Journal of Chromatography B, 947–948 (2014) 41–48

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Preparative separation and purification of rosmarinic acid from perilla seed meal via combined column chromatography Weizhuo Tang a,b , Baoshan Sun b,c , Yuqing Zhao a,b,∗ a b c

Key Laboratory of Structure-Based Drug Design & Discovery Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China Department of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China INIA Dois Portos, Instituto Nacional de Investigac¸ão Agrária e Veterinária, I.P.2565-191 Dois Portos, Portugal

a r t i c l e

i n f o

Article history: Received 4 September 2013 Received in revised form 29 November 2013 Accepted 5 December 2013 Available online 17 December 2013 Keywords: Combination chromatography Macroporous resin Perilla seed meal Polyamide resin Rosmarinic acid

a b s t r a c t In this study, the preparative separation and purification of rosmarinic acid (RA) from perilla seed meal (PSM), which is a by-product of edible oil production, was achieved using combined column chromatography over macroporous and polyamide resins. To optimize the RA enrichment process, the performance and separation characteristics of nine selected macroporous resins with different chemical and physical properties were investigated. SP825 resin was the most effective: the content of RA increased from 0.27% in the original extract to 16.58% in the 50% ethanol fraction (a 61.4-fold increase). During further purification treatment on polyamide resin, 90.23% pure RA could be obtained in the 70% ethanol fraction. RA with a higher purity (>95%) could also be easily obtained using one crystallization operation. The proposed method is simple, easily operated, cost-effective, and environmentally friendly and is suitable for both large-scale RA production and waste management. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Perilla frutescens (L) Britt is an aromatic herb that is cultivated worldwide and used extensively as a food garnish in some Asian countries, including China and Japan [1]. Perilla seeds possess large amounts of oil rich in alpha-linoleic and omega-3 fatty acids. Perilla seed meal (PSM) is a by-product of perilla seed oil production. Most PSM is used as a protein source for animal feed. However, after the main constituents (proteins) are isolated from the PSM, the residue still contains many bioactive compounds, which makes it a cheap, undervalued material that has been largely ignored until now. Although many reports have been published describing the activities of natural antioxidants and the evaluations of the stability of the different phenolic acids in P. frutescens L. [2–5], very few studies have qualitatively or quantitatively analysed the polyphenol and phenolic acids in PSM. Recently, RA (Fig. 1) has attracted research interest due to its various biological activities, including its antioxidant [6,7], antiinflammatory [8], anticancer [9], and anti-allergenic activities [10]. In our previous work [11], several phenolic compounds and organic

∗ Corresponding author at: Department of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenhe District, Shenyang 110016, Liaoning, China. Tel.: +86 24 23986521/+86 13 804053251; fax: +86 24 23986521. E-mail addresses: [email protected], [email protected] (Y. Zhao). 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.12.007

acids were isolated from PSM extracts, and the RA content was determined by HPLC. These results reveal the potential of PSM to provide antioxidant compounds in addition to polysaccharides and proteins. However, the preparation of RA is not easy because it readily degrades, particularly at high temperatures, under direct sunlight, and in aqueous solutions with high pH. To date, the methods for the preparative separation and purification of high-purity RA include high-speed counter-current chromatography (HSCCC) [12–17], biotechnological production [18], and total synthesis [19]. However, these established methods possess several disadvantages, such as wasted organic solvent, expensive media, time-consuming procedures, and necessary special instruments. Therefore, the broad use of these methods in large-scale industrial production is limited, particularly in some developing areas. Synthetic adsorbents have been successfully used to adsorb valuable solutes, such as drugs and polyphenols [20]. Recently, macroporous resin chromatography has been used to separate and enrich bioactive components from herbal crude extracts [21–24] because they have several advantages, such as stable chemical and physical properties, inertness toward toxic solvents, simple regeneration, low cost, and long service life [25]. Therefore, various functionally enhanced commercially available macroporous resins are designed and produced in increasing amounts and varieties to improve the separation industry. Moreover, polyamide (Nylon-6) can be used with “green solvents” during the separation and purification process of some secondary metabolites, including

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For the standard solution, an appropriate amount of RA was dissolved in methanol to obtain a concentration of 0.2 mg/mL. All of the solutions prepared for HPLC analysis were filtered through 0.45-␮m nylon membranes before injection. Fig. 1. Chemical structure of RA.

