Enrichment and purification of polyphenols in pine cone extracts of Pinus koraiensis Sieb. et Zucc. using a novel multi-channel parallel–serial chromatographic system packed with macroporous resin

Abstract

The present study uses a novel multi-channel parallel–serial chromatographic system packed with macroporous resin in the enrichment and purification of polyphenols in the pine cone extracts of Pinus koraiensis. The performance and separation characteristics of five macroporous resins have been evaluated, and the adsorption and desorption properties compared. According to the analysis results, AB-8 resin offers higher adsorption and desorption capacities than other resins. The experimental data are described by the pseudo second-order kinetic model and Langmuir and Freundlich isotherm. The multi-channel parallel–serial chromatographic system based on distinguishment between the loading zone and the separating zone packed with AB-8 resin is used to perform dynamic adsorption and desorption tests to optimize the enrichment process. The optimum conditions are: ethanol concentration, elute rate, sample volume and the concentration of polyphenols are 60% (v/v), 30 mL min⁻¹, 80 mL and 1.2 mg mL⁻¹, respectively. After one run treatment, the purity of polyphenols from SC-3 separation columns increased from 5.32% to 38.39%, 7.22 times to extracts; and its antioxidant capacity also enhanced significantly. The results show that the multi-channel parallel–serial chromatographic system reveals a high ability in the enrichment and purification of polyphenols.

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1 Introduction

Pinus koraiensis is distributed mainly over northeast China and surrounding countries and regions. The studies of Pinaceae extracts have found that the pine bark extracts have antioxidant,¹ anti-carcinogenic,² anti-inflammatory,³ anti-neurodegenerative disease,⁴ anti-atherosclerosis⁵ and anti-UV radiation⁶ activity. However, pine barks are non-renewable resources, the excessive exploitation of which would cause serious environmental damages. Pine cones are annual pine globular fruits, which can be exploited without causing environmental damages. The pine cones extracts, like pine barks extracts, thus have a variety of biological utilizing potentials.

The composition analysis of pine barks extracts finds that the main ingredient is proanthocyanidins, followed by the catechin-based flavonoids and phenolic acids.⁷ In order to make clear its composition and functions, the enrichment and purification of functional components is needed. Previous reports show that polyphenols can be separated by adsorption chromatography on columns packed with macroporous resin.^8–12 The adsorption process of macroporous resins is mainly through intermolecular hydrogen bonds and Van der Waals' force. Macroporous resin is inconsistent with pore size, and has macropores, mesopores, and micropores. The adsorption process therefore could be divided into three stages according to the capillary effects occurring at the two intersections: macropores, mesopores, and micropores. Adsorption process is a multi-layer adsorption, and the inductive effect transmitted to the first layer is passed on to a higher layer and can be transmitted to the multilayers.^13–15

The single column chromatographic system has a long operating time and low separating efficiency, and it is difficult to track the strong antioxidant components; multi-column chromatographic systems have made significant progresses in recent years.^16,17 Particularly, simulated moving bed chromatographic system can effectively pre-separate sugars, organic acids, natural products,^18–21 etc. Another example of the rapid development of multi-column chromatographic system is a two-dimensional or multidimensional liquid chromatographic system, which can analyze and identify target components.^22–24 Based on the researches of recent years, our laboratory has designed a novel multi-channel parallel–serial chromatographic system which can efficiently enrich and purify polyphenols and track the strong antioxidant components. The purpose of the study is to screen the optimal macroporous resin, optimize the enrichment conditions of polyphenols and track the strongest antioxidant components. The result of the present study can be taken as a reference for the enrichment and purification of other natural products from herbal raw materials.

2 Multi-channel parallel–serial chromatographic system

2.1 Basic principles

Traditional long chromatographic system is usually a single column, the top of which is used to load samples, and the bottom used to gather target components. However, the basic principles of a multi-channel parallel–serial chromatographic system are to distinguish between the loading zone and the separating zone. The loading zone adsorbs and congregates samples; the separating zone separates and collects target components. In order to further improve separation efficiency, multiple separation columns in the separating zone would be used. In this study, one short loading column and four short separation columns were adopted, as shown in Fig. 1.

