CN104764846B - A kind of extraction, purification, method of identification anthocyanidin from fresh leaf of Camelliae sinensis - Google Patents
A kind of extraction, purification, method of identification anthocyanidin from fresh leaf of Camelliae sinensis Download PDFInfo
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Abstract
The invention discloses a kind of from fresh leaf of Camelliae sinensis extraction, purification, identification anthocyanidin method it is characterised in that:Comprise the following steps:A. sample broke;B. extract;C. concentration;D. filtration treatment;E. Solid-Phase Extraction;F. concentration;G. acidolysis;H.HPLC detects.Present invention advantage compared with prior art is:1st, Folium Camelliae sinensis contain very high aldehydes matter, easily aoxidize anthocyanin.This method can obtain highly purified anthocyanin efficiently against the interference of other aldehydes matters.2nd, the method extract from fresh leaf of Camelliae sinensis anthocyanin loss rate low, favorable reproducibility.3rd, the method is applied to the qualitative and quantitative analysis of the anthocyanin of all tea tree breeds.
Description
Technical field
The present invention relates to extract in a kind of Folium Camelliae sinensis, the method for purification health-care components, more particularly, to a kind of from fresh leaf of Camelliae sinensis
Extraction, purification, the method for identification anthocyanidin.
Background technology
Camellia sinensis are Theaceae (Theaceae) Camellia (Camellia) plant, are shrub or dungarunga, Camellia sinensis bud-leaf can
Tea making, Folium Camelliae sinensis are one of big beverages in the world today three, containing more than the 450 kinds of chemical compositions beneficial to human body in Folium Camelliae sinensis, main bag
Include tea polyphenols, caffeine, theanine, anthocyanin, vitamin etc., therefore Folium Camelliae sinensis have good health-care effect, can play antiinflammatory
The good actions such as sterilization, lowering blood pressure and blood fat, antioxidation, radioprotective, anticancer.
Anthocyanin (anthocyanin), also known as anthocyanidin, are class water colo(u)rs.Extensive just because of anthocyanin
Exist, higher plant just presents the multicoloured color world.Anthocyanin belongs to Flavonoid substances, is by anthocyanidin
And the class compound that is combined into glycosidic bond of glycan molecule (anthocyanidin).Identify natural anthocyanin at present
Up to thousand of kinds of species, the multiformity of this position mainly due to glycosidic bond and sugared substituent group, and in addition to glycosylation
Other modify cause, be such as acylated, methylate.Higher plant is due to the synthesis component of anthocyanin and the difference of concentration
And assume a series of gay colours such as pink colour, redness, blueness, purple, black.
Anthocyanin is important natural pigment, safe and nontoxic, is widely used in food service industry.Anthocyanin also has multiple guarantors
Health-care function.Many research shows, anthocyanin non-oxidizability, reduces the glycerol lipid level of hyperlipemia rat, improves high glycerine fat fat
The catabolism of albumen, suppresses cholesterol absorption, reduces low-density lipoprotein cholesterol content etc..Additionally, anthocyanin has disappearing
The effects such as inflammation, reduction canceration incidence rate.Therefore, the correlated traits of anthocyanin synthesis is directly connected to agriculture, the commodity through crop.
Because Folium Camelliae sinensis are extremely important beverage source, and anthocyanin pole is dissolved in water, and therefore having tea is the reason that human body takes in anthocyanin
Think mode.At present, the Folium Camelliae sinensis containing anthocyanin are big focuses of industry class.
The conventional extraction of anthocyanin has:Solvent extraction method, supercritical extraction, microwave method, enzymatic isolation method and ultrasound wave
Extraction method.Main isolation and purification method has:High performance countercurrent chromatography, membrane separation technique, gel chromatography, macroporous adsorbent resin
Method.Abroad many scholars utilize C18 post, reversed-phase high-performance liquid chromatography (RP-HPLC), high speed adverse current chromatogram and high performance liquid chromatography
Purification anthocyanin.