2.3. Preparation of crude PSM extract flavonoids, and can be cost-effective and environmentally friendly, which facilitates its extensive application. However, according to previous reports [26–28], it is hard to obtain highly pure RA (>90%) using only macroporous or polyamide resins because the impurities in the crude extracts of herbal raw materials are complex. Therefore, combined column chromatography over macroporous and polyamide resins may be a viable strategy for removing impurities and obtaining highly pure RA. Additionally, the re-use of agricultural wastes has become more important because of the increasing shortage of natural resources and the development of serious environmental problems. Studies have widely reported the conversion of these waste materials into food ingredients, bio-fuels, and other value-added applications [29]. Therefore, utilising PSM as a source for RA production may help maximise the available resources and solve waste disposal problems. Consequently, this work investigates the adsorption and desorption properties of RA on various macroporous resins with different polarities to maximise its enrichment and to subsequently develop an efficient and inexpensive technology to produce high-purity RA from crude PSM extracts. We disclose the first systematically investigated combined column chromatography procedure using both macroporous and polyamide resins and the first reported use of PSM as a new source for RA. 2. Material and methods

Dry PSM (100 g) was extracted with 1 L of 50% ethanol (v/v) using ultrasound for 2 h in a sonication water bath (KQ2200B, Kunshan Ultrasonic Equipment Co., Ltd., Kunshan, China) three times. The extracts were combined, filtered, and centrifuged at 3000 rpm for 10 min. The supernatant was concentrated with a rotary evaporator (RE52AA, Yarong Equipment Co., Shanghai, China) under reduced pressure at 50 ◦ C to eliminate any volatile alcohol. This PSM extract (8 g) was used during the following experiments.

2.4. HPLC analysis of RA in PSM A L2000 series reversed-phase HPLC system (Hitachi, Japan) was employed to determine the RA content. The chromatographic separation was performed with a Kromasil C18 reversed-phase column (250 mm × 4.6 mm, 5 ␮m). The mobile phases were 0.1% orthophosphoric acid in water (v/v) (eluent A) and methanol (eluent B). A mixture of 50% A and 50% B over 40 min was used for isocratic elution at a flow rate of 1.0 mL/min; the detection wavelength was 330 nm. The injection volume was 10 ␮L. The quantification was conducted using the RA standard solution. The calibration curve (five data points) was linear with R2 = 0.9996. All of samples used for analysis were filtered through a 0.45-␮m membrane before injection.

2.1. Samples The PSM was obtained from Liaoning Jiashi Nutritional Plant Oil Development Corporation Limited (Shenyang, China), and the materials were stored at room temperature in a desiccator until use. 2.2. Chemicals and reagents RA (purity >98.0%) was purchased from Jianfeng Natural Products Research Co. Ltd. (Tianjin, China). Methanol (chromatography grade) was purchased from Concord Chemical Reagents Co. (Tianjin, China). Orthophosphoric acid was of chromatography grade and was obtained from Shenyang NO. 5 chemical agent factory (Shenyang, China). Ethanol and petroleum ether were of analytical grade and obtained from Bodi Chemical Reagents Co. (Tianjin, China). The water used during the HPLC analysis and for sample preparation was obtained from Wahaha Group Co. Ltd. (Hangzhou, China).

