CN108117558B - Method for separating teddenox A and teddenox B from fermented tea - Google Patents
Method for separating teddenox A and teddenox B from fermented tea Download PDFInfo
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- CN108117558B CN108117558B CN201611061352.3A CN201611061352A CN108117558B CN 108117558 B CN108117558 B CN 108117558B CN 201611061352 A CN201611061352 A CN 201611061352A CN 108117558 B CN108117558 B CN 108117558B
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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 invention provides a method for separating teddenox A and teddenox B from fermented tea, which comprises the following steps: A) providing fermented tea comprising teddenox a and teddenox B; B) extracting the fermented tea with boiled water or an organic solvent to obtain an extract; C) extracting the extract with an organic solvent to obtain an extract containing teddenox a and teddenox B; D) subjecting the extract to silica gel column separation to obtain a crude isolate; and E) carrying out macroporous resin column separation on the crude isolate, wherein 5-20% ethanol or methanol aqueous solution is adopted for elution, and an eluent is collected, so that the teddenox A is obtained. The method can obtain the teddenox A and the teddenox B with high purity, and has the advantages of simple operation, low cost, few steps and easy mass production.
Description
Technical Field
The invention belongs to the technical field of chemistry, and particularly relates to a method for separating tyldino A and tyldino B from fermented tea.
Background
Camellia plant tea and its processed products such as green tea, black tea, Pu her tea, etc. all contain catechin compounds with good biological activity, such as gallocatechin 3-O-gallic acid (EGCG) separated from green tea, and theaflavin components separated from black tea, all have good activities of resisting oxidation and aging, etc. Besides, tea products contain many biological components with lipid-lowering, antibacterial and anticancer catechin derivatives, which are hot spots in agricultural and medical research.
Taidenool A (Teadenol A) and Taidenool B (Teadenol B) are white powders, and molecular formula is C14H12O6The molecular weight is 276.0638, and the structural formula is shown as the following formula (I) and formula (II) respectively:
teddenox a and teddenox B are catechin derivatives originally separated from fermented tea by japanese scholars, and have remarkable weight-losing and blood lipid-lowering effects. However, there is still a lack in the prior art of an effective method for the resolution and isolation of tednor a and tednor B from fermented tea.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for separating tednor A and tednor B from fermented tea.
Specifically, the present invention provides:
(1) a method for resolving teddenox A and teddenox B from fermented tea, wherein the teddenox A is shown as a formula (I) in the specification, and the teddenox B is shown as a formula (II) in the specification:
wherein the method comprises the steps of:
A) providing fermented tea comprising teddenox a and teddenox B;
B) extracting the fermented tea with boiled water or an organic solvent to obtain an extract;
C) extracting the extract with an organic solvent to obtain an extract containing teddenox a and teddenox B;
D) subjecting the extract to silica gel column separation to obtain a crude isolate; and
E) and (3) carrying out macroporous resin column separation on the crude isolate, wherein ethanol or methanol water solution with the volume of 5% to less than 20% is adopted for elution, and collecting eluent, so that the teddenox A is obtained.
(2) The method according to (1), wherein the method further comprises F) eluting the macroporous resin column treated in the step E) with 20 to 60 volume% of ethanol or methanol aqueous solution, and collecting the eluent, thereby obtaining teddenox B.
(3) The method according to (1) or (2), wherein in step D), elution is carried out using a chloroform: methanol eluent at a volume ratio of (10-30):1 to obtain the crude isolate.
(4) The process according to (1) or (2), wherein in step D), the silica gel column is a 200-mesh 300-mesh silica gel column.
(5) The process according to (1) or (2), wherein in the step B), the extraction with boiling water or the extraction with an organic solvent is carried out 2 to 4 times each for 10 to 50 minutes under ultrasonic waves of 30000-50000 HZ.
(6) The method according to (1) or (2), wherein in the step B), the fermented tea and the boiled water are mixed according to the mass ratio of 1 (1-3) to the fermented tea to the boiled water so as to carry out the boiled water extraction; wherein the temperature of the boiled water is 80 ℃ to 100 ℃.
(7) The process according to (1) or (2), wherein in step B), the fermented tea and the organic solvent are mixed in a ratio of the fermented tea to the organic solvent of 1 (1-3) by mass to carry out the organic solvent extraction; wherein the organic solvent is selected from methanol, ethanol and acetone.
(8) The method according to (1) or (2), wherein step C) comprises:
C1) extracting the leach solution with 1, 2-dichloroethane or chloroform to obtain an aqueous phase and an organic phase; wherein the volume ratio of the 1, 2-dichloroethane or chloroform to the leaching solution is 1 (1-3);
C2) extracting the aqueous phase obtained in step C1) with ethyl acetate to obtain said extract containing tyldinol a and tyldinol B; wherein the volume ratio of the ethyl acetate to the water phase is 1 (1-3).
