- Email: [email protected]
Isolation and purification of glycyrrhizic acid with solvent extraction
Separation and Purification Technology 44 (2005) 189–196
Isolation and purification of glycyrrhizic acid with solvent extraction Guo-guang Niu, Yu-chun Xie, Jian-feng Lou, Hui-zhou Liu∗ Laboratory of Separation Science and Engineering, State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, China Received 11 December 2003; accepted 5 May 2004
Abstract Solvent extraction of glycyrrhizic acid (GA) from liquorice leaching solution was studied with tributyl phosphate, trialkylphosphine oxide, n-hexanol and 2-ethyl-hexanol as extractants. The results showed that GA was easily extracted into the organic phase in molecular state by the formation of hydrogen bond. pH was the key factor influencing the selectivity during extraction and stripping. The extraction mechanism was studied with transmitting electron microscopy and analysis of solute distribution. It was deduced that glycyrrhizic acid was extracted into the organic solvent in the form of molecular aggregation rather than as single molecules. © 2004 Elsevier B.V. All rights reserved. Keywords: Liquorice; Glycyrrhizic acid; Solvent extraction; Purification
1. Introduction Liquorice, derived from Glycyrrhiza glabra, is one of the traditional medical herbs used in China. Glycyrrhizic acid (GA) is an active ingredient of liquorice. Pharmacological researches show that GA has an anti-inflammatory activity [1] and is a scavenger of oxygen-free radicals [2,3]. GA possesses immune stimulation [4] and is well known of its clinical applications in the treatment of spleen, sore throat, bronchitis, liver [5], kidney and ulcer. Because of above reasons, there is considerable interest in the extraction and isolation of GA from the aqueous solution with different methods, such as with high performance liquid chromatography (HPLC) [6,7], with resin column and adsorbent [8,9], with supercritical fluid extraction [10] and with foam separation [11]. Solvent extraction method, with advantages of its welldeveloped technology, easy to operating, high efficiency and time saving, has been widely used for the preparation and separation of active compounds in pharmaceutical scopes [12,13]. Compared with other extractants, the neutral organic phosphorous extracting-reagent and alcohols are preferably used because of their lower toxicity. For example, the ex-
∗
Corresponding author. Tel.: +86 10 62555005; fax: +86 10 62554264. E-mail address: [email protected] (H.-z. Liu).
1383-5866/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2004.05.011
traction of leucine and isoleucine with di-(2-ethylhexyl)phosphoric acid (P204) [14], recovery of malic acid, fumaric acid and hydroxyacetic acid with trialkylphosphine oxide (TRPO) [15–17], isolation of penicillin G with tributyl phosphate (TBP) [18], the recovery of theophylline with 2-ethyl-hexanol [19] and the extraction of spriamycins with capryl alcohol or alcohol from coconut oil [20] have been reported. GA, one of weak acids and composed of glycyrrhitinic acid and glucuronic acid, has three carboxyls and five hydroxyl groups. Those polar function groups are capable of interacting with functional groups of the extracting-reagent through hydrogen bond. Therefore, the molecules of GA can be possibly extracted into the organic phase from aqueous solution and selectively separated from impurities. In this paper, the isolation and purification of GA was performed with neutral organic phosphorous extractants and alcohols while the solvent extraction mechanism was also investigated. 2. Experimental 2.1. Materials and methods Crude and naturally dried liquorice roots were purchased from Inner-Mongolia, China, and cut in our laboratory into small pieces. Standard samples of glycyrrhizic acid and its
190
G.-g. Niu et al. / Separation and Purification Technology 44 (2005) 189–196
mono-ammonium salt were purchased from the Chinese Institute of Drugs and Biological Products Identification. Commercial glycyrrhizic acid mono-ammonium salt (UV, 98%, Yu Jiu Company, Shanghai) was used without further purification in our initial stage solvent extraction experiments. Water and acetonitrile were used in the HPLC analysis. Acetic acid, anhydrous ethanol, 2-ethyl-hexanol, n-hexanol, TBP, TRPO and petroleum ether were of analytical grade and obtained commercially. Liquorice leaching solution was obtained using the method of microwave-assisted extraction. A household microwave oven (National brand, Japan) used in this work was modified first in our laboratory [21]. Liquorice root small pieces (about 5 g total) were mixed with distilled water (100 ml) and then were irradiated with microwaves (700 W) at ambient pressure with operation time of about 4 min at boiling point (about 100 ◦ C). Testing solution was prepared by dissolving the commercial GA directly in distilled water. Solvent extraction was carried out in a beaker equipped with magnetic stirring (about 500 rpm). Liquorice leaching solution or testing solution was mixed with extractant and the pH of the aqueous-organic mixture was adjusted with hydrochloric acid solution (2 mol/l) to an appropriate level. The separation of aqueous phase from organic phase was easily obtained with centrifuge. Aqueous/organic phase volumes were measured and contents of GA in each phase were analyzed with the method of HPLC. Stripping process was carried out in a beaker equipped with magnetic stirring (about 500 rpm), in which the organic phase containing GA was mixed with distilled water. The pH of the aqueous-organic mixture solution was adjusted with sodium hydroxide solution (1 mol/l). The separation between the aqueous and organic phase was carried out by gravitational sedimentation. The volumes of aqueous and organic phases were measured and contents of GA in each phase were analyzed with HPLC.
