CN107698548A - A kind of extracting method of Tea Polyphenols - Google Patents
A kind of extracting method of Tea Polyphenols Download PDFInfo
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- CN107698548A CN107698548A CN201710751045.6A CN201710751045A CN107698548A CN 107698548 A CN107698548 A CN 107698548A CN 201710751045 A CN201710751045 A CN 201710751045A CN 107698548 A CN107698548 A CN 107698548A
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- tea polyphenols
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
- C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
- C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
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Abstract
The invention belongs to field of natural product extraction, more particularly to a kind of extracting method of Tea Polyphenols, comprise the following steps：Stock, pre-treatment, immersion, extraction, dilution, separation；Fresh tea leaf is by liquid nitrogen flash freezer, crushing, powder is pre-processed with Alternaria LJX27 fermentation crude extracts, the permeability of tea cell is changed, while there is the effect for suppressing Tea Polyphenols oxidation, pretreated tealeaves is soaked with mixed solvent, Microwave Extraction, so that the recovery rate of crude extract is up to 65.7%, the content of Tea Polyphenols reaches 95.2% in crude extract, separates to obtaining high purity 99.3%, wherein catechin content is up to 92.3%, and EGCG content is up to 62.5%.
Description
Technical field
The invention belongs to field of natural product extraction, more particularly to a kind of extracting method of Tea Polyphenols.
Background technology
The drying tender leaf or leaf bud of tealeaves system Theaceae Camellia Plants tea, Tea Polyphenols be in tealeaves it is main effectively into
Point, Tea Polyphenols is also known as tea tan or tea tannin, is one of main component to form tealeaves color, smell and taste, and have healthcare function in tealeaves
One of main component.Content of the Tea Polyphenols in tealeaves is typically 20%~35%.Tea Polyphenols mainly includes flavanol compound, hydroxyl
Base -4- flavanol compounds, anthocyanin class, flavonoids and phenolic acid class, wherein with flavanols compounds (mainly catechin) most
To be important, its content accounts for 80% or so of Polyphenols total amount, catechin mainly include epicatechin (epicatechin,
EC), epigallocatechin (epigallocatechin, EGC), L-Epicatechin gallate
(epicatechingallate, ECG) and Epigallo-catechin gallate (EGCG) (epigallo catechin
Gallate, EGCG).Tea Polyphenols has antitumor, reducing blood lipid, antiatherosclerosis, cardiac stimulant, anti-arrhythmia, anti-aging
With the multiple biological activities such as antibacterial, and largely to disclose these bioactivity relevant with its antioxidation for report.
Since the 1960s, domestic and foreign scholars are to the extraction and separation technology and its biology of Tea Polyphenols and physiologically active etc.
Aspect has done research extensively and profoundly.Traditional extracting method mainly has solvent extraction method, remains recovery rate is low, toxic solvent
Residual, the shortcomings of production cost is high；Ion catch electron microscopy, wherein heavy metal basic salt easily bring bad shadow to the security of product
Ring, hydroxide category highly basic, production equipment is corrosive；Ultrasonic extraction and supercritical fluid extraction, are equally also deposited
The shortcomings that reagent is too many, and cost is higher is consumed in elution, equipment investment is costly, is difficult to input large-scale production for the moment.
The content of the invention
The present invention is in order to solve the above technical problems, provide a kind of extracting method of Tea Polyphenols, particular by following skill
Art scheme is realized.
A kind of extracting method of Tea Polyphenols, comprises the following steps：
1) stock up：It is raw material to choose fresh tealeaves, and addition liquid nitrogen flash freezer, crushing are standby；
2) pre-treatment：By the tealeaves of crushing and solvent in mass ratio 5:1 wetting tea-leaf power, obtains moistening material, moistening material with
The 40.7-41.5 in mass ratio of solution 1:1.3 mixing, 35-40 DEG C of reaction 45-50 min, obtain pre-treatment materials；
3) soak：By pre-treatment materials and solvent in mass ratio 1:20 mixing, 32-35 DEG C of immersion 27-30min；
4) extract：Tealeaves Microwave Extraction after immersion, obtains Microwave Extraction thing liquid；
5) dilute：The clear water for adding 5.6-6 times of Microwave Extraction liquid quality mixes with Microwave Extraction liquid, is stood at 25-27 DEG C
23-27min, 12000-13200r/min centrifuge 30-40min, layer of fetching water, obtain dilution；
6) separate：Dilution is subjected to Dynamic Adsorption to Tea Polyphenols with HPD-600 macroporous absorbent resins, and washed with eluent
It is de-.
