Adsorption Chromatography Separation of Baicalein and Baicalin

Article pubs.acs.org/IECR

Adsorption Chromatography Separation of Baicalein and Baicalin Using Collagen Fiber Adsorbent Qi-xian Zhang,†,‡ Juan Li,† Wen-hua Zhang,†,‡ Xue-pin Liao,† and Bi Shi*,‡ †

Department of Biomass Chemistry and Engineering and ‡Key Laboratory of Leather Chemistry and Engineering of Ministry of Education, Sichuan University, Chengdu 610065, Sichuan, China ABSTRACT: The separation of baicalein and baicalin, two major flavonoids in Scutellaria, by using collagen fiber adsorbent (CFA), was investigated. Static adsorption and adsorption kinetics indicated that baicalin had stronger hydrogen-bond interaction with CFA in high ethanol concentration solution, while baicalein presented stronger hydrophobic interaction with CFA in low ethanol concentration solution. In CFA column chromatography separation, baicalein and baicalin could be well separated by stepwise elution of 90% and 70% aqueous ethanol solutions, and their recoveries were 83.42% and 94.31%, respectively. The maximum loading mass for separating baicalein and baicalin on a CFA column was higher than 90 mg/g CFA, which is much more than the loading mass on a D101 macroporous resin column. Applications repeated 10 times indicated that the CFA column has satisfactory reusability. Furthermore, the separation of baicalein and baicalin from Scutellaria extracts using CFA was investigated. The purity of baicalein and baicalin obtained was both higher than 98%.

1. INTRODUCTION Baicalein and baicalin are two major flavonoids in Scutellaria, a well-known Chinese medicine herb.1 Compared with baicalein, baicalin has one more glucosyl, as shown in Figure 1, and therefore these two natural compounds present different physiological activities.2,3 Baicalin is more hydrophilic than baicalein, so it is more suitable to take orally and could stay a longer time in the humoral and cellular environment to enhance the immune function.4 Baicalein is more fat-soluble and hydrophobic than baicalin, and thus it could be more effectively absorbed in the human body,5−7 and exhibits higher activities of scavenging free radicals, inhibiting the activity of HIV reverse transcriptase, and chelating metal ions.8,9 However, baicalein and baicalin usually coexist in plant extracts, so the separation and purification of baicalein and baicalin is a significant task for pharmacological research and fine utilization of these two compounds. Some methods for separating flavonoids from plant extracts have been developed, such as fractional crystallization, preparative high performance liquid chromatography, solvent extraction, and column chromatography,10 among which column chromatography is the most efficient way that can simultaneously purify the compounds with relatively low cost. The current packing materials of column chromatography include polyamide, silica, dextran, and macroporous resin,11−15 and macroporous resin is most frequently used for the preparative scale. Recently many natural separation materials with good biocompatibility have been promoted, such as cellulose, chitosan, agarose, and silk fibroin.16−19 Obviously, research and development of new natural separation material with high efficiency and low cost is still a significant work. Collagen fiber, one of the most abundant renewable biomasses, can be easily obtained from skins of domestic animals, and it consists of an ordered arrangement of a collagen molecule. A collagen molecule is composed of three polypeptide chains with a triple helical structure, and it is stabilized by the hydrogen-bonds formed among the amino © 2013 American Chemical Society

acids, especially the hydroxyprolines on each polypeptide chains. These molecules assemble into collagen fibrils in a staggered manner, which imparts good mechanical strength and stability to the collagen fiber with uniform morphology.20,21 The peptide chain in the collagen molecule has approximately 1052 amino acid residues, and Gly-X-Y is the characteristic repeat unit, where X and Y are often proline and hydroxyproline, respectively. So collagen fiber has a large number of functional groups, including hydrophobic groups, peptide bond, −OH, −NH2, and −COOH.22 Therefore, it has potential to be used as an adsorbent to plant extracts based on both the hydrophobic and hydrogen bond interactions. The abundance of polar groups in collagen fiber also promises that it is strongly hydrophilic, so that the affinity between the collagen fiber and plant extracts can be adjusted by changing the content of water. Moreover, as we know, compared with powder or spherical particles, the material with fibrous morphology usually offers better mass transfer characteristics and lower pressure drop. Another important reason for our interest in using collagen fiber as adsorbent is its low-cost as compared with other materials. In our previous works, the adsorbent made from collagen fiber exhibited excellent adsorption selectivity to tannins23−25 and was effective in separation of flavonoids and alkaloids.26 The difference in molecular structure between baicalein and baicalin is obviously smaller than that between flavonoids and alkaloids. But this difference may still lead to different affinity of these two compounds to collagen fiber. Thus it is expected that collagen fiber may be used as an adsorbent for separation and purification of baicalein and baicalin. Received: Revised: Accepted: Published: 2425

November 5, 2012 January 12, 2013 January 19, 2013 January 19, 2013 dx.doi.org/10.1021/ie303031j | Ind. Eng. Chem. Res. 2013, 52, 2425−2433

Industrial & Engineering Chemistry Research

Article

Figure 1. Molecular structure of baicalein (A) and baicalin (B).