(9) The process of (1), wherein the process further comprises G) purifying the teddenox A separated by the macroporous resin column by High Performance Liquid Chromatography (HPLC), wherein the HPLC adopts a C-18 column, and the mobile phase is a methanol/water mixed solution with a volume ratio of methanol to water of 11:9 to 3: 7.
(10) The method according to (2), further comprising H) performing HPLC purification on the teddnoc B separated by the macroporous resin column, wherein the HPLC adopts a C-18 column, and a mobile phase is a methanol/water mixed solution with a volume ratio of methanol to water of 1:1 to 3: 7.
(11) The method according to (1) or (2), wherein the step A) comprises the steps of wetting raw tea to a water content of more than 40 to 65 wt% based on the dry weight of the raw tea, sterilizing the wet raw tea, inoculating a single strain of Aspergillus niger, and performing fermentation culture to obtain the fermented tea containing the teddenox A and the teddenox B.
(12) The method of (11), wherein the fermentation culture is carried out at 28-40 ℃ for 10-25 days.
(13) The method according to (11), wherein the raw tea is selected from sun-dried raw tea, raw green tea, raw white tea, raw yellow tea and raw oolong tea.
Compared with the prior art, the invention has the following advantages and positive effects:
1. the invention optimizes the process and conditions for separating and separating the teduo A and the teduo B from the fermented tea, has simple operation, low cost, few steps and easy mass production, and the obtained teduo A and the teduo B have high purity and the used organic reagent is easy to recycle.
2. Further, the invention optimizes the process and conditions for fermenting the tea by using the microorganisms, so that the fermented tea contains higher teddenox A and teddenox B, thereby further facilitating the separation to obtain a product with high purity.
Biological material preservation information
The Aspergillus niger (Aspergillus niger) CGMCC NO.12763 strain of the invention is preserved in China general microbiological culture Collection center (CGMCC) in 2016, 7 and 5 days, and the preservation address is as follows: west road No.1, north chen, chaoyang district, beijing, zip code: 100101, accession number: CGMCC NO. 12763.
Detailed Description
The present invention is further described in the following description of the specific embodiments, which is not intended to limit the invention, but various modifications and improvements can be made by those skilled in the art according to the basic idea of the invention, within the scope of the invention, as long as they do not depart from the basic idea of the invention.
The inventor optimizes the process and conditions for separating and separating the tednor A and the tednor B from the fermented tea through a large amount of theoretical research and experimental groping, so that the obtained product has high purity, the process operation is simple, and the process is easy for large-scale production.
Specifically, the invention provides a method for separating tyldinonol A and tyldinonol B from fermented tea, wherein the structure formula of the tyldinonol A is shown as the following formula (I), and the structure formula of the tyldinonol B is shown as the following formula (II):
wherein the method comprises the steps of:
A) providing fermented tea comprising teddenox a and teddenox B;
B) extracting the fermented tea with boiled water or an organic solvent to obtain an extract;
C) extracting the extract with an organic solvent to obtain an extract containing teddenox a and teddenox B;
D) subjecting the extract to silica gel column separation to obtain a crude isolate; and
E) and (3) carrying out macroporous resin column separation on the crude isolate, wherein 5-20% of ethanol or methanol is adopted for elution, and collecting eluent, thereby obtaining the teddenox A.
Thus, the present invention realizes the resolution of tedunox a and B contained in fermented tea.
For further separation to obtain teddenox B, preferably, the method further comprises F) eluting the macroporous resin column treated in step E) with 20 to 60 vol% ethanol or methanol, and collecting the eluate, thereby obtaining teddenox B.
The analysis of nuclear magnetic resonance C spectrum, H spectrum, DEPT spectrum, HMBC, HMQC and TOCOSY spectrum proves that the teddenox A and the teddenox B obtained by the invention are the same as the teddenox A (Teadenol A) and the teddenox B (Teadenol B) found by Japanese scholars.
As will be appreciated by those skilled in the art, a step of pulverizing the fermented tea may be added between the step A) and the step B). Among them, it is preferably pulverized to 30 to 60 mesh.
The inventors of the present invention further optimized the process conditions of the process of the present invention.
In step E), preferably elution is 2-3 column volumes; most preferably, 10% by volume ethanol is used for elution. In step F), preferably 2 to 3 column volumes are eluted; most preferably 20% by volume ethanol is used for elution.
Preferably, in step D), elution is carried out with a chloroform: methanol eluent in a volume ratio of (10-30: 1, more preferably 15:1, to obtain the crude isolate. Also preferably, the silica gel column is a 200-mesh 300-mesh silica gel column. In a preferred embodiment, the crude isolate is obtained by eluting 1 to 1.5 column volumes with the above eluent to remove impurities and then continuing the elution 2 to 3 column volumes. It will be appreciated by those skilled in the art that the resulting crude isolates may be combined and concentrated.
