Puerarin(4，7-dihydroxy-8-β-D-glucosylisoflavone)is a major isoflavone glycoside isolated from the roots of Pueraria lobata(Willd)Ohwi(Figure 1)which has been used as traditional Chinese medicinal herb for centuries. Its comprehensive biological actions have been well-documented by numerous studies，regarding the protective effects on pancreatic islet cells(Xiong et al，2006)，myocardial fibrosis(Chen et al，2012)，liver fibrosis(Li et al，2013; Zhang et al，2006)，liver injury(Liu et al，2011; 2012)， and retinal micro vascular dysfunction(Kim et al，2012). Puerarin could attenuate the endothelial insulin resistance through inhibiting the inflammatory response(Huang et al，2012). Therefore， puerarin has attracted a lot of attention and the preparation of puerarin is of significance for not only the further pharmacological and clinical effects research(Zhang et al，2012)，but also improvement of the quality. However，the separation and purification of puerarin using the conventional methods，such adsorption chromatography(He et al，2004)， require several steps resulting in a low recovery.

Figure 1 Chemical structure of puerarin

Countercurrent chromatography(CCC)is a liquid-liquid partition chromatography that uses no solid stationary phase support. The stationary phase is retained inside the column due to the centrifugal field，while the mobile phase is pumped through the stationary phase. The solutes are separated through the column according to their respective partition coefficient. Therefore，the CCC has some advantages:(1)irreversible adsorption，(2)massive amounts of sample injection，(3)less solvent consumption， and (4)time saving(Berthod，2002). Two types of CCC apparatuses were used in CCC separation:(1)apparatuses containing coiled tubes，the hydrodynamic CCC machines， and (2)those with rotary seals and channels，the hydrostatic CCC machines. The hydro- dynamic machines would be called high speed countercurrent chromatography(HSCCC)using a variable gravity field(Hu et al，2013a; 2013b). The hydrostatic machines would be called centrifugal partition chromatography(CPC)using a constant gravity field(Foucault，1995). Puerarin was isolated and purified from the roots of P. lobata by HSCCC(Cao et al，1999)，while CPC has not been used to separate puerarin yet. In this study，fast centrifugal partition chromatography(FCPC)with analytical column was used to separate and purify puerarin from the roots of P. lobata in one step. The analytical CPC column allowed for quick and easy optimization of the separation conditions， and comparison with HSCCC in separation effects.

All organic solvents used for FCPC were of analytical grade and purchased from Sinopharm Chemical Reagent Co.，Ltd.(Shanghai，China). Methanol of HPLC grade from J & K Scientific Ltd.(Beijing，China)was used in the HPLC analyses.

The puerarin reference substance(Batch No. 752-200108)was purchased from The National Institutes for Food and Drug Control(Beijing，China). The roots of Pueraria lobata(Willd)Ohwi(Batch No. HP120511)were purchased from Ankang Health Element Pharmtech Co.，Ltd.(Shaanxi，China).

Fast centrifugal partition chromatography(FCPC)was from Kromaton Technologies(Rousselet Robatel，France). The experimental FCPC was equipped with Shimadzu LC–10AD Pump，UV monitor operating at 254 nm，N2000 Work Station， and sample injection valve with 1 mL sample loop. The FCPC total column was 38 mL which is measured by two-phase liquid system of heptane-methanol-water(10:9:1)for three times. The rotation speed was adjustable from 3000 to 0 r/min. The flow rate was from 1.0 to 10 mL/min. And the sample injection was from 10 to 500 mg.

HSCCC was manufactured by Zhejiang University， and its total capacity is 47 mL. The rotation speed was adjustable from 1000 to 0 r/min.

HPLC analysis was performed on an Aglient Series 1260 HPLC Instrument(USA)equipped with a quaternary pump，a diode array detector，an autosampler， and a column compartment. Samples were separated on a Capcell Pak C₁₈ column(250 mm × 4.6 mm，5 μm，Shiseido，Japan). The mobile phase consisted of methanol-water(25:75). The flow rate was 1.0 mL/min. The effluent was monitored using a UV detector at 250 nm. The data were processed with Aglient 2.0 ChemStation software.

Ultrasonic and circulated extraction device CTGXZ–2B with the total capacity of 2 L was manufactured by Hongxianglong Biotechnology Co.，Ltd.(Beijing，China).

The st and ard puerarin was accurately weighed and dissolved in 30% ethanol to make the st and ard solution with concentration of 79 μg/mL.

The dried roots of P. lobata were crushed over 20 mesh and extracted twice by ultrasound and circulated extraction(800 W)with 40% ethanol for 30 min. The combined extracts were evaporated in vacuum to recover the solvent and then dried in vacuum at 60 ^oC to obtain the crude sample，in which the purity of puerarin was determined to be 15.821% by HPLC.

The sample solution was prepared by dissolving the crude sample in the solvent(mobile phase-stationary phase 4:1)for FCPC separation.

