CN104764846A - Method for extracting, purifying and identifying anthocyanin from fresh tea leaves - Google Patents

Abstract

The invention discloses a method for extracting, purifying and identifying anthocyanin from fresh tea leaves. The method is characterized by comprising the following steps: A, crushing a sample; B, leaching; C, concentrating; D, filtering; E, performing solid-phase extraction; F, concentrating; G, performing acidolysis; and H, performing HPLC detection. Compared with the prior art, the method disclosed by the invention has the advantages that 1, the tea leaves contain extremely high phenolic substances, and anthocyanin is easily oxidized, according to the method, the interference of other phenolic substances can be effectively overcome, and high-purity anthocyanin can be obtained; 2, according to the method, the loss rate of the anthocyanin extracted from the fresh tea leaves is low, and the reproducibility is high; and 3, the method is applicable to qualitative and quantitative detection of the anthocyanin of all tea tree varieties.

Description

A kind of method of extraction from fresh leaves of tea plant, purifying, qualification anthocyanidin

Technical field

The present invention relates to a kind of method of extraction, purifying health-care components in tealeaves, particularly relate to a kind of method of extraction from fresh leaves of tea plant, purifying, qualification anthocyanidin.

Background technology

Tea tree is Theaceae (Theaceae) Camellia (Camellia) plant, for shrub or dungarunga, tea tree bud-leaf can tea making, tealeaves is one of large beverage in the world today three, containing more than the 450 kind of chemical composition useful to human body in tealeaves, mainly comprise Tea Polyphenols, caffeine, theanine, anthocyanin, vitamin etc., therefore tealeaves has good health-care effect, can play anti-inflammation and sterilization, hypotensive, reducing blood lipid, anti-oxidant, radioresistance, the good action such as anticancer.

Anthocyanin (anthocyanin), has another name called anthocyanidin, is a class water colo(u)r.Just because of the extensive existence of anthocyanin, higher plant just presents the multicoloured color world.Anthocyanin belongs to Flavonoid substances, is the compounds be combined into glycosidic bond by anthocyanidin (anthocyanidin) and glycan molecule.Identified at present the kind nearly several thousand kinds of natural anthocyanin, this is mainly due to position and the substituent diversity of sugar of glycosidic bond, and other modification except glycosylation except causes, and as acyl group, methylates.Higher plant presents a series of gay colours such as pink colour, redness, blueness, purple, black due to the synthesis component of anthocyanin and the difference of concentration.

Anthocyanin is important natural colouring matter, safe, nontoxic, is widely used in food service industry.Anthocyanin also has plurality of health care functions.Many research shows, anthocyanin inoxidizability, reduces the glyceride level of hyperlipemia rat, improves the kalabolism of high glyceride lipoprotein, suppresses cholesterol absorption, reduces LDL-C content etc.In addition, anthocyanin has anti-inflammatory, reduces the effects such as canceration incidence.Therefore, the correlated traits of anthocyanin synthesis is directly connected to agriculture, commodity through crop.Because tealeaves is extremely important beverage source, and anthocyanin is extremely water-soluble, and therefore having tea is the ideal style that human body takes in anthocyanin.At present, containing the tealeaves of anthocyanin be a large focus of industry class.

The conventional extraction of anthocyanin has: solvent extraction method, supercritical extraction, microwave method, enzymatic isolation method and ultrasonic extraction.Main isolation and purification method has: high performance countercurrent chromatography, membrane separation technique, gel chromatography, Flavonoids by Macroporous Adsorption Resin.External many scholars utilize C18 post, reversed-phase high-performance liquid chromatography (RP-HPLC), high speed adverse current chromatogram and high-efficient liquid phase chromatogram purification anthocyanin.

