CN108117558A - The method that Taide promise A and Taide promise B is split from fermented tea - Google Patents
The method that Taide promise A and Taide promise B is split from fermented tea Download PDFInfo
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- CN108117558A CN108117558A CN201611061352.3A CN201611061352A CN108117558A CN 108117558 A CN108117558 A CN 108117558A CN 201611061352 A CN201611061352 A CN 201611061352A CN 108117558 A CN108117558 A CN 108117558A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/04—Ortho-condensed systems
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
- A23F3/00—Tea; Tea substitutes; Preparations thereof
- A23F3/06—Treating tea before extraction; Preparations produced thereby
- A23F3/08—Oxidation; Fermentation
- A23F3/10—Fermentation with addition of microorganisms or enzymes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B57/00—Separation of optically-active compounds
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
Abstract
The present invention provides a kind of methods that Taide promise A and Taide promise B is split from fermented tea, comprise the following steps：A) fermented tea containing Taide promise A and Taide promise B is provided；B the fermented tea) is subjected to boiling water extraction or organic solvent extracts, to obtain leaching liquor；C) leaching liquor is extracted with organic solvent, to obtain the extract containing Taide promise A and Taide promise B；D silica gel post separation) is carried out to the extract, to obtain crude separation object；And E) macroreticular resin post separation is carried out to the crude separation object, wherein being eluted using 5% volume to the ethyl alcohol or methanol aqueous solution for being less than 20% volume, eluent is collected, so as to obtain Taide promise A.The method of the present invention can obtain purity high Taide promise A and Taide promise B, and easy to operate, and at low cost, step is few, is easy to mass production.
Description
Technical field
The invention belongs to technical field of chemistry, in particular to the fractionation Taide promise A and Taide promise B from fermented tea
Method.
Background technology
Containing in Camellia Plants tea and its converted products such as green tea, black tea, Pu'er tea has good biological activity
Catechin compounds, nutgall catechin 3-O- gallic acids (EGCG) such as isolated from green tea, from black tea point
The theaflavins ingredient separated out is respectively provided with good anti-oxidant, anti-aging isoreactivity.In addition, also containing perhaps in tea product
Have lipid-loweringing, antibacterial, anticancer catechin-derived object biotic component, be all agricultural and medical research hot spot.
Taide promise A (Teadenol A) and Taide promise B (Teadenol B) is white powder, and molecular formula is
C14H12O6, accurate molecular weight 276.0638, structural formula is respectively as shown in following formula (I) and (II)：
Taide promise A and Taide promise B is initially by Japanese scholars catechin derivative isolated from fermented tea, tool
There are significant weight-reducing and blood fat reducing function.But still lack Taide promise is effectively split and separated from fermented tea in the prior art
The method of A and Taide promise B.
The content of the invention
For solve it is above-mentioned the problems of in the prior art, the present invention provides split from fermented tea Taide promise A and
The method of Taide promise B.
Specifically, the present invention provides：
(1) a kind of method that Taide promise A and Taide promise B is split from fermented tea, the structural formula such as following formula of the Taide promise A
(I) shown in, shown in the structural formula such as following formula (II) of Taide promise B：
Wherein it the described method comprises the following steps：
A) fermented tea containing Taide promise A and Taide promise B is provided；
B the fermented tea) is subjected to boiling water extraction or organic solvent extracts, to obtain leaching liquor；
C) leaching liquor is extracted with organic solvent, to obtain the extract containing Taide promise A and Taide promise B；
D silica gel post separation) is carried out to the extract, to obtain crude separation object；With
E macroreticular resin post separation) is carried out to the crude separation object, wherein using 5% volume to the second for being less than 20% volume
Alcohol or methanol aqueous solution are eluted, and eluent are collected, so as to obtain Taide promise A.
(2) according to the method described in (1), wherein the method further includes F) utilize the ethyl alcohol of 20% volume to 60% volume
Or methanol aqueous solution to pass through step E) processing macroporous resin column elute, collect eluent, so as to obtain Taide promise B.
(3) method according to (1) or (2), wherein in step D) in, volume ratio is used as (10-30):1 chloroform:
Meoh eluate is eluted, to obtain the crude separation object.
(4) method according to (1) or (2), wherein in step D) in, the silicagel column is the silica gel of 200-300 mesh
Column.
(5) method according to (1) or (2), wherein in step B) in, the boiling water extraction or organic solvent extraction exist
It is carried out 2-4 times under the ultrasonic wave of 30000-50000HZ, it is 10-50 minutes each.
