expert software, according to
On liquid-to-solid ratio, extraction time and concentration of hydrochloric acid, these influence the factor that Amino Acid of Glossy ganoderma extracts to Box-Behnken design principle
It optimizes, obtains in the step (1), acid-hydrolyzed optimal conditions are as follows: liquid-to-solid ratio is 20:1 (mL/g), concentration of hydrochloric acid solution
For 7.0mol/L, being heated to reflux the time is 9.5h.
In step (1), the ganoderma powder is that ganoderma lucidum is placed in at 60~80 DEG C drying to constant weight, is crushed to 5~10
Mesh obtains.
In step (1), the temperature being heated to reflux is usually 100~110 DEG C.
In step (1), the residue is recommended to add the water dissolution of 6~9 mass times.
In step (2), recommend the active carbon quality dosage be step (1) described in ganoderma powder quality 5%~
25%, preferably 15%.The optimal conditions of the activity carbon decoloring are as follows: decolourize 45min at 80 DEG C.
In step (2), the filtrate decompression is recommended to be concentrated into the 1/6~1/10 of initial volume, can be observed have crystal analysis
Out.The dehydrated alcohol of 1~3 times of volume of addition in the filtrate of recommendation after concentration, preferably 2 times.
In step (1) or step (2), the adjusting of pH is usually using sodium hydroxide and/or hydrochloric acid, preferably adjusting pH to 3.
The present invention is using PNPG as substrate, using 96 orifice plates as reaction carriers, measures the Amino Acid of Glossy ganoderma extract to α-
The inhibiting rate of glucuroide, it is demonstrated experimentally that Amino Acid of Glossy ganoderma extract of the present invention is preparing health-caring product capable of reducing blood sugar and drug side
Face has application prospect.
The present invention is using DPPH as substrate, using 96 orifice plates as reaction carriers, measures the Amino Acid of Glossy ganoderma extract pair
The residual rate of DPPH free radical after the Scavenging activity and addition Amino Acid of Glossy ganoderma extract of DPPH free radical, it is demonstrated experimentally that this hair
Bright Amino Acid of Glossy ganoderma extract equally has application prospect preparing antioxidant health-care product and pharmaceutical preparations.
Present invention beneficial effect outstanding and advantage are:
1, Box-Behnken (BBD) center combination design model that the present invention uses is in response to face method, compared with Orthogonal Method
This method can reduce test number (TN), while evaluate the interaction relationship between multiple parameters；It is obtained by response phase method optimal
Extraction conditions: liquid-to-solid ratio 20:1, extraction time 9.5h, concentration of hydrochloric acid 7.0moL/L；The ganoderma lucidum amino that final gained purifies
Its amino acid content of sour extract is up to 17.78%；
2, the extraction process of the first public Amino Acid of Glossy ganoderma of the present invention, and gained Amino Acid of Glossy ganoderma extract can be applied to make
It makes preparation for dropping blood glucose and oxidation-resisting health-care product and drug etc.；Present invention process process is short, and method is simple, and it is convenient to operate, and has preferable
Economic benefit and social benefit, for the health care product and drug of Amino Acid of Glossy ganoderma exploitation provide certain theory and practice according to
According to.