In step B), the extraction with boiled water or the extraction with an organic solvent is preferably carried out 2 to 4 times each for 10 to 50 minutes, more preferably 3 times each for 30 minutes under ultrasonic waves of 30000-50000 HZ.
When the boiled water extraction is carried out, the fermented tea and the boiled water are preferably mixed according to the mass ratio of 1 (1-3) to the boiled water, and the ratio is more preferably 1: 3.
In the present invention, the boiled water preferably means water having a temperature of 80 to 100 ℃.
When the organic solvent extraction is carried out, the fermented tea and the organic solvent are mixed according to the mass ratio of 1 (1-3) of the fermented tea to the organic solvent, and the ratio is more preferably 1: 3. The organic solvent used in this step is preferably selected from methanol, ethanol and acetone.
Preferably, the step C) includes:
C1) extracting (preferably 3 times) the leach solution with 1, 2-dichloroethane or chloroform to obtain an aqueous phase and an organic phase; wherein the volume ratio of the 1, 2-dichloroethane or chloroform to the leaching solution is 1 (1-3), more preferably 1: 1; and
C2) extracting (preferably 3 times) the aqueous phase obtained in step C1) with ethyl acetate to obtain said extract containing tyldinol a and tyldinol B; wherein the volume ratio of the ethyl acetate to the water phase is 1 (1-3), and more preferably 1: 1.
In a specific embodiment of the invention, step C) comprises filtering the leaching solutions obtained in each of steps B) and combining the filtrates, adding 1, 2-dichloroethane to the filtrate in a 1:1 volume ratio of 1, 2-dichloroethane to the filtrate for extraction, extracting 3 times to obtain an aqueous phase and an organic phase; and continuously extracting the obtained water phase with ethyl acetate according to the volume ratio of 1:1 for 3 times, combining the organic phases and concentrating to obtain the extract containing the teddenox A and the teddenox B.
Preferably, the method of the present invention further comprises G) purifying the tedunox A and B separated by the macroporous resin column by High Performance Liquid Chromatography (HPLC), wherein the HPLC uses a C-18 column; for teddenox a, the mobile phase is a methanol/water mixed solution with a volume ratio of methanol to water of 11:9 to 3:7, more preferably 5: 5; for teddenox B, the mobile phase is a methanol/water mixed solution in a volume ratio of 1:1 to 3:7, more preferably 4: 6.
The invention further optimizes the process and conditions for fermenting the tea by using the microorganisms, so that the fermented tea contains higher teddenox A and teddenox B.
Preferably, the step A) comprises the steps of wetting the raw tea to a water content of more than 40 to 65 weight percent based on the dry weight of the raw tea, sterilizing the wet raw tea, inoculating a single aspergillus niger strain, and performing fermentation culture to obtain the fermented tea containing the teddenox A and the teddenox B.
The term "flush" as used herein means the artificial addition of water to tea leaves to a certain moisture content.
The terms "sun-dried raw tea" and "raw tea" are common terms in the art, and the meaning of these terms is understood and known by those skilled in the art, and thus, will not be described in detail.
Preferably, the raw tea is moisturized to a moisture content of 50 to 60% by weight based on the dry weight of the tea leaves. The water content is such that the resulting fermented tea has the highest content of tedno A and tedno B.
Preferably, it may be greater than or equal to 105Inoculating said species of Aspergillus niger at a spore count of, for example, 105Inoculation with spore amount of order of magnitude.
The fermentation culture is preferably carried out at 28-40 ℃ for 10-25 days. The fermentation temperature is more preferably 30-37 ℃ (more preferably 35-37 ℃), and the aspergillus niger grows most vigorously in the temperature interval. The fermentation time is more preferably 15-20 days, and the fermentation is completed in the time, so that the beneficial ingredients are more accumulated.
Preferably, the sterilization is carried out at 110-121 ℃ for 10-30 minutes, preferably 15-20 minutes. The incomplete sterilization is easily caused by too short sterilization time, and the beneficial components in the tea are easily damaged by too long sterilization time.
Preferably, the tea leaves and the thalli are mixed uniformly once in the fermentation culture process at 3-10 days.
Preferably, after the fermentation culture, the resulting fermented tea is dried at 40-60 ℃. Preferably to a moisture content of less than 10% based on the dry weight of the tea.
