色谱  2014, Vol. 32 Issue (3): 235-241  PDF (1285KB)    
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本文作者相关文章
开桂青
刘柳
王焕明
齐墩果烷型和乌苏烷型五环三萜同分异构体配位色谱法分离机理（英文）
开桂青1, 刘柳1, 王焕明2     
1. 安徽大学生命科学学院, 安徽 合肥 230601;
2. 安徽友勇生物科技有限公司, 安徽 六安 231300
收稿日期：2013-9-11
通讯作者：E-mail：gqkai2008@gmail.com.
摘要：本文主要研究了配位色谱法分离齐墩果烷型和乌苏烷型五环三萜同分异构体的分离机理。基于计算模拟分析，β-环糊精（β-CD）和其衍生物为适宜的配位剂。采用HPLC法测定了包合物的表观形成常数，并制备了asiaticoside-B与β-CD包合物。实验结果显示:流动相中添加葡萄糖基-β-环糊精（Glu-β-CD）时，同分异构体的分离度为11.95，比添加β-CD或添加甲基-β-环糊精（DM-β-CD）时（分别为9.61和9.89）都略高些。假定五环三萜类化合物与β-CD形成1：1的包合物，对于asiaticoside-B，流动相中添加Glu-β-CD时，表观形成常数（KF）为2534 L/mol，比添加β-CD或添加DM-β-CD时（分别为1467和1373 L/mol）都略大些。根据asiaticoside-B与β-CD包合物的红外光谱解析及计算模拟，推测asiaticoside-B的E环上甲基部分进入了β-CD的空腔内，而其羰基基团没有进入β-CD的空腔内，其糖苷部分与亲水性的β-CD空腔外部形成氢键作用力。因此，配位色谱法分离齐墩果烷型和乌苏烷型五环三萜同分异构体的分离机理可以推测如下：齐墩果烷型和乌苏烷型五环三萜同分异构体E环上甲基的不同空间位阻导致了同分异构体的不同色谱分离行为。
关键词： 高效液相色谱     羟基积雪草苷     asiaticoside-B     同分异构体     环糊精    
Separation mechanism of oleanane and ursane pentacyclic triterpenoid isomers by coordination chromatography
KAI Guiqing1, LIU Liu1, WANG Huanming2    
1. School of Life Science, Anhui University, Hefei 230601, China;
2. Anhui Youyong Biotechnology Co., Ltd., Liuan 231300, China
Received: 2013-9-11
Corresponding author：E-mail：gqkai2008@gmail.com.
Abstract: This paper focuses on the study of the separation mechanism of oleanane and ursane pentacyclic triterpenoid isomers by a coordination chromatographic method. Based on the calculation analysis,β-cyclodextrin (β-CD) and its derivatives were selected as the suitable agents. The experimental results showed that the resolution of madecassoside isomers with the addition of glucosyl-β-cyclodextrin (Glu-β-CD) to the mobile phase (11.95) was higher than that of the addition of β-CD (9.61) or dimethyl-β-cyclodextrin (DM-β-CD) (9.89). The formation of 1:1 inclusion complexes was assumed. The apparent formation constants (KF) of pentacyclic triterpenes with β-CD were determined by HPLC method. For asiaticoside-B, the KF value with the addition of Glu-β-CD (2534 L/mol) was larger than that with the addition of β-CD (1467 L/mol) or DM-β-CD (1373 L/mol). According to the infrared characteristic absorbing peaks and simulation, the methyl part of asiaticoside-B might enter into β-CD cavity while the carbonyl group of asiaticoside-B might not enter into the cavity of β-CD, and the large glycoside parts were kept outside, forming hydrogen-bond interaction with the exterior of β-CD cavity. The results of the separation of oleanane and ursane pentacyclic triterpenoid isomers by coordination chromatography indicated that, the separation mechanism might be attributed to the steric differences (the place of methyl group in E cyclic), which lead to different chromatographic behaviors.
Key words: high performance liquid chromatography (HPLC)     madecassoside     asiaticoside-B     isomeric compound     cyclodextrin    

There are many oleanolic and ursolic pentacyclic triterpenoid isomers in natural plants,such as Centella Asiatica (Fig. 1),Fructus Ligustri Lucidi and Folium Eriobotryae. The previous studies reported that these isomers showed therapeutic effects [1]. Centella Asiatica increases collagen synthesis in vitro and extracellular matrix accumulation in vivo. It was found to attenuate anxiety-related disorders and reduce stress phenomenon in clinical studies [2, 3].