(4) Detailed description of the invention
Fig. 1 a is the canonical plotting that ninhydrin colorimetry measures amino acid content in embodiment 1；
Fig. 1 b is liquid-to-solid ratio in embodiment 1 to the influence diagram of Amino Acid of Glossy ganoderma yield；
Fig. 1 c is extraction time in embodiment 1 to the influence diagram of Amino Acid of Glossy ganoderma yield；
Fig. 1 d is concentration of hydrochloric acid in embodiment 1 to the influence diagram of Amino Acid of Glossy ganoderma yield；
Fig. 2 a is that the response surface that liquid-to-solid ratio and extraction time extract yield influence to Amino Acid of Glossy ganoderma in embodiment 1 is three-dimensional
Figure；
Fig. 2 b is that liquid-to-solid ratio and concentration of hydrochloric acid extract the response surface three-dimensional that yield influences on Amino Acid of Glossy ganoderma in embodiment 1
Figure；
Fig. 2 c is that extraction time and concentration of hydrochloric acid extract the response surface three-dimensional that yield influences on Amino Acid of Glossy ganoderma in embodiment 1
Figure；
Fig. 3 a is Amino Acid of Glossy ganoderma extract in embodiment 5 to the inhibiting rate figure of alpha-glucosidase；
Fig. 3 b is acarbose in embodiment 5 to the inhibiting rate figure of alpha-glucosidase；
Fig. 4 a is Amino Acid of Glossy ganoderma extract in embodiment 5 to the Scavenging activity figure of DPPH free radical；
Fig. 4 b is vitamin C in embodiment 5 to the Scavenging activity figure of DPPH free radical；
Fig. 5 a is that the Amino Acid of Glossy ganoderma extract of various concentration in embodiment 5 removes DPPH free radical curve graph；
Fig. 5 b is that the vitamin C of various concentration in embodiment 5 removes DPPH free radical curve graph.
(5) specific embodiment
Below by specific embodiment, the present invention is further illustrated, but protection scope of the present invention is not limited in
This.
Embodiment 1
The determination of the optimal extraction conditions of Amino Acid of Glossy ganoderma
A kind of method that Amino Acid of Glossy ganoderma is extracted in response phase method optimization, using design expert software, according to Box-
On liquid-to-solid ratio, extraction time and concentration of hydrochloric acid, these factors for influencing that Amino Acid of Glossy ganoderma extracts carry out Behnken design principle
Optimization, method includes the following steps:
(1) preparation of samples
(1) it pre-processes: ganoderma lucidum 80.0g is placed at 60~80 DEG C to drying to constant weight, crush, obtain ganoderma powder 76.4g；
(2) sour water solution: taking ganoderma powder 2.0g obtained by step (1), by liquid-to-solid ratio be 10~30:1 (mL/g) addition 4~
In the hydrochloric acid solution of 10mol/L, it is heated to reflux 4~12h, filters, obtains amino acid crude extract；
(3) it adjusts pH: using the pH to 3 of sodium hydroxide solution regulating step (2) resulting amino acid crude extract；
(4) constant volume: the resulting amino acid crude extract of step (3) is settled to 50mL, obtains Amino Acid of Glossy ganoderma sample liquid
It is spare.
(2) ninhydrin colorimetry measures Amino Acid of Glossy ganoderma content
(1) preparation of amino acid standard curve
Measure respectively 0 μ L, 100 μ L, 150 μ L, 200 μ L, 250 μ L, 300 μ L, 350 μ L, 400 μ L, 450 μ L, 500 μ L it is sweet
For propylhomoserin solution in test tube, being mended with phosphate buffer to volume is 1500 μ L, is separately added into 1mL ninhydrin solution, is shaken,
15min is reacted at 100 DEG C, cooling 10min under room temperature is settled to 10mL with 50% ethyl alcohol, detects absorbance under 570nm wavelength
(X), three parallel groups of each group of setting.With absorbance (X) for abscissa, amino acid weight (Y) is ordinate, draws amino acid
The standard curve of standard curve, ninhydrin colorimetry measurement amino acid content is shown in Fig. 1 a.Its equation of linear regression is Y=
0.1201X+0.0209, R2=0.9989.
(2) assay of Amino Acid of Glossy ganoderma
It takes certain density 100 μ L of Amino Acid of Glossy ganoderma sample liquid in test tube respectively, is mended with phosphate buffer to volume
For 1500 μ L, it is separately added into 1mL ninhydrin solution, is shaken, 15min, cooling 10min under room temperature, with 50% second are reacted at 100 DEG C
Alcohol is settled to 10mL, and absorbance, three parallel groups of each group of setting are detected under 570nm wavelength.Pass through amino acid standard curve meter
Calculate the content of amino acid in sample liquid.Amino Acid of Glossy ganoderma yield=amino acid content/ganoderma lucidum weight × 100%.
