Article pubs.acs.org/IECR
Crystallization of Asiaticoside from Total Triterpenoid Saponins of Centella Asiatica in a Methanol + Water System Jie Fu, Xingfang Zheng, and Xiuyang Lu* Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, Zhejiang, China S Supporting Information *
ABSTRACT: In this contribution, a novel solvent system for the crystallization of asiaticoside from total triterpenoid saponins of Centella asiatica was established by utilizing the difference between the induction periods of madecassoside and asiaticoside. Asiaticoside could be separated from the mixture of asiaticoside and madecassoside by crystallization with about 80% yield and 95% purity. The mechanism behind the significantly different induction periods of asiaticoside and madecassoside in the methanol + water system is also proposed. Crystallization of asiaticoside from total triterpenoid saponins of Centella asiatica achieved a maximum yield of 60% with 70% purity. A recrystallization was carried out to obtain 76% yield with 91% purity. The optimized conditions for the crystallization of asiaticoside from total triterpenoid saponins of Centella asiatica were determined.
1. INTRODUCTION Crystallization, a traditional separation process in chemical engineering, is an important treatment for the separation of pharmaceutical products such as antibiotics, due to its advantages of high purity of product, simplicity, efficiency, and environmental benignity. However, it is difficult to apply crystallization in the extraction of pure active components from natural resources because of the variety of components, similarity in structure for active components, and a low content of single active components.1 Further research on crystallization for the separation of pure active components from natural resources is critical for industries related to the environment. Asiaticoside,2,3 a terpenoid component extracted from Centella asiatica, has been shown to increase collagen synthesis and antiwrinkle activity.4,5 There are, however, a considerable number of homologous compounds with similar structures that possess the same sugar chain units but differ in their agylcone skeletons. These include asiaticoside, madecassoside,6 scheffoleoside A, asiaticoside B, 2α,3β,23-trihydroxyurs-20-en-28-oic acid O-α-L-rhamnopyranosyl-(1→4)-O-β-D-glucopy-ranosyl(1→6)-O-β-D-glucopyranosyl ester, centellasaponin A, centellasaponin C, and centellasaponin D. Among them, asiaticoside A (madecassoside, urs-12-en) and asiaticoside B (terminoloside, olea-12-en) are isomers that differ only by the hydroxy group on C-29. Other isomers, centellasaponin A, centellasaponin C, centellasaponin D, and asiaticoside, differ in the location of the hydroxyl (−OH) group at either C-2, C-6, or C-23, or the locations of double bonds. Thus, the separation of asiaticoside from total triterpenoid saponins of Centella asiatica is made challenging by the high structural similarity of these isomers. Chromatography has been developed as a mainstream method for the separation of these compounds,7−11 but it consumes a large amount of absorbents and solvents, limiting its application in industry. For this reason, there has been great demand for the separation of asiaticoside by crystallization.1 © 2014 American Chemical Society
In our previous work, the solubilities and induction periods12−15 of asiaticoside and madecassoside were determined. They have similar solubilities in the methanol + water system but have induction periods that are orders of magnitude apart.16,17 The scale of this disparity suggests that this difference in induction periods can be employed to separate asiaticoside and madecassoside. In this Article, a 3D molecular model was employed to analyze the mechanism behind the similar solubilities and different induction periods between asiaticoside and madecassoside in the methanol + water system. The crystallization of asiaticoside from the mixture of asiaticoside and madecassoside was conducted in the methanol + water system. In addition, asiaticoside was also separated from total triterpenoid saponins of Centella asiatica by crystallization in the methanol + water system.