The partition coefficient(K_D)value of puerarin and the selectivity(α = K₁/K₂，where K₁ > K₂)were used as evaluation parameters for selecting the solvent system. According to the golden rules in selecting solvent system(Ito，2005)，the suitable K values for CCC should be in the range of 0.5–2.0 and the α value should be greater than 1.5. According to the literature(Cao，1999)，the ethyl acetate-n-butanol-water system was used to separate and purify puerarin by FCPC.

The FCPC separation was performed with a two-phase solvent system composed of ethyl acetate-n-butanol-water(2:1:3). When used for FCPC separation，the solvent system was prepared in a separatory funnel，allowing for full mixing and equilibration. The two phases were separated and sonicated for 15 min.

The FCPC column(38 mL)was fully filled in ascending mode with lower phase(stationary phase，64 mL)at a flow rate of 8 mL/min，while the apparatus was rotated at 600 r/min. Then，the FCPC was filled in descending mode with lower phase(64 mL)at a flow rate of 8 mL/min in order to eliminate air bubbles. The upper phase(mobile phase)was pumped into the column at a flow rate of 2.0 mL/min with a rotation speed of 2200 r/min. After the hydrostatic equilibrium was established，the sample solution was injected into the column. Puerarin fraction was collected from 11.5 to 12.5 min at a wavelength of 254 nm.

The flow rate and rotation speed were optimized on the FCPC A. The optimization was to select the operating conditions for high stationary phase retention(S_f)，the stationary phase retention equation must be adapted as follows

where the Vs represents the volume of stationary phase inside the FCPC column， and Vc is the FCPC column.

The flow rates were set as 2.0，3.0，5.0， and 7.0 mL/min， and the rotation speed was increased from 1600 to 2300 r/min to optimize the flow rate and rotation speed for high stationary phase retention.

Sample loading was optimized from 10 to 30 mg using FCPC A. At a constant sample volume of 1 mL，the sample loading was optimized from 10 to 30 mg in ascending mode with the rotation speed of 2200 r/min and the flow rate of 2.0 mL/min. Sample size was selected for good separation and high purity of puerarin.

The column of HSCCC was entirely filled with the lower phase as stationary phase and the elution mode was in tail to head mode. Then the apparatus was rotated at 800 r/min，while the upper organic phase was pumped at a flow rate of 2.0 mL/min. After the hydrodynamic equilibrium was established，the sample solution(10 mg/mL)was injected into the 1 mL injection valve.

The purities of puerarin and the separation resolution by FCPC were compared with that by HSCCC. In that case，the resolution equation must be adapted as follows:

where V_R1 and V_R1 are the retention volumes of solutes 1 and 2， and W₁ and W₂ are the peak widths of solutes 1 and 2 at the base.

Different volume ratios of the ethyl acetate-n-butanol- water system were tested for selecting a suitable solvent system. The solvent system of ethyl acetate-n-butanol-water(2:1:3)had better K，α， and S_f values. The results are shown in Tables 1 and 2. The solvent system of ethyl acetate-n-butanol- ethanol-water(4:1:0.5:5)had a suitable K value，while the selectivity(α = K_puerarin / K₃)was less than 1.5.

Figure 2 shows HPLC separation of crude extract from the roots of P. lobata. The compounds 1–5 were the main compounds in the crude extract and the peak 2 was puerarin.

Figure 3 and Table 3 show the effect of mobile phase flow rate and rotation speed on S_f. It is observed a linear relationship with the highest rotation speed giving the highest stationary phase retention: 1600 r/min: Y = 58.397 – 3.305X，

表 1(Table 1)

Table 1 Solute distribution ratios(KD)of five main compounds from roots of P. lobata in different solvent systems ethyl acetate-n-butanol- ethanol-water KD 1 2 3 4 5 4:1:0:5 0.42 0.76 0.59 0.18 1.92 3:2:0:5 1.13 1.46 1.13 0.40 2.40 2:1:0:3 0.70 1.84 1.19 0.54 4.19 4:1:0.5:5 0.44 0.99 0.71 0.29 2.29 4:0.6:0.6:5 0.34 0.71 0.48 0.18 1.68

Table 1 Solute distribution ratios(KD)of five main compounds from roots of P. lobata in different solvent systems

表 2(Table 2)

Table 2 Separation factors for puerarin between compounds 3 and 5 ethyl acetate-n-butanol-ethanol-water Ki / Kj Kpuerarin / K3 K5 / Kpuerarin 4:1:0:5 1.29 2.53 3:2:0:5 1.29 1.64 2:1:0:3 1.55 2.28 4:1:0.5:5 1.39 2.31 4:0.6:0.6:5 1.48 2.37

Table 2 Separation factors for puerarin between compounds 3 and 5

Figure 2 HPLC separation of crude extract from P. lobata roots

Figure 3 Relationships between Sf and flow rate with different rotation speeds

表 3(Table 3)