Fig. 1 Schematic of traditional chromatographic system (A), L is loading zone, S-1 to S-4 are separating zone; and multi-channel parallel–serial chromatographic system (B), L is loading zone and loading column, SC-1 to SC-4 are separating zone and four separation columns.

Here, it is assumed that a mixture contains components a, b, c, and d, whose order of antioxidant capacity is d < c < a < b; the target is component b. In the traditional long chromatographic system, when b arrives at S-3 zone, it is necessary to continue to elute the long column for the enrichment of component b, and components a, b, c, and d are sequentially eluted, but it takes a long operation time; in the multi-channel parallel–serial chromatographic system, when the component a reaches SC-4 column, the compounds b, c, d sequentially arrive at SC-3, 2 and 1 columns. Then, the target is only in SC-3 column; at this time, elute SC-3 column only for the enrichment of component b. These processes significantly shorten the operation time. Components a, b, c, and d may be a substance or a mixture of classes. In this study, the mixture was pine cone extracts of Pinus koraiensis, and components a, b, c, and d were fractions eluted from separation columns. The purpose is to find the high-purity polyphenols and verify the antioxidant capacity.

2.2 Operational programs

Depending on the switch combination, the operation of series and parallels on a loading column and four separation columns could be achieved. Operational program 1 is switched on 1, 3, 6, 9, 12, and 14, and off 2, 4, 5, 7, 8, 10, 11, and 13, which can link loading column and four separation columns in series; operational program 2 is switched on 4, 5, 7, 8, 10, 11, 13, and 14, and off 1, 2, 3, 6, 9, and 12, which can link four separation columns in parallel, etc. When operational program 1 starts, polyphenols loaded in loading column are eluted, and sent into SC-1, 2, 3 and 4 separation columns in order. When the elution volume reaches a total column bed volumes (equal to one loading column volume and four separation column volumes), operational program 1 will be stopped. According to the difference of the distribution coefficient of polyphenols between mobile phase and stationary phase, components are distributed in different separation columns. Then, operational program 2 starts to elute components from separation columns. When the program 2 starts, switch 4 and 5 were turned on. At this point, polyphenols from SC-1 column is eluted and 2 BV of separation column are collected in switch 5; other three columns are operated the same way.

3 Materials and methods

3.1 Chemicals and reagents

Analytical-grade 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) were purchased from Sigma (America). 1 M Folin–Ciocalteu reagent was obtained from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). HPLC-grade methanol was purchased from Dikma (China). Food-grade 95% ethanol was purchased from a local reagents corporation. Macroporous resins including AB-8, D101, D140, X-5 and NKA-9 were obtained from the Chemical Plant of Nankai University (Tianjin, China). HPLC-grade catechin, epicatechin, taxifolin, rutin, quercetin and gallic acid were purchased from Shanghai Yuanye Biological Technology co., Ltd. (Shanghai, China).

3.2 Preparation of extracts

Pine cones (without pine nut) of Pinus koraiensis are obtained from Yi Chun, China and are authenticated by Professor Zhenyu Wang from the College of Food science and engineering, Harbin Institute of Technology, Harbin, China. The People's Republic of China issued the specific permissions are required from authority of plant collection in a protected area of land. The location we collect our plant materials is a national forest farm and the author was not obliged to have any permissions. Acquisition process did not disrupt the normal growth of Pinus koraiensis and other trees. Dried materials of pine cones are powdered by a disintegrator and then sieved (30 meshes). One kilogram of Pine cones powder is extracted with 20 L of ethanol–water (60%, v/v) solution²⁵ in an ultrasonic bath for 60 min, repeated for one time. The filtered solutions are gathered and concentrated by removing the ethanol using a rotary evaporator. The concentration of polyphenols in concentrated extracts is 2.0 mg mL⁻¹. The extracts obtained are diluted to appropriate concentrations with deionized water as sample solution for the subsequent experiments.

