Article pubs.acs.org/IECR
Enrichment and Purification of Total Flavonoid C-Glycosides from Abrus mollis Extracts with Macroporous Resins Huibin Du,†,‡ Hao Wang,*,†,‡ Jia Yu,†,‡ Chunyi Liang,†,‡ Wencai Ye,‡,§ and Ping Li† †
State Key Laboratory of Natural Medicines and ‡Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China § Institute of Traditional Chinese Medicine and Natural Products, Jinan University, 601 Huangpu Road West, Guangzhou 510632, China ABSTRACT: In the present study, the performance and separation characteristics of six macroporous resins for the enrichment and purification of total flavonoid C-glycosides (vicenin-2, 1; isoschaftoside, 2; schaftoside, 3) from Abrus mollis extracts have been evaluated. The adsorption and desorption properties of total flavonoid C-glycosides were studied on macroporous resins, including HPD-100, HPD-450, AB-8, HPD-600, DA-201, and HPD-417. According to the results, HPD-100 resin showed the best adsorption and desorption capacity for these three flavonoid C-glycosides among six resins. Adsorption isotherms were constructed on HPD-100 resin and fitted well to the Langmuir (R2 > 0.99) and Freundlich (R2 > 0.89, 0.1 < 1/n < 0.5) models. After the treatment with gradient elution on HPD-100 resin packed chromatography column, the contents of the flavonoid Cglycosides (1, 2, and 3) increased from 2.05, 2.90, and 2.30% in the extracts to 14.3, 21.9, and 15.1%, respectively, in the product. The recoveries of compounds 1, 2, and 3 were 83.8, 90.3, and 78.7%, respectively.
1. INTRODUCTION The genus of Abrus (Leguminosae) includes seventeen species mainly distributed in subtropics. Two species, Abrus cantoniensis, and Abrus mollis are commonly used traditional Chinese medicines, which called “Jigucao” and mainly distribute in southwestern of China.1 With obvious bioactivities including clearing heat, removing dampness, detoxification, and analgesic, the whole plants of A. cantoniensis and A. mollis are used as a folk remedy for hepatitis, pediatric dyspepsia, as well as born injuries. These two herbs were also used in cooking soup and herbal tea in Guangxi and Guangdong province.2 The aerial part of A. mollis is main herbal source of some well-known Chinese patent medicines, such as “Jigucao capsules”, for treating acute and chronicle hepatitis, and cholecystitis diseases in China.3 The major bioactive compounds of A. mollis are three flavonoid C-glycosides, including vicenin-2 (apigenin-6, 8-di-Cβ-D-glucopyranoside, 1), isoschaftoside (apigenin 6-C-α-Larabinopyranosyl-8-C-β-D-glucopyranoside, 2), and schaftoside (apigenin 6-C-β-D-glucopyranosyl-8-C-α-L-arabinopyranoside, 3)4 (Figure 1). It was reported that the flavonoid C-glycosides possess the biological activity of antioxidant, antiflammatory, and antiplatelet.5−7 In our previous studies, compounds 1, 2,
and 3 isolated from A. mollis show potent hepatoprotective activities evaluated their effects on carbon tetrachloride (CCl4), Bacillus Calmette-Guerin (BCG) + lipopolysaccharide (LPS), and ethanol induced hepatocytes damage in vitro and may be developed as a new natural drug for the prevention and treatment of hepatitis and alcoholic liver disease.8,9 The conventional separation method of flavonoid compounds is normally carried out from the herbal extracts by means of solid−liquid extraction from natural resources, followed by liquid−liquid extraction by using different solvents, and then followed by a column chromatography (silica gel, polyamide, Sephadex LH-20, ODS) with gradient solvent system. However, all of them have the same disadvantages, such as consuming more solvent and taking a long time, and result in lower recovery of the products. Macroporous resin, with the property of highly