The molecular structure of anthocyanin can be obtained by LC-MS-MS.But require high, with high costs to experimental apparatus.Another practical method decomposes glycosidic bond by acidolysis, then identify the type of aglycone by the method for high performance liquid chromatography (HPLC).Although the anthocyanin that nature exists is of a great variety, the type of aglycone and anthocyanidin is very limited.The natural anthocyanidin overwhelming majority belongs to delphinidin (delphinidin), cyanidin (cyanidin), pelargonidin (pelargonidin), petunia pigment (petunidin), malvidin (malvidin), paeonidin (peonidin) six type.

The method of the extraction of tealeaves anthocyanin, purifying, qualification also relevant ripe, standard.One is because most of tea tree breed only synthesizes and synthesizes anthocyanin on a small quantity or not, as common greenery, yellow leaf kind.The tea tree breed that can synthesize anthocyanin is in a large number quite rare, only has domestic well-known as " purple cuckoo ", " the Sun Rouge " as Japan of famous foreign.Two is that the anthocyanin of fresh leaves of tea plant is purified and compares that other plant is more complicated more not easily purifies.Up to the present, have kind more than 700 through the known compound be separated, identify in tealeaves, polyphenols kind is many, and rich content, comprise catechin, flavonoids, flavonols, anthocyanin, leucoanthocyanidin class, phenolic acid and depside etc.

The people such as Shandong Agricultural University Wang Shuai study the extraction purification of tealeaves anthocyanin:

Get purple beautiful kind fresh leaves of tea plant sample 2Kg, be put in ultrasonic 10min in 1% methanol hydrochloride solution of 8L, cold soaking two h before harvest extract under 4 DEG C of low temperature, re-treatment twice, merge extract, extract rotary evaporation in vacuo at 30 DEG C, near dry, dissolved with a small amount of water, is used methenyl choloride, extraction into ethyl acetate respectively.Aqueous solution is gradient elution on the polyamide chromatography post handled well, and utilizes recrystallization and lead salt sedimentation to obtain different anthocyanin component.

The all extractions (food and fermentation science and technology, 2013, Vol.49, No.6, p38-42) waiting people to study tealeaves anthocyanin of Shandong Agricultural University's model skill:

Getting " purple beautiful " shines after dark green tea 2g pulverizes 2min, add 400mL extract (V methyl alcohol: V water: V formic acid: V trifluoroacetic acid=70: 27: 2: 1) room temperature lixiviate 1h, again with the centrifugal 5min of 5000r/min, after supernatant is evaporated to about 30mL after solvent micropore filtering film (accurate 50mm, 0.45 μm, organic system) filters under 50 ± 2 DEG C of conditions, freeze drying is to powder and be placed in-10 DEG C of refrigerator-freezers and preserve for pigment qualitative analysis.

The people such as Shandong Agricultural University Lu Hai Peng study the authentication method (Food Science, 2012, Vol.33, No.22, p203-206) of tealeaves anthocyanin component:

With " purple cuckoo " baked green tea sample for raw material, after extraction, purifying anthocyanidin, following condition is adopted to identify anthocyanin 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; Determined wavelength, 520nm; Standard model is pelargonin-3,5-diglucoside (pelargonidin-3,5-diglucoside chloride), corn flower-3-O-galactoside (cyanidin-3-Ogalactoside), pelargonidin (pelargonidinchloride), malvidin (malvidin chloride).

The people such as Shandong Agricultural University Wang Shuai disclose the method for one " purple beautiful " tea extraction purification anthocyanin, and the method mainly extracts with trichloroethanes, ethyl acetate, with polyamide chromatography post wash-out.Fan Yifan etc. (food and fermentation science and technology, 2013, Vol.49, No.6, p38-42) disclose the extracting method of one " purple beautiful " anthocyanin.The anthocyanin that said method extracts or degree of purification is not high or leaching process loss percentage is comparatively large, the anthocyanin of extracting according to these methods can not be used for qualitative, the Quantitative measurement in downstream.