(3) experimental design and statistical analysis
1, single factor experiment
Changing liquid-to-solid ratio, extraction time and concentration of hydrochloric acid, these influence the factor progress single factor test that Amino Acid of Glossy ganoderma extracts
Test.
(1) influence of yield is extracted in the variation of liquid-to-solid ratio to Amino Acid of Glossy ganoderma:
At 100 DEG C of Extracting temperature, extraction time 8h, under conditions of concentration of hydrochloric acid is 6moL/L, liquid-to-solid ratio is set as 10:
1, five changing values of 15:1,20:1,25:1,30:1.The influence result of yield is extracted in the variation of liquid-to-solid ratio to Amino Acid of Glossy ganoderma
As shown in Figure 1 b.
Through detecting, when liquid-to-solid ratio is 15:1, Amino Acid of Glossy ganoderma yield is 4.417%, with the increase of solid-liquid ratio, amino
Sour yield declines instead, and therefore, solid-liquid ratio takes 15:1 more appropriate.
(2) extraction time extracts the influence of yield to Amino Acid of Glossy ganoderma:
At 100 DEG C of Extracting temperature, liquid-to-solid ratio 15:1, under conditions of concentration of hydrochloric acid is 6moL/L, will set extraction time
For five changing values of 4h, 8h, 12h, 16h, 20h.Extraction time extracts influence result such as Fig. 1 c institute of yield to Amino Acid of Glossy ganoderma
Show.
Through detecting, upon extracting between be 8h when, Amino Acid of Glossy ganoderma yield be 4.324%, with the increase of extraction time, ammonia
Base acid yield declines instead, and therefore, extraction time takes 8h more appropriate.
(3) concentration of hydrochloric acid extracts the influence of yield to Amino Acid of Glossy ganoderma:
Under conditions of 100 DEG C of Extracting temperature, liquid-to-solid ratio 15:1, extraction time 8h, concentration of hydrochloric acid is set as 2moL/
L, five changing values of 4moL/L, 6moL/L, 8moL/L, 10moL/L.Concentration of hydrochloric acid extracts the influence knot of yield to Amino Acid of Glossy ganoderma
Fruit is as shown in Figure 1 d.
Through detecting, when concentration of hydrochloric acid is 6moL/L, Amino Acid of Glossy ganoderma yield is 4.384%, with the increasing of concentration of hydrochloric acid
Add, amino acid yield declines instead, and therefore, concentration of hydrochloric acid takes 6moL/L more appropriate.
2, the method that Amino Acid of Glossy ganoderma is extracted in the optimization of phase response phase method
(1) response phase method design optimization
More significant liquid-to-solid ratio, extraction time are influenced as a result, choosing and extracting on Amino Acid of Glossy ganoderma according to single factor experiment
And concentration of hydrochloric acid these three factors, using design expert software, according to Box-Behnken design principle design experiment,
Three levels of each factor design, setting the results are shown in Table 1.
Table 1: response surface analysis factor and water-glass
It is certainly with liquid-to-solid ratio (A), extraction time (B) and concentration of hydrochloric acid (C) according to response surface analysis factor and water-glass
Variable, using Amino Acid of Glossy ganoderma yield as response (Y), response surface experiments scheme and response are shown in Table 2.
Table 2: response surface experiments scheme and response
(2) foundation of model and statistical analysis
Secondary multiple regression analysis is carried out according to the data obtained, obtains relevant variable (liquid-to-solid ratio A, extraction time B and salt
Acid concentration C) and response (amino acid yield Y) between polynary quadratic regression equation.
Polynary quadratic regression equation: Y=-4.62331+0.51495A+0.48688B+0.73299C-2.71750 ×
10-3AB+0.013705AB+6.80938×10-3BC-0.016624A2- 0.025543B2- 0.076071C2
Variance analysis is carried out to secondary multiple regression equation items and significance test the results are shown in Table 3.The result shows that this time
Significance test P < 0.0001 for returning model shows that the model has statistical significance, the correction coefficient R of regression model2=
0.9743, coefficient of variation C.V.%=3.2% lose quasi- item P=0.073 > 0.005, and the quasi- property inspection result of mistake is not significant, illustrates this
Model is fitted with data that degree is higher, and experimental error is small, and the model analysis and prediction Hydrochloric Acid Hydrolysis Method can be used to extract ganoderma lucidum ammonia
The result of base acid.