cross-linked and a large number of permanent pores, is one of the most efficient separation materials.10 It was studied that macroporous resin has the properties of surface adsorption, sieve classification, surface electrical property, hydrogen bonding interactions, and so on.11 Because of its advantages of high adsorption capacity, low operational expense, easy regeneration after the adsorption, environmental friendliness, and long service life, macroporous resins have be successfully applied in industry for separation and preparation of flavonoids, glycosides, saponins, carotenoids, fatty alcohols, and so on.12−14 To achieve efficient large-scale separation of the three flavonoid C-glycosides from A. mollis extracts, a detail investigation on suitable macroporous resin and its adsorption Received: Revised: Accepted: Published:
Figure 1. Structures of active constituents in A. mollis: vicenin-2, 1; isoschaftoside, 2; schaftoside, 3. © 2012 American Chemical Society
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Table 1. Properties of the Six Macroporous Resins name
polarity
particle diameter (mm)
surface area (m2/g)
average pore diameter (nm)
moisture content (%)
HPD-100 HPD-450 HPD-417 HPD-600 AB-8 DA-201
nonpolar weak polar hydrogen bond polar weak polar polar
0.30−1.20 0.30−1.20 0.30−1.25 0.30−1.20 0.30−1.25 0.30−1.25
650−700 500−550 90−150 550−600 480−520 250−300
8.5−9.0 9.0−11.0 25.0−30.0 8.0 13.0−14.0 20.0−30.0
61.2 62.5 58.9 68.4 68.3 65.3
column (250 × 4.6 mm, 5 μm, Agilent Technologies) at 40 °C. Elution was performed by using mobile phase A (2% acetic acid aqueous solution) and mobile phase B (methanol), samples (20 μL) were eluted at a flow rate of 1 mL/min, and the detective wavelength was 272 nm. The solvent gradient in volumetric ratios was as follows: 19% B over 30 min, The gradient was held at 90% B for an additional 5 min to clean up the column, followed by reequilibration of the column for 15 min with 19% B before the next run. 2.5. Static Adsorption and Desorption Tests. In order to investigate the adsorptive and desorptive properties of six resins to the compounds 1, 2, and 3, the test was performed as follows: A certain amount of wet resin (equal to 1 g dry resin) was put into a 50 mL Erlenmeyer flask with a lid. The crude extracts were dissolved with Milli-Q water as sample solution and the concentrations of compounds 1, 2, and 3 were 1.88, 2.71, and 1.73 mg/mL, respectively. Twenty-five milliliters (25 mL) of the above solution was added into the flask, and then flask was shaken (200 rpm) in a constant temperature shaker at 25 °C, the concentration of the three flavonoid C-glycosides in the adsorption solution were detected at 2, 4, 6, 8, and 10 h during the adsorption process. The equilibrium can be reached within 8 h. The solution was gained after adsorption by filtration and the filtrate was monitored by HPLC analysis. Subsequently, the resin was washed with Milli-Q water for three times and then 25 mL 60% ethanol was added for desorption. The flask was shaken (200 rpm) in a constant temperature shaker at 25 °C for 8 h.16 The contents of 1−3 in the solution after desorption were analyzed by HPLC methods. The capacity of adsorption, capacity of desorption, and desorption ratio of the resins were calculated by the following equations. Candidate resin was screened out in term of the adsorption capacities, desorption capacities, and desorption ratios.17
properties are needed. The aim of the current work is to investigate the adsorption and desorption properties of the flavonoid C-glycosides on different macroporous resins, and develop an efficient method for the preparative separation of the flavonoid C-glycosides from A. mollis crude extracts with the optimal resin.