2.2. HPLC analysis The aqueous phase after extraction or stripping was diluted with distilled water and then was adjusted to pH 7 for HPLC analysis. Anhydrous ethanol was used as diluent for the organic phase. The contents of GA in diluted aqueous and organic samples were analyzed by HPLC with the Zorbax-SB guard column and Zorbax-ODS assay column (5 m, 150 mm × 4.6 mm, Du Pont). The mobile phase was composed of acetic acid:acetonitrile:water (3:41:59, v/v) at a flow rate of 1.0 ml min−1 . The HPLC system is the HP 1090 liquid chromatography, equipped with an automatic injector and a diode array detector (DAD). Ultraviolet detection at 248 nm was used for quantification at ambient temperature. The retention time of GA was 4.2 min. The amount of GA in the extracts was calibrated using the peak area and a calibration curve with good linearity generated by injecting standard GA solutions in mobile phase with mass from 0.10 g to 2.50 g. The extraction and stripping percentages of GA were defined respectively as follows: Extraction percentage of GA (%) =
V o Co × 100 V i Ci
(1)
Vo , Co —volume and concentration of GA in organic phase after solvent extraction, ml and mg/ml, respectively; Vi , Ci —initial volume and initial concentration of GA in liquorice leaching (or testing) solution before solvent extraction, ml and mg/ml, respectively. Stripping percentage of GA (%) =
V a Ca × 100 Vio Cio
(2)
Va , Ca —volume and concentration of GA in aqueous phase after stripping, ml and mg/ml, respectively; Vio , Cio —initial volume and initial concentration of GA in organic phase before stripping, ml and mg/ml, respectively.
Fig. 1. Effect of pH on the extraction of GA in model solution (conditions: organic phase/aqueous phase = 10 ml/10 ml; magnetic stirring, 3 min; for 2ethylhexanol and n-hexanol, concentration of GA in solution = 0.80 mg/ml, T = 29 ◦ C; for TBP, concentration of GA in solution = 0.87 mg/ml, T = 25.5 ◦ C; for TRPO, concentration of GA in solution = 0.92 mg/ml, T = 21.5 ◦ C).
G.-g. Niu et al. / Separation and Purification Technology 44 (2005) 189–196
191
Fig. 2. Effect of pH on the viscosity of model solution (conditions: concentration of glycyrrhizic acid mono-ammonia salt 0.75 mg/ml, T = 30 ◦ C).
2.3. Preparation of specimen for transmitting electron microscope (TEM)
2.4. Ultraviolet measurements and viscosity measurement
Preparation of specimen for TEM was performed with the technique of Freezing-Etch Replica [22,23]. Freezing was carried out by dripping organic reagent loaded with GA in an inert gas cooled and liquefied with liquid nitrogen. After fracturing the frozen specimen in a high vacuum chamber (JEE-4X vacuum evaporator), a sample was made by evaporating a thin platinum layer and also a carbon layer on the fractured organic reagent. The platinum layer was evaporated under an angle of 45◦ , while the carbon layer was made with an angle of 90◦ . Following the step of platinum and carbon evaporation, the frozen specimen underlying the replica was thawed with ethanol while the platinum and carbon layers are inert. After being washed by distilled water, the cleaned replica was finally recovered on a copper mesh screen identical to that used for picking up thin-sections and was ready for observation with TEM (JEM-100X electron microscope).
Ultraviolet Scan spectra with the slit of 1.0 nm were recorded on a Lambda Bio 40 UV/vis spectrophotometer (Perkin Elmer). The viscosity of aqueous solution of GA was measured with Ubbelohde viscosimeter.
3. Results and discussion 3.1. GA extraction in the testing solution with different pH Fig. 1 showed that pH had a marked effect on the extraction of GA in the testing solution. An increase of pH brought about a considerable decrease of extraction percentage of GA for all extractants tested. For 2-ethylhexanol, the extraction percentage of GA was above 90% at pH = 2, but
Fig. 3. Effect of pH on the stripping of GA in model solution (conditions: organic phase/aqueous phase = 10 ml/10 ml; magnetic stirring, 5 min; concentration of GA in 2-ethylhexanol, n-hexanol, TBP and TRPO being, respectively, 0.97 mg/ml, 1.01 mg/ml, 0.88 mg/ml and 1.13 mg/ml; T = 27.5 ◦ C; for TRPO, T = 18.0 ◦ C).
192
G.-g. Niu et al. / Separation and Purification Technology 44 (2005) 189–196
Fig. 4. Ultraviolet-scanning spectra of glycyrrhizic acid mono-ammonium salt in aqueous solution (0.18 mg/ml) in 200–300 nm region within pH of 1.3–11.0.
decreased to about 0 with pH > 6. For n-hexanol, GA could be almost completely extracted into organic phase with pH < 3, whereas the extraction percentage decreased quickly with the increase of pH and could be neglected at pH > 7. For TBP and TRPO as solvent, there were also similar results that the GA extraction percentage decreased from 90% to about 0 with increase of pH. But the pH ranges, in which the extraction percentage of GA changed rapidly, were of a little difference, for TBP pH = 4.5–6.5 and for TRPO pH = 5.5–7, respectively. Because GA is a weak acid, it was reasonable to deduce that under acidic conditions, GA existed as un-ionized molecular form, the solution has higher viscosity because of the formation of intermolecular hydrogen bonds through carboxylic groups. Similarly, under basic conditions, GA existed in ionized form, the solution has lower viscosity because of the disappearance of intermolecular hydrogen bonding. For this reason, the variation of viscosity of aqueous solution con-
taining GA mono-ammonium salt (about 0.75 mg/ml) with pH was investigated, as shown in Fig. 2 (the line was made by the method of B-Spline with the software of origin 7, so it did not pass through the highest point). It could be seen that when pH < 5, the viscosity of the solution clearly increased with the decrease of pH, and at pH equal to 2, the viscosity reached the maximum. These results implied that with solution pH from 2 to 5, the GA in the solution will be changed from molecular form to ionized form. In the molecular form (pH 2–5), because of the formation of hydrogen bond between GA and extractant, GA was extracted into the organic phase. No hydrogen bond could be formed between GA and the extractant with pH > 5, the hydrophilic GA was then re-extracted back to the aqueous phase (Fig. 3). Further study was carried out with ultraviolet scan of glycyrrhizic acid mono-ammonium salt in aqueous solution under different values of pH. It can be seen from Fig. 4 that
Fig. 5. Effect of pH on the maximal absorbing wavelength of glycyrrhizic acid mono-ammonium salt in aqueous solution (0.18 mg/ml).