The molecular structure of anthocyanin can be obtained by LC-MS-MS.But experimental apparatus are required
Height, with high costs.Another practical method is to decompose glycosidic bond by acidolysis, then passes through high performance liquid chromatography
(HPLC) method identifies the type of aglycone.Although the anthocyanin species that nature exists is various, aglycone is
The type of anthocyanidin is very limited.The natural anthocyanidin overwhelming majority belongs to delphinidin (delphinidin), cornflowerblue
Plain (cyanidin), pelargonidin (pelargonidin), petunia pigment (petunidin), enidin
(malvidin), paeonidin (peonidin) six type.
The extraction of Folium Camelliae sinensis anthocyanin, purification, identification also do not have the related ripe, method of standard.One is because most of tea
Tree kind only synthesizes a small amount of or does not synthesize anthocyanin, as common greenery, yellow leaf kind.The Camellia sinensis of anthocyanin can be synthesized in a large number
Kind is quite rare, only domestic well-known as " purple cuckoo ", famous foreign as Japanese " Sun Rouge ".Two is that Camellia sinensis are fresh
The anthocyanin purification of leaf is compared and more complicated for other plant is less easily purified.Up to the present, separated in Folium Camelliae sinensis, identification
Known compound has kind more than 700, and polyphenols species is many, and rich content, including catechin, flavonoid, flavonols,
Anthocyanin, leucoanthocyanidin class, phenolic acid and depside etc..
Shandong Agricultural University Wang Shuai et al. studies the extraction purification of Folium Camelliae sinensis anthocyanin:
Take purple beautiful kind fresh leaf of Camelliae sinensis sample 2Kg, be put in ultrasonic 10min in 1% methanol hydrochloride solution of 8L, 4 DEG C of low temperature
Lower merceration collected extracting solution after two hours, repeated to process twice, united extraction liquid, rotary evaporation in vacuo is extremely at 30 DEG C for extracting solution
Closely dry, dissolved with a small amount of water, used chloroform, ethyl acetate extraction respectively.Aqueous solution is in the polyamide chromatography handled well
Gradient elution on post, and obtain different anthocyanin components using recrystallization and lead salt sedimentation.
Shandong Agricultural University's model skill all et al. research Folium Camelliae sinensis anthocyanin extraction (food and fermentation science and technology, 2013,
Vol.49, No.6, p38-42):
After taking " purple beautiful " dry in sun green tea 2g to pulverize 2min, add 400mL extracting solution (V methanol: V water: V formic acid: V trifluoro second
Acid=70: 27: 2: 1) room temperature extraction 1h, then with 5000r/min be centrifuged 5min, supernatant through solvent micropore filtering film (quasi- 50mm,
0.45 μm, organic system) filter be evaporated under the conditions of 50 ± 2 DEG C 30mL about after, lyophilization to powder is placed in-
10 DEG C of refrigerator-freezers preserve for pigment qualitative analyses.
Shandong Agricultural University Lu sea roc et al. research Folium Camelliae sinensis anthocyanin component authentication method (Food Science, 2012,
Vol.33, No.22, p203-206):
With " purple cuckoo " baked green tea sample as raw material, after extracted, purification anthocyanidin, anthocyanin is identified using following condition
Component:Chromatographic column, YMC-Pack ODS-AQ C18 (150mm × 4.6mm);Mobile phase, 0.5% formic acid solution (A) and acetonitrile
(B);Column temperature, 35 DEG C;Flow velocity, 0.8mL/min;Sample size, 10 μ L;Detection wavelength, 520nm;Standard sample is pelargonin -3,
5- diglucoside (pelargonidin-3,5-diglucoside chloride), Centaurea cyanus -3-O- galactoside
(cyanidin-3-Ogalactoside), pelargonidin (pelargonidin chloride), enidin (malvidin
chloride).