In this study, collagen fiber adsorbent (CFA) was prepared by using glutaraldehyde as a cross-linking agent, which can improve the chemical stability and antibacterial property of collagen fiber during application, and it was used as a column packing material to separate baicalein and baicalin mixture. The adsorption behavior of baicalein and baicalin on the adsorbent was investigated via static adsorption, adsorption kinetics, and desorption researches. On the basis of these results, the column chromatography separation of baicalein and baicalin was conducted by stepwise elution. Furthermore, in comparison with D101 macroporous resin, the capacity of sample loading mass and the reusability of CFA for separation of baicalein and baicalin were evaluated. Finally, a CFA was applied in the separation and purification of baicalein and baicalin from a practical sample mixture of Scutellaria extracts. The elution system in the present investigation is an aqueous ethanol solution, so that the target products are not polluted.

E% =

C0 − C1 100 C0

(1)

where E is the adsorption extent (%) and C0 and C1 are the concentrations of each component in initial and equilibrium solutions, respectively. 2.5. Solubility Determination of Baicalein and Baicalin. A proper amount of baicalein and baicalin was added into aqueous ethanol solutions with ethanol concentrations of 10%−100%, respectively, so that the oversaturation solutions were obtained. The baicalein and baicalin solutions were allowed to stand for 24 h and then the supernatant was taken and analyzed by UV−vis after being diluted to an appropriate concentration. The solubility of baicalein and baicalin in different aqueous ethanol solutions was then obtained. 2.6. Verification of Adsorption Mechanism. Comparable to the method in our previous work,26 urea as a breaking agent of the hydrogen bond and n-propanol as a breaking agent of the hydrophobic bond were used to verify the adsorption mechanism between collagen fiber and flavonoids. Urea was dissolved in pure ethanol to prepare urea−ethanol solutions, and the concentrations of urea were 0, 0.1, 0.25, 0.5, and 0.75 mol/L. Simultaneously, the urea was dissolved in 10% aqueous ethanol solutions, and the concentrations were 0, 0.25, 0.5, 0.75, 1.0, 1.5, and 2.0 mol/L. Then baicalein and baicalin were respectively dissolved in these solutions, and their content in all of the solutions was 30 mg/L. The adsorption was performed under the conditions in section 2.4. The initial and equilibrium concentrations of each component in the solutions were determined by UV−vis after being diluted for 5 times with distilled water, and the extent of adsorption was calculated by eq 1. n-Propanol was respectively added into pure ethanol and 10% ethanol to prepare a series of solutions, and the concentrations of n-propanol in the solutions were 0%, 10%, 20%, 30%, and 40% (v/v). Comparable to the conditions and operations above, baicalein and baicalin were dissolved in these solutions and then the adsorption was conducted and monitored. 2.7. Adsorption Kinetics and Desorption. The singlecomponent solutions of baicalein and baicalin (20 mg/L) were prepared by dissolving them in pure ethanol, respectively. A 100 mL portion of the solution and 0.5g of CFA were added into a flask. Then the adsorption was conducted under the same conditions in section 2.4. The concentration of baicalein and baicalin in the solution was analyzed by UV−vis at a proper interval, and the adsorption quantity was calculated by eq 2. After the adsorption was completed, the baicalein adsorbed adsorbent was filtrated in vacuum and then desorbed. The desorption was performed in 100 mL of 90% aqueous ethanol solution with constant shaking at 25 °C. The concentration of baicalein in solution was analyzed by UV−vis at a proper

2. EXPERIMENTAL SECTION 2.1. Chemicals. Baicalein and Baicalin were bought from Shanxi Huike Botanical Research and Development Corp. Ltd., China, and their purity was over 98%. D101 macroporous resin (Chengdu Kelong Chemical Agent Company) was used for the comparison with CFA. Methanol was chromatographic grade, and other chemicals were analytical grade. Scutellaria root was bought from a Chinese traditional medicine shop. 2.2. Equipments. HPLC 1100 (Agilent Technologies, USA), equipped with ArchromBond-AQ C18 reversed-phase column (150 mm × 4.6 mm, I.D., 5 μm) and G1315B diode array detector, was used to analyze the components in solutions. UV−vis spectrometer (Perkin-Elmer Lambda 25, USA) was used to measure the UV absorbance of solutions. Glass chromatography column (φ1.6 cm), packed with CFA or macroporous resin, was connected with constant flow pump and automatic collector (Shanghai Huxi Analytical Instrument Factory, China) to conduct chromatography separation. 2.3. Preparation of Collagen Fiber Adsorbent. Collagen fiber and CFA were prepared according to the method established in our previous work.25,26 2.4. Effect of Ethanol Content on Adsorption. The single-component solutions of baicalein and baicalin were prepared by dissolving them in aqueous ethanol solutions. The ethanol concentrations (%, v/v) of the solutions were 10%∼100%. The content of each component in the solutions was 30 mg/L. For each experiment, 0.15 g of CFA (dry weight) and 15 mL of the sample solution were added into a flask, and then the adsorption was conducted with constant shaking at 25 °C for 12 h in order to achieve adsorption equilibrium. The concentration of each component in the initial solution and equilibrium solution was analyzed by UV−vis, and the adsorption extent was calculated by 2426