(6) method according to (1) or (2), wherein in step B) in, the mass ratio by the fermented tea and boiling water is
1:The ratio of (1-3) mixes the fermented tea and boiling water, to carry out the boiling water extraction；The temperature of wherein described boiling water is 80 DEG C
To 100 DEG C.
(7) according to the method described in (1) or (2), wherein in step B) in, by the fermented tea and the organic solvent
Mass ratio is 1:The ratio of (1-3) mixes the fermented tea and the organic solvent, to carry out the organic solvent extraction；Wherein
The organic solvent is selected from methanol, ethyl alcohol and acetone.
(8) method according to (1) or (2), wherein step C) include：
C1) leaching liquor is extracted with 1,2- dichloroethanes or chloroform, to obtain water phase and organic phase；Wherein institute
The volume ratio for stating 1,2- dichloroethanes or chloroform and the leaching liquor is 1:(1-3)；
C2) with ethyl acetate to step C1) obtained water mutually extracts, to obtain described containing Taide promise A and Taide
The extract of promise B；The volume ratio of wherein described ethyl acetate and the water phase is 1:(1-3).
(9) method according to (1), wherein the method further include G) to isolated through the macroporous resin column
Promise A in Taide carries out high performance liquid chromatography (HPLC) and purifies, wherein the HPLC uses C-18 columns, mobile phase is methanol and the body of water
Product is than being 11:9 to 3:7 methanol/water mixed solution.
(10) method according to (2), the method further include H) to through the isolated Thailand of the macroporous resin column
De-Nol B carries out HPLC purifying, wherein the HPLC uses C-18 columns, mobile phase is that the volume ratio of methanol and water is 1:1 to 3:7
Methanol/water mixed solution.
(11) method according to (1) or (2), wherein step A) include gross tea tidewater to water content being based on gross tea
Dry weight is calculated as being more than 40 weight % to 65 weight %, gross tea of the gained through tidewater is sterilized, then inoculated aspergillus niger single culture,
The fermented tea containing Taide promise A and Taide promise B is obtained after fermented culture.
(12) method according to (11), wherein the fermented and cultured carries out 10-25 days at 28-40 DEG C.
(13) method according to (11), wherein the gross tea is selected from solar dried green tea, hair green tea, hair white tea, hair yellow tea
And Mao Qingcha.
The present invention has the advantages that compared with prior art：
1. it is easy to operate present invention optimizes being split from fermented tea and separate the technique and condition of Taide promise A and B, into
This is low, and step is few, is easy to mass production, the purity of the Taide promise A and Taide promise B of acquisition are high, and used organic reagent is easy to
Recycling.
2. further, present invention optimizes the techniques and condition with microbial fermentation tea so that containing more in fermented tea
High Taide promise A and Taide promise B, so as to further be conducive to the high product of isolated purity.
Biomaterial preservation information
Aspergillus niger (Aspergillus niger) CGMCC NO.12763 bacterial strains of the present invention are July 5 in 2016
Day is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address：Beijing's southern exposure
The institute 3 of area North Star West Road 1, postcode：100101, deposit number is：CGMCC NO.12763.
Specific embodiment
The following describes the present invention further through the description of specific embodiments, but this is not the limit to the present invention
System, those skilled in the art's basic thought according to the present invention can make various modifications or improvements, but without departing from this
The basic thought of invention, is all within the scope of the present invention.
The present inventor is groped by substantial amounts of theoretical research and experiment, is optimized and is split and separate from fermented tea
The technique and condition of Taide promise A and B so that the product purity of acquisition is high, and technological operation is simple, is easy to the big production of technique.
Specifically, the present invention provides a kind of method that Taide promise A and Taide promise B is split from fermented tea, the Thailands
Shown in the structural formula of De-Nol A such as following formula (I), shown in the structural formula such as following formula (II) of Taide promise B：
Wherein it the described method comprises the following steps：
A) fermented tea containing Taide promise A and Taide promise B is provided；
B the fermented tea) is subjected to boiling water extraction or organic solvent extracts, to obtain leaching liquor；
C) leaching liquor is extracted with organic solvent, to obtain the extract containing Taide promise A and Taide promise B；
D silica gel post separation) is carried out to the extract, to obtain crude separation object；With
E macroreticular resin post separation) is carried out to the crude separation object, wherein using 5 volume % to the second for being less than 20 volume %
Alcohol or methanol are eluted, and eluent are collected, so as to obtain Taide promise A.
The present invention realizes the fractionation of Taide promise A and B contained in fermented tea as a result,.