2.5. Pretreatment of macroporous and polyamide resins Macroporous resins, including AB-8, ADS-7, ADS-17, HPD100, HPD400A, HPD450, HPD700, and HPD-826, were purchased from Cangzhou Baoen Co. Ltd. (Cangzhou, China), and the SP825 resin was obtained from Mitsubishi Chemical Corporation (Tokyo, Japan). The physical and chemical properties of the resins are summarized in Table 1. The polyamide resin (Nylon-6; particle size, 75–150 ␮m; surface area, 5–10 m2 /g) was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). The macroporous and polyamide resins were pre-treated by soaking in 95% ethanol for 8 h. After the ethanol was removed, the resins were washed twice with deionized water and subsequently soaked in 1 M NaOH for 5 h. The resins were then washed twice with deionized water. The washed resins were soaked in 1 M HCl for 5 h and then washed thoroughly with deionized water before use. The pre-treated resins were dried in an oven (Taisite Instrument Co. Ltd., Tianjing, China) at 60 ◦ C until their weight was constant [26].

Table 1 Physical and chemical properties of the tested macroporous resins. Macroporous resin

Surface area (m2 /g)

Average pore diameter (nm)

Particle diameter (mm)

Polarity

AB-8 ADS-7 ADS-17 HPD-100 HPD-400A HPD-450 HPD-700 HPD-826 SP825

480–520 >100 90–150 650–700 500–550 500–550 650–700 500–600 1000

130–140 25–30 25–30 8–9 8–9 9–11 8–9 9–10 5–6

0.3–1.25 0.3–1.25 0.3–1.25 0.3–1.2 0.3–1.2 0.3–1.2 0.3–1.2 0.3–1.25 1.25

Weak-polar Strong-polar Weak-polar Non-polar Middle-polar Weak-polar Nor-polar Weak-polar Weak-polar

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2.6. Static adsorption/desorption experiments

2.7. Dynamic adsorption and desorption tests

The static adsorption capacity of the PSM extract on macroporous resin was investigated using the following procedure: 1 g of the extract was accurately weighed and dissolved in 200 mL of deionized to yield a 5 mg/mL solution. Subsequently, 2 g (dry weigh) sample of the tested resin was placed into 100 mL conical flask with 100 mL of ethanol and soaked for 12 h. After removing the solvent, the resin was washed thoroughly with deionized water. After the deionized water was poured out, 40 mL of the PSM extract solution was added [26]. The flask was shaken (110 rpm) at 25 ◦ C. The variations in the RA concentration at different intervals up to 6 h were monitored using HPLC analysis. When the adsorption equilibrium was reached, the resin was washed twice with deionized water. After removing the deionized water, 20 mL of 70% ethanol solution was added, and then the flask was shaken at 110 rpm for 6 h at a constant temperature of 25 ◦ C. The desorbed RA was analysed by HPLC. To investigate the effects of different pH values on the adsorption of RA, 0.5 g (dry weight) of the selected resin was tested using sample solutions (5 mg/mL) with pH values between 3 and 7 by shaking for 6 h at 110 rpm. The following equations were used to quantify the adsorption and desorption capacities and the desorption ratio:

The dynamic adsorption and desorption tests were performed by loading the sample (1 g of the PSM extract) onto glass columns (1.5 × 30 cm) that were previously wet-packed with 4 g (dry weight) of the selected resin. The bed volume was 10 mL, and the packed length of the resin bed was 6 cm. The sample solutions (8 mg/mL) flowed through the glass columns at 1 mL/min. When the adsorption equilibrium was reached, the resin column was washed with deionized water and subsequently desorbed using 30 mL (3 BV) of solutions with different ethanol concentrations (10%, 20%, 30%, 40%, 50%, 70%, 80%, and 90% (v/v)). Each eluent was evaporated to dryness under vacuum, and the RA in each eluent was detected by HPLC.