2. EXPERIMENTAL SECTION 2.1. Chemicals and Reagents. HPLC-grade methanol (Merck, Germany), acetic acid (Merck, Germany), and Milli-Q water were used for HPLC analysis. Ethanol of analytical grade was used for preparation of herbal extracts and column chromatography. Other chemical regents were all analytical grades and purchased from Nanjing Chemical Reagent Co., Ltd. Vicenin-2 (1), isoschaftoside (2), and schaftoside (3) were isolated from A. mollis in our lab and their structures were established by means of 1H, 13C NMR, and MS spectroscopic analysis, and comparisons with the literature data.15 The purities of these compounds were all proven to be above 98% by HPLC methods. 2.2. Adsorbents. Macroporous resins including HPD-100, HPD-450, AB-8, HPD-600, DA-201, and HPD-417 were purchased from Cangzhou Bon Adsorber Technology Co., Ltd. (Hebei, China). The physical properties of these resins were summarized in Table 1. According to the manufacturer’s recommendation, the resins were soaked in 95% ethanol for 24 h and washed to remove the monomers and porogenic agents trapped inside the pores during the synthesis process and then washed by Milli-Q water thoroughly in glass chromatography columns before use. Each kind of resin was weighted precisely, and then placed in a drying oven, dried at 105 °C to constant weight. The moisture content of each resin was also summarized in Table 1. 2.3. Preparation of A. mollis Extracts. The aerial parts of A. mollis were collected from Yulin city of Guangxi province and identified by Senior Engineer Zhiwen Ye, Guangxi Yulin Pharmaceutical Company. Dry aerial parts of A. mollis (removing the seeds) were grounded into powder. The powdered plant (300 g) was extracted with 3000 mL 70% ethanol under reflux at 90 °C for two hours, repeated two times. The filtrates were combined and concentrated to a crude extracts by removing the ethanol solvent in a rotary evaporator at 60 °C. Part of the obtained crude extracts was accurately weighted and then diluted in 50% methanol (HPLC grade). This sample was filtered and the filtration was injected into HPLC for analysis. The contents of compounds 1, 2, and 3 in the crude extracts were 2.05, 2.90, and 2.30%, respectively. 2.4. HPLC Conditions. HPLC-UV analysis was performed on an Agilent 1100 series HPLC system equipped with a G1312A binary pump, a G1329A autosampler, and a G1314A variable wavelength detector (Agilent Technologies). HPLC separation was achieved by using a Zorbax analytical SB-C18
(C0 − Ce)Vi W CV Qd = d d W CdVd D= × 100% (C0 − Ce)Vi Qe =
Qe is the adsorption capacity (mg/g), C0 and Ce are the initial and equilibrium concentration of flavonoid C-glycosides (mg/ mL), Vi is the volume of the initial sample solution (mL), W is the weight of the tested dry resin (g), Qd is the desorption capacity (mg/g), Cd is the concentration of flavonoid Cglycoside in desorption solution (mg/mL), Vd is the volume of the desorption solution (mL), D is the desorption ratio (%). 2.6. Adsorption Isotherms. The Langmuir and Freundlich theoretical equations were used to describe the interaction between sorbent and adsorbed material.18 Adsorption isotherm experiments on HPD-100 resin (1 g of dry resin) were 7350
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Figure 2. Adsorption capacity and desorption capacity of (A) vicenin-2, (B) isoschaftoside, (C) schaftoside, and (D) the sum of the three flavonoid C-glycosides on the six macroporous resins.
conducted by bringing into contact five aliquots of 25 mL sample solutions with the pH value of 5 at different concentrations (1, 0.37, 0.77, 1.14, 1.53, and 1.85 mg/mL; 2, 0.54, 1.12, 1.69, 2.31, and 2.82 mg/mL; 3, 0.43, 0.89, 1.37, 1.84, and 2.25 mg/mL) in a constant temperature shaker, and then shaking at 25, 30, and 35 °C for 8 h, respectively.19 The initial and equilibrium concentrations at different temperatures were determined by HPLC. 2.7. Effect of Sample Solution pH Value on the Adsorption Capacity. The effect of the initial pH on adsorption of compounds 1, 2, and 3 was studied in the range of 3.0−9.0 (pH 3, 5, 7, and 9) which were adjusted using concentrated hydrochloric acid or ammonia. For these experiments, 25 mL of sample solution were taken in flask and 1 g of HPD-100 resin was added. The solution was agitated in the constant temperature shaker (200 rpm) for 8 h. The adsorption solution was analyzed by HPLC. 2.8. Dynamic Adsorption and Desorption Tests on the Chromatography Column. The adsorption and desorption properties of the HPD-100 resin packed chromatography column under different conditions, including sample loading amount and concentration were evaluated before the chromatography tests. The influence of the initial concentration of sample solution for the HPD-100 resin dynamic adsorption procedure was investigated by using different concentrations of A. mollis extracts solutions (0.5, 0.2, 0.1, 0.04 g/mL) of the same quantities loading onto four resin columns (2 cm × 20 cm), respectively. Optimum amount of sample loaded onto the column was carried out in a glass column (2 cm × 20 cm) wet packed with HPD-100 resin (15 g of dry resin). The prepared sample solution (0.1 g/mL) was applied to the column bed thoroughly at a flow rate of 0.5 BV/hour. The effluents during the sample
loading were collected, every 5 mL for a fraction. These fractions were monitored by HPLC method. The chromatography tests were performed on a glass column (2.5 cm × 25 cm) wet-packed with selected resin (1 BV = 120 mL, BV). The A. mollis extracts (48.5 g) were dissolved in distilled water evenly by stirring and adjust the pH value to 5 with concentrated hydrochloric acid. Then, the liquid was centrifuged (4000 rpm, 10 min) to remove the insoluble part. The soluble part as sample solution was applied to the column bed at the rate of 0.5 BV/hour which referred to the manufacturer’s recommendation. The gradient elution tests were taken as follows: after adsorptive saturation, the column was first washed by Milli-Q water, and then eluted by ethanol− water (20, 40, 60%, v/v) solution. The eluted fractions were collected and detected by HPLC, and then concentrated fractions of ethanol−water (40%, v/v) to dryness under vacuum. The contents of compounds 1, 2, and 3 in the dry product were detected by HPLC.