dx.doi.org/10.1021/ie303031j | Ind. Eng. Chem. Res. 2013, 52, 2425−2433


Article

ethanol to deposit the protein and polysaccharide in the extract. Then the Scutellaria extract was obtained after the supernatant was freeze-dried. The loading solution was prepared by dissolving 500 mg of the extract in 50 mL of ethanol, and 27 mL of the solution was loaded on 3g of the CFA column. The separation was conducted by stepwise elution of 90% and 70% aqueous ethanol solutions. Then, the fraction eluted by 90% aqueous ethanol solution was further separated on 12 g of the CFA column by stepwise elution of 100%−50% aqueous ethanol solutions, and the fraction that was eluted by 70% aqueous ethanol solution was further separated on a 6 g CFA column by a stepwise elution of 100%−50% aqueous ethanol solutions. 2.12. Analysis Conditions of HPLC. HPLC detection was performed at 280 nm and 30 °C with an injection volume of 20 μL. The mobile phases H3PO4 aqueous solution (0.5%, v/v) (A) and CH3OH (B) ran in the following gradient mode: the volume of solvent (B) increased from 20% to 60% during 0−3 min, from 60% to 70% during 3.01−13 min, and was kept at 100% during 13.01−15 min. The flow rate was 0.8 mL/min.

interval, and the desorption extent was calculated by eq 3. Similarly, the baicalin adsorbed adsorbent was desorbed in 100 mL of 90% aqueous ethanol solution and 70% aqueous ethanol solution, respectively. Qe =

(C0 − Ce)V m

De% =

(Ce − C0)V 100 mQ e

(2)

(3)

where Qe is the adsorption quantity of unit CFA (mg/g); De is the desorption extent (%); C0 is the initial concentration of baicalein or baicalin before adsorption or desorption (mg/L); Ce is the concentration of baicalein or baicalin after adsorption or desorption (mg/L); V is the volume of solution for adsorption or desorption (L); m is the mass of CFA used (g). 2.8. Column Chromatography Separation. The mixture sample solution of baicalein and baicalin was prepared by dissolving them in pure ethanol and the concentration of each component was 5 mg/mL. An amount of 3.0 g of CFA was soaked in ethanol for 30 min and then packed into a glass column (φ1.6 cm). The column length was 7.7 cm, and the bed volume (BV) was 15.5 mL. The column was equilibrated with 90% aqueous ethanol solution (the first eluent). Then, 1 mL of the sample solution was loaded on the top of column followed by stepwise elution of 90% and 70% aqueous ethanol solution at a constant flow rate of 20 mL/h. A 10 mL portion of effluent solution was collected every 30 min, and the concentration of each component in the effluent solution was analyzed by HPLC. All the fractions eluted by 90% or 70% aqueous ethanol solution were mixed and diluted into 250 mL for HPLC analysis. Simultaneously, before separation, 1 mL of sample solution prepared was diluted into 250 mL and analyzed by HPLC. The purity and recovery were calculated by eq 4 and eq 5. P% =

M1 100 M1 + M 2

(4)

R% =

M1 100 M0

(5)

3. RESULTS AND DISCUSSION 3.1. Characterization of Collagen Fiber Adsorbent. The size of the ground collagen fiber is 0.1−0.25 mm. The specific surface area of collagen fiber adsorbent (CFA) is only 2.05m2/g, in the same level as that of typical polymer adsorbents.26,29 But the adsorbents made from collagen fiber always show high adsorption capacities.30−32 The denaturation temperature of natural collagen fiber is 60−65 °C, but as for CFA, the denaturation temperature is increased to 80−86 °C due to the cross-linking reaction of glutaraldehyde, which favors the practical application of this adsorbent.26 As shown in our previous work, CFA well keeps the uniform fibrous structure of collagen fiber.26 It has been reported that the adsorbents with fibrous structure usually have a higher mass transfer rate and a lower back pressure in comparison with conventional porous materials, such as porous silica gel and macroporous resin,33 which promise that CFA can be used in high flow rate though it has lower mechanical strength than silica gel and macroporous resin. So the fibrous structure of CFA may be beneficial to large-scale application. 3.2. Static Adsorption and Mechanism. Considering the ecological and dietary restraints, water and ethanol were used for all the adsorption and separation processes in this research. The static adsorptions of baicalein and baicalin on CFA in different aqueous ethanol solutions were shown in Figure 2. It was found that the adsorption extents of these two compounds were obviously influenced by the concentration of ethanol. The