(C0 − Ce )Vi qe = W

D=

Cd Vd × 100% (C0 − Ce )Vi

The Langmuir and Freundlich equations are the two most popular models because these are currently the simplest theoretical models and are frequently applied to describe the monolayer adsorption equilibrium [30]. The Langmuir isotherm is the bestknown model for describing the adsorption of solutes from a solution and is written as follows: qe =

(1)

where qe is the adsorption capacity at the adsorption equilibrium (mg/g resin), C0 and Ce are the initial and equilibrium concentrations (mg/mL) of the solute in the solution, respectively, Vi is the volume of the sample solution (mL), and W is the weight of dry resin (g). The desorption was evaluated as follows: C V qd = d d W

2.8. Equilibrium models

(2)

(3)

where qd is the desorption capacity after the adsorption equilibrium (mg/g resin), Cd is the concentration of the solution in the desorption solution (mg/mL), Vd is the volume of the desorption solution (mL), D is the desorption ratio (%), and Ce and Vi are the same as described above.

qm Ce K + Ce

(4)

where qe is the adsorption capacity at the adsorption equilibrium, Ce is the concentration of the solute at equilibrium, K is the adsorption equilibrium constant, and qm is the maximum adsorption needed to form a monolayer (mg/g). The Freundlich isotherm is an empirical equation for non-ideal adsorptions and is expressed using the following formula: 1/n

qe = KCe

(5)

where K is the Freundlich constant, 1/n is an empirical constant, and qe and Ce are the same as described above. The adsorption isotherms were obtained according to the following procedures: the optimal resin (0.5 g) was placed into a 100-mL conical flask with a stopper. The procedures undertaken before adding the sample solution were identical to those described above. The concentrations of the PSM extract solutions were 5, 8, 10, 16, and 20 mg/mL. Subsequently, 10 mL of each different solution was poured into flasks that were shaken in a thermostatic oscillator at 25, 30, and 35 ◦ C. The initial and equilibrium concentrations at the different temperatures were determined by HPLC.

Fig. 2. Adsorption and desorption capacities and desorption ratio of RA on different resins.

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Fig. 4. Linear correlation of RA on the SP825 resin at 25, 30, and 35 ◦ C based on the Langmuir and Freundlich models.

Fig. 3. Adsorption kinetics for RA on the SP825, ADS-17, and HPD700 resins (A). Adsorption isotherms on the SP825 resin at 25, 30, and 35 ◦ C for RA (B).

2.9. Purification of RA using polyamide resin The RA fraction enriched by the optimized macroporous resin was dissolved in deionized water and subsequently centrifuged at 3000 rpm for 10 min. The supernatant was loaded into the polyamide column (1.5 × 30 cm, 20 g) for RA purification. To optimize the RA desorption condition, polyamide column was eluted using a gradient composed of different ethanol concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% (v/v)). The desorbed RA in each elute was analysed by HPLC.

resins with different polarities exhibited similar adsorption capacities. However, the RA desorption ratios for these resins were notably distinct. The polar ADS-7 and HPD400A resins possessed a strong affinity for the solute, resulting in a poor desorption behaviour, whereas the other non or weakly polar resins exhibited a higher desorption ratio for RA. The differences in their desorption performances were determined by their different physical properties, such as surface area and average pore diameter. The weakly polar SP825 resin demonstrated the best desorption capabilities with RA because it had a higher surface area, and the ADS-17 resin also exhibited a higher desorption ratio due to its larger average pore diameter. In summary, compared with the other resins, SP825, ADS-17, and HPD700 exhibited better adsorption and desorption capabilities and were thus selected for the adsorption kinetics experiments. 3.2. Adsorption kinetics of RA on the SP825, ADS-17, and HPD700 resins