3.3 Determination of the concentration of polyphenols

The concentration of polyphenols is determined by the method of Lin²⁶ with some modifications. Five hundred microliters of samples are transferred to a 10 mL of test tube, to which 0.5 mL of 1 M Folin–Ciocalteu reagent is added. After 5 min, 1.0 mL of 10% (w/v) Na₂CO₃ is added and the volume is added to 3 mL with deionized water. After 2 h of incubation at room temperature, the absorbance is measured at 760 nm in a SPECORD® 200 Plus UV/VIS Spectrophotometers. The concentration of polyphenols is then calculated on the basis of the standard curve for gallic acid and expressed as mg of gallic acid equivalents per mL of samples.

3.4 Selection of macroporous resins

3.4.1 Pretreatment of macroporous resins. Macroporous resins including AB-8, D101, D140, X-5 and NKA-9 are screened. Their physical and chemical properties provided by manufacturers are shown in Table 1. The resins are pre-treated according to the manufacturer's recommendation so as to remove the monomers and porogenic agents trapped inside the pores during the synthesis process. In brief, prior to use, the resins are soaked in 4% NaOH for 4 h and then washed by deionized water. Washed resins are soaked in 2% HCl for 4 h and washed by deionized water. Finally, resins are leached with 95% ethanol for 24 h and washed several times with deionized water thoroughly. The dry-basis weight of resins is measured by weighing the resins after drying for 12 h at 80 °C. All calculations are made on the basis of dry resin.

Table 1 Physical and chemical properties of the test macroporous resins

Type	Polarity	Structrue	Particle size (mm)	Specific surface area (m^2 g^−1)	Average pore size (nm)
X-5	Non	SDVB	0.3–1.25	500–600	29–30
D101	Non	SDVB	0.25–0.84	480–520	9–10
AB-8	Weak	SDVB	0.3–1.25	480–520	13–14
D140	Weak	SDVB	0.315–1.25	500–600	95
NKA-9	Strong	SDVB	0.315–1.25	250–290	155–165

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3.4.2 Adsorption and desorption experiments. The adsorption experiment of polyphenols is performed as follows: 1.0 g (dry weight) of pretreated resins is put into conical flasks loaded with 50 mL of 2.0 mg mL⁻¹ extracts solution, and the conical flasks are shaken in a SHA-C incubator for 8 h at 298.15 K. The static desorption experiments are carried out as follows: when the adsorption time is reached, resins are washed by deionized water for six times and desorbed with 50 mL (80 : 20, v/v) ethanol–water solutions in the conical flask in a incubation shaker at 298.15 K for 2 h. The raffinate and desorption solutions are first evaporated to remove ethanol, and then the concentrations of polyphenols are determined.

3.4.3 Adsorption kinetics. The adsorption kinetics of polyphenols on the optimum resin is investigated on the basis of the following operation mode: 1 g of pretreated resins and 50 mL of 2.0 mg mL⁻¹ extracts solution are added into a 150 mL conical flask and shaken at 298.15 K. Then, the raffinates at different time intervals from the start to equilibration are analyzed.

3.4.4 Adsorption isotherm. Extracts solutions (50 mL) with different concentrations of polyphenols are contacted with 1 g of pretreated resins in conical flasks, continually shaken for 3 h at temperatures of 298.15, 308.15, and 318.15 K, respectively. Then, the raffinates are analyzed.

3.5 Determination of purity of polyphenols

A certain amount of concentrated samples are placed in an oven for 24 h at 50 °C, drying to a constant weight; the dry weight was measured, and the purity of polyphenols is calculated. The equation is as follows:

P = C_p/C_s × 100% (1)

where P is the purity of polyphenols (%); C_p and C_s are the concentrations of polyphenols and solids (mg mL⁻¹).

3.6 Optimization of multi-channel parallel–serial chromatographic system

Five short glass columns (100 × 40 mm) were used and the medium pressure chromatographic pump was served as the power producer. On the basis of preliminary studies, ethanol concentration, elute rate, sample volume, and the concentration of polyphenols significantly affected the purity of polyphenols in the dynamic adsorption and desorption separation process. In order to obtain high purity of polyphenols, ethanol concentration, elute rate, sample volume, and the concentration of polyphenols were investigated in order within the ranges: 30–80% (v/v), 10–60 mL min⁻¹, 40–140 mL and 0.8–1.8 mg mL⁻¹, respectively.