Lv Haipeng etc. (food and fermentation science and technology, 2013, Vol.49, No.6, p38-42) disclose the method for " purple cuckoo " anthocyanin component.The first, the method only could be implemented when learning anthocyanin molecular structure.Because tea tree genetic background is complicated, the anthocyanin type of current known Different Tea Varieties synthesis is made a world of difference.The second, the standard model that the method uses buys difficulty, and the cost for purification as the anthocyanin molecule of standard specimen is high even cannot be bought from market.3rd, synthesize polytype anthocyanin when a certain tea tree and divide the period of the day from 11 p.m. to 1 a.m, the object of HPLC is too much, and background is too complicated, may the close anthocyanin of isolated molecule structure.

Summary of the invention

The present invention is to solve above-mentioned deficiency, provides a kind of method of extraction from tea fresh leaves, purifying, qualification anthocyanidin.

Above-mentioned purpose of the present invention is realized by following technical scheme: a kind of method of extraction from tea fresh leaves, purifying, qualification anthocyanidin, is characterized in that: comprise the following steps:

A. sample broke: precise plucks the two leaves and a bud 5g of fresh purple tea tree, goes to 50ml centrifuge tube after adding liquid nitrogen grinding;

B. lixiviate: the HCL-MeOH adding 20ml 1%, to sample, is placed in the static lixiviate of refrigerator 4 casees reefers; Centrifugal after 2h, rotating speed 9000r/min, centrifugation time 10min; Sucking-off supernatant moves to new 50ml centrifuge tube; Add 20ml 1%HCL-MeOH again and carry out secondary lixiviate to sample, centrifugal after 2h, rotating speed 9000r/min, centrifugation time 10min, transfer supernatant; Finally adding 10ml 1%HCL-MeOH again carries out third time lixiviate, centrifugal after 12h, transfer supernatant;

C. concentration: utilize Rotary Evaporators Rotavapour R-300 (Buchi, Switzerland) to concentrate the leaching liquor obtained, revolving and steaming instrument rotational speed is 4-7, and temperature is 37 °; Finally obtain the concentrate that volume is 25 ~ 30ml;

D. filtration treatment: by concentrate through 0.45 μm of organic phase needle cylinder type filter membrane metre filter;

E. Solid-Phase Extraction: use trans solid-phase extraction column c18 cartridge (Waters, the U.S.) extracts anthocyanin.Extraction procedure is as follows: post activates, and 5ml 0.01%HCL-MeOH, repeats once; Balance, 5ml 0.01%HCL; Loading; Drip washing, 5ml 0.01%HCL, repeats once, then adds 5ml normal hexane, repeats once; Wash-out, adds 5ml 0.01%HCL-MeOH, collects eluent, wash-out 3 times;

F. concentration: utilize Rotary Evaporators Rotavapour R-300 (Buchi, Switzerland) to be concentrated by the extract that Solid-Phase Extraction obtains, concentrates rear volume with 0.01%HCL-MeOH constant volume to 10ml;

G. acidolysis: remove 200ul sample to 1.5ml centrifuge tube, add 400ul 5mol/LHCl, seals with Parafilm sealed membrane.Constant temperature oven 90 ° process 30min, puts 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; Determined wavelength 520nm;

Standard model: delphinidin (delphinidin), cyanidin (cyanidin), pelargonidin (pelargonidin), malvidin (malvidin), paeonidin (peonidin) be purchased from American ChromaDex company all.

The present invention's advantage is compared with prior art:

1, tealeaves contains very high aldehydes matter, is very easily oxidized anthocyanin.This method can overcome the interference of other aldehydes matter effectively, obtains highly purified anthocyanin.As shown in Figures 2 and 3, adopt the Anthocyanin-rich Extract that obtains of the method to identify through HPLC, background material or interfering material few.

2, from fresh leaves of tea plant, extract the loss percentage of anthocyanin low for the method, favorable reproducibility.In " purple handsome " strain anthocyanidin leaching process, add paeonidin as interior mark, detect through HPLC and find, average loss rate is 7.24%.

3, the method is applicable to the qualitative and quantitative analysis of the anthocyanin of all tea tree breeds.As shown in Figures 2 and 3, " purple handsome " is identical with the anthocyanin type that " purple cuckoo " synthesizes, but component ratio is different.