where P is purity (%); R is recovery (%); M0 is the mass of objective component in solution before separation (mg); M1 is the mass of objective component after separation (mg); M2 is the mass of nonobjective component after separation (mg). 2.9. Effect of Loading Mass on Separation. The volume range of 0.5−27 mL of the sample solution prepared in section 2.8 was loaded on the column respectively, and then the separation was conducted under the same conditions in section 2.8 so as to investigate the effect of loading mass on the separation. Meanwhile, 27 mL of the sample solution was loaded on the D101 macroporous resin column, and the separation was performed by stepwise elution of 0%−100% aqueous ethanol solutions. 2.10. Reusability of Collagen Fiber Adsorbent. Under the same conditions in section 2.8, the separation was performed for 10 times to test the reusability of CFA. 2.11. Application in Separation of Scutellaria Extract. A 10 g powder sample of Scutellaria root was extracted in 100 mL of 60% aqueous ethanol solution at 90 °C for 2 h, according to reported methods.27,28 The extract liquid was dried by rotary evaporator, followed by adding 1 L of pure

Figure 2. Adsorption extents of baicalein and baicalin in different aqueous ethanol solutions. 2427



Article

by their difference in taking hydrogen bond and hydrophobic interactions. Comparable to the method in our previous work,26 urea, the hydrogen-bond breaking agent,34 and n-propanol, the hydrophobic interaction breaking agent,35−37 were used to further verify the mechanism. The adsorption extents of baicalein and baicalin on CFA in ethanol in the presence of urea and npropanol were shown in Figure 4A,B. It was found that in pure

adsorption extent of baicalein was lower than 35% when the concentration of ethanol varied from 70% to 90%, while it increased in pure ethanol or in 10%−60% ethanol solutions. As for baicalin, the adsorption extent was higher than 80% when the ethanol concentration was higher than 90%, and significantly decreased when ethanol concentration was below 70%. Compared with baicalein, the adsorption extent of baicalin was always higher in 80%−100% ethanol solutions, and lower in 10%−70% ethanol solutions. To explain this phenomenon, the solubility of baicalein and baicalin was investigated, and the results were shown in Figure 3. It was found that the solubility of both baicalein and baicalin

Figure 3. Solubility of baicalein and baicalin in different aqueous ethanol solutions.

was very low in 10% and 30% aqueous ethanol solutions and was high in pure ethanol, which indicates that both baicalein and baicalin are hydrophobic compounds to some extent. In 70%-90% aqueous ethanol solutions, the solubility of baicalein was much higher than that of baicalin, indicating that baicalein has stronger hydrophobic property than baicalin. The weaker hydrophobic property of baicalin should be due to the fact that it contains a hydrophilic glucosyl. On the basis of the discussed results, it is surmised that adsorption of CFA to baicalein and baicalin in a high ethanol concentration is mainly through the hydrogen bond and in a low ethanol concentration is mainly by hydrophobic interaction. Ethanol has smaller polarity and weaker ability to form hydrogen bond compared with water. Therefore, the environment of high ethanol concentration favors the formation of the hydrogen bond between CFA and flavonoids, while this kind of hydrogen bond is weakened in the low ethanol concentration solution due to the competition between water molecules and flavonoids in forming the hydrogen bond interaction with CFA. Baicalin has a stronger ability to form a hydrogen bond because it has one more glucosyl, so its adsorption extent is higher than that of baicalein in high ethanol concentration where hydrogen bond adsorption of flavonoids on the adsorbent is favored. With the decrease of ethanol concentration, the hydrogen bond between adsorbent and flavonoids is weakened due to the competitive reaction of water, and thus the adsorption extent of baicalein and baicalin decreased. With a further decrease of ethanol concentration, the hydrophobic property of flavonoids leads to hydrophobic interaction between CFA and flavonoids although the hydrogen bond interaction is weakened, and as a result, the adsorption extent is again increased. Baicalein has stronger hydrophobic property than baicalin, so its adsorption extent is higher than baicalin in low ethanol concentration solution. Therefore, the difference of adsorption behaviors of baicalein and baicalin on the adsorbent in different ethanol concentrations is determined

Figure 4. Effect of urea (A) and n-propanol (B) on adsorption extent of flavonoids in ethanol and effect of urea (C) and n-propanol (D) on adsorption extent of flavonoids in 10% aqueous ethanol solution.

ethanol, the adsorption extents of baicalein and baicalin were decreased by 40% and 70%, respectively, when the concentration of urea increased from 0 to 0.75 mol/L, suggesting a hydrogen bond plays a key role on the adsorption of baicalein and baicalin in ethanol. On the contrary, when the concentrations of n-propanol increased from 10% to 40% (v/ v), there was no obvious change in adsorption extents of baicalein and baicalin, showing a small effect of hydrophobic force on the adsorption in ethanol. The effects of urea and npropanol on the adsorption extents of baicalein and baicalin on CFA in 10% aqueous ethanol solution were illustrated in Figure 4C,D. The adsorption extents of baicalein and baicalin almost remained unchanged in 10% aqueous ethanol solution when the concentration of urea was increased, while the adsorption extents of baicalein and baicalin were reduced by 20% and 40%, respectively, as the concentration of n-propanol increased from 10% to 40%, demonstrating that the adsorption of baicalein and baicalin is mainly influenced by the hydrophobic interaction breaking agent. So it is hydrophobic interaction that primarily contributes to the adsorption of baicalein or baicalin on CFA in 10% aqueous ethanol solution. Anyway, the static adsorption behaviors of baicalein and baicalin and the adsorption mechanism imply that they could be separated on CFA by changing the ethanol concentration in elution. The conditions of column chromatography separation can be suggested by the result of static adsorption. When the concentration of ethanol was 90%, the greatest difference in adsorption extent between baicalein and baicalin was achieved, where the adsorption extent of baicalin was 62% higher than that of baicalein. When the concentration of ethanol was below 70%, the adsorption extent of baicalin was quite low. 2428