Further, described solvent, it is by ethanol, ethyl acetate, acetone, methanol by volume 20.3:5:7:1.7 mixing
And obtain.
Further, described solution 1, it is Alternaria LJX27 fermentation crude extracts.
Further, described crude extract, including following extraction step：
1) Alternaria LJX27 is cultivated 7-8 days under conditions of pH=7.5,27-28 DEG C, 180-200r/min,
12000-12400rpm, centrifugation 10min, take precipitation；
2) liquid nitrogen flash freezer is precipitated, thalline, 4-6 DEG C, 20-25KHZ is resuspended in the phosphate buffer for adding 5-6 times of thalline quality
Ultrasonic extraction 30-35min, obtain ultrasonic extraction liquid；
3) add 1/5 extract solution absolute ethyl alcohol, 180-220r/min vibration 25-30min, 12000-13300rpm, from
Heart 10min, takes supernatant；
4) ether for adding 1/2 supernatant volume mixes with supernatant, takes ether layer, is evaporated under reduced pressure recovery ether, must consolidate
Body 1, the water dissolved solid 1 for adding the 5-6 times of mass of solid 1 both obtained.
Further, described phosphate buffer, phosphate dihydrogen sodium concentration 0.3mol/L, disodium hydrogen phosphate concentration are
0.04mol/L, sodium chloride concentration 0.5mol/L, glycerol concentration 12.5%, pH=8.0.
Further, described Microwave Extraction, microwave power 650w-700w, extraction time 8-10min.
Further, described Dynamic Adsorption, eluent flow rate 0.8-1.1ml/min, dilution mass concentration are 0.8-
1.1mg/ml。
Further, described eluent is 65-70% ethanol solution.
The beneficial effects of the present invention are：The Tea Polyphenols that the present invention extracts, its fresh tea leaf pass through liquid nitrogen flash freezer, crushed,
Powder is pre-processed with Alternaria LJX27 fermentation crude extracts, changes the permeability of tea cell, while have suppression tea
The effect of polyphenol oxidase, pretreated tealeaves mixed solvent (ethanol, ethyl acetate, acetone, methanol by volume 20.3:
5:7:1.7) soaked, Microwave Extraction so that the recovery rate of crude extract is up to 65.7%, and the content of Tea Polyphenols reaches in crude extract
To 95.2%.
Catechin is separated with HPD-600 macroporous absorbent resins, the ethanol solution by the use of 65% is used as eluent, separation
To obtaining high purity 99.3%, wherein catechin content is up to 92.3%, and EGCG content is up to 62.5%.
Embodiment
The embodiment of the present invention is described in further detail below, but the invention is not limited in these realities
Mode is applied, any improvement or replacement on the present embodiment essence spirit, it is claimed to still fall within the claims in the present invention
Scope.
Embodiment 1
A kind of extracting method of Tea Polyphenols, comprises the following steps：
1) stock up：It is raw material to choose fresh tealeaves, and addition liquid nitrogen flash freezer, crushing are standby；
2) pre-treatment：By the tealeaves of crushing and solvent in mass ratio 5:1 wetting tea-leaf power, obtains moistening material, moistening material with
Solution 1 in mass ratio 40.7:1.3 mixing, 35 DEG C of reaction 45min, obtain pre-treatment materials；
3) soak：By pre-treatment materials and solvent in mass ratio 1:20 mixing, 32 DEG C of immersion 27min；
4) extract：Tealeaves Microwave Extraction after immersion, obtains Microwave Extraction thing liquid；
5) dilute：The clear water for adding 5.6 times of Microwave Extraction liquid quality mixes with Microwave Extraction liquid, is stood at 25 DEG C
23min, 12000r/min centrifuge 30min, layer of fetching water, obtain dilution；
6) separate：Dilution is subjected to Dynamic Adsorption to Tea Polyphenols with HPD-600 macroporous absorbent resins, and washed with eluent
It is de-.
Described solvent, it is by ethanol, ethyl acetate, acetone, methanol by volume 20.3:5:7:1.7 it is obtained by mixing.
Described solution 1, it is Alternaria LJX27 fermentation crude extracts.