2. EXPERIMENTAL SECTION 2.1. Materials. Total triterpenoid saponins of Centella asiatica (80% purity, content of asiaticoside B11 = 15.6%, content of madecassoside = 32.6%, content of asiaticoside = 29.7%), asiaticoside (90% purity), and madecassoside (90% purity) were obtained from Guangxi Changzhou Natural Products Development Co., Ltd. Asiaticoside standard (>0.985 mass fraction purity) was purchased from Sigma. Madecassoside standard (0.95 mass fraction purity) was purchased from Shanghai Tauto Biotech Co., Ltd. (Shanghai, China). Analytical reagent (AR) grade methanol (≥0.995 mass fraction purity) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). High-performance liquid chromatography (HPLC) grade methanol and acetonitrile (≥0.999 mass fraction purity) were obtained from Merck Received: February 10, 2014 Accepted: August 23, 2014 Published: August 24, 2014 14022
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(Darmstadt, Germany). Distilled water was obtained from Hangzhou Wahaha Group Co., Ltd. (Hangzhou, China). 2.2. Molecular Energy Calculations. A semiempirical quantum chemistry PM3 method18 was employed to calculate the optimized relative total molecular energies for asiaticoside and madecassoside. The calculations were conducted by a Gaussian calculation software, and the details on the calculations of relative total molecular energy for asiaticoside and madecassoside are shown in the Supporting Information. 2.3. Crystallization Procedure. For crystallization of asiaticoside from a mixture of asiaticoside and madecassoside, 3.5 g of asiaticoside (90% purity) and 3.5 g of madecassoside (90% purity) were dissolved in 100 g of methanol in a water bath. For the crystallization of asiaticoside from total triterpenoid saponins of Centella asiatica, 11.67 g of total triterpenoid saponins of Centella asiatica was dissolved in 100 g of methanol in a water bath. A certain amount of water was then added to each solution. The solution was stirred at 200 r/ min at 298.15 K for a given amount of time. The obtained sample was filtered to retrieve the crystallization sample. After drying, 0.5 g of the crystallization sample was dissolved in 50 g of methanol using ultrasound; then, 2 g of the solution was diluted to 10 mL with methanol. The diluted solution was filtered by a 0.45 μm membrane and analyzed by HPLC. 2.4. Analysis. The samples were analyzed by HPLC (Agilent 1100). A Synergi 4 μm Hydro-RP 80A reverse-phase column (4.6 mm × 250 mm, 4 μm, Phenomenex) was used. The mobile phase was acetonitrile and water at a volumetric ratio of 21:79. The flow rate was 0.7 mL·min−1; the wavelength was 205 nm, and the column temperature was 298.15 K. The equations for the yield and purity of asiaticoside and the mass ratio of asiaticoside in the asiaticoside−madecassoside mixture are shown below: m X= 1 m0 (1) Y=
Z=
m1 mp
(2)
m1 m1 + m2
(3)
Figure 1. Chemical structure of asiaticoside (RH) and madecassoside (ROH).
This suggests that the polarities of asiaticoside and madecassoside are very similar, resulting in similar solubilities. The crystal lattice of asiaticoside obtained in methanol favors the chain conformation19 but can be transformed into the folded conformation by rotating the C−O−C bonds. As far as we know, there are no reported data on the crystal lattice of madecassoside. The semiempirical quantum chemistry PM3 method was employed to calculate the relative total molecular energy of asiaticoside and madecassoside in chain and folded conformations. Figures 2 and 3 show the optimized conformations of asiaticoside and madecassoside, respectively, and their relative total molecular energies are listed in Table 1. The details on the optimized conformation of asiaticoside and madecassoside are shown in the Supporting Information. The molecular models of asiaticoside and madecassoside in the folded conformation have similar relative total molecular energies as those in the chain conformation, indicating that both asiaticoside and madecassoside exist as an equilibrium of the two conformations (though both can only nucleate in the chain conformation). The reason that asiaticoside has a shorter induction period, however, may be because it is more readily able to break its intramolecular hydrogen bonds, transfer from folded conformation to chain conformation, and thus nucleate out of the solution. On the other hand, for madecassoside the two additional intramolecular hydrogen bonds between the −OH group on C-6 and the −OH groups of C-39 and C-42 (shown in Figure 3c), which are more likely to form than other potential hydrogen bonds, make it much more difficult for madecassoside to transfer from the folded conformation to the chain conformation. Thus, madecassoside has a slower nucleation rate and an induction period that is 10 times longer than that of asiaticoside. Since intramolecular hydrogen bonds influence crystallization kinetics, including nucleation rate and crystal growth rate, this difference provided us the opportunity to separate asiaticoside and madecassoside by crystallization. In summary, the difference in the intramolecular hydrogen bonds of asiaticoside and madecassoside, caused by the minor −OH difference in their aglycone skeletons, might lead to orders of magnitude of difference in their induction periods.
where X is the yield of asiaticoside, m1 is the mass of asiaticoside, m0 is the mass of asiaticoside in the raw material, Y is the purity of asiaticoside, mp is mass of the crystal product, Z is the mass ratio of asiaticoside in the asiaticoside−madecassoside mixture, and m2 is the mass of madecassoside.
3. RESULTS AND DISCUSSION 3.1. Separation Mechanism Analysis of Asiaticoside and Madecassoside. In our previous work,17 the induction period of asiaticoside was found to be 2000 s. In contrast, the induction period of madecassoside was much longer, 21 600 s. This indicates that the induction period of madecassoside was at least 10 times longer than that of asiaticoside, meaning that the nucleation rate of madecassoside was much lower than that of asiaticoside. The structures of asiaticoside and madecassoside are shown in Figure 1. The only difference between asiaticoside and madecassoside is the −OH group on C-6. The number of −OH groups on these two compounds is very similar (12 −OH groups for asiaticoside and 13 −OH groups for madecassoside). 14023
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Figure 2. Conformations of asiaticoside: chain (a) and folded (b).
