CN107973826B - Method for separating and purifying plant leaf anthocyanin by adopting mixed adsorption resin - Google Patents

Method for separating and purifying plant leaf anthocyanin by adopting mixed adsorption resin Download PDF

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CN107973826B
CN107973826B CN201711287802.5A CN201711287802A CN107973826B CN 107973826 B CN107973826 B CN 107973826B CN 201711287802 A CN201711287802 A CN 201711287802A CN 107973826 B CN107973826 B CN 107973826B
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杨淑红
张江涛
朱镝
高福玲
董玉山
赵蓬晖
马永涛
晏增
徐惠鸽
田丽
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Henan Academy Of Forestry
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Abstract

The invention belongs to the technical field of separation and purification of effective components of plant leaves, and particularly relates to a method for separating and purifying anthocyanin of plant leaves by using mixed adsorption resin. The invention adopts nonpolar and medium polar resins to mix according to a certain mass proportion, and regulates the hydrophobic acting force of the resin by regulating and controlling the content of different polar components in the resin skeleton, thereby weakening the adsorption acting force of the resin and anthocyanin, simultaneously not influencing the adsorption capacity of the resin to impurities, and finally forming the mixed macroporous resin filter body suitable for the separation and purification of plant leaf anthocyanin.

Description

Method for separating and purifying plant leaf anthocyanin by adopting mixed adsorption resin
Technical Field
The invention belongs to the technical field of separation and purification of effective components of plant leaves, and particularly relates to a method for separating and purifying anthocyanin of plant leaves by using mixed adsorption resin.
Background
Anthocyanins, the most important and widely distributed group of coloured substances in plants, are the basis of almost all colours in the petals, leaves and fruits of plants. Anthocyanins have the basic structure of flavonoids, i.e. a (C6-C3-C6) C15 compound consisting of two aromatic rings and one oxygen-containing heterocycle, which has the following structure:
Figure BDA0001498811220000011
anthocyanins are largely classified into six classes according to the location and number of hydroxylations and methylations of the B-ring: r1 ═ R2 ═ H is pelargonidin (pelargonidin); when R1 ═ OH and R2 ═ H, cyanidin (cyanidin) is obtained; r1 ═ OMe, R2 ═ H, peoniflorin (peonidin) (3' -methyl cyanidin); r1 ═ R2 ═ OH, delphinidin (delphinidin); r1 ═ OMe and R2 ═ OH are petunia (petuniain) (3',5' -methyldelphinidin); r1 and R2 are both malvidin (malvidin) (3',5' -dimethyl delphinidin). Free anthocyanin is rare under natural conditions, exists mainly in a glucoside form, has different anthocyanin types and components, can make tissues and organs such as flowers, leaves, fruits and the like of plants show various colors, and is from red, blue to purple, yellow and the like. In addition, anthocyanin is a natural pigment with biological activity, has the functions of antioxidation and free radical scavenging, and can improve the stress resistance of the plant. The anthocyanin-rich plant and vegetable can be used as a good source of natural pigment, so that the purification and separation process of the anthocyanin is also widely concerned in foods, medicines and cosmetics.
The macroporous resin adsorption method has the characteristics of various resin varieties, strong adsorption force, adjustable structure, simple separation process, wide application range, high selectivity, high efficiency and the like. The macroporous resin in different types has different polarities due to different molecular structures of the selected monomers, is generally divided into four types of non-polarity, medium polarity, polarity and strong polarity, is respectively suitable for separating and purifying different effective components and effective parts, and can reach the optimal level of separating and purifying the effective components only by selecting when in use.