Fig. 1 Molecular structures of (a) asiaticoside-B and (b) madecassoside

The conventional analysis methods of the triterpenoids in plants include liquid chromatography [4, 5, 6, 7],gas chromatography after silylation and methylation [8, 9]. The capillary supercritical fluid chromatography and high performance thin-layer chromatography combined with densitometry have also been used for the plant analysis [10, 11, 12, 13].

Many of the oleanolic and ursolic pentacyclic triterpenoid isomers often exist together,which might be resulted from the same biosynthesis pathway of them. The place of methyl group in E cyclic is the only difference in their structure,which results in the similar physicochemical property. They are difficult to be separated by the common analytical methods.

The separation of asiaticoside,madecassoside,madecassic acid and asiatic acid was studied in the literature [14, 15]. However,few work has been done on the separation of asiaticoside-B and madecassoside [16, 17]. The separation of oleanolic and ursolic acids was studied in several papers with special HPLC [18, 19, 20].

The theory of coordination chromatography separation is based on the differences in the structures of the target substances and impurities. This separation method has high selectivity in target substances and impurities,which can reach high resolution. Some studies reported that betulinic,oleanolic and ursolic acids can achieve a good separation by coordination chromatography [21]. However the separation mechanism was not disclosed. In this paper,based on the coordination chromatography theory and the characteristic of cyclodextrins (CD),suitable CDs were selected by the chemical calculation and the separation mechanisms were proposed.

1 Experimental section
1.1 Reagents and equipment

The reference standard of madecassoside was purchased from National Institute for the Control of Pharmaceutical and Biological Products of China (Beijing,China). Asiaticoside-B was kindly offered by Hefei Tuofeng Biotechnology Co.,Ltd. The extract powder of 70% (w/w) total Centella triterpenes was bought from Guangxi Changzhou Natural Products Development Co.,Ltd. (Guangxi,China). β -CD was obtained from the Fine Chemicals Factory,Nankai University (Tianjin,China). Glucosyl- β -cyclodextrin (Glu- β -CD) and dimethyl- β -CD (DM- β -CD) were bought from Shandong Xinda Group Co.,Ltd. (China).

The D-2000 Elite HPLC system consists of a high-pressure constant flow pump (L-2130),a UV spectrophotometric detector (L-2420) and an autosampler (L-2200). The analytical column is an Agilent Zorbax SB-C18 (250 mm×4.6 mm,5 μ m). The flow rate of the mobile phase was 0.8 mL/min. The wavelength of UV detector was set at 204 nm. The temperature of the column was 25 ℃. A 10 mL volume of sample was injected into the column. Nicolet 5700 Fourier transform infrared spectrometer (Thermo Fisher Scientific,Inc.).

1.2 Sample preparation

The extract powder of Centella (70% (w/w) of total Centella triterpenes) was accurately weighed (40 mg),and dissolved in methanol to 10 mL solution. The solution was filtered through a 0.45- μ m filter and the clear filtrate was used for HPLC analysis. The reference standards of madecassoside and asiaticoside-B were processed by the same way.

1.3 Mobile phase

The mobile phases for the separation of madecassoside and asiaticoside-B consisted of acetonitrile and water solution (25 ∶ 75,v/v) with the addition of 4 mmol/L of DM- β -CD,β -CD or Glu- β -CD. The solutions were filtered through 0.45- μ m filters and the clear filtrates were used as the HPLC mobile phases.

1.4 Screening coordination agents

The space sizes of the isomeric compounds (only around the place of methyl group in E cyclic) were simulated and calculated by Chemoffice 2008. The minimum energies between β -CD and the guest were also calculated before and after inclusion respectively.