Accompanying drawing explanation

Fig. 1 is anthocyanidin standard items chromatograms.

Fig. 2 is the chromatogram of tea strain " purple handsome " anthocyanin acid hydrolysate.

Fig. 3 is the chromatogram of " purple cuckoo " variety design thuja acid hydrolysis products.

Embodiment

Below in conjunction with 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 method of extraction from fresh leaves of tea plant, purifying, qualification anthocyanidin, comprises the following steps:

A. sample broke: precise plucks the two leaves and a bud 5g of fresh purple tea tree strain " purple handsome ", goes to 50ml centrifuge tube after adding liquid nitrogen grinding;

B. lixiviate: the HCL-MeOH adding 20ml 1%, to sample, is placed in the static lixiviate of refrigerator 4 casees reefers; Centrifugal after 2h, rotating speed 9000r/min, centrifugation time 10min; Sucking-off supernatant moves to new 50ml centrifuge tube; Add 20ml 1%HCL-MeOH again and carry out secondary lixiviate to sample, centrifugal after 2h, rotating speed 9000r/min, centrifugation time 10min, transfer supernatant; Finally adding 10ml 1%HCL-MeOH again carries out third time lixiviate, centrifugal after 12h, transfer supernatant;

C. concentration: utilize Rotary Evaporators Rotavapour R-300 (Buchi, Switzerland) to concentrate the leaching liquor obtained, revolving and steaming instrument rotational speed is 4-7, and temperature is 37 °; Finally obtain the concentrate that volume is 25 ~ 30ml;

D. filtration treatment: by concentrate through 0.45 μm of organic phase needle cylinder type filter membrane metre filter;

E. Solid-Phase Extraction: use trans solid-phase extraction column c18 cartridge (Waters, the U.S.) extracts anthocyanin.Extraction procedure is as follows: post activates, and 5ml 0.01%HCL-MeOH, repeats once; Balance, 5ml 0.01%HCL; Loading; Drip washing, 5ml 0.01%HCL, repeats once, then adds 5ml normal hexane, repeats once; Wash-out, adds 5ml 0.01%HCL-MeOH, collects eluent, wash-out 3 times;

F. concentration: utilize Rotary Evaporators Rotavapour R-300 (Buchi, Switzerland) to be concentrated by the extract that Solid-Phase Extraction obtains, concentrates rear volume with 0.01%HCL-MeOH constant volume to 10ml;

G. acidolysis: remove 200ul sample to 1.5ml centrifuge tube, add 400ul 5mol/LHCl, seals with Parafilm sealed membrane.Constant temperature oven 90 ° process 30min, puts 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; Determined wavelength 520nm;

Standard model: delphinidin (delphinidin), cyanidin (cyanidin), pelargonidin (pelargonidin), malvidin (malvidin), paeonidin (peonidin) be purchased from American ChromaDex company all.

Embodiment 2: as shown in Figure 1, Figure 3, a kind of method of extraction from fresh leaves of tea plant, purifying, qualification anthocyanidin, comprises the following steps:

A. sample broke: precise plucks the two leaves and a bud 5g of fresh purple tea tree strain " purple beautiful ", goes to 50ml centrifuge tube after adding liquid nitrogen grinding;

B. lixiviate: the HCL-MeOH adding 20ml 1%, to sample, is placed in the static lixiviate of refrigerator 4 casees reefers; Centrifugal after 2h, rotating speed 9000r/min, centrifugation time 10min; Sucking-off supernatant moves to new 50ml centrifuge tube; Add 20ml 1%HCL-MeOH again and carry out secondary lixiviate to sample, centrifugal after 2h, rotating speed 9000r/min, centrifugation time 10min, transfer supernatant; Finally adding 10ml 1%HCL-MeOH again carries out third time lixiviate, centrifugal after 12h, transfer supernatant;

