Fingerprinting Analysis of Rhizoma Chuanxiong of Commercial Types using ¹H Nuclear Magnetic Resonance Spectroscopy and High Performance Liquid Chromatography Method

Nuclear magnetic resonance (NMR) spectroscopy was first described and measured in molecular beams by Isidor Rabi in 1938 (Martin and Zekter 1988). It is now one of the principal techniques used to obtain chemical structural information about organic molecules in solution and the solid state due to the chemical shift and Zeeman effect on the resonant frequencies of the nuclei. Recently, NMR has been used to meta-bolomically profile several types of plants and plant functional genomes, as well as in a fingerprinting tool for authenticating and assessing the quality of natural products (Rycroft 1996; Choi et al. 2004; Shin et al. 2007; Su et al. 2008).

High performance liquid chromatography (HPLC) is a method now used to separate and analyze the chemical constituents of plants, which combines column efficiency with speed of analysis. It provides both quantitative and qualitative data on plant substances, since measurements of the area under the peaks shown on the HPLC trace are directly related to the concentration of different components in the original mixture. Like NMR, HPLC has also been used as a fingerprinting tool for authenticating and assessing the quality of natural products (Qin et al. 2004; Xie et al. 2006; Jin et al. 2006).

Methodological studies on the establishment of the system of the exclusive control substance for a plant drug (CSPD, including CSPD A, B, and C, etc. according to the different metabolites from fractionation of plant drug) and on authenticating plant species by ¹H NMR and HPLC fingerprinting analysis of CSPD have been reported previously by the authors (Qin et al. 2004, 2005; Su et al. 2008). It was concluded that the ¹H NMR and HPLC fingerprinting techniques are rapid, reproducible and stable with time for the authentication of medicinal plant species. But, it should be pointed out that fingerprinting analysis of medicinal plant using ¹H NMR spectroscopy and the HPLC method must be based on the detailed investigation of chemical constituents of the corresponding CSPD for the purpose of clearly exhibiting the existence of their characteristic constituents. In the present paper, we report on the application of ¹H NMR and HPLC fingerprinting analysis of specific metabolites for the identification of Ligusticum chuanxiong Hort. species.

L. chuanxiong belongs to the family Umbelliferae and is mainly distributed in southwest China, especially in Sichuan Province. In traditional Chinese medicine (TCM), the commercial soaked, sliced, and dried form of Rhizoma chuanxiong, the rhizomes of L. chuanxiong, has long been used for activating blood circulation and acesodyne, as well as for the treatment of cerebro- and cardio-vascular diseases. Previous investigations on chemical constituents of the plant led to the isolation of aromatic compounds, terpenoids, organic acids, sugars, alkaloids, and phthalides (Cao et al. 1983; Xiao et al. 2002). The rhizomes of L. chuanxiong contain up to 1.2% volatile oil in which alkyl phthalides are the major components with up to 32%–76% (Zhong et al. 1996). In the present paper, the non-polar and neutral CSPD A from Rhizoma chuanxiong of seven commercial types collected from different regions in China were obtained by the procedure reported previously (Qin et al. 2005; Su et al. 2008). The ¹H NMR spectra and HPLC profiles of these CSPD A were recorded on an Fourier transform-nuclear magnetic resonance (FT-NMR) spectrometer and on a Shimadzu 10A HPLC instrument, respectively, for the purpose of analyzing and comparing the characteristics and the differences in the major constituents. The signals of the ¹H NMR and HPLC fingerprints of the samples were also analyzed and assigned mainly by comparing them with each other and with the spectra and profiles of the isolated pure compounds.

Results

The investigation of the chemical constituents showed that the neutral compounds, phthalide derivatives, were the main and characteristic constituents of CSPD A from the rhizomes of L. chuanxiong. Repeated silica gel column chromatographic processes on the CSPD A allowed the isolation of six phthalides, (Z)-ligustilide (1) (Beck and Stermitz 1995), senkyunolide A (2) (Naito et al. 1992), Z-Levistolide A (3) (Su et al. 2005), (Z)-6,8′,7,3′-diligustilide (6) (Kaouadji et al. 1986), senkyunolide H (7) (Naito et al. 1992), Senkyunolide I (8) (Naito et al. 1992), along with pregnenolone (4) (Terada et al. 1978), 4-((E)-3-ethoxyprop-1-enyl)-2-methoxyphenol (5) (Wang et al. 2006), and eicosanoic acid (9) (Liang et al. 2005) (Figure 1). These structures were identified by comparison of their signals with published data, and compound 5 was isolated from L. chuanxiong for the first time.