3. Results and discussion 3.1. Adsorption and desorption capacities and desorption ratio of different resins To maximize the adsorptive capacity of RA, nine macroporous resins, namely AB-8, ADS-7, ADS-17, HPD100, HPD400A, HPD450, HPD700, HPD-826, and SP825, were selected. As illustrated in Fig. 2, the RA adsorption performance of these tested resins was nearly identical, whereas their desorption behaviours were different. The desorption capacities of the SP825, ADS-17, and HPD700 resins were considerably higher than those of the other tested resins possibly due to the physical and chemical properties of these resins in combination with the features of the solute. The resins with polarities similar to RA demonstrated better adsorption abilities. Additionally, different physical features, including the surface area, functionality, porosity, internal pore, and particle sizes of the selected resins, should also be considered. Phenol molecules form hydrogen bonds with the surface functional groups, and the aromatic ring modulates the hydrophobic interactions [31]. RA contains benzene rings, hydrogens, and a carboxyl group, which enable its adsorption both to polar and nor-polar resins with the proper physical features because these functionalities within RA may interact with the benzene ring and the polar groups in the resins. As shown in Fig. 2, the nine selected

As observed in Fig. 3A, the adsorption capabilities of RA increased with an increase in the adsorption time before the adsorption equilibrium of the three tested resins was reached. The equilibrium time for RA on the SP825 resin was 70 min, which is shorter than that obtained with ADS-17 and HPD700 resins (nearly 180 min). Its higher surface area and smaller average pore diameter made the SP825 resin more suitable for the rapid adsorption of RA during our experiment. The comprehensive study of the adsorption time and capacity and the desorption ratio showed that SP825 was the best resin for the enrichment of the RA from the PSM extract and was thus used in the subsequent tests. 3.3. Adsorption isotherms and thermodynamics of RA To investigate and characterize the adsorption behaviour of RA on the SP825 resin, sample solutions of 20, 16, 10, 8, and 5 mg/mL, which correspond RA concentrations of 0.9004, 0.6618, 0.5951, 0.4121, 0.2163, and 0.0904 mg/mL, respectively, were prepared and shaken at 25, 30, and 35 ◦ C, successively. As depicted in Fig. 3B, the adsorption capacity of RA increased with an increase in the initial concentration to gradually reach the saturation plateau. Table 2 reveals the effects of differences in the initial concentration on the adsorption rate at 25, 30, and 35 ◦ C. The table indicates that the adsorption rate decreased with an increase

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Table 2 Effects of changes in the initial concentration on the adsorption rates of RA at 25, 30, and 35 ◦ C. 25 ◦ C

Initial concentration (mg/mL)

5 8 10 16 20

30 ◦ C

35 ◦ C

Adsorption capacity (mg/g)

Adsorption rate (%)

Adsorption capacity (mg/g)

Adsorption rate (%)

Adsorption capacity (mg/g)

Adsorption rate (%)

4.16 8.11 11.73 12.82 17.32

99.5 98.4 96.2 91.5 86.2

4.32 8.23 11.75 12.82 16.07

99.9 99.8 98.7 91.1 89.2

4.32 8.13 11.48 12.31 15.41

99.9 98.6 96.4 92.9 85.6

Table 3 Langmuir and Freundlich parameters of RA on SP825 resin at different temperatures (◦ C). Compound

Temperature (◦ C)

Langmuir equation

R2

qm (mg/g)

Freundlich equation

R2

1/n

RA

25 30 35

Ce /qe = 0.0566Ce +0.0002 Ce /qe = 0.0649Ce +0.0002 Ce /qe = 0.0633Ce +0.0004

0.9976 0.9892 0.9918

17.32 15.66 15.41

qe = 27Ce 0.1935 qe = 21Ce 0.1454 qe = 23Ce 0.1974

0.9189 0.9255 0.9949

0.1935 0.1454 0.1974

Table 4 Effect of changes in the pH of the sample solution on the adsorption capacities of RAa . pH value

Adsorption capacity (mg/g)