For further isolated Taide promise B, it is preferable that the method further includes F) utilize 20 volume % to 60 bodies
Product % ethyl alcohol or methanol to pass through step E) processing macroporous resin column elute, collect eluent, so as to obtain Taide
Promise B.
Pass through nuclear magnetic resonance C spectrums, H spectrums, DEPT spectrums, HMBC, HMQC and TOCOSY spectrum analysis, it was demonstrated that the Thailand that the present invention obtains
De-Nol A and Taide promise B is identical with Taide promise A (Teadenol A) and Taide promise B (Teadenol B) that Japanese scholars are found.
It will be appreciated by persons skilled in the art that in step A) and step B) between, it can also increase the fermentation
The broken step of tea powder.Wherein, preferably it is crushed to 30-60 mesh.
The present inventor further optimizes the process conditions of the method for the present invention.
In step E) in, preferably elute 2-3 times of pillar volume；Most preferably eluted using the ethyl alcohol of 10 volume %.
Step F) in, preferably elute 2-3 times of pillar volume；Most preferably eluted using the ethyl alcohol of 20 volume %.
Preferably, in step D) in, volume ratio is used as (10-30):1, more preferable 15:1 chloroform:Meoh eluate into
Row elution, to obtain the crude separation object.It is further preferred that the silicagel column is the silicagel column of 200-300 mesh.Preferably implementing
In scheme, first with above-mentioned 1-1.5 times of pillar volume of elution to remove impurity, 2-3 times of pillar volume of elution is then proceeded to,
So as to obtain the crude separation object.It is concentrated it will be appreciated by persons skilled in the art that obtained crude separation object can be merged.
In step B) in, boiling water extraction or organic solvent extraction preferably under the ultrasonic wave of 30000-50000HZ into
Row 2-4 times, it is 10-50 minutes each, more preferably carry out 3 times, every time 30 minutes.
It is 1 preferably by the mass ratio of the fermented tea and boiling water when carrying out boiling water extraction:The ratio mixing institute of (1-3)
Fermented tea and boiling water are stated, which is more preferably 1:3.
In the present invention, boiling water preferably refers to the water that temperature is 80 DEG C to 100 DEG C.
It is 1 by the mass ratio of the fermented tea and the organic solvent when carrying out organic solvent extraction:The ratio of (1-3)
Example mixes the fermented tea and the organic solvent, the ratio are more preferably 1:3.This step organic solvent used is preferably selected from first
Alcohol, ethyl alcohol and acetone.
Preferably, the step C) include：
C1) leaching liquor is extracted with 1,2- dichloroethanes or chloroform and (is preferably extracted 3 times), with obtain water phase and
Organic phase；The volume ratio of wherein described 1,2- dichloroethanes or chloroform and the leaching liquor is 1:(1-3), more preferably 1:1；With
C2) with ethyl acetate to step C1) obtained water mutually extracted and (preferably taken 3 times), and it is described containing Thailand to obtain
The extract of De-Nol A and Taide promise B；The volume ratio of wherein described ethyl acetate and the water phase is 1:(1-3), more preferably 1:
1。
In a specific embodiment of the invention, step C) include, by step B) in each leaching liquor mistake obtained
Simultaneously merging filtrate is filtered, the volume ratio that 1,2- dichloroethanes and filtrate are pressed into filtrate is 1:1 addition 1,2- dichloroethanes is extracted
It takes, coextraction 3 times obtains water phase and organic phase；Gained water mutually continues with ethyl acetate by volume 1:1 is extracted, co-extraction
It takes 3 times, merge organic phase and concentrates, obtain the extract containing Taide promise A and Taide promise B.
Preferably, method of the invention further includes G) to being carried out through the macroporous resin column isolated Taide promise A and B
High performance liquid chromatography (HPLC) purifies, wherein the HPLC uses C-18 columns；For Taide promise A, mobile phase is methanol and water
Volume ratio is 11:9 to 3:7th, more preferable 5:5 methanol/water mixed solution；For Taide promise B, mobile phase is that volume ratio is 1:1
To 3:7th, more preferable 4:6 methanol/water mixed solution.
The present invention also further optimizes the technique and condition with microbial fermentation tea so that containing higher in fermented tea
Taide promise A and Taide promise B.
Preferably, step A) include gross tea tidewater to water content being to be calculated as being more than 40 weight % to 65 based on gross tea dry weight
Weight % sterilizes gross tea of the gained through tidewater, and then inoculated aspergillus niger single culture, obtains after fermented culture containing Taide
The fermented tea of promise A and Taide promise B.
The term as used herein " tidewater " refers to artificially add water to tealeaves certain water content.