Table 3: variance analysis and significance test result
(3) test result analysis and optimization
Using Design Expert software, plot analysis is carried out according to regression equation, obtains the response surface three of regression equation
Dimension figure.Response surface three-dimensional figure such as Fig. 2 a~2c institute that liquid-to-solid ratio, extraction time, concentration of hydrochloric acid influence Amino Acid of Glossy ganoderma yield
Show.
The optimum extraction condition of Amino Acid of Glossy ganoderma is extracted by the Hydrochloric Acid Hydrolysis Method that Design Expert software obtains are as follows: liquid
Ratio is expected for 20:1, extraction time 9.41h, concentration of hydrochloric acid 7.04moL/L, and Amino Acid of Glossy ganoderma yield is with this condition
4.60%.For the simplicity of operation, the optimal conditions of practical operation are as follows: liquid-to-solid ratio 20:1, extraction time 9.5h, hydrochloric acid
Concentration 7.0moL/L, Amino Acid of Glossy ganoderma yield is 4.78% with this condition.
Embodiment 2
The preparation of Amino Acid of Glossy ganoderma extract and its Contents of Amino Acids:
(1) it pre-processes: ganoderma lucidum 40g is placed at 70 DEG C to drying to constant weight, crush, obtain ganoderma powder 38.3g；
(2) sour water solution: taking ganoderma powder 30.0g obtained by step (1), is that 20:1 (mL/g) addition 600mL is dense by liquid-to-solid ratio
Degree is to be heated to reflux 9.5h in the hydrochloric acid solution of 7.0moL/L at 100 DEG C, filter to obtain hydrolyzate, gained hydrolyzate is depressurized
It is evaporated, adds water 15mL to dissolve enriched product, and adjust pH to 3 with sodium hydroxide, obtain crude extract；
(3) purify: 4.5g active carbon is added into crude extract obtained by step (2), and (additive amount of active carbon is ganoderma powder
The 15% of quality), decolourize 45min at 80 DEG C, filters and removes active carbon, takes filtrate decompression to be concentrated into the 1/8 of initial volume, have
Crystal is precipitated, and 2 times of volume dehydrated alcohols are added, with sodium hydroxide and salt acid for adjusting pH to 3,4 DEG C of standings 22h, Zhi Hou
It is centrifugated 15min under the revolving speed of 3000r/min, removes supernatant liquor, precipitating is collected, in freeze-day with constant temperature in 50 DEG C of baking ovens to perseverance
Weight, the Amino Acid of Glossy ganoderma extract 1.6818g purified；
(4) the ganoderma lucidum amino extract purified obtained by step (3) Contents of Amino Acids: is made into the sample of 4mg/mL
Liquid measures amino acid content by ninhydrin colorimetry, calculates Amino Acid of Glossy ganoderma content.Amino Acid of Glossy ganoderma content=amino acid contains
Amount/Amino Acid of Glossy ganoderma extract weight × 100%.
Through detecting, the Amino Acid of Glossy ganoderma content of the Amino Acid of Glossy ganoderma extract of the extracted purifying of the present embodiment is
17.78%.