3.7 ABTS˙⁺ radical scavenging activity (TEAC)

The free radical scavenging activity using ABTS˙⁺ radical is also carried out according to the method of Wang.²⁵ Briefly, an ABTS solution (7 mM) is mixed with potassium persulfate (140 mM) with a ratio of 62.5 : 1 for 16 h in the darkness at room temperature to produce ABTS radical cation (ABTS˙⁺) stock solutions. The ABTS˙⁺ stock solution is diluted to an absorbance of 0.70 ± 0.05 at 734 nm as a working solution. An aliquot (5 μL) of aqueous solution containing 1 mg mL⁻¹ of extracts or fractions is mixed thoroughly with the ABTS˙⁺ solution (200 μL) and after 6 min in darkness at room temperature; the absorbance is read at 734 nm in a gene5 microplate reader. Different levels (0–500 μg mL⁻¹) of Trolox standard solution are prepared and assayed under the same conditions. Results are expressed in Trolox equivalent antioxidant capacity (TEAC), i.e., μg Trolox per mg samples.

3.8 HPLC

Analytical experiments are performed using a HPLC system (Waters, 2695 Quatpump, 2489 ultraviolet detector, Waters, U.S.A.) with an ODS column (150 mm × 4.6 mm, 5 μm, Extend, Agilent, U.S.A.) and a chromatography data system (Empower 2, U.S.A.). The mobile phase A is methanol and B is water containing formic acid (0.1%, w/v). The mobile phase condition: 0–40 min, 20 A/80 B–40 A/60 B; 40 min–60 min, 40 A/60 B–60 A/40 B. Its flow rate is 0.8 mL min⁻¹. The absorbance wavelength is 280 nm.

3.9 Statistical analysis

All tests are performed in triplicate and the results are presented as mean ± standard deviation (SD). The coefficients of determination (R²) are calculated using Origin 7.5. Differences between mean values are compared by Student t-test using the SPSS 18 software. Difference significance with P values of <0.05 has been taken into consideration.

4 Results and discussions

4.1 The selection of macroporous resins

The selection of macroporous resins is evaluated based on the adsorption and desorption capacities, and ratio of desorption. The equations are:

Adsorption evaluation:²⁶

Q_e = (C₀ − C_e)V_i/W (2)

where Q_e is the adsorption capacity at adsorption equilibrium (mg g⁻¹ resin); C₀ and C_e are the initial and equilibrium concentrations of polyphenols in the solutions, respectively (mg mL⁻¹). V_i is the volume of sample solution, and W is the weight of the dry resin.

Desorption evaluation:²⁵

Q_d = C_dV_d/W (3)

D = C_dV_d/(C₀ − C_e)V_i × 100% (4)

where Q_d is the desorption capacity after adsorption equilibrium (mg g⁻¹ resin); D is the desorption ratio (%); C_d is the concentration of polyphenols in the desorption solution (mg mL⁻¹); V_d is the volume of the desorption solution (mL); C₀, C_e, W and V_i are the same as described above.

The optimal resin is screened through static adsorption and desorption experiments at 298.15 K. The results are shown in Fig. 2. The order of adsorption capacities is AB-8 > X-5 > NKA-9 > D101 > D140. The adsorption capacity of AB-8 resin to polyphenols has attained 42.93 mg g⁻¹; the order of desorption efficiency is AB-8 > X-5 > D101 > D140 > NKA-9. The adsorption characteristics of polyphenols on AB-8 resin are not only in close relation to the polarity of polyphenols, but also to the dimensional structure (specific surface area, pore diameter and pore volume) of resin. Weak polar and nonpolar resins can easily adsorb and desorb polyphenols. Contrary to the specific surface area and average pore diameter of different resins, resins which have smaller a specific surface area and a larger average pore diameter absorbs more polyphenols. The strong polar NKA-9 resin also adsorbs polyphenols easily, but has the lowest desorption efficiency in 80% solutions, because the strong polarity gives more binding force.

Fig. 2 Adsorption and desorption capacities, and ratio of desorption of polyphenols on five macroporous resins.