3. RESULTS AND DISCUSSION 3.1. Adsorption Properties of the Resins for the Flavonoid C-Glycosides. Six macroporous resins with different physical properties were employed for the separation of the flavonoid C-glycosides from A. mollis extracts, and the results are shown in Figure 2. The adsorption capacity of compounds 1, 2, and 3 on HPD-100 resin was 49.5 mg/g, and considerably higher than other resins (HPD-450: 25.5, HPD417: 13.1, HPD-600: 25.3, AB-8: 26.7, and DA-201: 23.2 mg/ g). The desorption radio of HPD-100 resin (64.9%) was also the highest among the six resins (HPD-450, 27.5; HPD-417, 23.5; HPD-600, 34.5; AB-8, 56.2; and DA-201, 57.6%). Therefore, HPD-100 resin was selected for the further study of adsorption process of the compounds 1, 2, and 3. 7351
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Table 2. Langmuir and Freundlich Parameters of Vicenin-2, Isoschaftoside, and Schaftoside on HPD-100 Resin at Different Temperatures vicenin-2
isoschaftoside
schaftoside
T (°C)
Langmuir equation
R2
25 30 35 25 30 35 25 30 35
Ce/Qe = 0.110Ce + 0.004 Ce/Qe = 0.107Ce + 0.003 Ce/Qe = 0.106Ce + 0.004 Ce/Qe = 0.055 Ce + 0.002 Ce/Qe = 0.054 Ce + 0.002 Ce/Qe = 0.053 Ce + 0.002 Ce/Qe = 0.021Ce + 0.001 Ce/Qe = 0.023Ce + 0.001 Ce/Qe = 0.022Ce + 0.001
0.997 0.999 0.998 0.995 0.996 0.995 0.992 0.998 0.997
T (°C) vicenin-2
isoschaftoside
schaftoside
25 30 35 25 30 35 25 30 35
Freundlich equation Qe Qe Qe Qe Qe Qe Qe Qe Qe
= = = = = = = = =
0.207
9.511Ce 10.07Ce0.222 9.996Ce0.231 18.44Ce0.235 19.61Ce0.239 19.58Ce0.235 19.09Ce0.380 15.78Ce0.33 16.07Ce0.348
Figure 5. Dynamic leakage curves of vicenin-2, isoschaftoside, and schaftoside on HPD-100 resin packed chromatography column.
R2 0.963 0.891 0.935 0.991 0.964 0.974 0.997 0.952 0.969
sorbent and adsorbed material in the adsorption isotherms test,20,21 suitable for revealing the linearity fitting and to describe how the solutes interact with the resin. The models of Langmuir and Freundlich can be expressed by the following mathematical formulas: Ce C 1 = e + Qe Qm KQ m
(Langmuir model)
where Qe is the adsorption capacity, Ce is the equilibrium concentration of flavonoid C-glycoside, Qm is the empirical constant, K is the Langmuir constant. Q e = kCe1/ n
(Freundlich model)
where k is the Freundlich constant that is an indicator of adsorption capacity, 1/n is an empirical constant related to the magnitude of the driving force. The Langmuir and Freundlich parameters were summarized and listed in Table 2. The K and 1/n value could be obtained from the intercept and slope, respectively. The correlation coefficients of two models were relatively high, and specifically the correlation coefficients of Langmuir model at different temperatures were rather higher (R2 > 0.99), indicating that the adsorption process was mainly a monomolecular layer adsorption. In the Freundlich, the adsorption can easily take place when the 1/n is between 0.1 and 0.5, and not easy to happen if 1/n value is between 0.5 and 1.22 In Table 2, the 1/n values were all between 0.1 and 0.5, which indicated that the adsorption of the flavonoid C-glycosides on HPD-100 resin could take place easily. All these results indicated that the HPD100 resin was appropriate for the separation of flavonoid Cglycosides from A. mollis. 3.3. Effect of Initial Sample Solution pH Value on Adsorption Capacity. The pH value of sample solution is very important for the adsorption and desorption properties of the resins, since the pH value determines the extent of ionization of solute molecules, thereby affecting their adsorption affinity.23 The flavonoid C-glycosides are weakly acidic compounds owning to the existence of phenolic hydroxyl groups. According to the results shown in Figure 3, the adsorption capacities of HPD-100 resin for compounds 1, 2, and 3 were higher at the pH value of 3 and 5. At higher pH values, the adsorption capacity of compounds 1, 2, and 3 on HPD-100 resin decreased linearly. These observations suggested that the hydrogen bonding might play a key role in the adsorption process of HPD-100 resins. At higher pH values,
Figure 3. Effects of pH value on adsorption capacity of vicenin-2, isoschaftoside, and schaftoside.