Article

separation conditions in section 3.2. That is, 90% aqueous ethanol solution might be suitable for the first step of elution to elute out baicalein, then 70% aqueous ethanol solution could be used for the second step of elution to elute out baicalin. 3.4. Chromatography Separation. The chromatography separation of baicalein and baicalin was conducted under the stepwise elution of 90% and 70% aqueous ethanol solutions. As shown in Figure 7, only baicalein was eluted out in the first step

Consequently, 90% aqueous ethanol solution can be used for the first step of elution to elute baicalein, and then 70% aqueous ethanol solution is used for the second step of elution to elute baicalin. Thus, baicalein and baicalin might be successfully separated. 3.3. Adsorption Kinetics and Desorption. Adsorption kinetics of baicalein and baicalin on CFA in pure ethanol were shown in Figure 5. The adsorption was fast during the first 90

Figure 5. Adsorption kinetics of baicalein and baicalin in pure ethanol. Figure 7. Chromatogram of baicalein−baicalin mixture on CFA column.

min, and then tended to dynamic equilibrium during 90−140 min. The equilibrium adsorption quantity of baicalin was 2.5 times more than that of baicalein, indicating that the adsorbent has good adsorption selectivity to baicalin in ethanol. Desorption curves of baicalein and baicalin in 90% aqueous ethanol solution were shown in Figure 6A. It was found that the desorption went fast and desorption equilibrium occurred in only 50−100 min. The desorption extent of baicalein reached 85% after equilibrium, while that of baicalin was below 20%, indicating that an apparent desorption selectivity to baicalein was obtained by using 90% aqueous ethanol solution. Therefore, it is reasonable to speculate that, in column chromatography separation, the baicalein adsorbed on adsorbent would be easily eluted out by 90% ethanol. Simultaneously, a small part of baicalin would be desorbed by 90% ethanol, but they may be readsorbed by the CFA during flowing in column. That is, baicalin would not be easily eluted out. The desorption curve of baicalin in 70% aqueous ethanol solution was shown in Figure 6B. The desorption went fast during the first 100 min, and the desorption equilibrium was achieved in 300 min. After equilibrium, the desorption extent of baicalin was 82%. So, if 70% aqueous ethanol solution is used as eluent, the baicalin adsorbed on CFA would be desorbed and elute out quickly. These results are in agreement with those in static adsorption research in optimizing chromatography

elution using 90% aqueous ethanol solution, and then baicalin was eluted out in the second step elution using 70% aqueous ethanol solution. There is no cross and overlap between their elution peaks, which indicates that the mixture of baicalein and baicalin are separated overall. Figure 8 shows the chromatograms of HPLC analysis for baicalein and baicalin before and after separation, which can be used to calculate the purity and recovery of baicalein and baicalin. As a result, their purities are higher than 98%, and their recoveries are 83.42% and 94.31%, respectively. Therefore, CFA exhibits an effective performance on separation and purification of baicalein and baicalin mixture. The elution order follows the adsorption mechanism described in sections 3.2 and 3.3. When the concentration of ethanol is higher than 70%, affinity of baicalein to CFA is weaker than baicalin since it has less active group (−OH) for hydrogenbond reaction, and therefore, baicalein is eluted out earlier than baicalin. The adsorption behaviors of baicalein and baicalin on CFA in the solutions with low ethanol concentration are also very different due to their difference in hydrophobic property. As observed in static adsorption, the adsorption extent of baicalein was greater than that of baicalin when the concentration of

Figure 6. Desorption curves of baicalein and baicalin in 90% aqueous ethanol solution (A) and desorption curve of baicalin in 70% aqueous ethanol solution (B). 2429



Article

Figure 8. Chromatograms of HPLC analysis for baicalein and baicalin before and after separation (diluted to 250 mL).