Described crude extract, including following extraction step：
1) Alternaria LJX27 is cultivated 7 days under conditions of pH=7.5,27 DEG C, 180r/min, 12000rpm, from
Heart 10min, take precipitation；
2) liquid nitrogen flash freezer is precipitated, thalline, 4 DEG C, 20KHZ ultrasonic extractions is resuspended in the phosphate buffer for adding 5 times of thalline quality
30min, obtain ultrasonic extraction liquid；
3) absolute ethyl alcohol of 1/5 extract solution is added, 180r/min vibrations 25min, 12000rpm, centrifugation 10min, is taken
Clearly；
4) ether for adding 1/2 supernatant volume mixes with supernatant, takes ether layer, is evaporated under reduced pressure recovery ether, must consolidate
Body 1, the water dissolved solid 1 for adding 5 times of mass of solid 1 both obtained.
Described phosphate buffer, phosphate dihydrogen sodium concentration 0.3mol/L, disodium hydrogen phosphate concentration are 0.04mol/L,
Sodium chloride concentration is 0.5mol/L, glycerol concentration 12.5%, pH=8.0.
Described Microwave Extraction, microwave power 650w, extraction time 8min.
Described Dynamic Adsorption, eluent flow rate 0.8ml/min, dilution mass concentration are 0.8mg/ml.
Described eluent is 65% ethanol solution.
Embodiment 2
A kind of extracting method of Tea Polyphenols, comprises the following steps：
1) stock up：It is raw material to choose fresh tealeaves, and addition liquid nitrogen flash freezer, crushing are standby；
2) pre-treatment：By the tealeaves of crushing and solvent in mass ratio 5:1 wetting tea-leaf power, obtains moistening material, moistening material with
Solution 1 in mass ratio 40.7:1.3 mixing, 35 DEG C of reaction 45min, obtain pre-treatment materials；
3) soak：By pre-treatment materials and solvent in mass ratio 1:20 mixing, 32 DEG C of immersion 27min；
4) extract：Tealeaves Microwave Extraction after immersion, obtains Microwave Extraction thing liquid；
5) dilute：The clear water for adding 5.6 times of Microwave Extraction liquid quality mixes with Microwave Extraction liquid, is stood at 25 DEG C
23min, 12000r/min centrifuge 30min, layer of fetching water, obtain dilution；
6) separate：Dilution is subjected to Dynamic Adsorption to Tea Polyphenols with HPD-600 macroporous absorbent resins, and washed with eluent
It is de-.
Described solvent, it is by ethanol, ethyl acetate, acetone, methanol by volume 20.3:5:7:1.7 it is obtained by mixing.
Described solution 1, it is Alternaria LJX27 fermentation crude extracts.
Described crude extract, including following extraction step：
1) Alternariaspp Alternaria LJX27 is cultivated 10 days under conditions of pH=7.0,25 DEG C, 180r/min,
12000rpm, centrifugation 10min, take precipitation；
2) liquid nitrogen flash freezer is precipitated, thalline, 4 DEG C, 20KHZ ultrasonic extractions is resuspended in the phosphate buffer for adding 5 times of thalline quality
30min, obtain ultrasonic extraction liquid；
3) absolute ethyl alcohol of 1/5 extract solution is added, 180r/min vibrations 25min, 12000rpm, centrifugation 10min, is taken
Clearly；
4) ether for adding 1/2 supernatant volume mixes with supernatant, takes ether layer, is evaporated under reduced pressure recovery ether, must consolidate
Body 1, the water dissolved solid 1 for adding 5 times of mass of solid 1 both obtained.
Described phosphate buffer, phosphate dihydrogen sodium concentration 0.3mol/L, disodium hydrogen phosphate concentration are 0.04mol/L,
Sodium chloride concentration is 0.5mol/L, glycerol concentration 12.5%, pH=8.0.
Described Microwave Extraction, microwave power 650w, extraction time 8min.
Described Dynamic Adsorption, eluent flow rate 0.8ml/min, dilution mass concentration are 0.8mg/ml.
Described eluent is 65% ethanol solution.
Embodiment 3
A kind of extracting method of Tea Polyphenols, comprises the following steps：
1) stock up：It is raw material to choose fresh tealeaves, and addition liquid nitrogen flash freezer, crushing are standby；
2) pre-treatment：By the tealeaves of crushing and solvent in mass ratio 5:1 wetting tea-leaf power, obtains moistening material, moistening material with
Solution 1 in mass ratio 40.7:1.3 mixing, 35 DEG C of reaction 45min, obtain pre-treatment materials；
3) soak：By pre-treatment materials and solvent in mass ratio 1:20 mixing, 32 DEG C of immersion 27min；
4) extract：Tealeaves Microwave Extraction after immersion, obtains Microwave Extraction thing liquid；
5) dilute：The clear water for adding 5.6 times of Microwave Extraction liquid quality mixes with Microwave Extraction liquid, is stood at 25 DEG C
23min, 12000r/min centrifuge 30min, layer of fetching water, obtain dilution；
6) separate：Dilution is subjected to Dynamic Adsorption to Tea Polyphenols with HPD-600 macroporous absorbent resins, and washed with eluent
It is de-.