Table 3 Variety of Sf with different flow rates and rotation speeds in ascending mode(n = 3 ) Rotation speeds/(r·min–1) Flow rates /(mL∙min–1) Elution volumes / mL Back pressures / bar Sf / % 1600 2.0 18.5 25 51 3.0 20 24 47 5.0 22.5 23 41 7.0 25 23 34 1800 2.0 16.2 32 57 3.0 17.8 30 53 5.0 20.3 29 47 7.0 23 28 39 2000 2.0 17 36 55 3.0 18.5 35 51 5.0 21 34 45 7.0 23.9 33 37 2200 2.0 16 47 58 3.0 17 45 55 5.0 19.8 44 48 7.0 22 42 42 2300 2.0 16 52 58 3.0 18 49 53 5.0 18.5 25 51 7.0 20 24 47 r2 = 0.996; 1800 r/min: Y = 64.286 – 3.305X，r2 = 0.997; 2000 r/min: Y = 62.442 – 3.586X，r2 = 0.999; 2200 r/min: Y = 64.422 – 3.217X，r2 = 0.998; 2300 r/min: Y = 62.956 – 3.466X，r2 = 0.994. The operating parameters were as follows: flow rate of 2.0 mL/min，rotation speed of 2200 r/min， and 58% of stationary phase retention.

Table 3 Variety of Sf with different flow rates and rotation speeds in ascending mode(n = 3 )

The effects of sample concentration are shown in Table 4. With the sample concentration increasing，the retention time of puerarin would be delayed due to some loss of stationary phase. This would result in lower resolution of puerarin. As the concentration was 10 mg/mL，the purity and the recovery of puerarin were better than those at other concentration. While the concentration increased to 30 mg/mL，the resolution of puerarin fell down，with the purity of 93.353% and recovery of 34.6%，respectively. Therefore，the optimal concentration of 10 mg/mL was chosen.

Figure 4 shows a chromatogram of puerarin separated by FCPC with flow rate of 2.0 mL/min，rotation speed of 2200 r/min， and the sample size of 10 mg/mL. The puerarin(1 mg)was obtained from 10 mg extract with the recovery rate of 65%.

表 4(Table 4)

Table 4 Results of separation in different sample loadings Sample loadings / mg Purities / % Recovery rates / % Retention time / min 10 99.396 65.9 12.721 15 99.567 50.0 13.072 20 93.385 45.6 14.239 25 93.381 41.1 14.472 30 93.353 34.6 13.538

Table 4 Results of separation in different sample loadings

Figure 4 FCPC of puerarin separation

The HPLC of purified target and puerarin reference substances separated by FCPC are shown in Figure 5，which indicated that the purified target was puerarin. For further identification of the target，the TOF-MS mass spectrum gave the ions of m/z of 439.2 [M + Na]⁺ and 417.2 [M + H]⁺ with a molecular mass of 416.2.

Figure 5 HPLC of puerarin(A) and reference substance(B) 1 : puerarin 2: puerarin reference substance

Figure 6 shows a chromatogram of puerarin separation by HSCCC. The separation parameters of HSCCC were similar to those of FCPC: flow rate: 2.0 mL/min，rotation speed: 800 r/min， and sample loading: 10 mg/mL(1 mL injection valve)，which gave 47% of stationary phase retention.

Figure 6 HSCCC of puerarin separation

Table 5 shows the results of HSCCC compared with FCPC on resolution(R_s)，the purity of puerarin，S_f and retention time. Puerarin fraction was collected from 25 to 29 min using HSCCC， and the purity of puerarin was below 90% due to lower R_s. Therefore，FCPC is a time-saving and effective method for separating puerarin compared with HSCCC.

表 5(Table 5)

Table 5 Results of puerarin separation using FCPC and HSCCC Methods Purity of puerarin / % Retention time / min Sf / % Rs FCPC > 99 13.306 58 0.90 HSCCC < 90 27.502 47 < 0.5

Table 5 Results of puerarin separation using FCPC and HSCCC

A time-saving and effective separating method has been developed for the preparation of puerarin from the roots of P. lobata. Fast centrifugal partition chromatography A(FCPC A)with total capacity of 38 mL can be helpful for the quickly optimizing experimental conditions as follows: flow rate of 2.0 mL/min，rotation speed of 2200 r/min，ascending mode，sample concentration of 10 mg/mL，S_f of 58%，purity above 99%， and recovery rate above 60% within 20 min.

The large polar solvent ethyl acetate-n-butanol-water system can be better retained on hydrostatic CPC column with S_f of 58% and resolution of 0.90 while obtained S_f of 47% and resolution below 0.50 by HSCCC. Therefore，FCPC is more suitable for separating puerarin with large polar solvent system(ethyl acetate-n-butanol-water)compared with HSCCC.

Overall，FCPC is an efficient method for puerarin preparation from the crude extract at one step. The results of the research can provide the reference for scaling up on a larger CPC column， and can be helpful for improving the quality of puerarin.