G.-g. Niu et al. / Separation and Purification Technology 44 (2005) 189–196
193
Fig. 6. Effect of concentration of GA on the maximal absorption wavelength at pH about 3.5 and 10.5, respectively.
with the increase of pH, the maximal absorption wavelength H2 O of solution (λmax ) has a little bathochromic shift and the abH2 O sorbency will be much stronger. A plot of pH versus λmax is shown in Fig. 5. The maximal absorption wavelength
Fig. 7. Comparison of HPLC chromatogram after extraction: (a) organic phase, (b) aqueous phase (conditions: extractant, 10% (v/v) TRPO, petroleum ether as diluent; concentration of GA in liquorice leaching solution = 2.05 mg/ml; magnetic stirring 3 min, organic/aqueous phase = 10 ml/10 ml).
started to increase at pH 2.1 and reached the maximum at pH 6.5, with a red shift of about 6.7 nm from 251.5 nm to about 258.2 nm. The pH range, in which the maximal absorption wavelength occurred to shift, was coincident with the pH range in which the viscosity of glycyrrhizic acid mono-
Fig. 8. Comparison of HPLC chromatogram after stripping: (a) aqueous phase, (b) organic phase (conditions: concentration of GA in 10% TRPO = 1.95 mg/ml, T = 8.0 ◦ C; magnetic stirring time = 3 min; extraction pH = 4.50; organic/aqueous phase = 10 ml/10 ml).
194
G.-g. Niu et al. / Separation and Purification Technology 44 (2005) 189–196
ammonium salt aqueous solution markedly changed. Therefore, it is proposed that red shift of the maximal absorption wavelength with pH was possibly caused by the change of intermolecular hydrogen bonding between the different GA molecules. The relationship between the GA intermolecular hydrogen bonding with the maximal absorption wavelength shifting could be further elucidated by the effect of GA concentration on the absorption wavelength, as shown in Fig. 6. Under acidic condition of pH 3.5, the intermolecular hydrogen bond will be weaker with the decrease of GA concentration resulting to the maximal absorption wavelength bathochromically shifted. Under basic condition of pH 10.5, no intermolecular hydrogen bond can be formed with the ionized GA, and the maximal absorption wavelength remained unchanged with concentration.
3.2. Effect of pH on the selectivity of extraction and stripping of GA in liquorice leaching solution The selectivity of the extractants is one of the most important factors in solvent extraction. The separation and purification of the main ingredients from other components can be easily found by using selective extractants or by changing the operational conditions based on the properties of different ingredients. Fig. 7(a and b) showed that when using 10% TRPO as extractant, GA was selectively extracted into the organic phase and impurities, especially with retention time of 1–2 min, were concentrated in the aqueous phase. Adjusting the acidity of the solution to a higher values of pH, about 4.5, the selectivity of extraction was increased. The peaks of impurities, with retention time of 2.0 min and 3.0–3.2 min in the organic phase,
Fig. 9. Effect of extractant concentration on distribution ratio: (a) extractant, TBP; concentration of GA in liquorice leaching solution = 0.75 mg/ml; T = 28.5 ◦ C; pH = 2.0–3.0; organic/aqueous = 10 ml/10 ml, petroleum ether as diluent; magnetic stirring time = 5 min; results based on the triplicated experiments and analysis; (b) extractant, TRPO; concentration of GA in liquorice leaching solution = 0.88 mg/ml; T = 21.5 ◦ C; pH = 2.5–3.5; organic/aqueous = 10 ml/10 ml, petroleum ether as diluent; magnetic stirring time = 3 min; results based on the triplicated experiments and analysis.
G.-g. Niu et al. / Separation and Purification Technology 44 (2005) 189–196
195
were clearly reduced compared with the highest peak of GA. Fig. 8(a and b) shows the HPLC chromatograms of aqueous phase and organic phase after stripping, respectively. Fig. 8(a) showed that the impurity peaks with retention time less than 4 min were remarkably reduced after stripping, with pH adjusted from 11.0 to 7.1. The impurities with retention time less than 4 min were left in the organic phase after stripping and the stripped GA remaining in the aqueous phase was purified. Similar results were obtained when 2-ethyl-hexanol, n-hexanol and TBP were used as extractants, and in all cases, GA could also be selectively extracted or stripped from aqueous phase or organic phase, with a small amount shifting of pH. 3.3. The mechanism of extraction To characterize the formation of molecular complexes in organic solvents, the dependence of the distribution ratio D of GA upon the organic reagent concentration was examined. If the general extraction equilibrium is assumed to be given by Eq. (3), and if the activity coefficients of organic reagents are constant when the concentration of extractant changes at the scope of experiments, the solvation number p can be determined from the distribution ratio measured as a function of concentration of the extractant. GA + pS + qH2 O ⇔ GA · pS · qH2 O
(3)
GA: glycyrrhizic acid; S: extractant; p: the solvation number; q: the number of water molecules interacting with one GA molecule. As given in literature [24], a plot of log D versus log [S] should be linear, and its slope should be equal to the number of ligand molecules per GA in the extraction species. The log D versus log [S] plots are shown in Fig. 9(a and b), respectively, for the extraction of GA with TBP and TRPO as extractants in testing solution, respectively. Surprisingly, the plots were not linear, the slopes decreased with the increase of GA concentration. Similar results were also obtained with hexanol and 2-ethyl-hexanol as extractants. These results strongly suggested that GA was extracted into organic solvent in the aggregated form rather than in the single molecular form. With the increase of concentration, the aggregation of GA would become more severe; more functional groups of GA would be deleted for the intermolecular actions of GA. As a result, the number of the functional groups of each GA molecule left for the combination with extractant would become less and the consequence is the decrease of the slope of log D versus log [S] plot. If GA concentration increased to a certain value, for example, 88 mg/ml in TBP, the GA extracted in the organic phase would be more viscous and will be moved to the bottom of the aqueous phase. This phenomenon was possibly attributed to the fact that a polymer phase of GA-extractant was formed at high GA concentration.
Fig. 10. The TEM of GA extracted into the organic phase (concentration of GA in TBP after extraction: (a) 20 mg/ml, (b) 75 mg/ml).