Described solvent, it is by ethanol, ethyl acetate, acetone, methanol by volume 15:2:3:1 is obtained by mixing.
Described solution 1, it is Alternaria LJX27 fermentation crude extracts.
Described crude extract, including following extraction step：
1) Alternariaspp Alternaria LJX27 is cultivated 7 days under conditions of pH=7.5,27 DEG C, 180r/min,
12000rpm, centrifugation 10min, take precipitation；
2) liquid nitrogen flash freezer is precipitated, thalline, 4 DEG C, 20KHZ ultrasonic extractions is resuspended in the phosphate buffer for adding 5 times of thalline quality
30min, obtain ultrasonic extraction liquid；
3) absolute ethyl alcohol of 1/5 extract solution is added, 180r/min vibrations 25min, 12000rpm, centrifugation 10min, is taken
Clearly；
4) ether for adding 1/2 supernatant volume mixes with supernatant, takes ether layer, is evaporated under reduced pressure recovery ether, must consolidate
Body 1, the water dissolved solid 1 for adding 5 times of mass of solid 1 both obtained.
Described phosphate buffer, phosphate dihydrogen sodium concentration 0.3mol/L, disodium hydrogen phosphate concentration are 0.04mol/L,
Sodium chloride concentration is 0.5mol/L, glycerol concentration 12.5%, pH=8.0.
Described Microwave Extraction, microwave power 650w, extraction time 8min.
Described Dynamic Adsorption, eluent flow rate 0.8ml/min, dilution mass concentration are 0.8mg/ml.
Described eluent is 65% ethanol solution.
Embodiment 4
A kind of extracting method of Tea Polyphenols, comprises the following steps：
1) stock up：It is raw material to choose fresh tealeaves, and addition liquid nitrogen flash freezer, crushing are standby；
2) soak：By tea-leaf power and solvent in mass ratio 1:20 mixing, 32 DEG C of immersion 27min；
3) extract：Tealeaves Microwave Extraction after immersion, obtains Microwave Extraction thing liquid；
4) dilute：The clear water for adding 5.6 times of Microwave Extraction liquid quality mixes with Microwave Extraction liquid, is stood at 25 DEG C
23min, 12000r/min centrifuge 30min, layer of fetching water, obtain dilution；
5) separate：Dilution is subjected to Dynamic Adsorption to Tea Polyphenols with HPD-600 macroporous absorbent resins, and washed with eluent
It is de-.
Described solvent, it is by ethanol, ethyl acetate, acetone, methanol by volume 20.3:5:7:1.7 it is obtained by mixing.
Described Microwave Extraction, microwave power 650w, extraction time 8min.
Described Dynamic Adsorption, eluent flow rate 0.8ml/min, dilution mass concentration are 0.8mg/ml.
Described eluent is 65% ethanol solution.
Experimental example
1st, effect assessment
Method according to embodiment 1-4 is extracted to Tea Polyphenols, while with application number CN201710213904.6《Tea
Polyphenol extracting method and its application》The method of announcement is extracted as a control group, calculates the recovery rate of 6 groups of Tea Polyphenols respectively,
After purifies and separates, the method that the detection of the purity of Tea Polyphenols, wherein Tea Polyphenols is announced according to GB/T 8313-2008 is carried out, as a result
As shown in table 1.
The tea polyphenol extract rate of table 1
As known from Table 1, embodiment 1 is extracted according to the method for the present invention to Tea Polyphenols, and fresh tea leaf is by liquid nitrogen speed
Freeze, crush, powder is pre-processed with Alternaria LJX27 fermentation crude extracts, changes the permeability of tea cell, has simultaneously
There is the effect for suppressing Tea Polyphenols oxidation, (ethanol, ethyl acetate, acetone, methanol press volume to pretreated tealeaves with mixed solvent
Than 20.3:5:7:1.7) soaked, Microwave Extraction so that the recovery rate of crude extract is up to 65.7%, Tea Polyphenols in crude extract
Content reach 95.2%.
Catechin is separated with HPD-600 macroporous absorbent resins, the ethanol solution by the use of 65% is used as eluent, separation
To obtaining high purity 99.3%, wherein catechin content is up to 92.3%, and EGCG content is up to 62.5%.