3 4 5 6 7

2.58 ± 0.18 3.01 ± 0.14 3.04 ± 0.16 2.88 ± 0.17 2.84 ± 0.14

a

indicates that the adsorption behaviours followed monomolecular layer adsorption kinetics. In addition, the qm for RA decreased with an increase in the temperature, which demonstrates that the adsorption behaviour was an exothermic process. In summary, the SP825 resin, an initial sample concentration of 8 mg/mL, and a temperature of 25 ◦ C were selected for the enrichment of RA from the PSM extract. 3.4. Effect of the pH of the sample solution on the adsorption capacity

The results are reported as the means ± SD (n = 3).

in the initial concentration. The RA adsorption rate was obviously lowered when the initial concentration of the extract solution was increased to 8 mg/mL. Therefore, the initial RA concentration in the sample solutions used for adsorption was 0.2163 mg/mL. The equilibrium experiment described the affinity between the solutes and the adsorbent. The Langmuir isotherm and Freundlich model are commonly used to reveal the linearity fit of the adsorption performance. Moreover, the Langmuir equation (Eq. (4)) can be converted to its linearized form (Eq. (6)), which has Ce and Ce /qe as independent variables. The experimental data were statistically analysed, and R2 was obtained. Ce Ce K = + qe qm qm

RA is a phenolic compound and is thus acidic due to the phenolic hydroxyl and carboxyl groups within its structure. The pH influences the ionization of the solutes and therefore affects the affinity between the solutes and solutions. Consequently, sample solutions with different pH values were investigated to study the RA adsorption capacity on the SP825 resin. As displayed in Table 4, a relatively high adsorption capacity for RA was found for sample solutions at pH values ranging from 4 to 5, which allow RA to exist in a stable form. As the pH was

(6)

Fig. 4 illustrates the resultant model fits for the RA experimental data, and Table 3 lists the two common isotherm equations at different temperatures. Table 3 reveals that the linear correlations for the adsorption equilibrium on the SP825 resin were statistically significant. Therefore, both the Freundlich and Langmuir models can be used to describe the adsorption equilibrium of RA, which Table 5 Results of the gradient elution of RA on the SP825 resin. Concentration of ethanol (%)

Mass of dried residue (mg)

Mass of RA (mg)

Content of RA (%)

0 10 20 30 40 50 60 70 80 90 Original crude extract of PSM

41.2 21.7 22.0 30.2 30.7 38.3 24.5 13.9 12.4 7.6

– – 0.13 2.07 4.36 6.35 0.35 0.02 0.01 –

– – 0.58 6.86 14.20 16.58 1.42 0.16 0.06 0.27

Fig. 5. Dynamic leakage curve (A) and dynamic desorption curve of RA during gradient elution (B) on the SP825 resin.

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Fig. 6. HPLC chromatograms of the extracts before and after purification using the macroporous gel and polyamide resins: extract before treatment (A), extract after purification using the SP825 resin (B), and fraction after purification with polyamide resin (C). The peak indicated with an asterisk (*) if the RA peak.

increased, the adsorption capacity decreased because the phenolic hydroxyl and carboxyl groups readily lost H+ at higher pH values, which enhanced the water solubility and polarity and lead to the poor adsorption behaviour on the weakly polar SP825 resin. Consequently, the pH value for the RA sample solution was adjusted to between 4 and 5 during the following tests.

3.5. Dynamic adsorption and desorption tests The dynamic leakage curve for RA on the SP825 resin was obtained based on the eluted volume and the solute concentration (Fig. 5A). As shown in Fig. 5A, the RA in the sample solution was nearly adsorbed before 100 mL when the leak process started

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Table 6 Results of the gradient elution of RA on polyamide column. Ethanol concentration (%)

0

10

20

30

40

50

60

70

80

90

Mass of dried residue (mg) RA content (%)