The method that Shandong Agricultural University Wang Shuai et al. discloses a kind of " purple beautiful " tea extraction purification anthocyanin, the method is main
It is to be extracted with trichloroethane, ethyl acetate, with polyamide chromatography post eluting.Fan Yifan etc. (food and fermentation science and technology,
2013, Vol.49, No.6, p38-42) disclose the extracting method of a kind of " purple beautiful " anthocyanin.The anthocyanin that said method extracts
Or degree of purification is not high or extraction process loss rate is larger, cannot be used for determining of downstream according to the anthocyanin that these methods are extracted
Property, Quantitative measurement.
Lv Haipeng etc. (food and fermentation science and technology, 2013, Vol.49, No.6, p38-42) discloses " purple cuckoo " anthocyanin group
The method divided.First, the method is only it has been found that could implement in the case of anthocyanin molecular structure.Due to Camellia sinensis heredity
Background is complicated, and the anthocyanin type being currently known Different Tea Varieties synthesis is made a world of difference.Second, the standard sample that the method uses
Product buy difficulty, and the cost for purification as the anthocyanin molecule of standard specimen is high or even cannot buy from market.3rd, when a certain tea
When tree synthesizes polytype anthocyanin molecule, the object of HPLC is excessive, and background is excessively complicated, possibly cannot separate molecule knot
The close anthocyanin of structure.
Content of the invention
The present invention is to solve above-mentioned deficiency, there is provided a kind of extraction, purification, side of identification anthocyanidin from tea fresh leaves
Method.
The above-mentioned purpose of the present invention is realized by following technical scheme:A kind of extraction, purification, identification from tea fresh leaves
The method of anthocyanidin it is characterised in that:Comprise the following steps:
A. sample broke:Precise plucks the two leaves and a bud 5g of fresh purple tea tree, plus goes to 50ml after liquid nitrogen grinding
Centrifuge tube;
B. extract:Plus the HCL-MeOH of 20ml 1% is to sample, it is placed in the static extraction of 4 casees cold rooms of refrigerator;It is centrifuged after 2h,
Rotating speed 9000r/min, centrifugation time 10min;Suction out supernatant and move to new 50ml centrifuge tube;Again plus 20ml 1%HCL-MeOH
Carry out secondary extraction to sample, be centrifuged after 2h, rotating speed 9000r/min, centrifugation time 10min, shift supernatant;Finally add again
10ml 1%HCL-MeOH carries out third time and extracts, and is centrifuged after 12h, shifts supernatant;
C. concentration:Using Rotary Evaporators Rotavapour R-300 (Buchi, Switzerland), the lixiviating solution obtaining is entered
Row concentrates, and revolving instrument rotary speed is 4-7, and temperature is 37 DEG C;Finally obtain the concentrated solution that volume is 25~30ml;
D. filtration treatment:Concentrated solution is filtered through 0.45 μm of organic faciess needle cylinder type filter membrane filter;
E. Solid-Phase Extraction:Using trans solid-phase extraction columnC18 cartridge (Waters, the U.S.) is to pattern
Glycosides is extracted.Extraction procedure is as follows:Post activates, and 5ml 0.01%HCL-MeOH is repeated once;Balance, 5ml 0.01%
HCL;Loading;Drip washing, 5ml 0.01%HCL, it is repeated once, then plus 5ml normal hexane, be repeated once;Eluting, plus 5ml
0.01%HCL-MeOH, collects eluent, eluting 3 times;
F. concentration:Using Rotary Evaporators Rotavapour R-300 (Buchi, Switzerland), Solid-Phase Extraction is obtained
Extracting solution is concentrated, volume 0.01%HCL-MeOH constant volume to 10ml after concentration;
G. acidolysis:Pipette 200 μ L sample to 1.5ml centrifuge tube, add 400 μ L 5mol/L HCl, sealed with Parafilm
Film phonograph seal.90 DEG C of process 30min of calorstat, put on ice after terminating immediately;
H.HPLC detects:Carry out HPLC detection, HPLC detection hardware condition is as follows:HPLC system is Agilent 1260
(Agilent, the U.S.), chromatographic column ZORBAX SB-C18 (Agilent, the U.S.);
Elution requirement:Mobile phase A, water/acetonitrile/formic acid, 87/3/10, v/v/v;Mobile phase B, acetonitrile;0min, 0%B;
5min, 5%B;10min, 14%B;30min, 30%B;
Testing conditions:Applied sample amount, 10 μ l;Column temperature, 35 DEG C;Detection wavelength 520nm;
Standard sample:Delphinidin (delphinidin), cyanidin (cyanidin), pelargonidin
(pelargonidin), enidin (malvidin), paeonidin (peonidin) are purchased from ChromaDex company of the U.S..