1.5 Inclusion complexes preparation

β -CD was accurately weighed and dissolved in a certain amount of distilled water. The equimolar amount of asiaticoside-B was accurately weighed and dissolved in ethanol,followed by adding into the β -CD solution. The mixed solution was stirred at 40 ℃ with a magnetic bar for 1 h,and then freeze-dried. Finally,the solid inclusion complexes were obtained.

2 Results and discussion
2.1 Screening coordination agents

CDs ( α ,β ,γ ) are torus-shaped,with the cyclic oligosaccharides composed of six to eight α -1,4 linked D-glucopyranose units per molecule ( α -,β - and γ -CD,respectively). The exterior of the CD molecule is hydrophilic,and its hydrophobic cavity may selectively include molecules with appropriately sized organic compounds by forming non-covalent inclusion complexes. First of all,the guest molecules must match the size of the CD cavity,which plays an important role in the stabilization of the complexes. α -CD,β -CD and γ -CD have different cavity diameters and selectively include the guest molecules with different sizes. Guest molecule is too big to enter the cavity. However,the inclusion compound is unstable if the guest molecule is too small. The space sizes of the isomeric compounds (only around the place of methyl group in E cyclic) were simulated and calculated.

The results showed that the space sizes of the isomeric compounds (only around the place of methyl group in E cyclic) were in the range of 0.377-0.560 nm (madecassoside: 0.444 nm,0.560 nm; asiaticoside-B: 0.377 nm,0.424 nm). The diameters of α -,β - and γ -CD cavities are 0.470-0.530 nm,0.600-0.650 nm,0.750-0.830 nm,respectively. Thereby,the space sizes of the isomeric compounds are bigger than the diameter of α -CD cavity,which might make it difficult to enter into the cavity. The space sizes of the isomers are much smaller than the diameter of γ -CD,which might result in the weak interactions between γ -CD and the guest,so that it could not form stable inclusion compound. Accordingly,β -CD should be the suitable agents. The appropriate interactions between β -CD and guest might be formed.

The minimum energies between β -CD and the guest were calculated before and after the inclusion respectively. As can be seen from Table 1,the total minimum energy in the inclusion of the guest and β -CD decreased more quickly compared with the energy in the simple mixture of the guest and β -CD,which indicates that the interactions between the guest and β -CD formed. The results validate that the calculation is correct for selecting β -CD as the suitable agents.

Table 1 Total minimum energies of the guest and β -CD under different conditions

According to the above analysis,β -CD and its derivatives were selected as the coordination agents. For studying the separation mechanisms,the β -CD derivatives (Glu- β -CD and DM- β -CD) were selected according to their different hydrophilicities which are in the following order: Glu- β -CD ＞ β -CD ＞ DM- β -CD.

2.2 Stoichiometry and apparent formation constants of the complexes

Several studies have theoretically examined the competing equilibria in the column of an HPLC system upon the introduction of the solute into a mobile-phase mixture consisting of β -CD and a primary organic modifier [22, 23, 24, 25, 26]. Based on the results of these studies,when isomeric compounds are introduced into the column in the presence of β -CD or its derivatives in the mobile phase,the equilibria presented in Fig. 2 will be established.

Fig. 2 Equilibria proposed for a 1 ∶ 1 isomers- β -CD inclusion complex AS or MA: asiaticoside-B or madecassoside; CD: β -cyclodextrin; AS or MA-CD: asiaticoside-B or madecassoside- β -CD complex; (AS or MA-CD)s: (asiaticoside-B or madecassoside- β -CD)s complex; m: mobile phase; s: stationary phases; M: organic modifier; Km: affinity constant of the modifier for the β -CD cavity; KF: apparent formation constant for the (asiaticoside-B or madecassoside- β -CD) complex in a 1 ∶ 1 model; K0: equilibrium constant of asiaticoside-B or madecassoside between mobile and stationary phases; K1: equilibrium constant of (asiaticoside-B or madecassoside- β -CD) complex between mobile and stationary phases; CD-M: β -CD-organic modifier interaction.