Figure 3. Conformations of madecassoside: chain (a), folded with one intramolecular hydrogen bond (b), and folded with two intramolecular hydrogen bonds (c).
Table 1. Relative Total Molecular Energy of Asiaticoside and Madecassoside in Different Conformations confirmation
asiaticoside (a.u.)
madecassoside (a.u.)
chain folded
−1.3763 −1.3787
−1.4352 −1.4371
Figure 5. Purity of asiaticoside separated from the mixture of asiaticoside and madecassoside at different xm values.
6 and 8 h. The maximum yield was highest at a mole fraction of 0.0588 and 0.1942, followed by an xm of 0.8351, which had a maximum yield clearly higher than at xm values of 0.36, 0.5676, and 0.6923. A relatively large xm (0.8351) was selected for further study of the efficiency of reusing raw materials in the solution. Figure 5 shows the purity of asiaticoside at different xm values. At the xm values of 0.0588, 0.1942, and 0.36, the purity of asiaticoside was always above 90% in the time range of 1−8 h. At the xm of 0.8351, the asiaticoside was not as pure, but the maximum purity was also above 90%, indicating that madecassoside at the xm of 0.8351 started to nucleate out
Figure 4. Yield of asiaticoside separated from the mixture of asiaticoside and madecassoside at different xm values.
3.2. Crystallization of Asiaticoside from the Mixture of Asiaticoside and Madecassoside. Figure 4 shows the yield of asiaticoside at different mole fractions of methanol in the mixture of methanol and water (xm). At the xm values of 0.0588 and 0.1942, the yield of asiaticoside reached about 80% after 1 h and then plateaued as time elapsed. At the xm values of 0.36, 0.5676, 0.6923 and 0.8351, the yield of asiaticoside increased as the time elapsed and then achieved the maximum yield between 14024
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Figure 6. Yield of asiaticoside from total triterpenoid saponins of Centella asiatica at different xm values.
Figure 9. Mass ratio of asiaticoside in the asiaticoside−madecassoside mixture.
Figure 7. Purity of asiaticoside from total triterpenoid saponins of Centella asiatica at different xm values.
3.3. Crystallization of Asiaticoside from Total Triterpenoid Saponins of Centella asiatica. Figure 6 shows the yield of asiaticoside from total triterpenoid saponins of Centella asiatica by crystallization at the xm values of 0.1942 and 0.8351. Due to the influence of other components in total triterpenoid saponins of Centella asiatica, the yield of asiaticoside was lower than that from the mixture of asiaticoside and madecassoside. At the xm of 0.1942, the maximum yield of asiaticoside was about 60%, compared to about 80% from the mixture of asiaticoside and madecassoside. At the xm of 0.8351, the maximum yield of asiaticoside was about 40% (compared to roughly 75% from the mixture of asiaticoside and madecassoside). Moreover, the crystallization time necessary to reach the maximum yield was also prolonged. At the xm of 0.1942, the time for the maximum yield was 2 h (1 h for the mixture of asiaticoside and madecassoside) and 6 h at the xm of 0.1942 (4 h for the mixture of asiaticoside and madecassoside). Figure 7 shows that purity remained stable at around 75%, compared to about 95% for the mixture of asiaticoside and madecassoside. The decrease in yield and delay in crystallization time were attributed to the large amount of pentacyclic triterpenoid saponin components resulting from product−impurity solvent interactions.20 For example, the structure of asiaticoside B, which makes up nearly 20% of the content in the raw material, was very similar to that of madecassoside, and their only difference is the −CH3 group on C-29. Thus, asiaticoside B probably has an induction period similar to that of madecassoside in the methanol + water system. The −OH groups on
Table 2. Peak Area Ratio of Each Triterpene Glycoside component
peak area
peak percentage/%
asiaticoside madecassoside asiaticoside B Component I scheffoleoside A centellasaponin A
4109 252 103 203 151 86
83.8 5.1 2.1 4.1 3.1 1.8
after 2 h and that, at an xm of 0.8351, crystallization time should be limited to 2 h or less.
Figure 8. HPLC chromatogram of the crystallization product (xm = 0.1942, time = 2 h). 14025
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Figure 10. HPLC chromatogram of total triterpenoid saponins of Centella asiatica.