Impurities contained in the crude anthocyanin extract are mainly components with stronger hydrophobicity, and hydrophilic anthocyanin usually adopts nonpolar or medium-polarity macroporous resin as a purification medium, so that when the adsorption acting force on anthocyanin is met to different degrees, the resin has stronger adsorption force on the impurities, so that the anthocyanin and the impurities are difficult to completely separate in the desorption process, and the quantity and the purity of the anthocyanin obtained are influenced.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for separating and purifying anthocyanin from crude extract of leaf anthocyanin by adopting nonpolar and medium-polarity mixed resin.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for separating and purifying plant leaf anthocyanin by adopting mixed adsorption resin, which comprises the following steps:
step 1: taking fresh plant leaves, shearing, adding an extracting solution, adding 10mL of the extracting solution into each 1g of sample, grinding until homogenate is obtained, carrying out ultrasonic extraction, then carrying out boiling water bath, cooling, diluting by 1.5-2 times with ethanol with the volume fraction of 60%, and then taking the supernatant and filtering with a 0.45 mu m filter membrane to obtain a crude anthocyanin extracting solution of the leaves;
step 2: respectively carrying out activation pretreatment on medium-polarity macroporous resin and nonpolar macroporous resin, and uniformly mixing the pretreated resins to obtain mixed resin;
and step 3: putting the mixed resin into a ground bottle with a plug, adding 30mL anthocyanin crude extract solution into every 1g of mixed resin, oscillating in a water bath at 25 ℃ for 1-2.5 h, and carrying out suction filtration to receive filtrate;
and 4, step 4: putting the mixed resin filtered out in the step 3 into an eluent, wherein the eluent is an ethanol solution with the volume fraction of 40% -90%, oscillating in a water bath at 25 ℃ for 1-5 h for desorption, performing suction filtration, and receiving filtrate;
and 5: and (4) mixing the filtrates obtained in the steps (3) and (4), and performing rotary evaporation and vacuum drying to remove the eluent to obtain the anthocyanin concentrated solution.
Further, the extracting solution in the step 1 is prepared from absolute ethyl alcohol: water: the concentrated hydrochloric acid is prepared according to the volume ratio of 2:1: 1.
Further, the medium-polarity macroporous resin in the step 2 comprises XAD-7, XAD-8, HPD500 and NKA-9; the non-polar macroporous resin comprises HPD100, HPD700 and X-5.
Further, the medium-polarity macroporous resin is NKA-9; the nonpolar macroporous resin is HPD 700.
Further, the mass ratio of the NKA-9 to the HPD700 is 1: 5-1: 2.
Further, the mass ratio of the NKA-9 to the HPD700 is 1: 4-1: 3.
Further, oscillating in water bath at 25 ℃ for 1.5-2 h in the step 3.
Further, the rotation speed of the 25 ℃ water bath oscillation in the steps 3 and 4 is 100 r/min.
Further, the eluent in the step 4 is ethanol solution with the volume fraction of 60%.
Further, in step 4, the mixture was shaken in a water bath at 25 ℃ for 3 hours.
The step 4 is a selection step, so that the process flow can be reduced if high-purity anthocyanin is obtained, the step 4 is omitted, and impurities brought by desorption are avoided. If the maximum amount of anthocyanin in the crude extract is desired, then step 4 is used to desorb the anthocyanin filtrate before it is concentrated.
Compared with the prior art, the invention has the beneficial effects that:
(1) the non-polar strong-hydrophobicity resin and the medium-polar weak-hydrophobicity resin which are mixed according to a certain mass ratio regulate the hydrophobic acting force of the resin by regulating the content of different polar components in the resin framework, weaken the adsorption acting force of the resin and anthocyanin, simultaneously do not influence the adsorption capacity of the mixed resin on impurities, and are suitable for separation and purification of plant leaf anthocyanin.
(2) The 'desorption' in the invention is a selection step, the mixed resin filter body can fully adsorb impurities and simultaneously ensure that the adsorption quantity of anthocyanin is minimum, a high-purity product can be directly obtained without the 'desorption' step, the process flow is reduced, and the impurities are prevented from being brought in again in the desorption process. If desorption is selected, the anthocyanin in the crude extract can be obtained to the maximum, the steric effect of the adsorption resin on the desorption of the anthocyanin can be effectively relieved by the ethanol eluent and the shorter desorption time, and the newly increased active points are prevented from re-adsorbing the anthocyanin, so that the aim of separating and desorbing the anthocyanin is fulfilled.
(3) The mixed resin in the invention has simple mixing treatment, high stability and repeated use.
(4) The static adsorption time of the mixed resin on impurities and the static desorption time of the adsorbed anthocyanin are shorter, and the purification and desorption efficiencies are obviously improved compared with the prior method.
(5) The purity, direct recovery rate and post-desorption recovery rate of the anthocyanin finally obtained by using the mixed resin are greatly improved compared with those of single resin.