In the method, the aspergillus niger used can be commercially available, preferably from traditional Pu' er tea pile fermentation, and is obtained after separation, purification and culture, so that safety and sanitation are guaranteed. The black spores are easily generated in the growth process of the Aspergillus niger, and the Aspergillus niger grows well at the temperature of 20-40 ℃. The Aspergillus niger can be Aspergillus niger strain which has been preserved in China general microbiological culture Collection center (CGMCC, No. 3 of No.1 Xilu-Chen-Yang district in Beijing) in 2016 (7/5) and has a preservation number of CGMCC NO. 12763. Preferably, the Aspergillus niger species used in the present invention is derived from a single strain, such as the CGMCC NO.12763 strain.
The species aspergillus niger may be provided by the following method: wetting raw tea with water to a water content of 10-30 wt% based on the dry weight of the raw tea, and then sterilizing, thereby providing a strain medium; aspergillus niger is inoculated into the strain culture medium and cultured for 3-7 days (optionally until the spore completely covers the strain culture medium), thereby providing Aspergillus niger species. Wherein, the inoculation amount of the Aspergillus niger can be more than or equal to 105Number of spores, e.g. 105Inoculation with spore amount of order of magnitude. The sterilization may be carried out at 115 ℃ and 121 ℃ for 10-30 minutes. Preferably, the green tea is moisturized to a moisture content of 20 to 30% by weight based on the dry weight of the tea leaves, which is more conducive to the growth and sporulation of Aspergillus niger. The culture time is preferably 4-5 days, so that the activity of the spores is best.
The raw tea is preferably selected from sun-dried raw tea, green tea, white tea, yellow tea and green tea, wherein the sun-dried raw tea is most preferred.
The fermentation mode of the method of the invention includes but is not limited to triangular flask fermentation, tray fermentation, fermentation tank fermentation and tank fermentation.
The invention also provides tyldino A and tyldino B separated by the method.
The present invention will be further explained or illustrated below by way of examples, which should not be construed as limiting the scope of the invention.
Examples
The materials and instruments used in the examples were sourced as follows:
aspergillus niger was obtained from the institute for microorganisms, Beijing, of the Chinese academy of sciences
Silica gel column is available from Lin Yihai Xiang chemical company, 200-300 mesh
The macroporous resin was obtained from Mitsubishi corporation under the model number HP20
Dubhe C-18 column from Hanbang
High performance liquid chromatography is available from Waters under model number 2695
Example one
A. Fermenting raw materials: weighing 1kg of sun-dried green tea, wetting until the water content is 55 wt% based on dry weight, sterilizing at 115 deg.C for 15 min, and inoculating 2 × 105Inoculating Aspergillus niger, culturing at 35 deg.C for 15 days, taking out tea, and oven drying at 50 deg.C until water content is 8%;
B. crushing: crushing the fermented tea leaves to 50 meshes;
C. leaching with boiled water: adding 90 deg.C boiled water into pulverized tea at a mass ratio of 1:3, performing 40000HZ ultrasonic extraction for 3 times, each for 30 min, filtering, and mixing filtrates;
D. 1, 2-dichloroethane extraction: adding 1, 2-dichloroethane into the filtrate obtained in the step C according to the volume ratio of 1:1 for extraction, extracting for 3 times in total, leaving a water phase, and recovering an organic phase;
E. and (3) ethyl acetate extraction: continuously extracting the water phase obtained in the step D with ethyl acetate according to the volume ratio of 1:1 for 3 times, taking organic phases, combining and concentrating to obtain 113g of ethyl acetate extract;
F. silica gel column separation: separating the ethyl acetate extract obtained in the step E by a silica gel column, eluting the ethyl acetate extract by chloroform-methanol (volume ratio) of 15:1 for 1 column volume, eluting the ethyl acetate extract by the volume ratio for 2 column volumes, collecting the eluent with the 2 column volumes, and concentrating and drying the eluent to obtain a crude silica gel isolate 25.2 g;
G. separating by macroporous resin: separating the silica gel crude isolate obtained in the step F by macroporous resin, eluting by 10% ethanol, 20% ethanol and 100% ethanol in sequence, eluting by 2.5 times of the volume of the column in each gradient, collecting the eluent of the 10% ethanol and the 20% ethanol, concentrating and drying to obtain a fine isolate R112.5 g (which represents 10% ethanol elution) and R23.8g (which represents 20% ethanol elution);
H. separating a preparation column: and (3) analyzing the fine isolate in the step G by using HPLC, wherein the conditions of the HPLC are as follows: the column was prepared using Dubhe C-18, using a methanol/water mixed solution of methanol to water in a volume ratio of 5:5 for R1 and a methanol/water mixed solution of methanol to water in a volume ratio of 4:6 for R2 as mobile phases, all at a flow rate of 60 ml/min. R1 and R2 were then further purified using a Dubhe C-18 preparative column, the mobile phase being the same as that used for HPLC analysis. Thus, tyldinor A and tyldinor B were obtained, and 9.47g of tyldinor A having a purity of 98.25% and 2.24g of tyldinor B having a purity of 97.55% were obtained after concentration.