Compared with Example 1, because embodiment 2 is different for Alternaria LJX27 condition of culture；Embodiment 3 uses
Extraction solvent ratio it is different；Embodiment 4 is without Alternaria LJX27 fermented tea crude extracts are used, so that crude extract contains
Amount is below embodiment 1.
Claims (8)
1. a kind of extracting method of Tea Polyphenols, it is characterised in that comprise the following steps：
1) stock up：It is raw material to choose fresh tealeaves, and addition liquid nitrogen flash freezer, crushing are standby；
2) pre-treatment：By the tealeaves of crushing and solvent in mass ratio 5:1 wetting tea-leaf power, obtains moistening material, moistening material and solution 1
40.7-41.5 in mass ratio:1.3 mixing, 35-40 DEG C of reaction 45-50min, obtain pre-treatment materials；
3) soak：By pre-treatment materials and solvent in mass ratio 1:20 mixing, 32-35 DEG C of immersion 27-30min；
4) extract：Tealeaves Microwave Extraction after immersion, obtains Microwave Extraction thing liquid；
5) dilute：The clear water for adding 5.6-6 times of Microwave Extraction liquid quality is mixed with Microwave Extraction liquid, and 23- is stood at 25-27 DEG C
27min, 12000-13200r/min centrifuge 30-40min, layer of fetching water, obtain dilution；
6) separate：Dilution is subjected to Dynamic Adsorption to Tea Polyphenols with HPD-600 macroporous absorbent resins, and uses elution.
2. the extracting method of Tea Polyphenols as claimed in claim 1, it is characterised in that described solvent, be by ethanol, acetic acid second
Ester, acetone, methanol by volume 20.3:5:7:1.7 it is obtained by mixing.
3. the extracting method of Tea Polyphenols as claimed in claim 1, it is characterised in that described solution 1, is Alternaria
LJX27 fermentation crude extracts.
4. the extracting method of Tea Polyphenols as claimed in claim 3, it is characterised in that described crude extract, including following extraction
Step：
1) Alternaria LJX27 is cultivated 7-8 days under conditions of pH=7.5,27-28 DEG C, 180-200r/min, 12000-
12400rpm, centrifugation 10min, take precipitation；
2) liquid nitrogen flash freezer is precipitated, the phosphate buffer for adding 5-6 times of thalline quality is resuspended thalline, 4-6 DEG C, 20-25KHZ ultrasounds carry
30-35min is taken, obtains ultrasonic extraction liquid；
3) add 1/5 extracting liquid volume absolute ethyl alcohol, 180-220r/min vibration 25-30min, 12000-13300rpm, from
Heart 10min, takes supernatant；
4) ether for adding 1/2 supernatant volume mixes with supernatant, takes ether layer, is evaporated under reduced pressure recovery ether, obtains solid 1,
The water dissolved solid 1 for adding the 5-6 times of mass of solid 1 both obtained.
5. the extracting method of Tea Polyphenols as claimed in claim 4, it is characterised in that described phosphate buffer, biphosphate
Na concn is 0.3mol/L, and disodium hydrogen phosphate concentration is 0.04mol/L, sodium chloride concentration 0.5mol/L, and glycerol concentration is
12.5%, pH=8.0.
6. the extracting method of Tea Polyphenols as claimed in claim 1, it is characterised in that described Microwave Extraction, microwave power are
650w-700w, extraction time 8-10min.
7. the extracting method of Tea Polyphenols as claimed in claim 1, it is characterised in that described Dynamic Adsorption, eluent flow rate
For 0.8-1.1ml/min, dilution mass concentration is 0.8-1.1mg/ml.
8. the extracting method of Tea Polyphenols as claimed in claim 1, it is characterised in that described eluent is 65-70% second
Alcoholic solution.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109096345A (en) * | 2018-08-16 | 2018-12-28 | 福建农林大学 | It is extracted in a kind of tealeaves and purifying tea polyphenols method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106943768A (en) * | 2017-04-01 | 2017-07-14 | 北京国康本草物种生物科学技术研究院有限公司 | Tea Polyphenols and its extracting method and application |
-
2017
- 2017-08-28 CN CN201710751045.6A patent/CN107698548A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106943768A (en) * | 2017-04-01 | 2017-07-14 | 北京国康本草物种生物科学技术研究院有限公司 | Tea Polyphenols and its extracting method and application |
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Application publication date: 20180216 |