The term as used herein " solar dried green tea " and " gross tea " are the essential terms of this field, those skilled in the art
Understand and know the meaning of these terms, therefore do not repeat.
Preferably, it is that 50 weight % are calculated as to 60 weight % based on dry weight of tea leaves by gross tea tidewater to water content.This is aqueous
Amount makes the content highest of the Taide promise A and Taide promise B of gained fermented tea.
It preferably, can be to be greater than or equal to 105A spore amount is inoculated with the aspergillus niger strain, such as with 105The order of magnitude
Spore amount inoculation.
The fermented and cultured carries out 10-25 days preferably at 28-40 DEG C.The fermentation temperature is more preferably 30-37 DEG C (more
It is preferred that 35-37 DEG C), in this temperature range, Aspergillus Niger Growth is the most vigorous.The fermentation time is more preferably 15-20 days,
Fermentation is complete in this time, and beneficiating ingredient aggregation is more.
Preferably, sterilize and carried out at 110-121 DEG C 10-30 minutes, preferably carried out 15-20 minutes.Sterilization time is too short
It is easy to cause sterilizing to be not thorough, overlong time is easily destroyed the beneficiating ingredient in tealeaves.
Preferably, during fermented and cultured, at the 3-10 days by tealeaves and thalline mixing once.
Preferably, after fermented and cultured, gained fermented tea is dried at 40-60 DEG C.It is preferred that it is low to be dried to water content
In being calculated as 10% based on dry weight of tea leaves.
In the method for the invention, aspergillus niger used can be commercially available, and be preferred from conventional Pu'er tea pile-fermentation,
Gained, safety and sanitation are secure after isolation and purification culture.Black spore is also easy to produce during the Aspergillus Niger Growth, in 20-
Well-grown at 40 DEG C.The aspergillus niger can use and be preserved in Chinese microorganism strain preservation pipe on July 5th, 2016
Reason committee common micro-organisms center (CGMCC, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), the deposit number of preservation are
Aspergillus niger (Aspergillus niger) bacterial strain of CGMCC NO.12763.Preferably, aspergillus niger strain used in the present invention comes from
Single bacterial strain is, for example, the CGMCC NO.12763 bacterial strains.
Aspergillus niger strain can provide by the following method：It is to be calculated as based on gross tea dry weight by gross tea tidewater to water content
10-30 weight %, then sterilize, so as to provide bacterium culture medium；Aspergillus niger is inoculated in the bacterium culture medium, cultivates 3-7
My god (can also treat that bacterium culture medium is completely covered in spore), so as to provide aspergillus niger strain.Wherein, the inoculum concentration of aspergillus niger can be with
To be greater than or equal to 105A spore amount, such as with 105The spore amount inoculation of the order of magnitude.Sterilizing can carry out at 115-121 DEG C
10-30 minutes.It is preferred that the, it is that 20-30 weight % are calculated as based on dry weight of tea leaves by gross tea tidewater to water content, this is more advantageous to black
The growth of aspergillus and generation spore.Incubation time is preferably 4-5 days, and the vigor of such spore is best.
The gross tea is preferably selected from solar dried green tea, hair green tea, hair white tea, hair yellow tea and Mao Qingcha, wherein most preferably sun withering
Gross tea.
The fermentation method of the method for the present invention includes but not limited to triangular flask fermentation, the fermentation of tray formula, fermentation tank fermentation, pot type
Fermentation.
The present invention also provides by method of the present invention isolated Taide promise A and Taide promise B.
Present disclosure is further explained and described below by way of the mode of example, but these examples are understood not to
Limitation to protection scope of the present invention.