To further investigate the possible GA aggregation, the profile of GA extracted in the organic phase was studied with Freezing-Etch Replica technique for TEM at different GA concentrations. Fig. 10(a and b) presented the pictures of GA extracted in TBP at concentrations of 20 mg/ml and 75 mg/ml, respectively. It can be seen that GA aggregation was observed for all concentrations. Under lower concentration (Fig. 10(a)), the aggregation particles were separated and under higher concentration (Fig. 10(b)), the aggregation particles were cross-linked to form a dense polymer phase which was more viscous and could even move to the bottom of aqueous phase as previously mentioned. Similar observations were obtained for other extractants such as n-hexanol and 2-ethyl-hexanol. These results consistently pointed out that due to the aggregation of GA, the complexes of GAextractants probably have no constant form and would be changed with GA concentration.
4. Conclusions GA can be efficiently extracted to the organic phase from acidic aqueous solution by the formation of hydrogen bond-
196
G.-g. Niu et al. / Separation and Purification Technology 44 (2005) 189–196
ing between GA and extractants used, such as n-hexanol, 2ethylhexanol, TBP and TRPO. When TRPO is used as the extractant, the extraction percentage of GA can reach about 90% at pH = 5.5. For other three extractants studied, more acidic conditions are needed to reach the similar extraction percentage with pH below 2.0 for 2-ethyl-hexanol as solvent as an example. In stripping process, GA can be easily stripped back to the aqueous phase in alkaline solution as the breakdown of the hydrogen bonding between ionized GA and extractants occurred. GA distribution and TME studies suggested that GA was probably extracted into the organic phase in the molecular aggregation form rather than as single molecule form. No constant ratio of extractant to GA in their complexes could be derived because the ratio probably changed with GA concentration to cause the change of GA molecular aggregation. By adjusting the pH condition, GA could be partially purified from the natural impurities in liquorice root by extraction and stripping process. The prospect of solvent extraction of glycyrrhizic acid from liquorice leaching solution could be applied to a wide variety of liquorice-containing products.
Acknowledgments The authors acknowledge support from the Outstanding Young Scientist Foundation of China (no. 29925617) and National Natural Science Foundation of China (no. 20221603 and no.20273075). The authors are grateful to Prof. Jiayong Chen for helpful discussion and major revision regarding the grammar.
References [1] R.S.H. Finney, G.F. Somers, J. Pharmacol. 10 (1958) 613–620. [2] H. Akamatsu, J. Komura, Y. Asada, Y. Niwa, Planta Med. 57 (1991) 119–121. [3] B. Furhman, S. Buch, J. Vaya, P.A. Belinky, T. Hayek, M. Aviram, Am. J. Clin. Nutr. 66 (1997) 267–275.
[4] M. Ito, H. Nakashima, M. Baba, R. Pauwels, E. De Clercq, E. Shigeta, N. Yamamoto, Antivir. Rec. 7 (1987) 127– 137. [5] H. Suzuki, Y. Ohta, T. Kakino, K. Fujisawa, D. Hirayama, Asian Med. J. 26 (1983) 423–438. [6] X.M. Zhou, Y.J. Chen, D.C. Wang, P.Y. Cheng, J. Chin. Pharm. Univ. 21 (1) (1990) 64 (in Chinese). [7] H.B.S. Thomas, W.Z. Howaed, D.B. Alexis, J. Chromatogr. 175 (1979) 350–355. [8] S.H. Yu, S.J. Wu, F.X. Jin, Y. Guo, Food Fermentation Ind. 25 (1) (1998) 40–43 (in Chinese). [9] F.S. Feng, C.S. Xuan, S.W. Chen, Y. Chang, B.Z. Qiao, Nat. Prod. Res. Dev. 10 (1) (1997) 60–64 (in Chinese). [10] Q.L. Li, M.H. Zhou, J.N. Chen, J. Instrumental Anal. 17 (1) (1998) 37–40 (in Chinese). [11] Y.X. Guan, S.J. Yao, Z.Q. Zhu, Proceedings of the Third China–Korea Conference on Separation Science and Technology, Hangzhou, China, October 31–November 2, 2001, pp. 575– 579. [12] X. Hu, R.S. Bai, X.J. Wang, C.R. Wan, J.W. Yang, X. Yang, J. Tsinghua Univ. (Sci. Tech.) 36 (12) (1996) 13–19 (in Chinese). [13] L.W. Bi, Z.S. Wu, J.H. Chen, Y.M. Wang, D.M. Wu, R.X. Gu, Chem. Ind. Forest Prod. 17 (3) (1997) 41–45 (in Chinese). [14] S.Y. Tan, R.S. Xue, Z.M. Zhou, H. Yang, J. Mod. Chem. Technol. 12 (1998) 23–26 (in Chinese). [15] D.W. Wang, Y.F. Su, J. East Chin. Inst. Chem. Technol. 18 (6) (1992) 718–723 (in Chinese). [16] D.W. Wang, X.G. Fan, J. East Chin. Univ. Sci. Technol. 21 (5) (1995) 548–551 (in Chinese). [17] P. Zhang, S.H. Cai, Q.H. Zhang, Z.C. Luo, J. East Chin. Univ. Sci. Technol. 20 (2) (1994) 148–153 (in Chinese). [18] Z.F. Yang, S.Q. Yu, J.Y. Cheng, G. Wan, R.F. Xu, Eng. Chem. Metall. 13 (1) (1992) 51–54 (in Chinese). [19] X.N. Hu, R.S. Bai, X.J. Wang, C.R. Wan, J.R. Yang, X. Yang, J. Tsinghua Univ. (Sci. Tech.) 36 (12) (1996) 13–19 (in Chinese). [20] Z.D. Tao, Z.N. Xu, P.L. Chen, J. Chem. Eng. Chin. Univ. 11 (3) (1997) 308–311 (in Chinese). [21] X.J. Pan, H.Z. Liu, G.H. Jia, Y.Y. Shu, Biochem. Eng. J. 5 (2000) 173–177. [22] L. Orici, A. Perrelet, Freeze-Etch Histology a Comparison between Thin Sections and Freeze-Etch Replicas, Springer-Verlag, Berlin, Heidelherg, New York, 1975. [23] J.H.M. Willison, A.J. Rowe, Replica Shadowing and Freeze-Etching Techniques, North-Holland publishing company, Amsterdam, New York, Oxford, 1980. [24] S. Tatsuya, H. Yuko, Solvent Extraction Chemistry, Marcel Dekker, Inc., New York, 1977.