The adsorption and desorption capacities, and ratio of desorption are compared; AB-8 resin is regarded as the optimal choice.

4.2 Adsorption kinetics

The adsorption kinetics of polyphenols on AB-8 resin is obtained at 298.15 K, as shown in Fig. 3. Generally speaking, the adsorption capacity rapid increases in the first hour because of prompt contact of polyphenols on macropores and then declines because of the diffusion of polyphenols from macropores into the mesopores and micropores.

Fig. 3 Adsorption kinetics curves of polyphenols on AB-8 resin at 298.15 K.

Finally, the adsorption capacity reaches the equilibrium after 3 h. The results show that the adsorption type of AB-8 resin belong to the intermediate adsorption (fast, t_a < 2 h; intermediate, 2 h < t_a < 3 h; slow, t_a > 3 h). Therefore, in order to complete the adsorption, adsorption time is set over 3 h.

To better understand the adsorption mechanism, the pseudo first-order kinetics and the pseudo second-order kinetic equations are used to fit the experimental data.

The pseudo first-order kinetic equation:

ln(q_e − q_t) = ln q_e − k₁t (5)

The pseudo second-order kinetic equation:

t/q_t = 1/(k₂q_e²) + t/q_e (6)

where q_e and q_t are the adsorption capacities at equilibrium and at any time t (mg g⁻¹ dry resin), respectively. k₁ (1/min), and k₂ [g mg⁻¹ min⁻¹] are the rate constants of pseudo first-order and pseudo second-order, respectively.

Drawing straight lines between ln(q_e − q_t) against t and t/q_t against t. The results are shown in Table 2. According to the values of the linear regression coefficient, the results have showed that the pseudo second-order kinetic equation provides a better correlation for the adsorption process. About the rate constants of the kinetic model for the adsorption process, the k₂ is a positive value, which indicates that q_e for polyphenols increases with the extension of adsorption time until equilibrium.

Table 2 Adsorption kinetics parameters of polyphenols on AB-8 resin at 298.15 K

Kinetics models	Kinetics parameters
Pseudo first-order equation
k1 (min^−1)	1.2464
qe (mg g^−1)	42.9871
R^2	0.9621
Pseudo second-order equation
k2 [g mg^−1 min^−1]	0.000328
qe (mg g^−1)	55.2486
R^2	0.9943

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4.3 Adsorption isotherms

The equilibrium adsorption isotherms are obtained at 298.15, 308.15, and 318.15 K, respectively, as seen in Fig. 4. Obviously, as the temperature increases, the adsorption capacity decrease, this may be considered an exothermic process. In addition, with increasing the concentrations of polyphenols, the adsorption capacity rapidly increase when the concentrations of polyphenols are less than 1.2 mg mL⁻¹ and then slows down.

Fig. 4 Equilibrium adsorption isotherms of polyphenols on AB-8 resin at different temperatures.

The adsorption isotherm is one of the most important approaches to illustrate the mechanism of the adsorption system. Langmuir and Freundlich isotherms¹² are adopted to analyze experimental data.

The Langmuir isotherm equation is represented as:

C_e/q_e = K_L/q_m + C_e/q_m (7)

The Freundlich isotherm equation is shown as:

log q_e = 1/n log C_e + log K_F (8)

where q_e and q_m are the equilibrium and maximum adsorption capacities (mg g⁻¹ dry resin), respectively. C_e is the equilibrium concentration of polyphenols solution (mg mL⁻¹). K_L is the parameter associated with the adsorption energy (g L⁻¹). K_F reflects the adsorption capacity of an adsorbent [(mg g⁻¹)(mL mg⁻¹)^1/n]. The parameter n represents the adsorption affinity of resin to polyphenols.