And confirming that the obtained products are tedunox A and tedunox B through nuclear magnetic resonance detection. The nuclear magnetic resonance results were as follows:
TABLE 1 of teddenono A1H、13C NMR data and 2D NMR analysis results (solvent: deuterated dimethyl sulfoxide (DMSO-D)6))
TABLE 2 of teddenox B1H、13C NMR data and 2D NMR analysis results (solvent: deuterated dimethyl sulfoxide (DMSO-D)6))
Example two
A. Fermenting raw materials: weighing 1kg of sun-dried green tea, wetting until the water content is 60% based on dry weight, sterilizing for 15 min at 115, and inoculating 2 × 105The amount of spores inoculated into blackCulturing Aspergillus at 35 deg.C for 15 days, taking out tea, and oven drying at 50 deg.C until water content is 8%;
B. crushing: crushing the fermented tea leaves to 50 meshes;
C. leaching with boiled water: adding 90 deg.C boiled water into pulverized folium Camelliae sinensis at a mass ratio of 1:3, ultrasonic extracting at 30000HZ for 3 times, each for 30 min, filtering, and mixing filtrates;
D. 1, 2-dichloroethane extraction: adding 1, 2-dichloroethane into the filtrate obtained in the step C according to the volume ratio of 1:1 for extraction, extracting for 3 times in total, leaving a water phase, and recovering an organic phase;
E. and (3) ethyl acetate extraction: continuously extracting the water phase obtained in the step D with ethyl acetate according to the volume ratio of 1:1 for 3 times, taking organic phases, combining and concentrating to obtain 118g of ethyl acetate extract;
F. silica gel column separation: separating the ethyl acetate extract obtained in the step E by a silica gel column, eluting the ethyl acetate extract by chloroform-methanol (volume ratio) of 15:1 for 1 column volume, eluting the ethyl acetate extract by the volume ratio for 2 column volumes, collecting the eluent with the 2 column volumes, and concentrating and drying the eluent to obtain a crude silica gel isolate 27.5 g;
G. separating by macroporous resin: separating the silica gel crude isolate obtained in the step F by macroporous resin, eluting by 10% ethanol, 20% ethanol and 100% ethanol in sequence, eluting by 2.5 times of the volume of each gradient, collecting the eluent of 10% ethanol and 20% ethanol, concentrating and drying to obtain a fine isolate R113.2g (which represents 10% ethanol elution) and R24.2g (which represents 20% ethanol elution);
H. separating a preparation column: and (3) analyzing the fine isolate in the step G by using HPLC, wherein the conditions of the HPLC are as follows: the column was prepared using Dubhe C-18, using a methanol/water mixed solution of methanol to water in a volume ratio of 5:5 for R1 and a methanol/water mixed solution of methanol to water in a volume ratio of 4:6 for R2 as mobile phases, all at a flow rate of 60 ml/min. R1 and R2 were then further purified using a Dubhe C-18 preparative column, the mobile phase being the same as that used for HPLC analysis. Thus, tyldinor A and tyldinor B were obtained, and after concentration, 10.53g of tyldinor A having a purity of 98.16% and 2.85g of tyldinor B having a purity of 97.52% were obtained. The products obtained were identified as tedno A and tedno B by NMR examination (same as example 1).
