A kind of method for producing low polarity ginsenoside
Technical field
The present invention relates to a kind of method for producing low polarity ginsenoside, belong to natural products manufacture field.
Background technology
Ginseng is the dry root of Araliaceae ginseng, is grown on small 500~1100 meters of mountain regions of height above sea level of day and night temperature more
In the theropencedrymion or shaw of gentle slope or slope land.Because root is loose, if shape spindle, often there is bifurcated, overall picture is quite like people
Head, hand, foot and four limbs, so referred to as ginseng.Ginseng is referred to as " kings of hundred grass ", be have won fame both at home and abroad, that old children all knows is rare
Medicinal material.For a long time, due to excessively excavation, resource exhaustion, the forest ecological environment that ginseng is depended on for existence is seriously damaged, mesh
Preceding northeast ginseng is also at endangered edge.The wild ginseng in northeast is also extremely rare, therefore, protects the natural resources of this kind and has
Its special significance.
Ginseng has a variety of physiology and pharmacological action, such as antitumor, enhancing is immune, improve microcirculation, calm the nerves, anti-aging,
Enhancing memory and learning ability etc..
In anti-tumor aspect, ginseng has:1. suppress growth of tumour cell, promote tumor stem cell differentiation;2. suppress swollen
The infiltration and transfer of oncocyte;3. the new vessels generation of tumor inducing is suppressed;4. the toxic side effect of chemotherapeutics is reduced.
Wild ginseng is to contain and cultivated ginseng identical ginsenoside (being referred to as natural ginseng saponin(e), again containing uniqueness
Ginsenoside (is referred to as rare ginsenoside and aglycon), and effect of wild ginseng comes from rare ginsenoside and aglycon.Edible people
After ginseng, the original ginsenoside of diol type therein only has 4% can be inhaled in enteral transformation into rare ginsenoside and aglycon, by body
Receive and utilize, remaining 96% is excluded with stool form.To improve the content of rare ginsenoside and aglycon in cultivated ginseng, people adopt
Ginseng is processed with a variety of methods.
1. first generation processing of Panax ginseng technology:By ginseng steaming, ginsenoside is thermally decomposed, lose part glycosyl, formed
Rare ginsenoside.Our common red ginsengs, and world-renowned import red ginseng are all the products obtained using this technology.
2. second generation processing of Panax ginseng technology:The glycosidic bond of ginsenoside is destroyed using organic acid, inorganic acid, alkali, is generated dilute
There is ginsenoside.
3. third generation processing of Panax ginseng technology:Various configuration, different types of glycosidic bond are degraded according to the selectivity of enzyme, reached
To directionally hydrolyzing purpose, the method maintains laboratory level.
4. forth generation processing of Panax ginseng technology:It is, by the antimicrobial composition catalytic decomposition of different degradation capabilities, substrate to be converted
For the process of target product, it is the enzymatic reaction occurred between microorganism and xenobiotic substrates at all.It is difficult to choose high activity engineering
Bacterium, the field after the development of more than 30 years, yet there are no the report of commercial application.
In the world, United States Patent (USP) (5776460) report through 120-180 DEG C processing 0.5-20 hours after, it is rare in ginseng
Content of ginsenoside increases.The patent is only control temperature, and the quality and quantity of ginseng thermal decomposition product can not be constant, product
Stability can not ensure, the content of rare ginsenoside is relatively low in product, batch wise differences are big.
The content of the invention
It is an object of the invention to provide a kind of method for producing low polarity ginsenoside.Processing of Panax ginseng method of the present invention can
Produce 25- hydroxyls dammarane and 25- hydroxyls reach agate alkene, the present invention extracts the production of saponin(e by finely controlling catalytic decomposition process
Amount and component are controllable, and the target product yield extracted from the raw materials such as the ginseng after processing is high.