was presented when the loading mass was increased to 40 mg. Obviously, the optimal loading mass is 30 mg, equal to 10 mg/g CFA. Our further experiments showed that even with a loading mass increase to 270 mg, the mixture of baicalein and baicalin could be completely separated. Therefore, as for preparative separation without considering the retention time, the maximum separable mass of baicalein and baicalin on a CFA column is as high as 270 mg, equal to 90 mg/g CFA. However, under the same loading mass, baicalein and baicalin could not be separated on the D101 macroporous resin column, a typical reverse-phase column, by stepwise elution of 0%−100% aqueous ethanol solutions,38 and baicalein and baicalin were eluted out simultaneously by 0% aqueous ethanol solution. 3.6. Reusability of CFA. It is economically required that an adsorbent should be reused for a separation process. Therefore, the reusability of the CFA for the separation of baicalein and baicalin was investigated, and the results were listed in Table 1. The mixture of baicalein and baicalin was completely separated in 10 times of repeated applications. The purities of both baicalein and baicalin were over 98%. The recovery of baicalein

ethanol was below 50% (Figure 2). It is inferred that baicalein and baicalin could be also separated by using eluent with low ethanol concentration. If so, baicalin with stronger polarity would be washed out early. However, the solubility of baicalein and baicalin is very low in low ethanol concentration solution, and therefore, most of baicalein and baicalin are likely to depose on the CFA column, leading to very low separation efficiency. 3.5. Effect of Loading Mass on Separation. The effect of the loading mass on the chromatography separation of the baicalein and baicalin mixture was shown in Figure 9. In the

Table 1. Purities and Recoveries of Baicalin and Baicalein in Repeated Applications of Collagen Fiber Adsorbent purity (%)

Figure 9. Chromatogram of baicalein−baicalin mixture on a CFA column with different loading masses of the mixture.

loading mass range of 5 to 40 mg, baicalein and baicalin were completely separated. As shown in Figure 9, the height of the elution peak of baicalein increased with the increase of sample loading mass, and the peak shape and retention time remained unchanged. As for baicalin, both the height and width of the elution peak increased as the sample loading mass increased, and its retention time was delayed. Particularly, a peak tailing 2430

recovery (%)

cycle

baicalein

baicalin

baicalein

baicalin

1 2 3 4 5 6 7 8 9 10

>98 >98 >98 >98 >98 >98 >98 >98 >98 >98

>98 >98 >98 >98 >98 >98 >98 >98 >98 >98

80.17 83.42 79.97 78.69 84.15 78.69 84.15 83.52 81.71 79.74

92.63 94.31 92.49 93.34 92.67 93.34 92.67 95.44 98.62 95.25



Article

Figure 10. Chromatograms of HPLC analysis of baicalein and baicalin in Scutellaria extract before separation (A); after the first separation of the extract on 3g of CFA column by stepwise elution of 90% aqueous ethanol solution (B) and 70% aqueous ethanol solution (C); after the second separation of baicalein and its analogues on 12g of CFA of column (D), and after the second separation of baicalin and its analogues on 6g of CFA column (E).

compounds.41 The molecular structure difference between baicalein or baicalin and their analogues is smaller than that between baicalein and baicalin. So, to further purify baicalein and baicalin, two longer CFA columns were used. In addition, baicalein and its analogues have less hydrogen-bond groups and weaker affinity to CFA compared with baicalin and its analogues, so a much longer CFA column was employed for further purification of baicalein from its analogues. As shown in Figure 10D, baicalein was well separated from its analogues on 12g of CFA column by stepwise elution of 100%−50% aqueous ethanol solutions. Baicalein was eluted out in the elution of 90% aqueous ethanol solution, and the recovery was 53.43%. Figure 10E showed that baicalin was purified on 6g of CFA column by stepwise elution of 100%−50% aqueous ethanol solutions. The baicalin was mainly eluted out by 70% aqueous ethanol solution, and the recovery was 38.92%. In summary, baicalein and baicalin could be separated from Scutellaria extract on a CFA column by adjusting the concentration of aqueous ethanol solution and the length of column.

was greater than 78%, and the recovery of baicalin was greater than 92%, indicating a better reusability of CFA compared with D101 macroporous resin.39,40 In addition, no operation on the CFA column is needed before every repeated application. 3.7. Application in Separation of Scutellaria Extract. The HPLC chromatograms of Scutellaria extract before and after separation using the CFA column a\were shown in Figure 10. As shown in Figure 10A, baicalein and baicalin were the main compounds in Scutellaria extract, and some analogues with approximate polarity with baicalein and baicalin coexisted in the extract. Following the experimental conditions in section 3.4, 270 mg of Scutellaria extract was loaded on 3g of CFA column, followed by stepwise elution of 90% and 70% aqueous ethanol solutions. Figure 10 panels B and C showed the HPLC analysis of the components in the fractions eluted by 90% and 70% aqueous ethanol solutions, which indicates that baicalein with its analogues and baicalin with its analogues were separated, and the elution order is consistent with the separation of baicalein and baicalin in section 3.4. That is, baicalein and its analogues were eluted earlier by 90% aqueous ethanol solution. Our former research has found that the increase of CFA column length benefited the purity of target 2431