Figure 4. Effects of sample concentration on adsorption capacity of vicenin-2, isoschaftoside, and schaftoside in HPD-100 resin packed chromatography column.
3.2. Adsorption Isotherms. The Langmuir and Freundlich equations were adopted to describe the interaction between 7352
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Figure 6. Effects of concentration and volume of ethanol solution on HPD-100 resin column separation of vicenin-2, isoschaftoside, and schaftoside.
Figure 7. Chromatograms of A. mollis extracts and the final product: vicenin-2, 1; isoschaftoside, 2; schaftoside, 3.
Therefore, the sample loading should be no more than 20% of the dry resin weight. The column chromatography test showed that the flavonoid C-glycosides and the impurities with different properties can be separated by gradient elution of the macroporous resin with different concentrations of ethanol solution. In gradient elution (Figure 6), large amount of impurities were eluted at 0−20% ethanol solution and only small amounts of 1 and 3 were detected. Most of the flavonoid C-glycosides were eluted by 40% ethanol solutions. Therefore, the 40% ethanol fractions were collected and dried under vacuum to gain 5.8 g yellow solid. The chromatograms of the final product and A. mollis extracts were shown in Figure 7. By comparison, it could be seen that some impurities in the crude extracts were removed and the relative peak areas of these three flavonoid C-glycosides in the final product had increased. After one-run HPD-100 resin packed column chromatography, the contents of the compounds 1, 2, and 3 increased from 2.05, 2.90, and 2.30% in the extracts to 14.3, 21.9, and 15.1% in the product, which were 6.0-, 6.5-, and 5.6-fold increased, respectively. The recoveries of compounds 1, 2, and 3 were 83.8, 90.3, and 78.7%, respectively. Therefore, the resin column chromatography method is efficient, environmentally friendly, and lower cost, which represents an excellent alternative for the conventional silica column chromatography method.
the hydrogen bonding interactions were reduced since that the phenolic hydroxyl groups in the three flavonoid C-glycosides dissociated to from H+ and their corresponding anions, resulting in the decrease of adsorption capacity. At lower pH values, the surface of the resins would be surrounded by the hydronium ions which enhanced the interaction of the unionized phenolic hydroxyl groups of the compounds 1, 2, and 3 with the HPD-100 resin by potent attractive forces. However, if the pH value was too low (pH ≤3), the solubility of the flavonoid C-glycosides would decrease to make the sample solution become muddy. Hence, the pH value of sample solution was adjusted to 5 for all subsequent experiments. 3.4. Dynamic Adsorption and Desorption Tests on the Chromatography Column. From Figure 4, it could be concluded that leakage of the three flavonoid C-glycosides in the effluent was lower at the sample concentration of 0.04 or 0.1 g/mL. However, too low sample concentration would make the sample volume much bigger, which would take much time during the sample loading process and be not helpful for promoting efficiency. Therefore, 0.1 g of extracts per 1 mL of solution was chosen for the optimum adsorption concentration. According to the result (Figure 5), while increasing sample loading from 0.5 to 2.5 g of herbal extracts on the separation, no target compound was detected by HPLC in the loading elution. However, target compound leakage was observed during sample loading of 3.5, 4.0, 4.5, and 5 g extracts. 7353