EXAMPLE III
A. Fermenting raw materials: weighing 1kg of sun-dried green tea, wetting until the water content is 60% based on dry weight, sterilizing for 15 min at 115, and inoculating 2 × 105Inoculating Aspergillus niger, culturing at 35 deg.C for 20 days, taking out tea, and oven drying at 50 deg.C until water content is 8%;
B. crushing: crushing the fermented tea leaves to 50 meshes;
C. leaching with boiled water: adding 90 deg.C boiled water into pulverized folium Camelliae sinensis at a mass ratio of 1:3, ultrasonic extracting at 50000HZ for 3 times, each for 30 min, filtering, and mixing filtrates;
D. 1, 2-dichloroethane extraction: adding 1, 2-dichloroethane into the filtrate obtained in the step C according to the volume ratio of 1:1 for extraction, extracting for 3 times in total, leaving a water phase, and recovering an organic phase;
E. and (3) ethyl acetate extraction: continuously extracting the water phase obtained in the step D with ethyl acetate according to the volume ratio of 1:1 for 3 times, taking organic phases, combining and concentrating to obtain 121g of ethyl acetate extract;
F. silica gel column separation: separating the ethyl acetate extract obtained in the step E by a silica gel column, eluting the ethyl acetate extract by chloroform-methanol (volume ratio) of 15:1 for 1 column volume, eluting the ethyl acetate extract by the volume ratio for 2 column volumes, collecting the eluent with the 2 column volumes, and concentrating and drying the eluent to obtain a silica gel crude isolate 28.6 g;
G. separating by macroporous resin: separating the silica gel crude isolate obtained in the step F by macroporous resin, eluting by 10% ethanol, 20% ethanol and 100% ethanol in sequence, eluting by 2.5 times of the volume of the column in each gradient, collecting the eluent of the 10% ethanol and the 20% ethanol, concentrating and drying to obtain a fine isolate R113.9g (which represents 10% ethanol elution) and R24.6g (which represents 20% ethanol elution);
H. and (3) analyzing the fine isolate in the step G by using HPLC, wherein the conditions of the HPLC are as follows: the column was prepared using Dubhe C-18, using a methanol/water mixed solution of methanol to water in a volume ratio of 5:5 for R1 and a methanol/water mixed solution of methanol to water in a volume ratio of 4:6 for R2 as mobile phases, all at a flow rate of 60 ml/min. R1 and R2 were then further purified using a Dubhe C-18 preparative column, the mobile phase being the same as that used for HPLC analysis. Thus, tyldinor A and tyldinor B were obtained, and after concentration, 10.93g of tyldinor A with a purity of 98.08% and 2.88g of tyldinor B with a purity of 97.46% were obtained. The products obtained were identified as tedno A and tedno B by NMR examination (same as example 1).
Example four
A. Fermenting raw materials: weighing 1kg of sun-dried green tea, wetting until the water content is 50% based on dry weight, sterilizing for 15 min at 115, and inoculating 2 × 105Inoculating Aspergillus niger, culturing at 35 deg.C for 20 days, taking out tea, and oven drying at 50 deg.C until water content is 8%;
B. crushing: crushing the fermented tea leaves to 50 meshes;
C. leaching with boiled water: adding 90 deg.C boiled water into pulverized tea at a mass ratio of 1:3, performing 40000HZ ultrasonic extraction for 3 times, each for 30 min, filtering, and mixing filtrates;
D. 1, 2-dichloroethane extraction: adding 1, 2-dichloroethane into the filtrate obtained in the step C according to the volume ratio of 1:1 for extraction, extracting for 3 times in total, leaving a water phase, and recovering an organic phase;
E. and (3) ethyl acetate extraction: continuously extracting the water phase obtained in the step D with ethyl acetate according to the volume ratio of 1:1 for 3 times, taking organic phases, combining and concentrating to obtain 114g of ethyl acetate extract;
F. silica gel column separation: separating the ethyl acetate extract obtained in the step E by a silica gel column, eluting the ethyl acetate extract by chloroform-methanol (volume ratio) of 15:1 for 1 column volume, eluting the ethyl acetate extract by the volume ratio for 2 column volumes, collecting the eluent with the 2 column volumes, and concentrating and drying the eluent to obtain a crude silica gel isolate 25.5 g;
G. separating by macroporous resin: separating the silica gel crude isolate obtained in the step F by macroporous resin, eluting by 10% ethanol, 20% ethanol and 100% ethanol in sequence, eluting by 2.5 times of the volume of the column in each gradient, collecting the eluent of 10% ethanol and 20% ethanol, concentrating and drying to obtain a fine isolate R112.8g (representing 10% ethanol elution) and R24.2g (representing 20% ethanol elution);
H. and (3) analyzing the fine isolate in the step G by using HPLC, wherein the conditions of the HPLC are as follows: the column was prepared using Dubhe C-18, using a methanol/water mixed solution of methanol to water in a volume ratio of 5:5 for R1 and a methanol/water mixed solution of methanol to water in a volume ratio of 4:6 for R2 as mobile phases, all at a flow rate of 60 ml/min. R1 and R2 were then further purified using a Dubhe C-18 preparative column, the mobile phase being the same as that used for HPLC analysis. Thus, tyldinor A and tyldinor B were obtained, and after concentration, tyldinor A with a purity of 98.36% was obtained in 9.63g, and tyldinor B with a purity of 97.82% was obtained in 2.35g. The products obtained were identified as tedno A and tedno B by NMR examination (same as example 1).
