Enrichment Daji macroporous resin of total flavonoids of Technology
【Abstract】 Objective To screen for separation and
purification of total flavonoids Daji macroporous resin and established the purification process parameters. Adsorption
and desorption method to study the rate of four kinds of models as an indicator of resin to determine the best resin purification Daji flavonoids. Through single-factor analysis
to study the resin separation and purification of total flavonoids Daji the optimum conditions. Results AB 8
adsorption rate and desorption rate are higher than other three kinds of resins. The specific process conditions for the control sample concentration 5.2 mg / ml, flow rate 0.5 ml / min, the sample size 3.5BV, elution concentration of 80%,
elution dosage 10BV. Conclusion AB-8 resin in the process
established under the conditions of purification Daji good effect of total flavonoids, total flavonoids yield up to nearly 90%.
Key words Daji flavonoids macroporous resin
Study on the Separation and Purification of Total Flavone from Herb Cirsii japonici by Macroporous Absorption Resin
Abstract: ObjectiveTo select the suitable resin and establish the optimal conditions for separation and purification of total flavone from herb Cirsii japonici. MethodsTo determine the optimal one by inspection of four types of macroporous resin. The optimal process of purification was studied by single factor analysis. ResultsThe static absorption capacity, the absorption ratio and the
static elution ratio of AB-8 resin was higher than the others. The optimal technological conditions were found in AB-8 as
follows: sample concentration was 5.2 mg / ml, flow rate was 0.5 ml / min, sample volume was 3.5BV, and 80% ethanol was used as eluting agent and its dosage was 10BV. ConclusionThe type of AB-8 showed comprehensive property in the condition and the yield of the total flavones achieved nearly 90%.
Key words: Herb Cirsii japonici; Total flavone; Macroporous absorption resin
For the thistle Compositae Cirsium japonicum Cirsium japonicum Fisch. Ex DC. The dry aerial parts or roots, with cooling blood to stop bleeding, eliminate carbuncle Removing silt function . Cirsium japonicum contains a variety of chemical composition : flavonoids, flavonoid glycosides, alkaloids, volatile oils and sitosterol, etc., in which flavonoid glycosides Linaria vulgaris leaves to stop bleeding as the main active ingredients .
Macroporous resin is a kind of three-dimensional porous
structure and selective adsorption function of polymer materials, macroporous adsorption resin separation technology from the late 20th century, 70 FENG Xiao Zhang, and others on the chemical constituents of Gastrodia beginning of the study, now widely used in traditional Chinese medicine active ingredients such as flavonoids, saponins, alkaloids, glycosides and other components of the separation and purification. Through this experiment four kinds of different types of macroporous resin screened to determine the AB-8 type
resin Daji flavonoids have a higher rate of adsorption and desorption experiments determined through use of the macroporous resin purified flavonoid Daji the best process conditions.
A reagent and instrument
Cirsium japonicum extract (homemade); rutin standard (identification of Pharmaceutical and Biological Products Institution, determination of use, batch 0080-9705); D101-
type, AB-8 type, RAR-9 type and HPD a 450 resin ( Deep Blue adsorption exchange materials in Shaanxi limited liability
company); U-3000 UV spectrophotometer (Japan HITACHI
companies). Chemical reagents were analytically pure, water
2 Methods and Results
2.1 Preparation of reference substance, said precision reference substance taking Rutin 0.020 14 g, set 100 ml flask, add 60% ethanol amount, Zhen Yao dissolved and diluted to the scale, shake derived.
2.2 Preparation of samples
Daji take appropriate herbs, with six times the amount of 70% ethanol was extracted twice, 2 h / times, recycling
ethanol, filtering, adding water constant volume to 1 000 ml, put it aside for 24 h, standby.
2.3 rutin standard curve drawing reference substance 
The standard solution of rutin were collected from 0,0.5,
1,2,3,4,5 ml in 10 ml flask, by adding 0.6 ml 5% NaNO2 solution, shake. Put it aside for 6 min, by adding 0.6 ml 10% Al (NO3) 3 solution, shaken, 6 min later by adding 3ml 8.6% NaOH solution and shake. With 60% ethanol, diluted to constant volume scale, at wavelength of 505 nm at colorimetric assay (with zero tube blank). The results shown in Figure 1.