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(11) Zhang, Z. F.; Liu, Y.; Luo, P.; Zhang, H. Separation and purification of two flavone glucuronides from Erigeron multiradiatus (Lindl.) Benth with macroporous resins. J. Biomed. Biotechnol. 2009, DOI: 10.1155/2009/875629. (12) Abrams, I. M. Macroporous Condensate Resins as Adsorbents. Ind. Eng. Chem. Prod. Res. Dev. 1975, 14, 108. (13) Li, J.; Chase, H. A. Development of adsorptive (non-ionic) macroporous resins and their uses in the purification of pharmacologically-active natural products from plant sources. Nat. Prod. Rep. 2010, 27, 1493. (14) Alexandratos, S. D. Ion-Exchange Resins: A Retrospective from Industrial and Engineering Chemistry Research. Ind. Eng. Chem. Res. 2008, 48, 388. (15) Xie, C.; Veitch, N. C.; Houghton, P. J.; Simmonds, M. S. Flavone C-glycosides from Viola yedoensis MAKINO. Chem. Pharm. Bull. 2003, 51, 1204. (16) Jia, G. G.; Lu, X. Y. Enrichment and purification of madecassoside and asiaticoside from Centella asiatica extracts with macroporous resins. J. Chromatogr. A 2008, 1193, 136. (17) Jia, D. D.; Li, S. F.; Gu, Z. P. Preparative isolation of Flavonoids from Mulberry (Morus Alba L.) Leaves by Macroporous Resin Adsorption. J. Food Process Eng. 2011, 34, 1319. (18) Kammerer, J.; Carle, R.; Kammerer, D. R. Adsorption and Ion Exchange: Basic Principles and Their Application in Food Processing. J. Agric. Food Chem. 2011, 59, 22. (19) Kong, Y.; Yan, M. M.; Chen, C. Y.; Zhao, B. S.; Zu, Y. G.; Fu, Y. J.; Luo, M.; Michael, W. Preparative enrichment and separation of astragalosides from Radix Astragali extracts using macroporous resins. J. Sep. Sci. 2010, 33, 2278. (20) Chao, L.; Hong, Z.; Li, Z.; Gang, Z. Study on Adsorption Characteristic of Macroporous Resin to Phenol in Wastewater. Can. J. Chem. Eng. 2010, 88, 417. (21) Liu, T.; Yang, M.; Wang, T.; Yuan, Q. Prediction Strategy of Adsorption Equilibrium Time Based on Equilibrium and Kinetic Results To Isolate Taxifolin. Ind. Eng. Chem. Res. 2011, 51, 454. (22) Liu, W.; Zhang, S.; Zu, Y. G.; Fu, Y. J.; Ma, W.; Zhang, D. Y.; Kong, Y.; Li, X. J. Preliminary enrichment and separation of genistein and apigenin from extracts of pigeon pea roots by macroporous resins. Bioresour. Technol. 2010, 101, 4667. (23) Huang, J. H.; Huang, K. L.; Wang, A. T.; Yang, Q. Adsorption characteristics of poly (styrene-co-divinylbenzene) resin functionalized with methoxy and phenoxy groups for phenol. J. Colloid Interface Sci. 2008, 327, 302.
4. CONCLUSIONS In this study, a method of separation and preparation of total flavonoid C-glycosides from A. mollis for industry production was established. HPD-100 resin was screened from the six macroporous resins for its better adsorption and desorption properties. The equilibrium experimental data of the adsorption of 1, 2, and 3 on HPD-100 resin at different temperatures were well fitted to the Langmuir and Freundlich isotherms. The contents of the flavonoid C-glycosides (1, 2, and 3) increased from 2.05, 2.90, and 2.30% in the extracts to 14.3, 21.9, and 15.1%, respectively, in the product. The recoveries of compounds 1, 2, and 3 were 83.8, 90.3, and 78.7%, respectively. The results evidenced a good adsorption and desorption chromatography, together with a noticeable selectivity for the preparative enrichment and separation of flavonoid from herbal materials.
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +86-25-86185376. Fax: +86-25-85301528. E-mail:
[email protected] Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support by the National Natural Science Foundation of China (81172955) and the Major National Science and Technology Projects of the Chinese eleventh five-year Plan (2009ZX09103-315).
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REFERENCES
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