Isolation and purification of glycyrrhizic acid with solvent extraction Guo-guang Niu, Yu-chun Xie, Jian-feng Lou, Hui-zhou Liu∗ Laboratory of Separation Science and Engineering, State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, China Received 11 December 2003; accepted 5 May 2004
Abstract Solvent extraction of glycyrrhizic acid (GA) from liquorice leaching solution was studied with tributyl phosphate, trialkylphosphine oxide, n-hexanol and 2-ethyl-hexanol as extractants. The results showed that GA was easily extracted into the organic phase in molecular state by the formation of hydrogen bond. pH was the key factor influencing the selectivity during extraction and stripping. The extraction mechanism was studied with transmitting electron microscopy and analysis of solute distribution. It was deduced that glycyrrhizic acid was extracted into the organic solvent in the form of molecular aggregation rather than as single molecules. © 2004 Elsevier B.V. All rights reserved. Keywords: Liquorice; Glycyrrhizic acid; Solvent extraction; Purification
1. Introduction Liquorice, derived from Glycyrrhiza glabra, is one of the traditional medical herbs used in China. Glycyrrhizic acid (GA) is an active ingredient of liquorice. Pharmacological researches show that GA has an anti-inflammatory activity [1] and is a scavenger of oxygen-free radicals [2,3]. GA possesses immune stimulation [4] and is well known of its clinical applications in the treatment of spleen, sore throat, bronchitis, liver [5], kidney and ulcer. Because of above reasons, there is considerable interest in the extraction and isolation of GA from the aqueous solution with different methods, such as with high performance liquid chromatography (HPLC) [6,7], with resin column and adsorbent [8,9], with supercritical fluid extraction [10] and with foam separation [11]. Solvent extraction method, with advantages of its welldeveloped technology, easy to operating, high efficiency and time saving, has been widely used for the preparation and separation of active compounds in pharmaceutical scopes [12,13]. Compared with other extractants, the neutral organic phosphorous extracting-reagent and alcohols are preferably used because of their lower toxicity. For example, the ex-
∗
Corresponding author. Tel.: +86 10 62555005; fax: +86 10 62554264. E-mail address: [email protected] (H.-z. Liu).
1383-5866/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2004.05.011
traction of leucine and isoleucine with di-(2-ethylhexyl)phosphoric acid (P204) [14], recovery of malic acid, fumaric acid and hydroxyacetic acid with trialkylphosphine oxide (TRPO) [15–17], isolation of penicillin G with tributyl phosphate (TBP) [18], the recovery of theophylline with 2-ethyl-hexanol [19] and the extraction of spriamycins with capryl alcohol or alcohol from coconut oil [20] have been reported. GA, one of weak acids and composed of glycyrrhitinic acid and glucuronic acid, has three carboxyls and five hydroxyl groups. Those polar function groups are capable of interacting with functional groups of the extracting-reagent through hydrogen bond. Therefore, the molecules of GA can be possibly extracted into the organic phase from aqueous solution and selectively separated from impurities. In this paper, the isolation and purification of GA was performed with neutral organic phosphorous extractants and alcohols while the solvent extraction mechanism was also investigated. 2. Experimental 2.1. Materials and methods Crude and naturally dried liquorice roots were purchased from Inner-Mongolia, China, and cut in our laboratory into small pieces. Standard samples of glycyrrhizic acid and its
190
G.-g. Niu et al. / Separation and Purification Technology 44 (2005) 189–196
mono-ammonium salt were purchased from the Chinese Institute of Drugs and Biological Products Identification. Commercial glycyrrhizic acid mono-ammonium salt (UV, 98%, Yu Jiu Company, Shanghai) was used without further purification in our initial stage solvent extraction experiments. Water and acetonitrile were used in the HPLC analysis. Acetic acid, anhydrous ethanol, 2-ethyl-hexanol, n-hexanol, TBP, TRPO and petroleum ether were of analytical grade and obtained commercially. Liquorice leaching solution was obtained using the method of microwave-assisted extraction. A household microwave oven (National brand, Japan) used in this work was modified first in our laboratory [21]. Liquorice root small pieces (about 5 g total) were mixed with distilled water (100 ml) and then were irradiated with microwaves (700 W) at ambient pressure with operation time of about 4 min at boiling point (about 100 ◦ C). Testing solution was prepared by dissolving the commercial GA directly in distilled water. Solvent extraction was carried out in a beaker equipped with magnetic stirring (about 500 rpm). Liquorice leaching solution or testing solution was mixed with extractant and the pH of the aqueous-organic mixture was adjusted with hydrochloric acid solution (2 mol/l) to an appropriate level. The separation of aqueous phase from organic phase was easily obtained with centrifuge. Aqueous/organic phase volumes were measured and contents of GA in each phase were analyzed with the method of HPLC. Stripping process was carried out in a beaker equipped with magnetic stirring (about 500 rpm), in which the organic phase containing GA was mixed with distilled water. The pH of the aqueous-organic mixture solution was adjusted with sodium hydroxide solution (1 mol/l). The separation between the aqueous and organic phase was carried out by gravitational sedimentation. The volumes of aqueous and organic phases were measured and contents of GA in each phase were analyzed with HPLC.
2.2. HPLC analysis The aqueous phase after extraction or stripping was diluted with distilled water and then was adjusted to pH 7 for HPLC analysis. Anhydrous ethanol was used as diluent for the organic phase. The contents of GA in diluted aqueous and organic samples were analyzed by HPLC with the Zorbax-SB guard column and Zorbax-ODS assay column (5 m, 150 mm × 4.6 mm, Du Pont). The mobile phase was composed of acetic acid:acetonitrile:water (3:41:59, v/v) at a flow rate of 1.0 ml min−1 . The HPLC system is the HP 1090 liquid chromatography, equipped with an automatic injector and a diode array detector (DAD). Ultraviolet detection at 248 nm was used for quantification at ambient temperature. The retention time of GA was 4.2 min. The amount of GA in the extracts was calibrated using the peak area and a calibration curve with good linearity generated by injecting standard GA solutions in mobile phase with mass from 0.10 g to 2.50 g. The extraction and stripping percentages of GA were defined respectively as follows: Extraction percentage of GA (%) =
V o Co × 100 V i Ci
(1)
Vo , Co —volume and concentration of GA in organic phase after solvent extraction, ml and mg/ml, respectively; Vi , Ci —initial volume and initial concentration of GA in liquorice leaching (or testing) solution before solvent extraction, ml and mg/ml, respectively. Stripping percentage of GA (%) =
V a Ca × 100 Vio Cio
(2)
Va , Ca —volume and concentration of GA in aqueous phase after stripping, ml and mg/ml, respectively; Vio , Cio —initial volume and initial concentration of GA in organic phase before stripping, ml and mg/ml, respectively.