Embodiment
The source of embodiment material therefor and instrument is as follows：
Aspergillus niger derives from Beijing institute of microbiology of the Chinese Academy of Sciences
Silicagel column derives from Linyi Hai Xiang chemical companies, 200-300 mesh
Macroreticular resin derives from Mitsubishi, model HP20
Dubhe C-18 columns derive from Han Bang companies
High performance liquid chromatography derives from Waters, model 2695
Embodiment one
A, fermenting raw materials：1kg solar dried green teas are weighed, tidewater to the water content based on dry weight is 55 weight %, and 115 DEG C sterilize
15 minutes, by inoculum concentration 2 × 105Spore amount access aspergillus niger, 35 DEG C cultivate 15 days, take out tealeaves, under conditions of 50 DEG C
It is dried to water content 8%；
B, crush：Tealeaves after fermentation is crushed to 50 mesh；
C, boiling water extracts：It is 1 that the tealeaves of crushing, which is pressed with the mass ratio of boiling water,:3 add in 90 DEG C of boiling water, and 40000HZ surpasses
Sound extraction 3 times, 30 minutes every time, merging filtrate after filtering；
D, 1,2- dichloroethanes extracts：By the filtrate in above-mentioned step C by volume 1:1, which adds in 1,2- dichloroethanes, carries out
Extraction, total coextraction 3 times leave water phase, organic phase recycling；
E, ethyl acetate extracts：Water in above-mentioned D steps is mutually continued with ethyl acetate by volume 1:1 is extracted,
Always coextraction 3 times, takes organic phase, is concentrated after merging, obtains ethyl acetate extract 113g；
F, silica gel post separation：Silicagel column on ethyl acetate extract will be obtained in above-mentioned E steps to separate, first with chlorine
It is imitative:Methanol=15:1 (volume ratio) elutes 1 pillar volume, then elutes 2 times of pillar volumes with this volume ratio again, collects this 2 times
The eluent of pillar volume obtains silica gel crude separation object 25.2g after concentration is dry；
G, macroreticular resin separates：The silica gel crude separation object obtained in above-mentioned F-step is separated through macroreticular resin, successively with
10% ethyl alcohol, 20% ethyl alcohol, 100% ethanol elution, each 2.5 times of pillar volumes of gradient elution collect 10% ethyl alcohol and 20%
The eluent of ethyl alcohol obtains smart isolate R1 12.5g (representing 10% ethanol elution) after concentration is dry, and R2 3.8g (represent 20%
Ethanol elution)；
H, post separation is prepared：Smart isolate in above-mentioned G steps is analyzed with HPLC, the wherein condition of HPLC is：Pillar
Column is prepared with Dubhe C-18, is 5 for the volume ratio of R1 methanol and water:5 methanol/water mixed solution as mobile phase,
It is 4 for the volume ratio of R2 methanol and water:For 6 methanol/water mixed solution as mobile phase, flow velocity is 60ml/ minutes.It
Column is prepared with Dubhe C-18 afterwards R1 and R2 is further purified, mobile phase and HPLC analyze used in it is identical.Thus prepare
Taide promise A and Taide promise B is obtained, the Taide promise A 9.47g that purity is 98.25% are obtained after concentration, it is 97.55% to obtain purity
Taide promise B 2.24g.
By magnetic resonance detection, confirmation products therefrom is Taide promise A and Taide promise B.Nuclear magnetic resonance result is as follows：
1. Taide promise A's of table1H、13Analysis result (the solvent of C NMR datas and 2D NMR：Deuterated dimethyl sulfoxide (DMSO-
d6))
2 Taide promise B's of table1H、13Analysis result (the solvent of C NMR datas and 2D NMR：Deuterated dimethyl sulfoxide (DMSO-
d6))
Embodiment two
A, fermenting raw materials：1kg solar dried green teas are weighed, tidewater to the water content based on dry weight is 60%, 115 15 points of sterilizings
Clock, by inoculum concentration 2 × 105Spore amount access aspergillus niger, 35 DEG C cultivate 15 days, take out tealeaves, dried under conditions of 50 DEG C
To water content 8%；
B, crush：Tealeaves after fermentation is crushed to 50 mesh；
C, boiling water extracts：It is 1 that the tealeaves of crushing, which is pressed with the mass ratio of boiling water,:3 add in 90 DEG C of boiling water, and 30000HZ surpasses
Sound extraction 3 times, 30 minutes every time, merging filtrate after filtering；
D, 1,2- dichloroethanes extracts：By the filtrate in above-mentioned step C by volume 1:1 adds in 1,2- dichloroethanes extraction
It takes, total coextraction 3 times, leaves water phase, organic phase recycling；
E, ethyl acetate extracts：Water in above-mentioned D steps is mutually continued with ethyl acetate by volume 1:1 is extracted,
Always coextraction 3 times, takes organic phase, is concentrated after merging, obtains ethyl acetate extract 118g；
F, silica gel post separation：Silicagel column on ethyl acetate extract will be obtained in above-mentioned E steps to separate, first with chlorine
It is imitative:Methanol=15:1 (volume ratio) elutes 1 pillar volume, then elutes 2 times of pillar volumes with this volume ratio again, collects this 2 times
The eluent of pillar volume obtains silica gel crude separation object 27.5g after concentration is dry；
G, macroreticular resin separates：The silica gel crude separation object obtained in above-mentioned F-step is separated through macroreticular resin, successively with
10% ethyl alcohol, 20% ethyl alcohol, 100% ethanol elution, each 2.5 times of pillar volumes of gradient elution collect 10% ethyl alcohol and 20%
The eluent of ethyl alcohol obtains smart isolate R1 13.2g (representing 10% ethanol elution) after concentration is dry, and R2 4.2g (represent 20%
Ethanol elution)；
H, post separation is prepared：Smart isolate in above-mentioned G steps is analyzed with HPLC, the wherein condition of HPLC is：Pillar
Column is prepared with Dubhe C-18, is 5 for the volume ratio of R1 methanol and water:5 methanol/water mixed solution as mobile phase,
It is 4 for the volume ratio of R2 methanol and water:For 6 methanol/water mixed solution as mobile phase, flow velocity is 60ml/ minutes.It
Column is prepared with Dubhe C-18 afterwards R1 and R2 is further purified, mobile phase and HPLC analyze used in it is identical.Thus prepare
Taide promise A and Taide promise B is obtained, the Taide promise A 10.53g that purity is 98.16% are obtained after concentration, obtaining purity is
97.52% Taide promise B 2.85g.By magnetic resonance detection (with embodiment 1), it is Taide promise A and Thailand to confirm products therefrom
De-Nol B.