Drawing straight lines between 1/q_e against 1/C_e and log q_e against log C_e. The values of the corresponding parameters can be calculated based on the intercept and slope. The results are shown in Table 3. The Langmuir isotherm is a good model and can explain the adsorption mechanism. By comparing the correlation coefficient, it is found that the Freundlich isotherm is also a good model for polyphenols adsorption. K_F could be described as adsorption or distribution coefficient. Therefore, the values of K_F correspond to the quantity of polyphenols adsorbed onto AB-8 resin. According to the Freundlich isotherm theory, the value of 1/n is to determine the adsorption intensity or surface heterogeneity, and the closer the value get to zero, the more heterogeneous the surface is. The value of 1/n below 1 indicates the normal adsorption, and that above 1 demonstrates the cooperative adsorption. In Table 3, the values of 1/n are below 1, which indicates a normal adsorption.

Table 3 Isotherm parameters for the adsorption process of polyphenols on AB-8 resin at different temperatures

Isotherm model	298.15 K	308.15 K	318.15 K
Langmuir isotherm
qm (mg g^−1)	121.95	106.38	86.21
KL (g L^−1)	3.3415	3.1064	2.6552
RL^2	0.9990	0.9993	0.9994
Freundlich isotherm
KF [(mg g^−1)(mL mg^−1)^1/n]	26.7547	24.6831	22.4233
1/n	0.8032	0.7949	0.7756
RF^2	0.9886	0.9883	0.9893

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4.4 Effect of ethanol concentration

Ethanol solution is adopted as the eluent, which can effectively desorb polyphenols from macroporous resin. Ethanol concentration significantly affects the purity of polyphenols,²¹ so its concentration was investigated within the range: 30–80% (v/v). Since water possesses more polarity than ethanol, the higher the concentration of ethanol, the more the desorption polyphenols. In the meantime, some impurities are desorbed at a higher ethanol concentration.¹⁰ As seen in Fig. 5(A), the 60% ethanol solution is the best elution for purity of polyphenols from SC-3 column (P < 0.01). In other SC columns, 30% ethanol solution is best for SC-1, 50% for SC-2 and 70% for SC-4.

Fig. 5 (A) Effect of ethanol concentration to the purity of polyphenols. Elute rate, sample volume and the concentration of polyphenols are 20 mL min⁻¹, 60 mL and 1.2 mg mL⁻¹, respectively. (B) Effect of elute rate to the purity of polyphenols. Ethanol concentration, sample volume and the concentration of polyphenols are 60% (v/v), 60 mL and 1.2 mg mL⁻¹, respectively. (C) Effect of sample volume to the purity of polyphenols. Ethanol concentration, elute rate, and the concentration of polyphenols are 60% (v/v), 30 mL min⁻¹ and 1.2 mg mL⁻¹, respectively. (D) Effect of the concentration of polyphenols to the purity of polyphenols. Ethanol concentration, elute rate and sample volume are 60% (v/v), 30 mL min⁻¹, and 80 mL, respectively.

4.5 Effect of elute rate

The elute rate also significantly affects chromatographic behaviors and the purity of polyphenols. According to the size and volume of the separation column, elute rate was investigated within the range: 10–60 mL min⁻¹. As seen in Fig. 5(B), the elute rate of 30 mL min⁻¹ is the optimal choice for the purity of polyphenols from SC-3 column. When the elute rate increases, polyphenols adsorbed in loading column are difficult to separate from impurities, which results in lower purity. The lower rate makes more concentrated products.²⁷ Generally speaking, a low elute rate impose a positive effect on the adsorption process because polyphenols have sufficient time to interact with active sites at the surface of resins and vice versa.²⁸ Therefore, because of the low rate, the contact time between polyphenols and the resin is longer enough to get products with high purity. In other SC columns, SC-1 first increases and then decreases; SC-2 first remains unchanged, and then increases; and SC-4 declines.

4.6 Effect of sample volume

The bed volume of the loading column is constant; however, the sample volume significantly affects the purity. The sample volume was investigated within the range: 40–140 mL. As seen in Fig. 5(C), the sample volume of 80 mL is the optimum selection for the purity of polyphenols from SC-3 column (P < 0.05). The more sample volume there is, the more polyphenols and impurities are adsorbed on the loading column, which results in lower purity. It is also attributed to the multiple partition equilibrium among various phases and the different affinity of AB-8 resin to polyphenols. When the sample volume is too small, the polyphenols in the loading column are few, and the surface coverage of AB-8 resin is low. Therefore, a large amount of acting loci would be naked after the partition equilibrium of polyphenols arrived. These naked acting loci would adsorb impurities to decrease their surface energy, and the number of impurities adsorbed on AB-8 resin is large too. Along with the increase of the sample volume, the polyphenols in the loading column greatly increases. At that time, partition equilibrium among various phases would play a dominant role in the adsorption process.^29,30 In other SC columns, SC-1 remains relatively unchanged; SC-2 first remains low, and then increases; and SC-4 declines.