Figure 11. HPLC chromatogram of the recrystallization product (xm = 0.8351, 4 h).
asiaticoside B increase the number of −OH groups in solution, raising the viscosity of the solution and decreasing the crystal nucleation rate of asiaticoside, which prolongs the crystallization time for maximum yield. Furthermore, the equilibrium state of asiaticoside in the solution was influenced, which decreased the yield of asiaticoside. The decrease in purity is attributed to nonpentacyclic triterpenoid saponins, which make up nearly 20% of the raw material. Table 2 and Figure 8 show the known triterpene glycoside components in the crystallization product. The peak percentages for asiaticoside, madecassoside, asiaticoside B, Component I, scheffoleoside A, and centellasaponin A were 83.8%, 5.1%, 2.1%, 4.1%, 3.1%, and 1.8%, respectively. Equation 3 was employed to calculate the ratios of asiaticoside and madecassoside, ignoring other impurities, and the results are shown in Figure 9. In all conditions, the Z values for asiaticoside were all around 90%, and the Z at an xm of 0.1942 was slightly larger than that at an xm of 0.8351. As the above results indicate, the first crystallization was influenced by the nontriterpene glycoside components of the raw material. Recrystallization was carried out to improve the purity. Since the interfacial energy at an xm of 0.8351 was higher than that at an xm of 0.1942,17 an xm of 0.8351 and reaction time of 4 h were chosen as the recrystallization conditions. The purity after recrystallization was 91% with a recrystallization yield of 76%. The chromatography of the raw material and recrystallization product are shown in Figures 10 and 11. 3.4. Crystallization Separation Analysis of Other Pentacyclic Triterpenoid Saponins. Besides asiaticoside and madecassoside, total triterpenoid saponins of Centella asiatica also contain asiaticoside B, Component I, centellasaponin A, centellasaponin B, scheffoleoside A, and some
unknown compounds. Just like madecassoside, asiaticoside B and centellasaponin B also have an −OH group on C-6, but Component I, centellasaponin A, and scheffoleoside A do not. As Figures 8 and 10 show, the raw material is composed of very little Component I, centellasponin A, and scheffoleoside A, and most of these components nucleated out during crystallization. The compounds have features similar to asiaticoside but without the −OH group on C-6. These results support the feasibility of the mechanism proposed in Section 3.1, that the minor −OH difference in the aglycone skeleton causes differences on the order of magnitude in the induction period, impacting the crystallization.
4. CONCLUSION A novel solvent system for the crystallization of asiaticoside from total triterpenoid saponins of Centella asiatica was established by utilizing the difference between the induction periods of madecassoside and asiaticoside. Asiaticoside could be separated from the mixture of asiaticoside and madecassoside by crystallization with about 80% yield and 95% purity. The presence of other pentacyclic triterpenoid saponins decreased the yield and purity of separating asiaticoside from total triterpenoid saponins of Centella asiatica by crystallization when compared to the separatation of asiaticoside from a mixture of asiaticoside and madecassoside. The yield and purity of asiaticoside from total triterpenoid saponins of Centella asiatica were lower than those from the mixture of asiaticoside and madecassoside. Recrystallization, however, could increase the purity to about 91%. The optimized conditions for the two-step crystallization of asiaticoside from total triterpenoid saponins of Centella asiatica are (1) methanol mole fraction (xm) of 0.1942 14026
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(16) Zheng, X.; Lu, X. Measurement and correlation of solubilities of asiaticoside in water, methanol, ethanol, n-propanol, n-butanol, and a methanol + water mixture from (278.15 to 343.15) K. J. Chem. Eng. Data 2011, 56, 674. (17) Zheng, X.; Fu, J.; Lu, X. Solubility and induction period study of asiaticoside and madecassoside in a methanol + water mixture. J. Chem. Eng. Data 2012, 57, 3258. (18) Zhao, Z.; Xu, X.; Chen, X.; Wang, X.; Lu, P.; Yu, G.; Liu, Y. Synthesis and characterization of deep blue emitters from starburst carbazole/fluorene compounds. Tetrahedron 2008, 64, 2658. (19) Mahato, S. B.; Sahu, N. P.; Luger, P.; Mueller, E. Stereochemistry of a triterpenoid trisaccharide from Centella asiatica. X-ray determination of the structure of asiaticoside. J. Chem. Soc., Perkin Trans. 2 1987, No. 10, 1509. (20) Wright, J. D. Molecular Crystals; Cambridge Univ. Press: Cambridge, 1995.
and crystallization time of 2 h; (2) methanol mole fraction (xm) of 0.8351 and crystallization time of 4 h.
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ASSOCIATED CONTENT
S Supporting Information *
The Gaussian calculation setting header and raw conformation and the optimized conformation of asiaticoside and madecassoside (both chain and folded conformations). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: +86 571 87952683. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 21306165, 21176218) and Zhejiang Provincial Natural Science Foundation of China (Nos. Z14B060009, Q13B060001).
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REFERENCES
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