Embodiment 3
The preparation of Amino Acid of Glossy ganoderma extract and its Contents of Amino Acids:
(1) it pre-processes: ganoderma lucidum 40.0g is placed at 60 DEG C to drying to constant weight, crush, obtain ganoderma powder 38.2g；
(2) sour water solution: taking ganoderma powder 30.0g obtained by step (1), is that 10:1 (mL/g) addition 300mL is dense by liquid-to-solid ratio
Degree is to be heated to reflux 4h in the hydrochloric acid solution of 4.0moL/L at 100 DEG C, filter to obtain hydrolyzate, and gained hydrolyzate is depressurized and is steamed
It is dry, add water 15mL to dissolve enriched product, and adjust pH to 3 with sodium hydroxide, obtains crude extract；
(3) purify: 1.5g active carbon is added into crude extract obtained by step (2), and (additive amount of active carbon is ganoderma powder
The 5% of quality), decolourize 55min at 50 DEG C, filters and removes active carbon, takes filtrate decompression to be concentrated into the 1/6 of initial volume, have
Crystal is precipitated, and 1 times of volume dehydrated alcohol is added, with sodium hydroxide and salt acid for adjusting pH to 3,2 DEG C of standings 20h, Zhi Hou
It is centrifugated 10min under the revolving speed of 3000r/min, removes supernatant liquor, precipitating is collected, in freeze-day with constant temperature in 60 DEG C of baking ovens to perseverance
Weight, the Amino Acid of Glossy ganoderma extract 1.3687g purified；
(4) the ganoderma lucidum amino extract purified obtained by step (3) Contents of Amino Acids: is made into the sample of 4mg/mL
Liquid measures amino acid content by ninhydrin colorimetry, calculates Amino Acid of Glossy ganoderma content.Amino Acid of Glossy ganoderma content=amino acid contains
Amount/Amino Acid of Glossy ganoderma extract weight × 100%.
Through detecting, the Amino Acid of Glossy ganoderma content of the Amino Acid of Glossy ganoderma extract of the extracted purifying of the present embodiment is
14.75%.
Embodiment 4
The preparation of Amino Acid of Glossy ganoderma extract and its Contents of Amino Acids:
(1) it pre-processes: ganoderma lucidum 40.0g is placed at 60 DEG C to drying to constant weight, crush, obtain ganoderma powder 38.5g；
(2) sour water solution: taking ganoderma powder 30.0g obtained by step (1), is that 30:1 (mL/g) addition 900mL is dense by liquid-to-solid ratio
Degree is to heat back 12h in the hydrochloric acid solution of 10moL/L at 100 DEG C, filter to obtain hydrolyzate, by gained hydrolyzate evaporated under reduced pressure,
Add water 15mL to dissolve enriched product, and adjust pH to 3 with sodium hydroxide, obtains crude extract；
(3) purify: 1.5g active carbon is added into crude extract obtained by step (2), and (additive amount of active carbon is ganoderma powder
The 5% of quality), decolourize 15min at 90 DEG C, filters and removes active carbon, takes filtrate decompression to be concentrated into the 1/10 of initial volume, have
Crystal is precipitated, and 3 times of volume dehydrated alcohols are added, and is stood for 24 hours with sodium hydroxide and salt acid for adjusting pH to 3,6 DEG C, Zhi Hou
It is centrifugated 15min under the revolving speed of 3000r/min, removes supernatant liquor, precipitating is collected, in freeze-day with constant temperature in 70 DEG C of baking ovens to perseverance
Weight, the Amino Acid of Glossy ganoderma extract 1.4881g purified；
(4) the ganoderma lucidum amino extract purified obtained by step (3) Contents of Amino Acids: is made into the sample of 4mg/mL
Liquid measures amino acid content by ninhydrin colorimetry, calculates Amino Acid of Glossy ganoderma content.Amino Acid of Glossy ganoderma content=amino acid contains
Amount/Amino Acid of Glossy ganoderma extract weight × 100%.
Through detecting, the Amino Acid of Glossy ganoderma content of the Amino Acid of Glossy ganoderma extract of the extracted purifying of the present embodiment is
15.44%.
Embodiment 5
The hypoglycemic activity of Amino Acid of Glossy ganoderma extract and the investigation of antioxidant activity are (with ganoderma lucidum amino made from embodiment 2
Sour extract is test sample):
For the hypoglycemic activity and antioxidant activity for investigating Amino Acid of Glossy ganoderma extract of the invention, following experiment is now carried out:
1, the hypoglycemic activity measurement of Amino Acid of Glossy ganoderma extract:
Using PNPG as substrate, using 96 orifice plates as reaction carriers, Amino Acid of Glossy ganoderma extract is measured to alpha-glucosidase
Inhibiting rate.