2.4 Determination of total flavonoids
Get an accurate volume of 1 ml sample in 10 ml flask, press "2.3" measured at the absorbance at 505 nm. Rutin
standard curve and then find out the corresponding flavonoid content. Test fluid content of total flavonoids calculated by the following formula:
The concentration of total flavonoids Po = y
Where: y is the corresponding standard curve of rutin
concentration (mg / ml).
2.5 Resin pretreatment
Washed with redistilled to small resin and broken resin, wet-loaded post-column with 10 cm above the resin layer of anhydrous ethanol, soaked 24 h, released extract and continue
to rinse with anhydrous ethanol to wash out the liquid, add water (1: 5) does not appear turbid, and then re-steam washed
with water to non-alcohol taste, reserve.
2.6 Resin Filter
2.6.1 Static adsorption behavior study
Were said to take four kinds of models have been handled
well in the resin: AB-8, RAR-9, D101, and HPD450 type
macroporous resin of 2.00 g (wet weight) in 100 ml flask with plug, by adding 30 ml sample solution , 20 ? under Zhenyao 2
h, put it aside for 24 h filter, absorb the filtered sample
solution by 2.4 Determination of adsorption equilibrium solution concentration of total flavonoids. Determined by the formula statically saturated adsorption capacity and adsorption rate. The results in Table 1.
Static adsorption capacity of E = (Po-Pa) Va / M (Unit:
mg / g)
Adsorption rate s = (Po-Pa) / Po
Where: Po --- sample solution in the initial
concentration of total flavonoids: mg / ml; Pa - adsorption
equilibrium solution concentration of total flavonoids: mg / ml; reposted elsewhere in the paper for free download http://www. hi138.com
Va: volume of sample solution (ml)
M: the amount of resin (g)
2.6.2 Static elution behavior study
After taking the above-mentioned static saturated
adsorption of total flavones of resin after the filter out, take 20 ml pure water washing, after 20 min dry surface moisture with filter paper, precision by adding 95% ethanol 30 ml, from time to time Zhen Yao, continuous 5 h, measured elution of concentration, determined by the formula of the
desorption rate of resin for each model. The results in Table 1. Table 1 four kinds of unsaturated resin static saturated adsorption capacity adsorption rate and desorption rate
Desorption rate = [eluent concentration × elution
volume] × 100% saturated adsorption amount
From the above results show that the four kinds of resin AB-8 has good adsorption rate and desorption rate are higher than the other three kinds of resin, but has good HPD450 desorption rate, but the absorption rate is too low, so in this second experiment, choose AB-8 resin for purification and
concentration of flavonoids in Daji.
Dynamic adsorption behavior of 2.7 visits
2.7.1 Determination of adsorption velocity
Take appropriate sample solution, respectively 0.5,1,2,3 ml / min flow rate on the samples collected under various flow rate of outflow of fluid, the outflow of fluid were measured total flavonoids, according to "2.6.1" absorption rate calculations. The results in Table 2. Table 2 Determination of adsorption velocity (abbreviated)
We can see from the above results: the impact absorption speed is an important factor in adsorption rate, slow favor adsorption. When the flow too fast, the flavone constituents
has not yet been fully adsorbed with the adsorption on the liquid flow in order to ensure complete absorption and therefore selective adsorption rate of 0.5 ml / min.
2.7.2 Adsorption of concentration in the choice of
Were collected from four concentrations of
6.33,5.20,3.76,1.25 mg / ml sample to 0.5 ml / min at the speed of samples were determined to be completely out of fluid absorption in the absorption rate of total flavonoids. The results shown in Figure 3.
We can see from the diagram: As the concentration
increases, the absorption rate increased. But the concentration of 6.33 mg / ml, the sample liquid is too thick, almost into the extract shape is not conducive to its spread and absorption. Have chosen to sample solution on the sample
concentration 5.20 mg / ml.
2.7.3 Leakage Curve Drawing
Taking a concentration of 5.2 mg / ml of the sample solution 200 ml, to 0.5 ml / min speed added to 200 g (wet weight) resin column were measured for the resin bed volume 40 ml. Sub-collection of outflow of fluid, 10 ml of 1 per cent, total flavonoids were determined, drawing leakage curve. Figure 3.