The method for the low polarity ginsenoside of production that the present invention is provided, comprises the following steps:By the plant containing ginsenoside,
The raw material of the tissue culture of the plant, the extract of the plant and/or natural ginseng saponin(e is through catalytic decomposition, Ran Houfen
From obtaining low polarity ginsenoside;
The low polarity ginsenoside is that 25- hydroxyls dammarane organizes at least one set of material in material and following (1)-(5)
Mixture:
(1) 25- hydroxyls reach agate alkene group:3 β, 12 β, 25- trihydroxies up to agate 20 (22) alkene, 3-O- β-D- glucopyranosyls-
3 β, 12 β, 25- trihydroxies reach agate 20 (22) alkene and 3-O- β-D- glucopyranosyls-(1 → 2) -3-O- β-D- glucopyranoses
The β of base -3,12 β, 25- trihydroxies reach at least one of agate 20 (22) alkene;
(2) rare diol saponin I groups:In 20- (S, R)-Rg3,20- (S, R)-Rh2,20- (S, R)-PPD and panoxadiol
At least one;
(3) rare diol saponin II groups:Rg5 and/or Rk3;
(4) triol I groups:20- (S, R)-protopanaxatriol and/or panaxatriol;
(5) triol II groups:At least one of 20- (S, R)-Rg2,20- (S, R)-Rg6, Rh4 and Rk5;
25- hydroxyls dammarane's group:3 β, 12 β, 20 (S, R), 25- tetrahydroxys dammarane, 3-O- β-D- glucopyranoses
Base -3 β, 12 β, 20 (S, R), 25- tetrahydroxys dammarane and 3-O- β-D- glucopyranosyls-(1 → 2) -3-O- β-D- pyrans Portugal
At least one of grape glycosyl -3 β, 12 β, 20 (S, R), 25- tetrahydroxy dammarane.
In above-mentioned method, the plant is at least one of ginseng, pseudo-ginseng, American Ginseng and gynostemma pentaphylla;
The plant is using at least one position in palpus, root, stem, leaf, flower, fruit and reed head;
The natural ginseng saponin(e is at least one of Rb1, Rb2, Rb3, Rc, Rd, Rg1, Re and R2;
The catalyst used that is catalytically decomposed is at least one of bacterium, enzyme, organic acid and organic base.
In above-mentioned method, the bacterium is in lactobacillus bulgaricus, streptococcus thermophilus, Paecilomyces hepiali chen and saccharomycete
At least one;The bacterial strain of the lactobacillus bulgaricus concretely CICC20244 models, the bacterial strain of the streptococcus thermophilus
Concretely CICC20365 models, the bacterial strain of the Paecilomyces hepiali chen concretely CICC14065 models, the saccharomycete
Bacterial strain concretely CICC1211 models, the lactobacillus acidophilus concretely CICC6083 models, the Bifidobacterium tool
Body can be CICC21711 models;
The enzyme is glycosidase, dehydrogenase, hydroxylase, oxidizing ferment, reductase, hydrolase, transferase, lyases and isomery
At least one of enzyme;
The organic acid is oxaloacetic acid, oxalic acid, malonic acid, succinic acid, butene dioic acid, tartaric acid, malic acid, lemon
Acid, adipic acid, phthalic acid, aspartic acid, glutamic acid, amino acid, uronic acid, formic acid, glacial acetic acid, lactic acid, propionic acid, butyric acid,
Valeric acid, benzoic acid, salicylic acid, sulfosalicylic acid, benzene sulfonic acid, a fluoroacetic acid, difluoroacetic acid, trifluoroacetic acid, chloroacetic acid, dichloro
At least one of acetic acid and trichloroacetic acid;
The organic base is organic amine.
In above-mentioned method, the glycosidase is glucan glycosides enzyme, glucuroide or high temperature resistant beta-glucosidase;
The dehydrogenase is nadh dehydrogenase or NADPH dehydrogenases;
The hydroxylase is ginsengenin hydroxylase;
The hydrolase is ginseng saponing-glycosidase;
The transferase is ginseng saponin glycosyl transferase
The lyases is ginseng saponin glycosyl lyases
The isomerase is ginsengenin isomerase;
The organic amine is at least one of pyridine, hyoscyamine, isoquinolin and indoles.
In above-mentioned method, the weight percent concentration of the enzyme can be 0.01~100%;
The weight percent concentration of the organic acid can be 0.01~100%, concretely 15%;
The weight percent concentration of the organic base can be 0.01~100%;
The number of bacterium described in raw material described in per g can be 102~1010Cuf, concretely 102cfu;
Described can be 1 with the enzyme activity of the enzyme per g raw materials:0.001U~108U, concretely 300U;
The mass ratio of the raw material and the organic acid can be 1:0.01~10, concretely 1:1;
The mass ratio of the raw material and the organic base can be 1:0.01~10.