Standard reaction system: the certain density Amino Acid of Glossy ganoderma extract solution of 40 μ L is added to the α-of 20 μ L 0.2U/mL
In glucose glucosides enzyme solutions, 10min is reacted at 37 DEG C, and 20 μ L 6.5mmol/L PNPG solution are added, react at 37 DEG C
30min, with 120 μ L 0.2mol/L Na2CO3Solution terminates reaction, measures the absorption of solution at 405nm wavelength with microplate reader
Value, three parallel groups of each group of setting.Amino acid concentration is shown in Table 4 to the inhibiting rate relationship of alpha-glucosidase.Experimental group ganoderma lucidum
Amino acid extract is shown in Fig. 3 a to the inhibiting rate of alpha-glucosidase；Suppression of the positive controls acarbose to alpha-glucosidase
Rate processed is shown in Fig. 3 b.
Inhibiting rate (%) of the sample to enzymatic activity: Inhibition rate (%)=[(A0Blank- ABlank)-(A0
A)]/(A0Blank- ABlank)
A0BlankFor α-glucose glucosides enzyme solutions and PNPG solution absorption value is only added；ABlankOnly to add PNPG solution
Absorption value；A0For testing sample solution, α-glucose glucosides enzyme solutions and PNPG solution absorption value is added；A is only plus to test sample
The absorption value of product solution and PNPG solution.
Table 4: inhibiting rate relationship of the amino acid concentration to alpha-glucosidase
Amino Acid of Glossy ganoderma extract has certain inhibiting effect to α-glucose glycosidase as seen from the above table, and IC50 value is
380.62mg/L。
2, the Antioxidative Activity Determination of Amino Acid of Glossy ganoderma extract:
(1) Scavenging activity of the Amino Acid of Glossy ganoderma extract to DPPH free radical
Using DPPH as substrate, using 96 orifice plates as reaction carriers, Amino Acid of Glossy ganoderma extract is measured to the clear of DPPH free radical
Removing solid capacity.
Take 0 μ L of solution of certain density Amino Acid of Glossy ganoderma extract, 10 μ L, 20 μ L, 30 μ L, 40 μ L, 50 μ L, 60 μ L,
70 μ L in 96 orifice plates, mend to 70 μ L of volume by dehydrated alcohol, adds 150 μ L of DPPH solution, using ethyl alcohol substitution DPPH solution as blank
Control.It is sufficiently mixed, after standing 30 minutes, surveys A value, three parallel groups of each group of setting at 516nm with microplate reader.Amino acid
Concentration and the clearance rate relationship of DPPH free radical are shown in Table 5.Removing energy of the experimental group Amino Acid of Glossy ganoderma extract to DPPH free radical
Power is shown in Fig. 4 a；Positive controls vitamin C is shown in Fig. 4 b to the Scavenging activity of DPPH free radical.
The calculation formula of free radical scavenging activity: clearance rate=(A0-A)/A0 × 100%
Table 5: the clearance rate relationship of amino acid concentration and DPPH free radical
Amino Acid of Glossy ganoderma extract has certain scavenging effect to DPPH as seen from the above table, and IC50 value is 484.54 μ
g/mL。
(2) preparation of the standard curve of DPPH
Take DPPH solution 0uL, 40uL, 80uL, 120uL, 160uL, 200uL in 96 orifice plates, dehydrated alcohol is mended to volume
200uL is sufficiently mixed, and absorbance, three parallel groups of each group of setting are surveyed at 516nm with microplate reader.It is vertical with absorbance (Y)
Coordinate is that transverse and longitudinal mark makes DPPH standard curve, equation of linear regression Y=with DPPH mass concentration ug/mL (X)
0.0192X-0.023, R2=0.9941.