Article

(12) Houghton, P. J. Chromatography of the chromone and flavonoid alkaloids. J. Chromatogr. A 2002, 967, 75. (13) Chavan, U. D.; Amarowicz, R.; Shahidi, F. Antioxidant activity of phenolic fractions of beach pea (Lathyrus maritimus L.). J. Food Lipids 1999, 6, 1. (14) Fu, B. Q.; Liu, J.; Li, H.; Li, L.; Lee, F. S. C.; Wang, X. R. The application of macroporous resins in the separation of licorice flavonoids and glycyrrhizic acid. J. Chromatogr. A 2005, 1089, 18. (15) Zhao, Z. Y.; Dong, L. L.; Wu, Y. L. Preliminary separation and purification of rutin and quercetin from Euonymus alatus (Thunb.) Siebold extracts by macroporous resins. Food Bioprod. Process 2011, 89, 266. (16) Zheng, L. Q.; Ding, Z. S.; Zhang, M.; Sun, J. C. Microencapsulation of bayberry polyphenols by ethyl cellulose: Preparation and characterization. J. Food Eng. 2011, 104, 89. (17) Xia, Y. Q.; Guo, T. Y.; Zhao, H. L.; Song, M. D.; Zhang, B. H.; Zhang, B. L. A novel solid phase for selective separation of flavonoid compounds. J. Sep. Sci. 2007, 30, 1300. (18) Xu, J.; Tan, T. W.; Janson, J. C. Mixed-mode retention mechanism for (−)-epigallocatechin gallate on a 12% cross-linked agarose gel media. J. Chromatogr. A 2006, 1137, 49. (19) Baycin, D.; Altiok, E.; Ulku, S.; Bayraktar, O. Adsorption of olive leaf (Olea europaea L.) antioxidants on silk fibroin. J. Agr. Food Chem. 2007, 55, 1227. (20) Smith, J. W. Molecular pattern in native collagen. Nature 1968, 219, 157. (21) Rabotyagova, O. S.; Cebe, P.; Kaplan, D. L. Collagen structural hierarchy and susceptibility to degradation by ultraviolet radiation. Mater. Sci. Eng., C 2008, 28, 1420. (22) Liao, L. L. Leather Chemistry and Technology; Science Press: Beijing, 2005. (23) Liao, X. P.; Ma, H. W.; Lu, Z. B.; Shi, B. Selective removal of tannins from Chinese medicinal herbs. Nat. Prod. Res. Dev. 2004, 16, 10. (24) Liao, X. P.; Tang, W.; Deng, H.; Shi, B. Selective removal of condensed of tannins from extracts of Chinese medicinal herbs. Nat. Prod. Res. Dev. 2004, 16, 111. (25) Li, J.; Liao, X. P.; Tang, R.; Shi, B. Selective removal of condensed tannins from ginkgo biloba using collagen fiber absorbent. Chin. Tradition. Herb. Drugs 2008, 39, 62. (26) Li, J.; Liao, X. P.; Liao, G. H.; He, Q.; Zhang, W. H.; Shi, B. Separation of flavonoid and alkaloid using collagen fiber adsorbent. J. Sep. Sci. 2010, 33, 230. (27) Zhang, C. Y.; Xie, W. G.; Zhang, Y. L.; Wu, D. Q.; Ren, X. F.; Yang, Z. R. Determination and optimization of extraction technology of flavonoids in Scutellaria Baicalensis Georgi. Chin. J. Spectrosc. Lab. 2011, 28, 1253. (28) Zhou, A. Z.; Yin, H.; Wang, Z. Q. Study on compatibility extraction technology of Rhei Radix et Rhizoma, Scutellariae Radix and Phellodendri Chinensis Cortex. China J. Chin. Mater. Med. 2012, 37, 461. (29) Marcus, R. K. Use of polymer fiber stationary phases for liquid chromatography separations: Part IIApplications. J. Sep. Sci. 2009, 32, 695. (30) Liao, X. P.; Lu, Z. B.; Du, X.; Liu, X.; Shi, B. Collagen fiber immobilized Myrica rubra tannin and its adsorption to UO22. Environ. Sci. Technol. 2004, 38, 324. (31) Liao, X. P.; Zhang, M. N.; Shi, B. Collagen-fiber-immobilized tannins and their adsorption of Au(III). Ind. Eng. Chem. Res. 2004, 43, 2222. (32) Huang, X.; Liao, X. P.; Shi, B. Hg(II) removal from aqueous solution by bayberry tannin-immobilized collagen fiber. J. Hazard. Mater. 2009, 170, 1141. (33) Marcus, R. K. Use of polymer fiber stationary phases for liquid chromatography separations: Part IPhysical and chemical rationale. J. Sep. Sci. 2008, 31, 1923. (34) Wang, J.; Somasundaran, P.; Nagaraj, D. R. Adsorption mechanism of guar gum at solid−liquid interfaces. Miner. Eng. 2005, 18, 77.