71.2 –

36.1 –

16.8 –

8.3 –

5.2 0.71

5.6 34.69

7.8 77.81

10.3 90.23

32.9 2.21

4.7 –

gradually and rapidly accelerates as the elution volume increased from 120 to 170 mL. There was a steady plateau up to 180 mL. In general, adsorption saturation is defined when the concentration in the effluent is 10% of the original concentration. Therefore, the feed volume of the sample solution with the selected RA concentration on the SP825 resin was determined to be 120 mL (12 BV). Dynamic desorption was performed using the gradient elution model at 1 mL/min. The gradient elution results (Fig. 5B) reveal that variations in the ethanol concentration during elution modulate the desorption ability. An increase in the ethanol concentration results in an increase in the desorption ability for RA, which reached a maximum at 50% ethanol. If the concentration of ethanol was continuously increased, the RA in the elution solution decreased sharply. Table 5 suggests that most of the RA adsorbed by the SP825 resin was eluted within ethanol concentrations from 30% to 50%, and the highest RA content was found in the 50% ethanol–water solution. During the separation and enrichment procedure with the SP825 resin, the RA content can reach 16.58%, which is a 61.4fold increase compared with the crude extract from the PSM. The HPLC analysis of the crude extract and the 50% fraction demonstrated that the most polar impurities were removed and that the relative peak areas for RA increased markedly after treatment with the SP825 resin (Fig. 6B).Therefore, 20% ethanol could be used to remove the impurities, and 60% ethanol was selected as the RA enrichment fraction (1 g of sample led to a enriched fraction of 207.2 mg). For RA purification on polyamide, the maximum capacity of RA desorption is prior considered in which the concentration of ethanol elution plays an important role. Therefore, to optimize this condition, a gradient elution with nine different ethanol concentrations was achieved. As observed in Table 6, low concentration of ethanol solution (50%) resulted in an increase in desorption ability for RA, which reached a maximum in 70% ethanol fraction with 90.23% purity of RA (Fig. 6C). If the concentration of ethanol was continuously increased, the RA in the elution solution decreased sharply and no RA was detected in the fraction of 90% ethanol fraction. The different capacities of RA desorption in different ethanol elution solutions partly because of the functional group within RA which may interact with the benzene ring and the polar groups in polyamide. Accordingly, these results demonstrated that the optimum condition for RA purification on polyamide was a gradient elution program with 50% of ethanol removing impurities and 70% of ethanol yielding high purity RA. Additionally, to avoid bubbles, which usually caused by a prominent changes of ethanol concentration in polyamide column, it is recommended to firstly elute with 10% of ethanol and then by 50% of ethanol in the impurity removal step. Moreover, purer RA (>95%) could be easily obtained after one crystallization in methanol to remove the phytochromes. In summary, the optimal preparative separation and purification conditions for RA from PSM using a combination of the SP825 and polyamide resins were confirmed as follows: RA enrichment on the SP825 resin: Adsorption: concentration of RA in the sample solution, 0.2163 mg/mL; feed volume, 12 BV; flow rate, 1 mL/min; pH, 4 to 5; temperature, 25 ◦ C.