Embodiment three
A, fermenting raw materials：1kg solar dried green teas are weighed, tidewater to the water content based on dry weight is 60%, 115 15 points of sterilizings
Clock, by inoculum concentration 2 × 105Spore amount access aspergillus niger, 35 DEG C cultivate 20 days, take out tealeaves, dried under conditions of 50 DEG C
To water content 8%；
B, crush：Tealeaves after fermentation is crushed to 50 mesh；
C, boiling water extracts：It is 1 that the tealeaves of crushing, which is pressed with the mass ratio of boiling water,:3 add in 90 DEG C of boiling water, and 50000HZ surpasses
Sound extraction 3 times, 30 minutes every time, merging filtrate after filtering；
D, 1,2- dichloroethanes extracts：By the filtrate in above-mentioned step C by volume 1:1 adds in 1,2- dichloroethanes extraction
It takes, total coextraction 3 times, leaves water phase, organic phase recycling；
E, ethyl acetate extracts：Water in above-mentioned D steps is mutually continued with ethyl acetate by volume 1:1 is extracted,
Always coextraction 3 times, takes organic phase, is concentrated after merging, obtains ethyl acetate extract 121g；
F, silica gel post separation：Silicagel column on ethyl acetate extract will be obtained in above-mentioned E steps to separate, first with chlorine
It is imitative:Methanol=15:1 (volume ratio) elutes 1 pillar volume, then elutes 2 times of pillar volumes with this volume ratio again, collects this 2 times
The eluent of pillar volume obtains silica gel crude separation object 28.6g after concentration is dry；
G, macroreticular resin separates：The silica gel crude separation object obtained in above-mentioned F-step is separated through macroreticular resin, successively with
10% ethyl alcohol, 20% ethyl alcohol, 100% ethanol elution, each 2.5 times of pillar volumes of gradient elution collect 10% ethyl alcohol and 20%
The eluent of ethyl alcohol obtains smart isolate R1 13.9g (representing 10% ethanol elution) after concentration is dry, and R2 4.6g (represent 20%
Ethanol elution)；
H, the smart isolate in above-mentioned G steps is analyzed with HPLC, the wherein condition of HPLC is：Pillar Dubhe C-18
Column is prepared, is 5 for the volume ratio of R1 methanol and water:5 methanol/water mixed solution is as mobile phase, for R2 methanol
Volume ratio with water is 4:For 6 methanol/water mixed solution as mobile phase, flow velocity is 60ml/ minutes.Dubhe C- are used afterwards
18, which prepare column, is further purified R1 and R2, mobile phase and HPLC analyze used in it is identical.Thus prepare Taide promise A and
Promise B in Taide obtains the Taide promise A 10.93g that purity is 98.08% after concentration, obtain the Taide promise B that purity is 97.46%
2.88g.By magnetic resonance detection (with embodiment 1), confirmation products therefrom is Taide promise A and Taide promise B.