Since the organic modifier competes with isomeric compounds for the β -CD cavity,the effective β -CD concentration in the mobile phase ([CD]m) is not the total analytical β -CD concentration ([CD]T). [CD]m must be calculated using the following equation [23, 25, 27]:

where K0 is the concentration of acetonitrile in the mobile phase.

Assuming that the interaction of the each complex (asiaticoside-B or madecassoside- β -CD) with the stationary phase is negligible [22],the relationship between the retention factor (k) and the effective β -CD mobile-phase concentration ([CD]m) can be established. An equation is proposed in taking into account the equilibria involving the formation of 1 ∶ 1 isomer- β -CD complexes [23, 24, 25]:

Where k0 is the solute retention factor in the absence of CD.

According to the reported studies [25, 28],the equilibria allow for 1 ∶ 2 asiaticoside-B or madecassoside- β -CD complexation. The equilibria presented in Fig. 3 show the formation of a 1 ∶ 2 asiaticoside-B or madecassoside- β -CD complex via a precursor 1 ∶ 1 complex.

As shown in both Fig. 2 and Fig. 3,when β -CD is added to the mobile phase,the isomer retention is governed by its partition between the mobile phase,the stationary phase and the isomer complexation with β -CD [25, 29]. Following the derivation of Mosheni et al [28],assuming that the interaction of the asiaticoside-B or madecassoside- β -CD complex with the stationary phase is negligible (K1 ≈ 0 and K2 ≈ 0) [22],and including the terms that account for the possibility of a 1 ∶ 2 asiaticoside-B or madecassoside- β -CD complex,a similar mathematical expression can be derived,which describes the dependence of k of asiaticoside-B or madecassoside on the effective concentration of β -CD in the mobile phase ([CD]m):

Equation 3 is an extension of Equation 2 and includes a second-order term that accounts for the possibility of a 1 ∶ 2 asiaticoside-B or madecassoside- β -CD complex formation. Equation 3 simplifies Equation 2 when a 1 ∶ 1 isomer- β -CD complex is the only complex formed; the apparent formation constant of the 1 ∶ 2 isomer- β -CD complex (KF2) is zero. In this case,a plot of the reciprocal of k versus [CD]m should give a straight line. In the case of a 1 ∶ 2 isomer- β -CD complex formation,a plot of reciprocal of k versus [CD]m should give a parabolic curve that fits Equation 3. The values of KF1 and KF2 can be obtained by performing a second-order polynomial fit to the data.

Fig. 3 Equilibria proposed for a 1 ∶ 2 asiaticoside-B or madecassoside- β -CD inclusion complex KF1: apparent formation constant for the asiaticoside-B or madecassoside- β -CD complex in a 1 ∶ 1 model; KF2: apparent formation constant for the asiaticoside-B or madecassoside-( β -CD)2 complex in a 1 ∶ 2 model; K2: equilibrium constant of asiaticoside-B or madecassoside-( β -CD)2 complex between mobile and stationary phases.

To determine the stoichiometric ratios for isomer- β -CD complexes formed,both Equations 2 and 3 were used. The reciprocal of k for asiaticoside-B was plotted as a function of [CD]m. The correlation coefficients arising from this plot were determined as shown in Table 2. The linear relationship that fits Equation 2 indicates that a 1 ∶ 1 guest-CD complex is preferred.

Table 2 Apparent formation constants of asiaticoside-B with Glu- β -CD,β -CD or DM- β -CD

For studying the separation mechanisms,Table 2 also reports the KF values of the complexes formed between asiaticoside-B and cyclodextrins (Glu- β -CD,β -CD,DM- β -CD). As expected,for asiaticoside-B,the KF values of Glu- β -CD (2534) were larger than that of β -CD (1467). To DM- β -CD,the relatively lower KF values can be attributed to their different hydrophilicities,which influence the host-guest interactions.