(6) The method improves the accuracy of qualitative and quantitative analysis of anthocyanin in the leaves of the colored-leaf plants, reduces the error influence caused by more impurities, and is an ideal method for extracting anthocyanin in the leaves of the colored-leaf plants.
Drawings
FIG. 1 shows the effect of static adsorption time on the adsorption capacity of 3 mixed resins, in which the mass ratios of NKA-9 and HPD700 are 1:4, 2:7 and 1:3, respectively;
FIG. 2 shows the influence of ethanol concentration in desorption solution on the desorption rate of 3 mixed resins, wherein the mass ratio of NKA-9 to HPD700 in the 3 mixed resins is 1:4, 2:7 and 1:3 respectively;
FIG. 3 shows the effect of desorption time on the desorption rate of 3 mixed resins, in which NKA-9 and HPD700 are present in the mass ratio of 1:4, 2:7 and 1:3, respectively.
Detailed Description
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example preparation of an anthocyanin extract
Taking fresh leaves of populus tremuloides, washing the leaves with distilled water, and airing. Removing petioles and veins, cutting, weighing 3.0g of a sample in a mortar, adding absolute ethyl alcohol: water: fully grinding 10mL of HCl 2:1:1 extracting solution until homogenate is obtained, transferring the homogenate into a 50mL test tube, adding the extracting solution, fixing the volume to 30mL, shaking up for 1min, and then carrying out ultrasonic extraction for 2 times, 20min each time. Then bathing in boiling water for 1h, taking out and cooling, then fixing the volume to 50mL by using an ethanol solution with the volume fraction of 60%, standing, taking the supernatant, and filtering by using a 0.45-micrometer filter membrane to obtain the crude extract of the anthocyanin in the red leaf poplar leaves.
EXAMPLE two optimization of macroporous resin type
According to the preparation method of the anthocyanin extract in the first embodiment, 300mL of the red leaf poplar leaf anthocyanin crude extract is prepared and is refrigerated for later use. Usually, anthocyanin has the maximum absorption peak in the visible light region of 520-560 nm, and a visible spectrophotometer is used for scanning in the range of 400-700 nm to measure the absorption wavelength of anthocyanin. The method comprises the following steps: taking 2mL of the crude extract, diluting to 10mL with an ethanol solution with a volume fraction of 60%, taking the ethanol solution with the volume fraction of 60% as a reference, immediately scanning with a visible spectrophotometer within a range of 400-700 nm, and determining that the maximum absorption wavelength of anthocyanin in the sample solution is 531 nm.
Selecting 8 kinds of macroporous resin including non-polar HPD700, HPD100, X-5, weak polar AB-8, medium polar XAD-7, XAD-8, HPD500 and NKA-9 for separating and purifying leaf anthocyanin.
Firstly, carrying out resin activation pretreatment: soaking the materials in absolute ethyl alcohol for 24 hours to fully swell the materials, then washing the materials with absolute ethyl alcohol until no white turbidity exists, washing the materials with distilled water until neutral and no alcohol smell exists, then soaking the materials with hydrochloric acid with volume fraction of 5% for 10 hours, washing the materials with distilled water until the materials are neutral, then soaking the materials with sodium hydroxide with mass fraction of 2% for 12 hours, and washing the materials with distilled water until the materials are neutral.
Weighing 1.0g of each of the treated 8 resins, respectively placing the resins into 50mL of ground conical flasks with plugs, and respectively adding 30mL of the crude extract of the anthocyanin of the red leaf poplar leaves; oscillating for 6h in water bath at 25 ℃ and at the rotating speed of 100r/min, filtering, and collecting filtrate; adding 20mL of ethanol solution with volume fraction of 60% into the filtered adsorption resin, oscillating in water bath at 25 ℃ at 100r/min for 3h for desorption, performing suction filtration, and collecting filtrate; and respectively measuring the absorbance of the crude anthocyanin extract, the filtrate after adsorption and the filtrate after desorption at 531 nm.
The adsorption amount, adsorption rate and desorption rate were calculated by the following formulas (refer to king girl et al, 2009):
adsorption amount ═ A0-A1)×V0(ii) a/M; adsorption rate (%) - (A)0-A1)/A0X is 100%; desorption rate (%) ═ A2/(A0-A1) X is 100%; wherein M is the mass of the resin; a. the0Is the crude extract absorbance value; v0The volume of the crude extract added to the mixed resin; a. the1The absorbance value of the filtrate after resin adsorption is obtained; a. the2Is the absorbance value of the filtrate of the mixed resin after ethanol desorption.