Fig. 1. Effect of pH on the extraction of GA in model solution (conditions: organic phase/aqueous phase = 10 ml/10 ml; magnetic stirring, 3 min; for 2ethylhexanol and n-hexanol, concentration of GA in solution = 0.80 mg/ml, T = 29 ◦ C; for TBP, concentration of GA in solution = 0.87 mg/ml, T = 25.5 ◦ C; for TRPO, concentration of GA in solution = 0.92 mg/ml, T = 21.5 ◦ C).
G.-g. Niu et al. / Separation and Purification Technology 44 (2005) 189–196
191
Fig. 2. Effect of pH on the viscosity of model solution (conditions: concentration of glycyrrhizic acid mono-ammonia salt 0.75 mg/ml, T = 30 ◦ C).
2.3. Preparation of specimen for transmitting electron microscope (TEM)
2.4. Ultraviolet measurements and viscosity measurement
Preparation of specimen for TEM was performed with the technique of Freezing-Etch Replica [22,23]. Freezing was carried out by dripping organic reagent loaded with GA in an inert gas cooled and liquefied with liquid nitrogen. After fracturing the frozen specimen in a high vacuum chamber (JEE-4X vacuum evaporator), a sample was made by evaporating a thin platinum layer and also a carbon layer on the fractured organic reagent. The platinum layer was evaporated under an angle of 45◦ , while the carbon layer was made with an angle of 90◦ . Following the step of platinum and carbon evaporation, the frozen specimen underlying the replica was thawed with ethanol while the platinum and carbon layers are inert. After being washed by distilled water, the cleaned replica was finally recovered on a copper mesh screen identical to that used for picking up thin-sections and was ready for observation with TEM (JEM-100X electron microscope).
Ultraviolet Scan spectra with the slit of 1.0 nm were recorded on a Lambda Bio 40 UV/vis spectrophotometer (Perkin Elmer). The viscosity of aqueous solution of GA was measured with Ubbelohde viscosimeter.
3. Results and discussion 3.1. GA extraction in the testing solution with different pH Fig. 1 showed that pH had a marked effect on the extraction of GA in the testing solution. An increase of pH brought about a considerable decrease of extraction percentage of GA for all extractants tested. For 2-ethylhexanol, the extraction percentage of GA was above 90% at pH = 2, but
Fig. 3. Effect of pH on the stripping of GA in model solution (conditions: organic phase/aqueous phase = 10 ml/10 ml; magnetic stirring, 5 min; concentration of GA in 2-ethylhexanol, n-hexanol, TBP and TRPO being, respectively, 0.97 mg/ml, 1.01 mg/ml, 0.88 mg/ml and 1.13 mg/ml; T = 27.5 ◦ C; for TRPO, T = 18.0 ◦ C).
192
G.-g. Niu et al. / Separation and Purification Technology 44 (2005) 189–196
Fig. 4. Ultraviolet-scanning spectra of glycyrrhizic acid mono-ammonium salt in aqueous solution (0.18 mg/ml) in 200–300 nm region within pH of 1.3–11.0.
decreased to about 0 with pH > 6. For n-hexanol, GA could be almost completely extracted into organic phase with pH < 3, whereas the extraction percentage decreased quickly with the increase of pH and could be neglected at pH > 7. For TBP and TRPO as solvent, there were also similar results that the GA extraction percentage decreased from 90% to about 0 with increase of pH. But the pH ranges, in which the extraction percentage of GA changed rapidly, were of a little difference, for TBP pH = 4.5–6.5 and for TRPO pH = 5.5–7, respectively. Because GA is a weak acid, it was reasonable to deduce that under acidic conditions, GA existed as un-ionized molecular form, the solution has higher viscosity because of the formation of intermolecular hydrogen bonds through carboxylic groups. Similarly, under basic conditions, GA existed in ionized form, the solution has lower viscosity because of the disappearance of intermolecular hydrogen bonding. For this reason, the variation of viscosity of aqueous solution con-
taining GA mono-ammonium salt (about 0.75 mg/ml) with pH was investigated, as shown in Fig. 2 (the line was made by the method of B-Spline with the software of origin 7, so it did not pass through the highest point). It could be seen that when pH < 5, the viscosity of the solution clearly increased with the decrease of pH, and at pH equal to 2, the viscosity reached the maximum. These results implied that with solution pH from 2 to 5, the GA in the solution will be changed from molecular form to ionized form. In the molecular form (pH 2–5), because of the formation of hydrogen bond between GA and extractant, GA was extracted into the organic phase. No hydrogen bond could be formed between GA and the extractant with pH > 5, the hydrophilic GA was then re-extracted back to the aqueous phase (Fig. 3). Further study was carried out with ultraviolet scan of glycyrrhizic acid mono-ammonium salt in aqueous solution under different values of pH. It can be seen from Fig. 4 that
Fig. 5. Effect of pH on the maximal absorbing wavelength of glycyrrhizic acid mono-ammonium salt in aqueous solution (0.18 mg/ml).