It is shown in Fig. 4 that the madecassoside isomers could not be separated without CD in the mobile phase. However,the resolution between madecassoside isomers was increased obviously by adding DM- β -CD,β -CD or Glu- β -CD into the mobile phase. Nevertheless,the resolution of the madecassoside isomers with the addition of Glu- β -CD into the mobile phase (11.95) was higher than that with the addition of β -CD (9.61) or DM- β -CD (9.89).

Fig. 4 Chromatograms of 70% (w/w) total Centella triterpenes Mobile phase: acetonitrile/water solution (25 ∶ 75,v/v) with the addition of 4 mmol/L DM- β -CD,β -CD or Glu- β -CD.
1. asiaticoside-B; 2. madecassoside.
2.3 Inclusion reaction
According to the theory of supramolecular chemistry,the infrared peaks of the characteristic absorbtion have changed when forming the guest-CD complexes. Hence,the asiaticoside-B- β -CD complexes were confirmed by IR. From the IR spectra,the carbonyl group was the characteristic absorption peak of asiaticoside-B,which was located at 1733.3 cm. The carbonyl group peak of asiaticoside-B- β -CD complexes was shifted to 1726.8 cm comparing with the peak of the mixture of asiaticoside-B and β -CD (1735.4 cm). This phenomenon indicated the forming of asiaticoside-B- β -CD complexes. However,the characteristic absorption peak did not vanish,suggesting that the carbonyl group of asiaticoside-B might not enter into the cavity of β -CD. The carbonyl group was influenced by the β -CD cavity. The peak at about 3400 cm was caused by the hydroxyl absorption. To asiaticoside-B- β -CD complex,this peak became wider,which can be attributed to the hydrogen-bond interaction between the glycoside group of asiaticoside-B and the hydrophilic group of β -CD cavity exterior. The results can explain why the KF values between asiaticoside-B and CDs are in the following sequence: Glu- β -CD＞ β -CD＞ DM- β -CD.

2.4 Simulation the host-guest complexes structure
Based on the above analysis,the optimum structure of host-guest coordination compound was simulated by the software (Chemoffice 2008). From the simulation results,the methyl part of madecassoside or asiaticoside-B can enter into the β -CD cavity,and the large glycoside parts are kept outside,forming hydrogen-bond interaction with the exterior of β -CD cavity. The results are consistent with the results of that in the IR spectra.

According to the above analysis,the proposed separation mechanism of coordination chromatography is schematically represented in Fig. 5.As shown in Fig. 5,the suitable agent was added to mobile phase. Following the mobile phase,the agent entered the stationary phase. There were many kinds of interaction occurred in the stationary phase between the isomers and the the agent,the isomers and the stationary phase microsphere,the isomers and the mobile phase,etc. The separation of oleanane and ursane pentacyclic triterpenoid isomers may be attributed to the collaborative effect of many kinds of interaction.

Fig. 5 Separation mechanism of chromatographic process
3 Conclusion
The experimental results showed that the resolution of madecassoside isomers with the addition of Glu- β -CD into the mobile phase (11.95) was higher than that with the addition of β -CD (9.61) or DM- β -CD (9.89). The formation of 1 ∶ 1 inclusion complexes was assumed. The KF values for asiaticoside-B with the addition of Glu- β -CD (2534 L/mol) were relatively larger than that with the addition of β -CD (1467 L/mol). For DM- β -CD,the low KF values can be attributed to their different hydrophilicities,which influence host-guest interactions. According to the infrared spectrum,the carbonyl group of asiaticoside-B might not enter into the cavity of β -CD,while the carbonyl group was influenced by the β -CD cavity. The hydrogen-bond interactions were formed between the glycoside group of asiaticoside-B and the hydrophilic group of β -CD cavity exterior.

In the molecular structures of oleanane and ursane pentacyclic triterpenoids,the large glycoside parts formed different hydrogen bonding interactions with the cavity exterior of Glu- β -CD,β -CD or DM- β -CD. The molecular structure of madecassoside belongs to ursane category while asiaticoside-B belongs to oleanane category. The separation of oleanane and ursane pentacyclic triterpenoids can be attributed to steric differences,which lead to different chromatography behaviors.

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