Gradient elution: 20% ethanol at 5 BV, followed by 60% ethanol at 10 BV; flow rate, 1 mL/min. RA purification on polyamide resin: Gradient elution: 10% ethanol at 5 BV, followed by 50% ethanol at 5 BV and 70% ethanol at 10 BV. 4. Conclusions In this study, RA was preparatively separated from PSM, which is a by-product of edible oil production, using a combination of macroporous and polyamide resins for the first time. To optimize the RA enrichment process, the performance and separation characteristics of nine macroporous resins were evaluated, and experiments were performed to characterize the adsorption/desorption dynamics. The most effective resin (SP825) that yielded a RA-enriched product that could be further purified on a polyamide resin was selected. During the gradient elution, 90.23% RA could be obtained from the 70% ethanol fraction, and higher-purity RA (>95%) could be obtained after one crystallization in methanol. This combined chromatography protocol could be used to prepare highly pure RA from the PSM extract. The proposed method is simple, cost-effective, and environmentally friendly and thus expands the applications of PSM and is suitable for large-scale RA production. Acknowledgements The authors gratefully acknowledge the financial support from the E&T Modern Center for Natural Products of Liaoning Province of China (No. 2008402021) and the Chinese Northeast Characteristic Nutritional Plant Oil Construction Foundation and Industrialization Item (No. 2008301026). References [1] Y.Y. Peng, J.N. Ye, J.L. Kong, J. Agric. Food Chem. 53 (2005) 8141–8147. [2] R. Raudonis, L. Bumblaukiene, V. Jakstas, A. Pukalskas, V. Janulis, J. Chromatogr. A 1217 (2010) 7690–7698. [3] H.Y. Park, M.H. Nam, H.S. Lee, W.J. Jun, S. Hendrich, K.W. Lee, Food Chem. 119 (2010) 724–730. [4] G. Schirrmacher, T. Skurk, H. Hauner, J. Grabmann, Plant Foods Hum. Nutr. 65 (2010) 71–76. [5] E.S. Lin, H.J. Chou, P.L. Kuo, Y.C. Huang, J. Med. Plants Res. 4 (2010) 477–483. [6] N. Troncoso, H. Sierra, L. Carvajal, P. Delpiano, G. Günther, J. Chromatogr. A 1100 (2005) 20–25. [7] A. Lamien-Meda, M. Nell, U. Lohwasser, J. Agric. Food Chem. 58 (2010) 3813–3819. [8] W.L. Jiang, X.G. Chen, G.W. Qu, Shock 32 (2009) 608–613. [9] K.A. Scheckel, S.C. Degner, D.F. Romagnolo, J. Nutr. 138 (2008) 2098–2105. [10] J. Lee, E. Jung, J. Koh, Y.S. Kim, D. Park, J. Dermatol. 35 (2008) 768–771. [11] W.Z. Tang, Y.Z. Liu, Y.Q. Zhao, Chin. Herb. Med. 4 (2012) 70–73. [12] A.F. Li, A.L. Sun, R.M. Liu, J. Chromatogr. A 1076 (2005) 193–197. [13] R.M. Liu, X. Chu, A.L. Sun, L.Y. Kong, J. Chromatogr. A 1074 (2005) 139–144. [14] S. Yao, J.G. Luo, X.F. Huang, L.Y. Kong, J. Chromatogr. B 864 (2008) 69–77. [15] Q.Z. Du, Z.G. Jiang, D.J. Wang, J. Chromatogr. A 1216 (2009) 4176–4180. [16] P. Xu, S. Guan, R. Feng, R. Tang, D. Guo, Phytochem. Anal. 23 (2012) 228–231. [17] Y. Wang, M. Liu, L. Zheng, L. Yin, Y. Qi, X. Ma, K. Liu, J. Peng, J. Sep. Sci. 35 (2012) 1977–1984. [18] V.P. Bulgakov, Y.V. Inyushkina, S.A. Fedoreyev, Crit. Rev. Biotechnol. (2011), doi:10.3109/07388551.2011.596804. [19] H. Yuan, L. Shan, Q.Y. Sun, W. Han, Acta Chim. Sin. 8 (2011) 945–948. [20] E.M. Silva, D.R. Pompeu, Y. Larondelle, H. Rogez, Sep. Purif. Technol. 53 (2007) 274–280. [21] Y. Fu, Y. Zu, W. Liu, T. Efferth, N. Zhang, X. Liu, Y. Kong, J. Chromatogr. A 1137 (2006) 145–152. [22] B. Zhang, R.Y. Yang, Y. Zhao, C.Z. Liu, J. Chromatogr. B 867 (2008) 253–258.

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Preparative separation and purification of rosmarinic acid from perilla seed meal via combined column chromatography.

In this study, the preparative separation and purification of rosmarinic acid (RA) from perilla seed meal (PSM), which is a by-product of edible oil p...
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