The anthocyanin content (%) in the crude extract and the recovered solution was calculated using the formula X-A.times.V/W (Zhangiang, 1990: 183-191.). In the formula: x is anthocyanin content (%); a is the absorbance value of the sample at the wavelength of 531 nm; v is the volume of the sample after dilution; w is the weight of the sample. Purity ═ X1(ii) a Recovery rate of (X)1×W1)/(X0×W0)；X1The anthocyanin content in the filtrate is shown; x0The anthocyanin content in the crude extract is shown; w1Weighing the obtained anthocyanin product; w0Is the weight of the crude anthocyanin extract.
TABLE 1 comparison of adsorption and desorption of crude extract of anthocyanin from red leaf poplar leaves by different macroporous resins
Figure BDA0001498811220000051
As can be seen from Table 1, the adsorption and desorption degrees of anthocyanin were different for different resins, depending on the polarity and type of the resin; the factors of adsorption rate, desorption rate, product purity and recovery rate are comprehensively considered, and the result shows that the nonpolar HPD700 and the medium-polarity NKA-9 resin have good effects of purifying and recovering anthocyanin.
EXAMPLES optimization of the mixing ratio of the triple HPD700 and NKA-9
According to the preparation method of the anthocyanin extract in the first embodiment, 200mL of the crude extract of the anthocyanin of the red leaf poplar leaves is prepared and is refrigerated for later use.
Respectively taking the processed macroporous resins NKA-9 and HPD700, uniformly mixing according to different mass proportions, respectively weighing 1.0g of each mixed resin, placing the mixed resins into 50mL of ground conical bottles with plugs, and respectively adding 30mL of the crude extract of anthocyanin of red leaf poplar leaves; oscillating for 2h in water bath at 25 ℃ and at the rotating speed of 100r/min, and receiving filtrate; putting the filtered resin into 20mL of ethanol solution with volume fraction of 60%, oscillating in water bath at 25 ℃ for 3h at 100r/min, and filtering to receive filtrate; and (3) respectively measuring the absorbance of the crude anthocyanin extract, the filtrate after adsorption and desorption at 531nm, and calculating the adsorption quantity, the adsorption rate, the desorption rate, the purity and the recovery rate.
TABLE 2 adsorption and desorption effects of mixed resins of different proportions on crude extract of anthocyanin from red leaf poplar leaf
Figure BDA0001498811220000052
In order to make the polarity and skeleton of the resin in the range most suitable for filtering and separating anthocyanin, and not reduce the adsorption force of the mixed resin on impurities, through the mixing of the nonpolar HPD700 and the medium polarity NKA-9 resin with different mass ratios, the following are found: when the NKA-9: when HPD700 is 1:5, the anthocyanins obtained by direct receiving and desorption are of highest purity, but recovery rates are relatively low; when the NKA-9: when the HPD700 is in a ratio of 1: 4-1: 3, the purity of the obtained anthocyanin is above 81.15%, the direct recovery rate is above 80.89%, and the recovery rate after desorption is above 91.82%; when the NKA-9: when HPD700 was 1:2, the purity of the obtained anthocyanin was significantly reduced. Therefore, the mass ratio of NKA-9 to HPD700 is optimally 1: 4-1: 3, the red poplar anthocyanin is separated and purified by using the mixed resin in the ratio, the finally obtained anthocyanin has the purity which is more than 1.43 times that of the anthocyanin extracted by single resin of NKA-9 and HPD700, the direct recovery rate is more than 80.89%, the recovery rate after desorption is more than 91.14%, and the recovery rate is improved by more than 4.86% compared with the single resin.
EXAMPLE optimization of the four static adsorption times
According to the preparation method of the anthocyanin extract in the first embodiment, 800mL of the crude extract of the anthocyanin of the red leaf poplar leaves is prepared and is refrigerated for later use.