In above-mentioned method, when being decomposed using the enzymatic, the temperature of the catalytic decomposition can be 15~85 DEG C, tool
Body can be 37 DEG C, and the time of the catalytic decomposition can be 4~168h, concretely 24h;
When being decomposed using the organic acid catalysis, the temperature of the catalytic decomposition can be 25~95 DEG C, the catalysis point
The time of solution can be 2~168h;
When being decomposed using the organic base catalytic, the temperature of the catalytic decomposition can be 25~95 DEG C, the catalysis point
The time of solution can be 2~168h;
In above-mentioned method, the step of also including heat inactivation after the catalytic decomposition;
The temperature of the heating can be 46~350 DEG C, concretely 100 DEG C, 121 DEG C or 100~121 DEG C;
The time of the heating can be 0.5~24h, concretely 0.5h, 2h, 5h or 0.5~5h;
The separation includes extraction and/or column chromatography;It is specific that the panaxan in product is gone out using extraction, column chromatography
Ginsenoside in the product is separated.
In above-mentioned method, the eluting solvent that the column chromatography is used is the acetonitrile and the water, the acetonitrile and institute
The volume ratio for stating water can be 3:1~9, concretely 3:1~9 is eluted according to gradient;
The eluting solvent that the column chromatography is used is the ethanol and the water, and the volume ratio of the ethanol and the water can
For 1:0.1~1.
The present invention has advantages below:
In the present invention, raw material is digested through ginsenoside during catalytic decomposition process, discharged, and is presented free state, desugar,
25 hydroxylation ginsenosides or aglycon are formed after hydroxylation, by finely controlling catalytic decomposition process, the yield and component of saponin(e are
Controllable.Particularly this process technology can obtain the dammarane containing 25-OH- of high level, up to ginsenosides such as agate alkene.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
In following embodiments, the bacterial strain of lactobacillus bulgaricus is CICC20244 models;The bacterial strain of streptococcus thermophilus is
CICC20365 models;The bacterial strain of Paecilomyces hepiali chen is CICC14065 models;The bacterial strain of saccharomycete is CICC1211 models;It is thermophilic
Lactobacillus lactis is CICC6083 models;Bifidobacterium is CICC21711 models.
Embodiment 1,
Raw cultivated ginseng root (the solarization trepang of purple stem haw ginseng, sun-dried ginseng 5000g, aqueous 5-12%) is added to 50L within 4 years
(lactobacillus bulgaricus, streptococcus thermophilus, Paecilomyces hepiali chen, the number of saccharomycete are respectively probiotics in per g raw materials
102cfu、102cfu、102cfu、102Cfu) in catalytic decomposition liquid, 37 DEG C are fermented 24 hours, and 100 DEG C are heated 30 minutes.Conversion production
Thing is through HPLC analysis shows, conversion ratio 90.3%.Principal product is Rg3, Rh2, protopanoxadiol, protopanaxatriol, panoxadiol,
Panaxatriol, 25-OH- dammarane, 25-OH- reaches rare ginsenoside and aglycon and the natural ginseng saponin(es such as agate alkene.Convert ginseng
Crush, extracted 3 times with methanol eddy, be removed under reduced pressure after methanol, be evaporated.The ethanol cyclic washing that 30% is added in thing to being evaporated,
Remove panaxan, drying precipitate.Dissolved with methanol, reverse phase preparative column is separated, and (mobile phase is acetonitrile and water, 1:3~
3:1 gradient elution) following ginsenoside is obtained respectively:
I group of glycol:
3 β, 12 β, 20 (S, R), 25- tetrahydroxy dammarane yield 3.5%;
3-O- β-D- glucopyranosyls -3 β, 12 β, 20 (S, R), 25- tetrahydroxy dammarane yield 4.1%;
3-O- β-D- glucopyranosyls-(1 → 2) -3-O- β-D- glucopyranosyls -3 β, 12 β, 20 (S, R), 25- tetra-
Hydroxyl dammarane yield 4.5%;
Glycol II groups:
3 β, 12 β, 25- trihydroxies reach agate 20 (22) alkene yield 3.3%;
The β of 3-O- β-D- glucopyranosyls -3,12 β, 25- trihydroxies reach agate 20 (22) alkene yield 4.6%;
3-O- β-D- glucopyranosyls-the β of (1 → 2) -3-O- β-D- glucopyranosyls -3,12 β, 25- trihydroxies reach agate
20 (22) alkene yields 4.7%;
Glycol III groups:
20- (S, R)-Rg3 yields 4.8%;
20- (S, R)-Rh2 yields 5.2%;
20- (S, R)-PPD yields 2.9%;
Panoxadiol yield 2.1%;
Glycol IV groups:
Rg5 yields 3.6%;Rk3 yields 3.3%;
Glycol V groups:
Natural diol type saponin productivity 6.2%, Rb1 yields 3.5%, Rb2 yields 0.5%, Rb3 yields 0.3%, Rc yields
0.5%, Rd yield 0.7%, Ra1 yields 0.4%, Ra2 yields 0.3%,
Triol I groups:
20- (S, R)-protopanaxatriol yield 2.7%;
Panaxatriol yield 12%.