Example IV
A, fermenting raw materials：1kg solar dried green teas are weighed, tidewater to the water content based on dry weight is 50%, 115 15 points of sterilizings
Clock, by inoculum concentration 2 × 105Spore amount access aspergillus niger, 35 DEG C cultivate 20 days, take out tealeaves, dried under conditions of 50 DEG C
To water content 8%；
B, crush：Tealeaves after fermentation is crushed to 50 mesh；
C, boiling water extracts：It is 1 that the tealeaves of crushing, which is pressed with the mass ratio of boiling water,:3 add in 90 DEG C of boiling water, and 40000HZ surpasses
Sound extraction 3 times, 30 minutes every time, merging filtrate after filtering；
D, 1,2- dichloroethanes extracts：By the filtrate in above-mentioned step C by volume 1:1 adds in 1,2- dichloroethanes extraction
It takes, total coextraction 3 times, leaves water phase, organic phase recycling；
E, ethyl acetate extracts：Water in above-mentioned D steps is mutually continued with ethyl acetate by volume 1:1 is extracted,
Always coextraction 3 times, takes organic phase, is concentrated after merging, obtains ethyl acetate extract 114g；
F, silica gel post separation：Silicagel column on ethyl acetate extract will be obtained in above-mentioned E steps to separate, first with chlorine
It is imitative:Methanol=15:1 (volume ratio) elutes 1 pillar volume, then elutes 2 times of pillar volumes with this volume ratio again, collects this 2 times
The eluent of pillar volume obtains silica gel crude separation object 25.5g after concentration is dry；
G, macroreticular resin separates：The silica gel crude separation object obtained in above-mentioned F-step is separated through macroreticular resin, successively with
10% ethyl alcohol, 20% ethyl alcohol, 100% ethanol elution, each 2.5 times of pillar volumes of gradient elution collect 10% ethyl alcohol and 20%
The eluent of ethyl alcohol obtains smart isolate R1 12.8g (representing 10% ethanol elution) after concentration is dry, and R2 4.2g (represent 20%
Ethanol elution)；
H, the smart isolate in above-mentioned G steps is analyzed with HPLC, the wherein condition of HPLC is：Pillar Dubhe C-18
Column is prepared, is 5 for the volume ratio of R1 methanol and water:5 methanol/water mixed solution is as mobile phase, for R2 methanol
Volume ratio with water is 4:For 6 methanol/water mixed solution as mobile phase, flow velocity is 60ml/ minutes.Dubhe C- are used afterwards
18, which prepare column, is further purified R1 and R2, mobile phase and HPLC analyze used in it is identical.Thus prepare Taide promise A and
Promise B in Taide obtains the Taide promise A 9.63g that purity is 98.36% after concentration, obtain the Taide promise B that purity is 97.82%
2.35g.By magnetic resonance detection (with embodiment 1), confirmation products therefrom is Taide promise A and Taide promise B.
Embodiment five
A, fermenting raw materials：1kg solar dried green teas are weighed, tidewater to the water content based on dry weight is 65%, 115 15 points of sterilizings
Clock, by inoculum concentration 2 × 105Spore amount access aspergillus niger, 35 DEG C cultivate 20 days, take out tealeaves, dried under conditions of 50 DEG C
To water content 8%；
B, crush：Tealeaves after fermentation is crushed to 50 mesh；
C, boiling water extracts：It is 1 that the tealeaves of crushing, which is pressed with the mass ratio of boiling water,:3 add in 90 DEG C of boiling water, and 40000HZ surpasses
Sound extraction 3 times, 30 minutes every time, merging filtrate after filtering；
D, 1,2- dichloroethanes extracts：By the filtrate in above-mentioned step C by volume 1:1 adds in 1,2- dichloroethanes extraction
It takes, total coextraction 3 times, leaves water phase, organic phase recycling；
E, ethyl acetate extracts：Water in above-mentioned D steps is mutually continued with ethyl acetate by volume 1:1 is extracted,
Always coextraction 3 times, takes organic phase, is concentrated after merging, obtains ethyl acetate extract 108g；
F, silica gel post separation：Silicagel column on ethyl acetate extract will be obtained in above-mentioned E steps to separate, first with chlorine
It is imitative:Methanol=15:1 (volume ratio) elutes 1 pillar volume, then elutes 2 times of pillar volumes with this volume ratio again, collects this 2 times
The eluent of pillar volume obtains silica gel crude separation object 23.5g after concentration is dry；
G, macroreticular resin separates：The silica gel crude separation object obtained in above-mentioned F-step is separated through macroreticular resin, successively with
10% ethyl alcohol, 20% ethyl alcohol, 100% ethanol elution, each 2.5 times of pillar volumes of gradient elution collect 10% ethyl alcohol and 20%
The eluent of ethyl alcohol obtains smart isolate R1 11.5g (representing 10% ethanol elution) after concentration is dry, and R2 2.8g (represent 20%
Ethanol elution)；
H, the smart isolate in above-mentioned G steps is analyzed with HPLC, the wherein condition of HPLC is：Pillar Dubhe C-18
Column is prepared, is 5 for the volume ratio of R1 methanol and water:5 methanol/water mixed solution is as mobile phase, for R2 methanol
Volume ratio with water is 4:For 6 methanol/water mixed solution as mobile phase, flow velocity is 60ml/ minutes.Dubhe C- are used afterwards
18, which prepare column, is further purified R1 and R2, mobile phase and HPLC analyze used in it is identical.Thus prepare Taide promise A and
Promise B in Taide obtains the Taide promise A 8.53g that purity is 97.56% after concentration, obtain the Taide promise B that purity is 97.22%
2.03g.By magnetic resonance detection (with embodiment 1), confirmation products therefrom is Taide promise A and Taide promise B.