Uniformly mixing pretreated NKA-9 and HPD700 macroporous resins according to the mass ratio of 1:4, 2:7 and 1:3, weighing 1.0g of each mixed resin and 8 parts of each mixed resin, respectively placing the mixed resins into 50mL of ground conical bottles with plugs, and respectively adding 30mL of crude anthocyanin extract of red leaf poplar leaves; oscillating for 0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 hours in water bath at 25 ℃ and at the rotating speed of 100r/min respectively, filtering, and collecting filtrate; and respectively measuring the absorbance of the crude anthocyanin extract and the filtrate adsorbed by the resin at 531nm, and calculating the adsorption quantity.
The result is shown in figure 1, 3 mixed resins all realize rapid adsorption of impurities within 1.5h, are in an adsorption equilibrium state within 1.5-2 h, and the adsorption amount is rapidly increased when the adsorption time is more than 2.5h, which indicates that the swelling strength of the extracting solution to the resins exposes more active sites after long-time extraction, and the adsorption effect on anthocyanin in the filtrate is increased; therefore, the static adsorption time of the crude anthocyanin extract is optimally 1.5-2 h.
EXAMPLE five optimum Desorption solution concentration screening
According to the preparation method of anthocyanin extract in the first embodiment, 700mL of crude extract of anthocyanin from red leaf poplar leaves is prepared and refrigerated for later use.
Uniformly mixing pretreated macroporous resins NKA-9 and HPD700 according to the mass ratio of 1:4, 2:7 and 1:3, weighing 1.0g of each mixed resin and 7 parts of each mixed resin, respectively placing the mixed resins into 50mL of ground conical bottles with plugs, and respectively adding 30mL of crude anthocyanin extract of red leaf poplar leaves; oscillating for 2h in 25 ℃ water bath at the rotating speed of 100r/min, filtering, and collecting filtrate; adding the filtered resin into 20mL of ethanol solutions with volume fractions of 40%, 50%, 60%, 70%, 80%, 90% and 95%, respectively, oscillating in a water bath at 25 ℃ at 100r/min for 3h, performing suction filtration, and collecting filtrate; and respectively measuring the absorbance of the crude anthocyanin extract, the filtrate after adsorption and the filtrate after desorption at 531nm, and calculating the desorption rate.
The results are shown in fig. 2, in the 3 mixed resins with mass ratio, the desorption rate of anthocyanin increases firstly and then decreases with the increase of the concentration of ethanol solution, and all reach the maximum value when the volume fraction of ethanol is 60%, which shows that the swelling strength of the ethanol solution with volume fraction of 60% to the mixed resins can greatly weaken the steric effect of desorbing anthocyanin so as to desorb the anthocyanin from the adsorbed impurities, and the desorption rate of the 3 mixed resins is higher than 54.3%; the swelling strength of the mixed resin is not enough due to the excessively low concentration of the ethanol, and the desorption is not sufficient; the swelling capacity of the mixed resin is increased due to the over-high concentration of the ethanol, the exposed active sites are increased, and the anthocyanin is adsorbed on the resin again.
EXAMPLES six preference for static Desorption time
According to the preparation method of the anthocyanin extract in the first embodiment, 600mL of the crude extract of the anthocyanin of the red leaf poplar leaves is prepared and is refrigerated for later use.
Uniformly mixing pretreated macroporous resins NKA-9 and HPD700 according to the mass ratio of 1:4, 2:7 and 1:3, weighing 1.0g of each mixed resin and 6 parts of each mixed resin, respectively placing the mixed resins into 50mL of ground conical bottles with plugs, and respectively adding 30mL of crude extract of anthocyanin of red leaf poplar leaves; oscillating for 6h in 25 ℃ water bath at the rotating speed of 100r/min, filtering and collecting filtrate. Respectively adding the filtered resin into 20mL of ethanol solution with volume fraction of 60%, respectively oscillating in water bath at 25 ℃ under 100r/min for 1, 2, 3, 4, 5 and 6 hours, carrying out suction filtration, and collecting filtrate; and respectively measuring the absorbance of the crude anthocyanin extract, the filtrate after adsorption and the filtrate after desorption at 531nm, and calculating the desorption rate.
The result is shown in fig. 3, the desorption rate of the ethanol with the volume fraction of 60% to 3 mixed resins is gradually increased within 3 hours, and slowly decreases within 3-4 hours and then rapidly decreases. The desorption time is less than 3h, and the 60 percent ethanol solvent has insufficient swelling strength to the resin, so that the desorption of the adsorbed anthocyanin in the holes is not facilitated; when desorbed for 3 hours, the resin swelling strength is suitable for desorption of anthocyanin and can keep balance basically in a certain time; when the desorption time exceeds 4 hours, the resin is excessively swelled to increase micropores and active points in the micropores, so that the adsorption effect of the resin is enhanced, anthocyanin is re-adsorbed on the resin, and therefore, the static desorption time is optimally 3 hours.