Present invention advantage compared with prior art is:
1st, Folium Camelliae sinensis contain very high aldehydes matter, easily aoxidize anthocyanin.This method can be efficiently against other phenol
The interference of class material, obtains highly purified anthocyanin.As shown in Figures 2 and 3, the Anthocyanin-rich Extract warp being obtained using the method
Cross HPLC identification, background material or interfering material are few.
2nd, the method extract from fresh leaf of Camelliae sinensis anthocyanin loss rate low, favorable reproducibility.In " purple handsome " strain anthocyanidin
In extraction process, add paeonidin as internal standard, detect through HPLC and find, average loss rate is 7.24%.
3rd, the method is applied to the qualitative and quantitative analysis of the anthocyanin of all tea tree breeds.As shown in Figures 2 and 3, " purple
Handsome " identical with the anthocyanin type that " purple cuckoo " synthesizes, but component ratio is different.
Brief description
Fig. 1 is anthocyanidin standard substance chromatogram.
Fig. 2 is the chromatogram of tea strain " purple handsome " anthocyanin acid hydrolysate.
Fig. 3 is the chromatogram of " purple cuckoo " variety design glycosides acid hydrolysate.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail.
Embodiment 1:As shown in Figure 1 and Figure 2, a kind of extraction, purification, method of identification anthocyanidin from fresh leaf of Camelliae sinensis, including
Following steps:
A. sample broke:Precise plucks the two leaves and a bud 5g of fresh purple tea tree strain " purple handsome ", plus liquid nitrogen grinding
After go to 50ml centrifuge tube;
B. extract:Plus the HCL-MeOH of 20ml 1% is to sample, it is placed in the static extraction of 4 casees cold rooms of refrigerator;It is centrifuged after 2h,
Rotating speed 9000r/min, centrifugation time 10min;Suction out supernatant and move to new 50ml centrifuge tube;Again plus 20ml 1%HCL-MeOH
Carry out secondary extraction to sample, be centrifuged after 2h, rotating speed 9000r/min, centrifugation time 10min, shift supernatant;Finally add again
10ml 1%HCL-MeOH carries out third time and extracts, and is centrifuged after 12h, shifts supernatant;
C. concentration:Using Rotary Evaporators Rotavapour R-300 (Buchi, Switzerland), the lixiviating solution obtaining is entered
Row concentrates, and revolving instrument rotary speed is 4-7, and temperature is 37 DEG C;Finally obtain the concentrated solution that volume is 25~30ml;
D. filtration treatment:Concentrated solution is filtered through 0.45 μm of organic faciess needle cylinder type filter membrane filter;
E. Solid-Phase Extraction:Using trans solid-phase extraction columnC18 cartridge (Waters, the U.S.) is to pattern
Glycosides is extracted.Extraction procedure is as follows:Post activates, and 5ml 0.01%HCL-MeOH is repeated once;Balance, 5ml 0.01%
HCL;Loading;Drip washing, 5ml 0.01%HCL, it is repeated once, then plus 5ml normal hexane, be repeated once;Eluting, plus 5ml
0.01%HCL-MeOH, collects eluent, eluting 3 times;
F. concentration:Using Rotary Evaporators Rotavapour R-300 (Buchi, Switzerland), Solid-Phase Extraction is obtained
Extracting solution is concentrated, volume 0.01%HCL-MeOH constant volume to 10ml after concentration;
G. acidolysis:Pipette 200 μ L sample to 1.5ml centrifuge tube, add 400 μ L 5mol/L HCl, sealed with Parafilm
Film phonograph seal.90 DEG C of process 30min of calorstat, put on ice after terminating immediately;