Example below is carried out according to the method for embodiment 1, difference and evaluation result are listed in Table 3 below.
Table 3
Claims (10)
1. a kind of method that Taide promise A and Taide promise B is split from fermented tea, structural formula such as following formula (I) institute of the Taide promise A
Show, shown in the structural formula such as following formula (II) of Taide promise B：
Wherein it the described method comprises the following steps：
A) fermented tea containing Taide promise A and Taide promise B is provided；
B the fermented tea) is subjected to boiling water extraction or organic solvent extracts, to obtain leaching liquor；
C) leaching liquor is extracted with organic solvent, to obtain the extract containing Taide promise A and Taide promise B；
D silica gel post separation) is carried out to the extract, to obtain crude separation object；With
E) to the crude separation object carry out macroreticular resin post separation, wherein using 5% volume to be less than 20% volume ethyl alcohol or
Methanol aqueous solution is eluted, and eluent is collected, so as to obtain Taide promise A.
2. according to the method described in claim 1, wherein the method further includes F) utilize the second of 20% volume to 60% volume
Alcohol or methanol aqueous solution are to passing through step E) macroporous resin column of processing elutes, eluent is collected, so as to obtain Taide promise
B。
3. method according to claim 1 or 2, wherein in step D) in, volume ratio is used as (10-30):1 chloroform:
Meoh eluate is eluted, to obtain the crude separation object；Preferably, in step D) in, the silicagel column is 200-300 mesh
Silicagel column.
4. method according to claim 1 or 2, wherein in step B) in, the boiling water extraction or organic solvent extraction exist
It is carried out 2-4 times under the ultrasonic wave of 30000-50000HZ, it is 10-50 minutes each.
5. method according to claim 1 or 2, wherein in step B) in, the mass ratio by the fermented tea and boiling water is
1:The ratio of (1-3) mixes the fermented tea and boiling water, to carry out the boiling water extraction；The temperature of wherein described boiling water is 80 DEG C
To 100 DEG C.
6. method according to claim 1 or 2, wherein in step B) in, by the fermented tea and the organic solvent
Mass ratio is 1:The ratio of (1-3) mixes the fermented tea and the organic solvent, to carry out the organic solvent extraction；Wherein
The organic solvent is selected from methanol, ethyl alcohol and acetone.
7. method according to claim 1 or 2, wherein step C) include：
C1) leaching liquor is extracted with 1,2- dichloroethanes or chloroform, to obtain water phase and organic phase；Wherein described 1,
The volume ratio of 2- dichloroethanes or chloroform and the leaching liquor is 1:(1-3)；
C2) with ethyl acetate to step C1) obtained water mutually extracts, and it is described containing Taide promise A's and Taide promise B to obtain
Extract；The volume ratio of wherein described ethyl acetate and the water phase is 1:(1-3).
8. according to the method described in claim 1, wherein the method further includes G) to isolated through the macroporous resin column
Taide promise A carry out high performance liquid chromatography (HPLC) and purify, wherein the HPLC uses C-18 columns, mobile phase is methanol and water
Volume ratio is 11:9 to 3:7 methanol/water mixed solution.
9. according to the method described in claim 2, the method further includes H) to through the isolated Thailand of the macroporous resin column
De-Nol B carries out HPLC purifying, wherein the HPLC uses C-18 columns, mobile phase is that the volume ratio of methanol and water is 1:1 to 3:7
Methanol/water mixed solution.
10. method according to claim 1 or 2, wherein step A) include gross tea tidewater to water content being based on gross tea
Dry weight is calculated as being more than 40 weight % to 65 weight %, gross tea of the gained through tidewater is sterilized, then inoculated aspergillus niger single culture,
The fermented tea containing Taide promise A and Taide promise B is obtained after fermented culture；Preferably, the fermented and cultured at 28-40 DEG C into
Row 10-25 days；It is highly preferred that the gross tea is selected from solar dried green tea, hair green tea, hair white tea, hair yellow tea and Mao Qingcha.
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