4. CONCLUSIONS In this study, we explored the possibility of separating baicalein and baicalin using collagen fiber adsorbent (CFA). Baicalein and baicalin presented different adsorption and desorption behaviors on the adsorbent due to their difference in forming hydrogen bond and hydrophobic interactions with adsorbent in aqueous ethanol solution. Therefore, the CFA can be used as column packing material for effective separation of baicalein and baicalin with satisfactory purity and recovery, as well as capacity of loading mass and reusability. On the basis of this result, CFA could be practically applied on the separation and purification of baicalein and baicalin from Scutellaria extract. Compared with macroporous resin, the most frequently used column packing material for separation of flavonoids, CFA is much cheaper (no more than $15/kg) and more easily obtained, and is more effective in the separation of baicalein and baicalin. On the basis of this work, the wider application of CFA in separation of flavonoids could be expected, and in fact, further research is undertaken in our laboratory.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86 28 85400356. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS This research is financially supported by the National Natural Science Foundation of China (20976111 and 21076130). REFERENCES

(1) Pharmacopoeia Commission. Chinese Pharmacopoeia; Chemical Industry Press: Beijing, 2005. (2) Woo, K. J.; Lim, J. H.; Suh, S. I.; Kwon, Y. K.; Shin, S. W.; Kim, S. C.; Choi, Y. H.; Park, J. W.; Kwon, T. K. Differential inhibitory effects of baicalein and baicalin on LPS-induced cyclooxygenase-2 expression through inhibition of C/EBPβ DNA-binding activity. Immunobiology 2006, 211, 359. (3) Zhang, Y. F.; Wu, H.; Li, L.; Li, J.; Jiang, Z. Y.; Jiang, Y. J.; Chen, Y.; Mol, J. Enzymatic conversion of baicalin into baicalein by βglucuronidase encapsulated in biomimetic core−shell structured hybrid capsules. Catal. B 2009, 57, 130. (4) Yuan, L. D.; Xu, H.; Du, Z. Z. Isolation and epidemiological study of Mycoplasma genitalium. J. Nanjing Railway Med. Coll. 1997, 16, 231. (5) Liang, Y.; Han, L. J. Advances in research of pharmacological activities of flavoniod compounds in Scutellaria baicalensis Georgi. J. China Agr. Univ. 2003, 8, 9. (6) Liu, T. M.; Jiang, X. H. Studies on the absorption kinetics of baicalin and baicalein in rats’ stomachs and intestines. China J. Chin. Mater. Med. 2006, 31, 999. (7) Liu, T. M.; Jiang, X. H. Investigation of the absorption mechanisms of baicalin and baicalein in rats. J. Pharm. Sci. 2006, 95, 1326. (8) Shieh, D. E.; Liu, L. T.; Lin, C. C. Antioxidant and free radical scavenging effects of baicalein, baicalin and wogonin. Anticancer Res. 2000, 20, 2861. (9) Shi, H.; Zhao, B.; Xin, W. Scavenging effects of baicalin on free radicals and its protection on erythrocyte membrane from free radical injury. Biochem. Mol. Biol. Int. 1995, 35, 891. (10) Liang, Y.; Han, L. J. Advance in extraction, isolation and purification studies of baicalin and baicalein in scutellaria baicaleinsis Georgi. Trans. Chin. Soc. Agr. Eng. 2003, 19, 218. (11) Wang, L.; Gong, L. H.; Chen, C. J.; Han, H. B.; Li, H. H. Column-chromatographic extraction and separation of polyphenols, caffeine and theanine from green tea. Food Chem. 2012, 131, 1539. 2432



Article

(35) Usha, R.; Ramasami, T. Influence of hydrogen bond, hydrophobic and electrovalent salt linkages on the transition temperature, enthalpy and activation energy in rat tail tendon (RTT) collagen fibre. Thermochim. Acta 1999, 338, 17. (36) Usha, R.; Maheshwari, R.; Dhathathreyan, A.; Ramasami, T. Structural influence of mono- and polyhydric alcohols on the stabilization of collagen. Colloids Surf. B 2006, 48, 101. (37) Usha, R.; Ramasami, T. The effects of urea and n-propanol on collagen denaturation: Using DSC, circular dicroism, and viscosity. Thermochim. Acta 2004, 409, 201. (38) Zhou, T. T.; Fan, G. R.; Hong, Z. Y.; Chai, Y. F.; Wu, Y. T. Large-scale isolation and purification of geniposide from the fruit of Gardenia jasminoides Ellis by high-speed counter-current chromatography. J. Chromatogr. A 2005, 1100, 76. (39) Yin, D.; Chen, K. L. Purification technology of total flavonoids from Selaginella moellendorf ii Hieron with macroporous adsorption resin. Chin. J. Pharm. Anal. 2010, 30, 2040. (40) Li, X. J.; Fu, Y. F.; Luo, M.; Wang, W.; Zhang, L.; Zhao, C. J.; Zu, Y. G. Preparative separation of dryofragin and aspidin BB from Dryopteris f ragrans extracts by macroporous resin column chromatography. J. Pharm. Biomed. Anal. 2012, 61, 199. (41) Zhang, Q. X.; Zhang, W. H.; Liao, X. P.; Shi, B. The study on separation and purification of apigenin using collagen fiber adsorbent. J. Funct. Mater. 2012, 43, 618.

2433


Adsorption Chromatography Separation of Baicalein and Baicalin

Recommend Documents