H.HPLC detects:Carry out HPLC detection, HPLC detection hardware condition is as follows:HPLC system is Agilent 1260
(Agilent, the U.S.), chromatographic column ZORBAX SB-C18 (Agilent, the U.S.);
Elution requirement:Mobile phase A, water/acetonitrile/formic acid, 87/3/10, v/v/v;Mobile phase B, acetonitrile;0min, 0%B;
5min, 5%B;10min, 14%B;30min, 30%B;
Testing conditions:Applied sample amount, 10 μ l;Column temperature, 35 DEG C;Detection wavelength 520nm;
Standard sample:Delphinidin (delphinidin), cyanidin (cyanidin), pelargonidin
(pelargonidin), enidin (malvidin), paeonidin (peonidin) are purchased from ChromaDex company of the U.S..
Embodiment 2:As shown in Figure 1, Figure 3, a kind of extraction, purification, method of identification anthocyanidin from fresh leaf of Camelliae sinensis, including
Following steps:
A. sample broke:Precise plucks the two leaves and a bud 5g of fresh purple tea tree strain " purple beautiful ", plus liquid nitrogen grinding
After go to 50ml centrifuge tube;
B. extract:Plus the HCL-MeOH of 20ml 1% is to sample, it is placed in the static extraction of 4 casees cold rooms of refrigerator;It is centrifuged after 2h,
Rotating speed 9000r/min, centrifugation time 10min;Suction out supernatant and move to new 50ml centrifuge tube;Again plus 20ml 1%HCL-MeOH
Carry out secondary extraction to sample, be centrifuged after 2h, rotating speed 9000r/min, centrifugation time 10min, shift supernatant;Finally add again
10ml 1%HCL-MeOH carries out third time and extracts, and is centrifuged after 12h, shifts supernatant;
C. concentration:Using Rotary Evaporators Rotavapour R-300 (Buchi, Switzerland), the lixiviating solution obtaining is entered
Row concentrates, and revolving instrument rotary speed is 4-7, and temperature is 37 DEG C;Finally obtain the concentrated solution that volume is 25~30ml;
D. filtration treatment:Concentrated solution is filtered through 0.45 μm of organic faciess needle cylinder type filter membrane filter;
E. Solid-Phase Extraction:Using trans solid-phase extraction columnC18 cartridge (Waters, the U.S.) is to pattern
Glycosides is extracted.Extraction procedure is as follows:Post activates, and 5ml 0.01%HCL-MeOH is repeated once;Balance, 5ml 0.01%
HCL;Loading;Drip washing, 5ml 0.01%HCL, it is repeated once, then plus 5ml normal hexane, be repeated once;Eluting, plus 5ml
0.01%HCL-MeOH, collects eluent, eluting 3 times;
F. concentration:Using Rotary Evaporators Rotavapour R-300 (Buchi, Switzerland), Solid-Phase Extraction is obtained
Extracting solution is concentrated, volume 0.01%HCL-MeOH constant volume to 10ml after concentration;
G. acidolysis:Pipette 200 μ L sample to 1.5ml centrifuge tube, add 400 μ L 5mol/L HCl, sealed with Parafilm
Film phonograph seal.90 DEG C of process 30min of calorstat, put on ice after terminating immediately;
H.HPLC detects:HPLC detection hardware condition is as follows:HPLC system is Agilent 1260 (Agilent, the U.S.),
Chromatographic column ZORBAX SB-C18 (Agilent, the U.S.);
Elution requirement:Mobile phase A, water/acetonitrile/formic acid, 87/3/10, v/v/v;Mobile phase B, acetonitrile;0min, 0%B;
5min, 5%B;10min, 14%B;30min, 30%B;
Testing conditions:Applied sample amount, 10 μ l;Column temperature, 35 DEG C;Detection wavelength 520nm;
Standard sample:Delphinidin (delphinidin), cyanidin (cyanidin), pelargonidin
(pelargonidin), enidin (malvidin), paeonidin (peonidin) are purchased from ChromaDex company of the U.S..