G.-g. Niu et al. / Separation and Purification Technology 44 (2005) 189–196
193
Fig. 6. Effect of concentration of GA on the maximal absorption wavelength at pH about 3.5 and 10.5, respectively.
with the increase of pH, the maximal absorption wavelength H2 O of solution (λmax ) has a little bathochromic shift and the abH2 O sorbency will be much stronger. A plot of pH versus λmax is shown in Fig. 5. The maximal absorption wavelength
Fig. 7. Comparison of HPLC chromatogram after extraction: (a) organic phase, (b) aqueous phase (conditions: extractant, 10% (v/v) TRPO, petroleum ether as diluent; concentration of GA in liquorice leaching solution = 2.05 mg/ml; magnetic stirring 3 min, organic/aqueous phase = 10 ml/10 ml).
started to increase at pH 2.1 and reached the maximum at pH 6.5, with a red shift of about 6.7 nm from 251.5 nm to about 258.2 nm. The pH range, in which the maximal absorption wavelength occurred to shift, was coincident with the pH range in which the viscosity of glycyrrhizic acid mono-
Fig. 8. Comparison of HPLC chromatogram after stripping: (a) aqueous phase, (b) organic phase (conditions: concentration of GA in 10% TRPO = 1.95 mg/ml, T = 8.0 ◦ C; magnetic stirring time = 3 min; extraction pH = 4.50; organic/aqueous phase = 10 ml/10 ml).
194
G.-g. Niu et al. / Separation and Purification Technology 44 (2005) 189–196
ammonium salt aqueous solution markedly changed. Therefore, it is proposed that red shift of the maximal absorption wavelength with pH was possibly caused by the change of intermolecular hydrogen bonding between the different GA molecules. The relationship between the GA intermolecular hydrogen bonding with the maximal absorption wavelength shifting could be further elucidated by the effect of GA concentration on the absorption wavelength, as shown in Fig. 6. Under acidic condition of pH 3.5, the intermolecular hydrogen bond will be weaker with the decrease of GA concentration resulting to the maximal absorption wavelength bathochromically shifted. Under basic condition of pH 10.5, no intermolecular hydrogen bond can be formed with the ionized GA, and the maximal absorption wavelength remained unchanged with concentration.
3.2. Effect of pH on the selectivity of extraction and stripping of GA in liquorice leaching solution The selectivity of the extractants is one of the most important factors in solvent extraction. The separation and purification of the main ingredients from other components can be easily found by using selective extractants or by changing the operational conditions based on the properties of different ingredients. Fig. 7(a and b) showed that when using 10% TRPO as extractant, GA was selectively extracted into the organic phase and impurities, especially with retention time of 1–2 min, were concentrated in the aqueous phase. Adjusting the acidity of the solution to a higher values of pH, about 4.5, the selectivity of extraction was increased. The peaks of impurities, with retention time of 2.0 min and 3.0–3.2 min in the organic phase,
Fig. 9. Effect of extractant concentration on distribution ratio: (a) extractant, TBP; concentration of GA in liquorice leaching solution = 0.75 mg/ml; T = 28.5 ◦ C; pH = 2.0–3.0; organic/aqueous = 10 ml/10 ml, petroleum ether as diluent; magnetic stirring time = 5 min; results based on the triplicated experiments and analysis; (b) extractant, TRPO; concentration of GA in liquorice leaching solution = 0.88 mg/ml; T = 21.5 ◦ C; pH = 2.5–3.5; organic/aqueous = 10 ml/10 ml, petroleum ether as diluent; magnetic stirring time = 3 min; results based on the triplicated experiments and analysis.
G.-g. Niu et al. / Separation and Purification Technology 44 (2005) 189–196
195
were clearly reduced compared with the highest peak of GA. Fig. 8(a and b) shows the HPLC chromatograms of aqueous phase and organic phase after stripping, respectively. Fig. 8(a) showed that the impurity peaks with retention time less than 4 min were remarkably reduced after stripping, with pH adjusted from 11.0 to 7.1. The impurities with retention time less than 4 min were left in the organic phase after stripping and the stripped GA remaining in the aqueous phase was purified. Similar results were obtained when 2-ethyl-hexanol, n-hexanol and TBP were used as extractants, and in all cases, GA could also be selectively extracted or stripped from aqueous phase or organic phase, with a small amount shifting of pH. 3.3. The mechanism of extraction To characterize the formation of molecular complexes in organic solvents, the dependence of the distribution ratio D of GA upon the organic reagent concentration was examined. If the general extraction equilibrium is assumed to be given by Eq. (3), and if the activity coefficients of organic reagents are constant when the concentration of extractant changes at the scope of experiments, the solvation number p can be determined from the distribution ratio measured as a function of concentration of the extractant. GA + pS + qH2 O ⇔ GA · pS · qH2 O
(3)
GA: glycyrrhizic acid; S: extractant; p: the solvation number; q: the number of water molecules interacting with one GA molecule. As given in literature [24], a plot of log D versus log [S] should be linear, and its slope should be equal to the number of ligand molecules per GA in the extraction species. The log D versus log [S] plots are shown in Fig. 9(a and b), respectively, for the extraction of GA with TBP and TRPO as extractants in testing solution, respectively. Surprisingly, the plots were not linear, the slopes decreased with the increase of GA concentration. Similar results were also obtained with hexanol and 2-ethyl-hexanol as extractants. These results strongly suggested that GA was extracted into organic solvent in the aggregated form rather than in the single molecular form. With the increase of concentration, the aggregation of GA would become more severe; more functional groups of GA would be deleted for the intermolecular actions of GA. As a result, the number of the functional groups of each GA molecule left for the combination with extractant would become less and the consequence is the decrease of the slope of log D versus log [S] plot. If GA concentration increased to a certain value, for example, 88 mg/ml in TBP, the GA extracted in the organic phase would be more viscous and will be moved to the bottom of the aqueous phase. This phenomenon was possibly attributed to the fact that a polymer phase of GA-extractant was formed at high GA concentration.
Fig. 10. The TEM of GA extracted into the organic phase (concentration of GA in TBP after extraction: (a) 20 mg/ml, (b) 75 mg/ml).