4.7 Effect of the concentration of polyphenols

According to static tests, the concentration of polyphenols was investigated within the range: 0.8–1.8 mg mL⁻¹. As shown in Fig. 5(D), the concentration of polyphenols of 1.2 mg mL⁻¹ is the best choice for the purity of polyphenols from SC-3 (P < 0.05). When the concentration of polyphenols is low, the adsorption capacity rises as the amount of polyphenols relative to active spots increases. However, together with further the increase of the concentration of polyphenols, more impurities are also adsorbed on AB-8 resin, resulting in the competition for active sites between polyphenols and impurities,¹⁰ thus reducing the purity. In other SC columns, the purity declines overall.

4.8 Purity of polyphenols and TEAC

From the above analysis, the multi-channel parallel–serial chromatographic system can effectively enrich and purify polyphenols. As shown in Table 4, polyphenols from SC-3 have higher purity, which increases to 7.22 times that of the extract. Polyphenols have a strong antioxidant capacity, and the ABTS˙⁺ radical scavenging capacity of polyphenols from SC-1, SC-2, SC-3, and SC-4 columns are analyzed. Polyphenols from SC-3 have the highest TEAC value, followed by SC-4. The purity of polyphenols has a significant impact on the ABTS˙⁺ radical scavenging capacity. The purer polyphenols are, the higher the ABTS˙⁺ radical scavenging capacity is.

Table 4 Purity and TEAC of polyphenols from extracts, SC-1, SC-2, SC-3 and SC-4 separation columns

	Purity of polyphenols (%)	n	TEAC (μg mg^−1)
a Value significantly different from extracts (P < 0.05).b Value very significantly different from extracts (P < 0.01).
Extracts	5.32 ± 0.37	1	288.71 ± 9.30
SC-1	10.01 ± 0.80^a	1.88	176.84 ± 5.84
SC-2	16.50 ± 0.57^b	3.10	350.33 ± 2.09^a
SC-3	38.39 ± 1.87^b	7.22	524.63 ± 3.62^b
SC-4	29.69 ± 1.99^b	5.58	507.91 ± 3.36^b

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4.9 HPLC

By comparison with HPLC chromatograms, as seen in Fig. 6, SC-3 contains catechin, epicatechin, procyanidin B2, rutin, taxifolin, and quercetin. Compared to the extracts, the purity of polyphenol from SC-3 column has improved significantly.

Fig. 6 Chromatogram of polyphenols from SC-3 separation columns. (1) Catechin, (2) procyanidin B2, (3) epicatechin, (4) taxifolin, (5) rutin, (6) quercetin.

5 Conclusion

The pine cone is kind of important alternative resource of pine bark. Pine cones of Pinus koraiensis contain polyphenols and its extracts had a strong antioxidant capacity. In this study, according to the results, AB-8 resin was the optimal choice. The multi-channel parallel–serial chromatographic system was used to perform the dynamic adsorption and desorption of polyphenols. After one run treatment, the purity of polyphenols from SC-3 column was increased from 5.32% to 38.39%. SC-3 had a higher TEAC value and contained catechin, epicatechin, procyanidin B2 and taxifolin. The multi-channel parallel–serial chromatographic system revealed a good ability in enriching and purifying polyphenols, and the method can be referred to for the enrichment of other natural products from herbal raw materials, because of its low cost, high efficiency and procedural simplicity.

Conflict of interest

The authors have declared no conflict of interest.

Acknowledgements

The authors gratefully acknowledge the financial support by National Natural Science Foundation of China (no. 31170510). The authors are very grateful to Professor Shujun Zhou guidance on language.

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