Curve we can see from the leak: Daji flavonoids from the first 14 Division started to leak out, this time moved to the
naked eye can see samples of ribbon at the bottom of column. Sample solution to 18 copies of Office to reach adsorption equilibrium. They can be used to determine the concentration of 5.2 mg / ml, the solution on the sample volume was 3.5-fold
when the resin bed volume was better than this proportion will leak.
2.7.4 the choice of elution concentration
Precision Weigh 5 copies of AB-8 resin 2g (wet weight),
respectively, with a plug shaped bottle set, plus the sample liquid 20 ml, the full adsorption, separation of resin,
drying, corresponding to adding concentration of 10%, 30 %, 60%, 80%, 95% ethanol of 20 ml, from time to time Zhen Yao, continuous 5 h, measured eluate concentration, washout rate calculation results in Table 3. Table 3 elution concentration
of the selection (abbreviated)
2.7.5 the choice of eluent volume
According to the above to determine the adsorption and elution conditions, the sample liquid to be completely adsorbed after washing with redistilled to colorless or pale
color is no longer up, take 80% ethanol solution 600 ml start-
eluting resin, sub-collection of eluate. 15 ml of each
collected a total of 40. Determination of Flavonoids in eluate to outflow of liquid volume of each abscissa, the corresponding concentration curve ordinate drawing elution. The results shown in Figure 4.
From Figure 4, we can see, the first 26 eluate beginning of flavonoids have been closer to eluting balance, can be considered the adsorption resin column-like flavonoids eluting
balance has been fully met and therefore determine the elution agent is 10BV ( The resin bed volume of 40 ml).
We can see from Table 3, with 95% ethanol eluted resin column, ethanol eluting materials of the highest content of flavonoids Daji, but the concentration of 80% ethanol eluate with more or less, from an economic point of view, it is selected in this experiment 80% ethanol as eluting agent.
2.7.6 Experimental verification
Daji sample solution obtained by the above method of
choice for adsorption and elution process conditions determined by the concentration of total flavonoids eluate to calculate the yield of total flavonoids of 89.53%.
In the dynamic adsorption process, the liquid resin, the first contact with the upper part of the column, and the first of the adsorption saturation, and then the saturated adsorption state of progressively down passage of time, constitute the chromatographic zone, when all the resin adsorption to a certain extent, started to be separated
leakage, stop adsorption, which is to achieve the best adsorption process .
In the experiment found that, in addition to selection of excellent performance macroporous resin, we must also meet the optimum conditions, such as the best adsorption flow rate,
sample solution on the sample concentration, which have a direct impact on the effect of concentration of flavonoids.
In order to achieve a better purification purposes, gradient elution may be better to play a role requires a
combination of TLC or HPLC to target compound for further investigation.
AB-8 resin as a weak polar resin, suitable for separation and purification of flavonoids from Cirsium japonicum. We can see from the experiment: when the sample concentration of 5.2
mg / ml, control sample flow rate of 0.5 ml / min, the sample volume of 3.5 BV when the adsorption is better with 10BV80% ethanol eluted with a total yield of nearly 90 flavone %. Therefore, this method can be used in separation and
purification of flavonoids Daji.
 Xiao Pei-Gen. A New Chinese medicine Zhi, Volume 3 [M]. Beijing: Chemical Industry Press, 2001:5.
 Zhi-Fei, KONG Ling-yi, PENG Si-qin. Medicine Daji
chemical and pharmacological research [J]. Chinese herbal
medicine, 2001,32 (7): 664.
 Ishida H. Chem Pharm Bull, 1987,35 (2): 861.
 Ni Xiao Ni, Zhao Hongyang, Hang Tai-Jun. Daji,
thistle flavonoids of different extraction methods and determination of [J]. Jiangsu Pharmaceutical and Clinical
Research, 2005,13 (1): 33.
 Feng-year-ping, Yu Wei. TCM Extraction Technology and Its Application [M]. Beijing: Chinese Medical Science and
Technology Publishing House, 2005:109. Reposted elsewhere in the paper for free download http://