(3) add Amino Acid of Glossy ganoderma extract after DPPH free radical residual rate
Using ethyl alcohol as solvent, the Amino Acid of Glossy ganoderma extract sample liquid of various concentration is prepared, takes variant concentration sample respectively
Product liquid 40uL in 96 orifice plates, mend to volume 70uL by dehydrated alcohol, adds DPPH solution 150uL, is with ethyl alcohol substitution DPPH solution
Blank control.It is sufficiently mixed, quickly measures the absorbance value of its different time at 516nm with microplate reader.According to standard curve
It is converted into [DPPH.] mass concentration, to calculate [DPPH.] residual rate.According to [DPPH.] residual rate and corresponding amino
Acid concentration draws out Amino Acid of Glossy ganoderma [DPPH.] free radical residual plots figure.The Amino Acid of Glossy ganoderma of experimental group various concentration extracts
Object removes the free base curves of DPPH and sees Fig. 5 a；The vitamin C of positive controls various concentration removes the free base curves of DPPH and sees figure
5b。
[DPPH.] residual rate calculation formula: [DPPH.]REM=[DPPH.]T/[DPPH.]T=0× 100%
Wherein [DPPH.]TFor moment [DPPH a certain during radicals scavenging.] mass concentration, [DPPH.]T=0For
[DPPH.] original concentration.
By Amino Acid of Glossy ganoderma extract known to Fig. 5 a to the dense of DPPH free radical scavenging ability and Amino Acid of Glossy ganoderma extract
Degree is positively correlated.
Claims (8)
1. a kind of Amino Acid of Glossy ganoderma extract, which is characterized in that the Amino Acid of Glossy ganoderma extract is prepared as follows
It arrives:
(1) it sour water solution: takes ganoderma powder to be added in hydrochloric acid solution, is heated to reflux 9.5h, filters to obtain hydrolyzate, by gained hydrolyzate
Evaporated under reduced pressure, residue are dissolved in water, and adjust pH to 2~4, obtain crude extract；The concentration of the hydrochloric acid solution is 7.0mol/L；
The volumetric usage of the hydrochloric acid solution is 20mL/1g ganoderma powder；
(2) it purifies: active carbon is added into crude extract obtained by step (1), decolourize 15~55min at 50~90 DEG C, and suction filtration removes
Deactivation charcoal, is added dehydrated alcohol after filtrate decompression is concentrated, adjust pH to 2~4, stand 20 in 2~6 DEG C~for 24 hours, Zhi Hou
It is centrifugated 10~15min under the revolving speed of 2000~3000r/min, removes supernatant liquor, solid precipitating is collected, in 50~70 DEG C
Freeze-day with constant temperature obtains the Amino Acid of Glossy ganoderma extract to constant weight in baking oven.
2. Amino Acid of Glossy ganoderma extract as described in claim 1, which is characterized in that the ganoderma powder is to be placed in ganoderma lucidum
Drying to constant weight at 60~80 DEG C, is crushed to the acquisition of 5~10 mesh.
3. Amino Acid of Glossy ganoderma extract as described in claim 1, which is characterized in that in step (1), the residue adds 6
The water dissolution of~9 mass times.
4. Amino Acid of Glossy ganoderma extract as described in claim 1, which is characterized in that in step (2), the quality of the active carbon
Dosage is 5%~25% of ganoderma powder quality described in step (1).
5. Amino Acid of Glossy ganoderma extract as described in claim 1, which is characterized in that in step (2), the filtrate decompression concentration
To the 1/6~1/10 of initial volume.
6. Amino Acid of Glossy ganoderma extract as described in claim 1, which is characterized in that in step (2), in filtrate after concentration
The dehydrated alcohol of 1~3 times of volume is added.
7. Amino Acid of Glossy ganoderma extract as described in claim 1, which is characterized in that in step (1) or step (2), the tune of pH
Section uses sodium hydroxide and/or hydrochloric acid.
8. a kind of Amino Acid of Glossy ganoderma extract as described in claim 1 is preparing hypoglycemic, oxidation-resisting health-care product and drug
In application.
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