The above-mentioned embodiments are merely preferred embodiments of the present invention, which are merely illustrative and not restrictive, and it should be understood that other embodiments may be easily implemented by those skilled in the art by means of replacement or modification according to the technical contents disclosed in the specification, and therefore, all changes and modifications that come within the spirit and technical conditions of the present invention should be included in the claims of the present invention.

Claims (5)

1. A method for separating and purifying plant leaf anthocyanin by adopting mixed adsorption resin is characterized by comprising the following steps:
step 1: taking fresh plant leaves, shearing, adding an extracting solution, adding 10mL of the extracting solution into each 1g of sample, grinding until homogenate is obtained, carrying out ultrasonic extraction, then carrying out boiling water bath, diluting 1.5-2 times by using ethanol with volume fraction of 60% after cooling, and then taking the supernatant and filtering the supernatant with a 0.45 mu m filter membrane to obtain a crude anthocyanin solution of the leaves; the plant is red-leaf poplar;
step 2: respectively carrying out activation pretreatment on medium-polarity macroporous resin and nonpolar macroporous resin, and uniformly mixing the pretreated resins to obtain mixed resin; the medium-polarity macroporous resin is NKA-9; the nonpolar macroporous resin is HPD 700; the mass ratio of the NKA-9 to the HPD700 is 1: 4-1: 3;
and step 3: putting the mixed resin into a ground bottle with a plug, adding 30mL of anthocyanin crude extract solution into every 1g of mixed resin, oscillating in a water bath at 25 ℃ for 1.5-2 h, and carrying out suction filtration to receive filtrate;
and 4, step 4: putting the mixed resin filtered out in the step 3 into an eluent, wherein the eluent is an ethanol solution with the volume fraction of 40% -90%, oscillating in a water bath at 25 ℃ for 1-5 h for desorption, performing suction filtration, and receiving filtrate;
and 5: mixing the filtrates obtained in the steps 3 and 4, and performing rotary evaporation and vacuum drying to remove the eluent to obtain an anthocyanin concentrated solution;
step 4 is a selection step, if high-purity anthocyanin is to be obtained, the step 4 is omitted, and impurities brought by desorption are avoided; if the maximum amount of anthocyanin in the crude extract is desired, then step 4 is used to desorb the anthocyanin filtrate before it is concentrated.
2. The method for separating and purifying plant leaf anthocyanin by using mixed adsorption resin as claimed in claim 1, wherein the extract in step 1 is prepared from absolute ethyl alcohol: water: the concentrated hydrochloric acid is prepared according to the volume ratio of 2:1: 1.
3. The method for separating and purifying plant leaf anthocyanin by using mixed adsorption resin as claimed in claim 1, wherein the rotation speed of the 25 ℃ water bath oscillation in the steps 3 and 4 is 100 r/min.
4. The method for separating and purifying plant leaf anthocyanin by using mixed adsorption resin as claimed in claim 1, wherein the eluent in the step 4 is ethanol solution with volume fraction of 60%.
5. The method for separating and purifying plant leaf anthocyanin by using mixed adsorption resin as claimed in claim 1, wherein the oscillating process is carried out in water bath at 25 ℃ for 3h in step 4.
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WO2009100165A2 (en) * 2008-02-06 2009-08-13 The Ohio State University Research Foundation High-purity fractionation of anthocyanins from fruits and vegetables
CN101659793A (en) * 2009-09-18 2010-03-03 华南师范大学 Method for extracting gynura bicolor purpurin

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Publication number Priority date Publication date Assignee Title
CN101120771A (en) * 2007-09-11 2008-02-13 展鹏远 Method for preparing cabbage pigment with cabbage leaves
WO2009100165A2 (en) * 2008-02-06 2009-08-13 The Ohio State University Research Foundation High-purity fractionation of anthocyanins from fruits and vegetables
CN101659793A (en) * 2009-09-18 2010-03-03 华南师范大学 Method for extracting gynura bicolor purpurin

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