C. concentration: utilize Rotary Evaporators Rotavapour R-300 (Buchi, Switzerland) to concentrate the leaching liquor obtained, revolving and steaming instrument rotational speed is 4-7, and temperature is 37 °; Finally obtain the concentrate that volume is 25 ~ 30ml;

D. filtration treatment: by concentrate through 0.45 μm of organic phase needle cylinder type filter membrane metre filter;

E. Solid-Phase Extraction: use trans solid-phase extraction column c18 cartridge (Waters, the U.S.) extracts anthocyanin.Extraction procedure is as follows: post activates, and 5ml 0.01%HCL-MeOH, repeats once; Balance, 5ml 0.01%HCL; Loading; Drip washing, 5ml 0.01%HCL, repeats once, then adds 5ml normal hexane, repeats once; Wash-out, adds 5ml 0.01%HCL-MeOH, collects eluent, wash-out 3 times;

F. concentration: utilize Rotary Evaporators Rotavapour R-300 (Buchi, Switzerland) to be concentrated by the extract that Solid-Phase Extraction obtains, concentrates rear volume with 0.01%HCL-MeOH constant volume to 10ml;

G. acidolysis: remove 200ul sample to 1.5ml centrifuge tube, add 400ul 5mol/LHCl, seals with Parafilm sealed membrane.Constant temperature oven 90 ° process 30min, puts on ice after terminating immediately;

H.HPLC detects: HPLC detection hardware condition is as follows: HPLC system is Agilent1260 (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; Determined wavelength 520nm;

Standard model: delphinidin (delphinidin), cyanidin (cyanidin), pelargonidin (pelargonidin), malvidin (malvidin), paeonidin (peonidin) be purchased from American ChromaDex company all.

The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize description of the present invention to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.

Claims (1)

1. a method for extraction from fresh leaves of tea plant, purifying, qualification anthocyanidin, is characterized in that: comprise the following steps:

A. sample broke: precise plucks the two leaves and a bud 5g of fresh purple tea tree, goes to 50ml centrifuge tube after adding liquid nitrogen grinding;

B. lixiviate: the HCL-MeOH adding 20ml 1%, to sample, is placed in the static lixiviate of refrigerator 4 casees reefers; Centrifugal after 2h, rotating speed 9000r/min, centrifugation time 10min; Sucking-off supernatant moves to new 50ml centrifuge tube; Add 20ml 1%HCL-MeOH again and carry out secondary lixiviate to sample, centrifugal after 2h, rotating speed 9000r/min, centrifugation time 10min, transfer supernatant; Finally adding 10ml 1%HCL-MeOH again carries out third time lixiviate, centrifugal after 12h, transfer supernatant;

C. concentration: utilize Rotary Evaporators to concentrate the leaching liquor obtained, revolving and steaming instrument rotational speed is 4-7, and temperature is 37 °; Finally obtain the concentrate that volume is 25 ~ 30ml;

D. filtration treatment: by concentrate through 0.45 μm of organic phase needle cylinder type filter membrane metre filter;

E. Solid-Phase Extraction: use trans solid-phase extraction column to extract anthocyanin; Extraction procedure is as follows: post activates, and 5ml 0.01%HCL-MeOH, repeats once; Balance, 5ml0.01%HCL; Loading; Drip washing, 5ml 0.01%HCL, repeats once, then adds 5ml normal hexane, repeats once; Wash-out, adds 5ml 0.01%HCL-MeOH, collects eluent, wash-out 3 times;

F. concentration: utilize Rotary Evaporators to be concentrated by the extract that Solid-Phase Extraction obtains, concentrates rear volume with 0.01%HCL-MeOH constant volume to 10ml;

G. acidolysis: remove 200ul sample to 1.5ml centrifuge tube, add 400ul 5mol/LHCl, seals with Parafilm sealed membrane; Constant temperature oven 90 ° process 30min, puts 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; Determined wavelength 520nm;

Standard model: delphinidin, cyanidin, pelargonidin, malvidin, paeonidin equal purchased from American ChromaDex company.