The foregoing is only embodiments of the invention, not thereby limit the present invention the scope of the claims, every using this
Equivalent structure or equivalent flow conversion that bright description is made, or directly or indirectly it is used in other related technology necks
Domain, is included within the scope of the present invention.
Claims (1)
1. a kind of from fresh leaf of Camelliae sinensis extraction, purification, identification anthocyanidin method it is characterised in that:Comprise the following steps:
A. sample broke:Precise plucks the two leaves and a bud 5g of fresh purple tea tree, plus goes to 50ml centrifugation after liquid nitrogen grinding
Pipe;
B. extract:Plus the HCL-MeOH of 20ml 1% is to sample, it is placed in the static extraction of 4 casees cold rooms of refrigerator;It is centrifuged after 2h, rotating speed
9000r/min, centrifugation time 10min;Suction out supernatant and move to new 50ml centrifuge tube;Again plus 20ml 1%HCL-MeOH is to sample
Product carry out secondary extraction, are centrifuged, rotating speed 9000r/min, centrifugation time 10min after 2h, shift supernatant;Finally add 10ml again
1%HCL-MeOH carries out third time and extracts, and is centrifuged after 12h, shifts supernatant;
C. concentration:Using Rotary Evaporators, the lixiviating solution obtaining is concentrated;Finally obtaining volume is 25~30ml's
Concentrated solution;
D. filtration treatment:Concentrated solution is filtered through 0.45 μm of organic faciess needle cylinder type filter membrane filter;
E. Solid-Phase Extraction:Using trans solid-phase extraction column, anthocyanin is extracted;Extraction procedure is as follows:Post activates, 5ml
0.01%HCL-MeOH, is repeated once;Balance, 5ml0.01%HCL;Loading;Drip washing, 5ml 0.01%HCL, it is repeated once, connect
Plus 5ml normal hexane, be repeated once;Eluting, plus 5ml 0.01%HCL-MeOH, collect eluent, eluting 3 times;
F. concentration:Using Rotary Evaporators, the extracting solution that Solid-Phase Extraction obtains is concentrated, after concentration, volume is used
0.01%HCL-MeOH constant volume is to 10ml;
G. acidolysis:Pipette 200 μ L sample to 1.5ml centrifuge tube, add 400 μ L5mol/L HCl, close with Parafilm sealed membrane
Envelope;90 DEG C of process 30min of calorstat, put on ice after terminating immediately;
H.HPLC detects:Carry out HPLC detection, HPLC detection hardware condition is as follows:
HPLC system is Agilent 1260, chromatographic column ZORBAX SB-C18;
Elution requirement:Mobile phase A, water/acetonitrile/formic acid, 87/3/10, v/v/v;Mobile phase B, acetonitrile;0min, 0%B;5min,
5%B;10min, 14%B;30min, 30%B;
Testing conditions:Applied sample amount, 10 μ l;Column temperature, 35 DEG C;Detection wavelength 520nm;
Standard sample:Delphinidin, cyanidin, pelargonidin, enidin, paeonidin are purchased from the U.S.
ChromaDex company.
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