EXAMPLE five
A. Fermenting raw materials: weighing 1kg of sun-dried green raw tea, wetting until the water content is 65% based on dry weight, sterilizing for 15 min at 115, and inoculating 2 × 105Inoculating Aspergillus niger, culturing at 35 deg.C for 20 days, taking out tea, and oven drying at 50 deg.C until water content is 8%;
B. crushing: crushing the fermented tea leaves to 50 meshes;
C. leaching with boiled water: adding 90 deg.C boiled water into pulverized tea at a mass ratio of 1:3, performing 40000HZ ultrasonic extraction for 3 times, each for 30 min, filtering, and mixing filtrates;
D. 1, 2-dichloroethane extraction: adding 1, 2-dichloroethane into the filtrate obtained in the step C according to the volume ratio of 1:1 for extraction, extracting for 3 times in total, leaving a water phase, and recovering an organic phase;
E. and (3) ethyl acetate extraction: continuously extracting the water phase obtained in the step D with ethyl acetate according to the volume ratio of 1:1 for 3 times, taking organic phases, combining and concentrating to obtain 108g of ethyl acetate extract;
F. silica gel column separation: separating the ethyl acetate extract obtained in the step E by a silica gel column, eluting the ethyl acetate extract by chloroform-methanol (volume ratio) of 15:1 for 1 column volume, eluting the ethyl acetate extract by the volume ratio for 2 column volumes, collecting the eluent with the 2 column volumes, and concentrating and drying the eluent to obtain a crude silica gel isolate 23.5 g;
G. separating by macroporous resin: separating the silica gel crude isolate obtained in the step F by macroporous resin, eluting by 10% ethanol, 20% ethanol and 100% ethanol in sequence, eluting by 2.5 times of the volume of the column in each gradient, collecting the eluent of the 10% ethanol and the 20% ethanol, concentrating and drying to obtain a fine isolate R111.5g (which represents 10% ethanol elution) and R22.8g (which represents 20% ethanol elution);
H. and (3) analyzing the fine isolate in the step G by using HPLC, wherein the conditions of the HPLC are as follows: the column was prepared using Dubhe C-18, using a methanol/water mixed solution of methanol to water in a volume ratio of 5:5 for R1 and a methanol/water mixed solution of methanol to water in a volume ratio of 4:6 for R2 as mobile phases, all at a flow rate of 60 ml/min. R1 and R2 were then further purified using a Dubhe C-18 preparative column, the mobile phase being the same as that used for HPLC analysis. Thus, tyldinor A and tyldinor B were obtained, and 8.53g of tyldinor A having a purity of 97.56% and 2.03g of tyldinor B having a purity of 97.22% were obtained after concentration. The products obtained were identified as tedno A and tedno B by NMR examination (same as example 1).
The following examples were conducted in accordance with the procedure of example 1, with the differences and evaluation results shown in Table 3.
TABLE 3
Claims (10)
1. A method for resolving teddenox A and teddenox B from fermented tea, wherein the teddenox A is shown as a formula (I) in the specification, and the teddenox B is shown as a formula (II) in the specification:
wherein the method comprises the steps of:
A) providing fermented tea comprising teddenox a and teddenox B;
B) leaching the fermented tea with boiled water to obtain a leaching solution;
C) extracting the extract with an organic solvent to obtain an extract containing teddenox a and teddenox B;
D) subjecting the extract to silica gel column separation to obtain a crude isolate; and
E) separating the crude isolate by using a macroporous resin column, wherein ethanol or methanol water solution with the volume of 5 percent to less than 20 percent is adopted for elution, and eluent is collected, so that the tyldinol A is obtained;
wherein the method further comprises F) eluting the macroporous resin column treated in the step E) with 20 to 60 volume percent ethanol or methanol aqueous solution, and collecting the eluent, thereby obtaining teddenox B;
wherein the step C) comprises the following steps:
C1) extracting the leach solution with 1, 2-dichloroethane or chloroform to obtain an aqueous phase and an organic phase; wherein the volume ratio of the 1, 2-dichloroethane or chloroform to the leaching solution is 1 (1-3); and
C2) extracting the aqueous phase obtained in step C1) with ethyl acetate to obtain said extract containing tyldinol a and tyldinol B; wherein the volume ratio of the ethyl acetate to the water phase is 1 (1-3).
2. The process according to claim 1, wherein in step D) elution is carried out with a chloroform: methanol eluent in a volume ratio of (10-30):1 to obtain the crude isolate.
3. The method as claimed in claim 1, wherein in step B), the boiling water leaching is performed 2-4 times for 10-50 minutes each time under the ultrasonic wave of 30000-50000 HZ.
4. The method according to claim 1, wherein in the step B), the fermented tea and the boiled water are mixed according to the mass ratio of 1 (1-3) to carry out the boiled water extraction; wherein the temperature of the boiled water is 80 ℃ to 100 ℃.
5. The process of claim 1, wherein the process further comprises G) purifying the teddenox a separated by the macroporous resin column by High Performance Liquid Chromatography (HPLC), wherein the HPLC employs a C-18 column and the mobile phase is a methanol/water mixed solution with a methanol to water volume ratio of 11:9 to 3: 7.
6. The process of claim 1, further comprising H) HPLC purification of teddenox B separated on the macroporous resin column, wherein the HPLC employs a C-18 column and the mobile phase is a methanol/water mixed solution with a methanol to water volume ratio of 1:1 to 3: 7.