Triol II groups:
20- (S, R)-Rg2 yields 6.7%;
20- (S, R)-Rg6 yields 7.6%;
Rh4 yields 7.3%;
Rk5 yields 7.4%.
III group of glycol:
Natural diol type saponin productivity 3.5%, Re yields 0.4%, Rh1 yields 2.1%, Rg1 yields 1%.
Embodiment 2,
Diol type ginsenoside (50g) (being available commercially from Jilin Zixin Chuyuan Pharmaceutical Co., Ltd.) is added to 50L 4 plants of lactic acid
(lactobacillus bulgaricus, lactobacillus acidophilus, streptococcus thermophilus, Bifidobacterium mix 10 to bacterium according to 1g mass ratioes2cfu、102cfu、
102cfu、102Cfu) mix, be catalytically decomposed 24 hours, 100 DEG C are heated 30 minutes.Converted product is through HPLC analysis shows, conversion
Rate 94%.Principal product is Rg3, and Rh2, protopanoxadiol, panoxadiol, 25-OH- dammarane, 25-OH- reaches the rare ginseng of agate alkene
Saponin(e and aglycon.Catalytic decomposition liquid is collected by centrifugation, methanol dissolving, centrifugation, supernatant is evaporated, to the ethanol for being evaporated addition 30% in thing
Cyclic washing, removes panaxan, drying precipitate.Dissolved with methanol, reverse phase preparative column separated (mobile phase be acetonitrile and
Water) following saponin(e is obtained respectively:
I group of glycol:
3 β, 12 β, 20 (S, R), 25- tetrahydroxy dammarane;(yield 15.2%)
3-O- β-D- glucopyranosyls -3 β, 12 β, 20 (S, R), 25- tetrahydroxy dammarane;(yield 9.5%)
3-O- β-D- glucopyranosyls-(1 → 2) -3-O- β-D- glucopyranosyls -3 β, 12 β, 20 (S, R), 25- tetra-
Hydroxyl dammarane;(yield 8.7%)
Glycol II groups:
3 β, 12 β, 25- trihydroxies reach agate 20 (22) alkene;(yield 8.4%)
The β of 3-O- β-D- glucopyranosyls -3,12 β, 25- trihydroxies reach agate 20 (22) alkene;(yield 8.7%)
3-O- β-D- glucopyranosyls-the β of (1 → 2) -3-O- β-D- glucopyranosyls -3,12 β, 25- trihydroxies reach agate
20 (22) alkene;(yield 9.5%)
Glycol III groups:
20-(S,R)-Rg3;(yield 10.5%)
20-(S,R)-Rh2;(yield 6.5%)
20-(S,R)-PPD;(yield 4.1%)
Panoxadiol;(yield 4.5%)
Glycol IV groups:
Rg5;(yield 4.5%)
Rk3.(yield 3.9%)
Embodiment 3,
Diol type ginsenoside (being available commercially from Jilin Zixin Chuyuan Pharmaceutical Co., Ltd.) (50g) and glucuroide (Shanghai
Good and bio tech ltd, enzyme activity is 300U/g) 5g is added in 500ml water, and 40 DEG C of reactions 16 are small
When, 100 DEG C of heating are inactivated for 30 minutes.The HPLC analysis shows of converted product, conversion ratio 72%, product master
It is that principal product is Rg3, Rh2, protopanoxadiol, 25-OH- dammarane and 25-OH- reach the rare ginseng of agate alkene
Saponin(e and aglycon.Add 5000mL water and suspension is presented, centrifugation is dissolved with methanol and precipitated, takes supernatant to be evaporated.