To further investigate the possible GA aggregation, the profile of GA extracted in the organic phase was studied with Freezing-Etch Replica technique for TEM at different GA concentrations. Fig. 10(a and b) presented the pictures of GA extracted in TBP at concentrations of 20 mg/ml and 75 mg/ml, respectively. It can be seen that GA aggregation was observed for all concentrations. Under lower concentration (Fig. 10(a)), the aggregation particles were separated and under higher concentration (Fig. 10(b)), the aggregation particles were cross-linked to form a dense polymer phase which was more viscous and could even move to the bottom of aqueous phase as previously mentioned. Similar observations were obtained for other extractants such as n-hexanol and 2-ethyl-hexanol. These results consistently pointed out that due to the aggregation of GA, the complexes of GAextractants probably have no constant form and would be changed with GA concentration.
4. Conclusions GA can be efficiently extracted to the organic phase from acidic aqueous solution by the formation of hydrogen bond-
196
G.-g. Niu et al. / Separation and Purification Technology 44 (2005) 189–196
ing between GA and extractants used, such as n-hexanol, 2ethylhexanol, TBP and TRPO. When TRPO is used as the extractant, the extraction percentage of GA can reach about 90% at pH = 5.5. For other three extractants studied, more acidic conditions are needed to reach the similar extraction percentage with pH below 2.0 for 2-ethyl-hexanol as solvent as an example. In stripping process, GA can be easily stripped back to the aqueous phase in alkaline solution as the breakdown of the hydrogen bonding between ionized GA and extractants occurred. GA distribution and TME studies suggested that GA was probably extracted into the organic phase in the molecular aggregation form rather than as single molecule form. No constant ratio of extractant to GA in their complexes could be derived because the ratio probably changed with GA concentration to cause the change of GA molecular aggregation. By adjusting the pH condition, GA could be partially purified from the natural impurities in liquorice root by extraction and stripping process. The prospect of solvent extraction of glycyrrhizic acid from liquorice leaching solution could be applied to a wide variety of liquorice-containing products.
Acknowledgments The authors acknowledge support from the Outstanding Young Scientist Foundation of China (no. 29925617) and National Natural Science Foundation of China (no. 20221603 and no.20273075). The authors are grateful to Prof. Jiayong Chen for helpful discussion and major revision regarding the grammar.
References [1] R.S.H. Finney, G.F. Somers, J. Pharmacol. 10 (1958) 613–620. [2] H. Akamatsu, J. Komura, Y. Asada, Y. Niwa, Planta Med. 57 (1991) 119–121. [3] B. Furhman, S. Buch, J. Vaya, P.A. Belinky, T. Hayek, M. Aviram, Am. J. Clin. Nutr. 66 (1997) 267–275.
[4] M. Ito, H. Nakashima, M. Baba, R. Pauwels, E. De Clercq, E. Shigeta, N. Yamamoto, Antivir. Rec. 7 (1987) 127– 137. [5] H. Suzuki, Y. Ohta, T. Kakino, K. Fujisawa, D. Hirayama, Asian Med. J. 26 (1983) 423–438. [6] X.M. Zhou, Y.J. Chen, D.C. Wang, P.Y. Cheng, J. Chin. Pharm. Univ. 21 (1) (1990) 64 (in Chinese). [7] H.B.S. Thomas, W.Z. Howaed, D.B. Alexis, J. Chromatogr. 175 (1979) 350–355. [8] S.H. Yu, S.J. Wu, F.X. Jin, Y. Guo, Food Fermentation Ind. 25 (1) (1998) 40–43 (in Chinese). [9] F.S. Feng, C.S. Xuan, S.W. Chen, Y. Chang, B.Z. Qiao, Nat. Prod. Res. Dev. 10 (1) (1997) 60–64 (in Chinese). [10] Q.L. Li, M.H. Zhou, J.N. Chen, J. Instrumental Anal. 17 (1) (1998) 37–40 (in Chinese). [11] Y.X. Guan, S.J. Yao, Z.Q. Zhu, Proceedings of the Third China–Korea Conference on Separation Science and Technology, Hangzhou, China, October 31–November 2, 2001, pp. 575– 579. [12] X. Hu, R.S. Bai, X.J. Wang, C.R. Wan, J.W. Yang, X. Yang, J. Tsinghua Univ. (Sci. Tech.) 36 (12) (1996) 13–19 (in Chinese). [13] L.W. Bi, Z.S. Wu, J.H. Chen, Y.M. Wang, D.M. Wu, R.X. Gu, Chem. Ind. Forest Prod. 17 (3) (1997) 41–45 (in Chinese). [14] S.Y. Tan, R.S. Xue, Z.M. Zhou, H. Yang, J. Mod. Chem. Technol. 12 (1998) 23–26 (in Chinese). [15] D.W. Wang, Y.F. Su, J. East Chin. Inst. Chem. Technol. 18 (6) (1992) 718–723 (in Chinese). [16] D.W. Wang, X.G. Fan, J. East Chin. Univ. Sci. Technol. 21 (5) (1995) 548–551 (in Chinese). [17] P. Zhang, S.H. Cai, Q.H. Zhang, Z.C. Luo, J. East Chin. Univ. Sci. Technol. 20 (2) (1994) 148–153 (in Chinese). [18] Z.F. Yang, S.Q. Yu, J.Y. Cheng, G. Wan, R.F. Xu, Eng. Chem. Metall. 13 (1) (1992) 51–54 (in Chinese). [19] X.N. Hu, R.S. Bai, X.J. Wang, C.R. Wan, J.R. Yang, X. Yang, J. Tsinghua Univ. (Sci. Tech.) 36 (12) (1996) 13–19 (in Chinese). [20] Z.D. Tao, Z.N. Xu, P.L. Chen, J. Chem. Eng. Chin. Univ. 11 (3) (1997) 308–311 (in Chinese). [21] X.J. Pan, H.Z. Liu, G.H. Jia, Y.Y. Shu, Biochem. Eng. J. 5 (2000) 173–177. [22] L. Orici, A. Perrelet, Freeze-Etch Histology a Comparison between Thin Sections and Freeze-Etch Replicas, Springer-Verlag, Berlin, Heidelherg, New York, 1975. [23] J.H.M. Willison, A.J. Rowe, Replica Shadowing and Freeze-Etching Techniques, North-Holland publishing company, Amsterdam, New York, Oxford, 1980. [24] S. Tatsuya, H. Yuko, Solvent Extraction Chemistry, Marcel Dekker, Inc., New York, 1977.