7. The method according to claim 1, wherein the step a) comprises the steps of wetting the raw tea to a water content of more than 40 to 65 wt% based on the dry weight of the raw tea, sterilizing the wet raw tea, inoculating a single strain of aspergillus niger, and performing fermentation culture to obtain the fermented tea containing teddenox a and teddenox B.
8. The method as claimed in claim 2, wherein in step D), the silica gel column is 200-300 mesh silica gel column.
9. The method of claim 7, wherein the fermentation culture is performed at 28-40 ℃ for 10-25 days.
10. The method of claim 7, wherein the raw tea is selected from the group consisting of sun-dried raw tea, raw green tea, raw white tea, raw yellow tea, and raw oolong tea.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1600755A (en) * | 2003-09-23 | 2005-03-30 | 南京宇道科技开发有限公司 | General flavones of hawthorn fruit, preparation method and application |
| CN1666627A (en) * | 2004-03-12 | 2005-09-14 | 日本合成化学工业株式会社 | Process for preparing fermented tea and composition |
| CN101643466A (en) * | 2009-06-02 | 2010-02-10 | 江苏天晟药业有限公司 | Epigallo-catechin gallate (EGCG) with high purity and preparation method thereof |
| CN101696201A (en) * | 2009-10-29 | 2010-04-21 | 浙江大学 | Method for preparing catechins |
| JP2011084560A (en) * | 2009-09-18 | 2011-04-28 | Riverson:Kk | Polyphenol derivative and method for producing the same |
| CN102047997A (en) * | 2009-11-10 | 2011-05-11 | 上海市新文达生物科技有限公司 | Wuyi Mountain rock tea extract, and preparation method and application thereof |
| CN102630142A (en) * | 2009-09-18 | 2012-08-08 | 株式会社里维松 | Functional microbial-fermented tea extract containing polyphenol derivative, and process for production thereof |
| WO2012140894A1 (en) * | 2011-04-12 | 2012-10-18 | 株式会社 レオロジー機能食品研究所 | Pharmaceutical composition |
| JP2015097511A (en) * | 2013-11-20 | 2015-05-28 | 株式会社 レオロジー機能食品研究所 | Production method of polyphenol derivative, and food and medicinal composition containing polyphenol derivative |
| CN105254642A (en) * | 2015-11-03 | 2016-01-20 | 云南农业大学 | Preparation method of hypolipidemic compound Fuzhuan tea element A |
-
2016
- 2016-11-28 CN CN201611061352.3A patent/CN108117558B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1600755A (en) * | 2003-09-23 | 2005-03-30 | 南京宇道科技开发有限公司 | General flavones of hawthorn fruit, preparation method and application |
| CN1666627A (en) * | 2004-03-12 | 2005-09-14 | 日本合成化学工业株式会社 | Process for preparing fermented tea and composition |
| CN101643466A (en) * | 2009-06-02 | 2010-02-10 | 江苏天晟药业有限公司 | Epigallo-catechin gallate (EGCG) with high purity and preparation method thereof |
| JP2011084560A (en) * | 2009-09-18 | 2011-04-28 | Riverson:Kk | Polyphenol derivative and method for producing the same |
| CN102630142A (en) * | 2009-09-18 | 2012-08-08 | 株式会社里维松 | Functional microbial-fermented tea extract containing polyphenol derivative, and process for production thereof |
| CN102648284A (en) * | 2009-09-18 | 2012-08-22 | 株式会社里维松 | Polyphenol derivative and method for producing the same |
| CN101696201A (en) * | 2009-10-29 | 2010-04-21 | 浙江大学 | Method for preparing catechins |
| CN102047997A (en) * | 2009-11-10 | 2011-05-11 | 上海市新文达生物科技有限公司 | Wuyi Mountain rock tea extract, and preparation method and application thereof |
| WO2012140894A1 (en) * | 2011-04-12 | 2012-10-18 | 株式会社 レオロジー機能食品研究所 | Pharmaceutical composition |
| JP2015097511A (en) * | 2013-11-20 | 2015-05-28 | 株式会社 レオロジー機能食品研究所 | Production method of polyphenol derivative, and food and medicinal composition containing polyphenol derivative |
| CN105254642A (en) * | 2015-11-03 | 2016-01-20 | 云南农业大学 | Preparation method of hypolipidemic compound Fuzhuan tea element A |
Non-Patent Citations (2)
| Title |
|---|
| 多酚的提取方法及应用研究进展;王玉春;《甘肃联合大学学报》;20080531;第22卷(第3期);第51-52页 * |
| 茶多酚提取与分离纯化研究进展;严志慧 等;《陕西农业科学》;20101231(第6期);第86-91页 * |
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