Thing is evaporated with after 30% ethanol rinse removal of impurities, is evaporated.Methanol is dissolved, and the inverted post for preparing is separated (flowing
Phase is 65% ethanol) Rg3 is respectively obtained, Rh2, protopanoxadiol, 25-OH- dammarane and 25-OH- reach agate
The rare ginsenosides such as alkene and aglycon.
I group of glycol:
3 β, 12 β, 20 (S, R), 25- tetrahydroxy dammarane;(yield 11.1%)
3-O- β-D- glucopyranosyls -3 β, 12 β, 20 (S, R), 25- tetrahydroxy dammarane;(yield 9.3%)
3-O- β-D- glucopyranosyls-(1 → 2) -3-O- β-D- glucopyranosyls -3 β, 12 β, 20 (S,
R), 25- tetrahydroxys dammarane;(yield 6.2%)
Glycol II groups:
3 β, 12 β, 25- trihydroxies reach agate 20 (22) alkene;(yield 7.6%)
The β of 3-O- β-D- glucopyranosyls -3,12 β, 25- trihydroxies reach agate 20 (22) alkene;(yield 6.5%)
3-O- β-D- glucopyranosyls-(1 → 2) -3-O- β-D- glucopyranosyls -3 β, 12 β, the hydroxyls of 25- tri-
Ji Dama 20 (22) alkene;(yield 8.4%)
Glycol III groups:
20-(S,R)-Rg3;(yield 7.2%)
20-(S,R)-Rh2;(yield 6.3%)
20-(S,R)-PPD;(yield 5.2%)
Panoxadiol;(yield 4.5%)
Glycol IV groups:
Rg5;(yield 2.5%)
Rk3.(yield 1.7%)
Embodiment 4,
Ginsenoside compound K (it is a kind of diol type ginsenoside, ethylmercuric cloride thing Science and Technology Ltd., catalog number is
156162-10-6) (1g) is dissolved in 50ml 15% glutamic acid solution (containing 1% Tween 80), and 40 DEG C are reacted 24 hours, and 100 DEG C add
Heat is inactivated for 30 minutes, the HPLC analysis shows of converted product, conversion ratio 45%, and product is mainly that principal product is Rg3, Rh2,25-
OH- dammarane and 25-OH- reach agate alkene rare ginsenoside and aglycon.Add 500mL water and suspension is presented, centrifugation is molten with methanol
Solution precipitation, takes supernatant to be evaporated.Thing is evaporated with after 30% ethanol rinse removal of impurities, is evaporated.Methanol dissolves, inverted to prepare post progress
Separation (mobile phase is 65% ethanol) respectively obtains Rg3, and Rh2,25-OH- reaches agate alkene rare ginsenoside and aglycon.
I group of glycol:
3 β, 12 β, 20 (S, R), 25- tetrahydroxy dammarane;(yield 4.4%)
3-O- β-D- glucopyranosyls -3 β, 12 β, 20 (S, R), 25- tetrahydroxy dammarane;(yield 3.9%)
3-O- β-D- glucopyranosyls-(1 → 2) -3-O- β-D- glucopyranosyls -3 β, 12 β, 20 (S,
R), 25- tetrahydroxys dammarane;(yield 5.6%)
Glycol II groups:
3 β, 12 β, 25- trihydroxies reach agate 20 (22) alkene;(yield 4.8%)
The β of 3-O- β-D- glucopyranosyls -3,12 β, 25- trihydroxies reach agate 20 (22) alkene;(yield 3.7%)
3-O- β-D- glucopyranosyls-(1 → 2) -3-O- β-D- glucopyranosyls -3 β, 12 β, the hydroxyls of 25- tri-
Ji Dama 20 (22) alkene;(yield 4.7%)
Glycol III groups:
20-(S,R)-Rg3;(yield 3.5%)
20-(S,R)-Rh2;(yield 2.1%)
20-(S,R)-PPD;(yield 5.8%)
Panoxadiol;(yield 1.3%)
Glycol IV groups:
Rg5;(yield 2.9%)
Rk3.(yield 2.3%)