The samples of L. chuanxiong used in this work showed good identity to each other in respect to the analysis of their characteristics. The ¹H NMR spectra of their CSPD A exhibited exclusive characteristic features in the composition of signals (Table 1, Figure 2). By comparing the ¹H NMR spectra with those of the pure compounds 1–9 isolated from CSPD A, it was found that many peaks of aliphatic, alkoxyl and aromatic regions could be detected without the need for chromatographic separation.

High performance liquid chromatography was applied to analyze and compare the differences in the major constituents of the CSPD A from these different samples of L. chuanxiong. As shown in Figure 3 and Table 2, the HPLC profiles mainly displayed 15 identical peaks under present chromatographic conditions. Compounds isolated in the present work were measured as reference substances with the same HPLC condition and their retention times were compared with those of the peaks in the HPLC profiles. As a result, some of the peaks were ascribed to their corresponding counterparts, that is, peaks at 5.27, 13.92, 16.90, 18.49 and 29.30 min corresponded to compounds 5, 3, 2, 6, 1, respectively, with (Z)-ligustilide (1) and senkyunolide A (2) predominated, and this analysis was identical to the ¹H NMR experiment. The compound corresponding to retention time at 25.03 min showed a considerable amount in all of the samples used. However, it hadn't been isolated as a monomer in the present work.

Discussion

The ¹H NMR spectra of CSPD A of the rhizomes of L. chuanxiong showed good identity in respect to the specificity for the authentication of L. Chuanxiong species to each other and were mainly formed by overlapping of the signals of phthalide derivatives, fatty compounds and 4-((E)-3-ethoxyprop-1-enyl)-2-methoxyphenol, in which the characteristic signals of (Z)-ligustilide (1) and senkyunolide A (2) predominated, and 4-((E)-3-ethoxyprop-1-enyl)-2-methoxyphenol (5) took third place. The spectra could be simply divided into three sections, that is, an aliphatic region with δ 0.8–2.8, an alkoxyl region with about δ 3.5–4.93 and an aromatic/alkenic region with δ 5.1–7.9, which were identical to the structural characteristics of the phthalide derivatives, fatty compounds and 4-((E)-3-ethoxyprop-1-enyl)-2-methoxyphenol. The first region included the overlapped signals of saturated aliphatic protons of phthalide derivatives. Apart from the well-defined signals from (Z)-ligustilide (1), that is, the signal of methyl group (CH₃-11) at δ 0.95 (t, J= 6.9 Hz) and the signals of two methylene groups (CH₂-9,10) on the butylidene side chain at δ 1.48 (sex, J= 7.2 Hz), 2.37 (q, J= 7.5 Hz), and those of the two methylene groups (CH₂-4,5) at δ 2.47 (m) and 2.60 (m), the remaining signals come mainly from senkyunolide A (2), including δ 0.90 (t, J= 6.6 Hz), 1.30–1.43 (m), 1.50–1.57 (m), 1.83–1.91 (m), and 2.43–2.48 (m) due to CH₃-11, CH₂-9 and CH₂-10, H-8a, H-8b, and CH₂-4 and CH₂-5, respectively. The signal of Me-2′ of 5 was also overlapped in the aliphatic region at δ–1.25 but could not be differentiated. The second region mainly included the signals at δ 4.12 (d, J= 6 Hz), 3.89 (s), and 3.54 (q, J= 6.6 Hz) from three alkoxyl groups of 5, CH₂-9, CH₃O, and CH₂-1′; and at δ 4.93 (dd, J= 6.5, 4 Hz) from CH-3 of senkyunolide A (2), and the other two strong signals at δ 3.91 (s) and 4.82 (d, J= 6.6 Hz) could not be assigned due to the failure of isolating corresponding compounds. Because of the relatively less content of 5 in some samples, the signals of CH₂-9 and CH₂-2′ couldn't be detected in their spectroscopy. The aromatic/alkenic region included the overlapped signals of unsaturated protons of phthalide derivatives and phenylpropenoids, in which the well-differentiated signals also come mainly from (Z)-ligustilide (1), senkyunolide A (2) and 4-((E)-3-ethoxyprop-1-enyl)-2-methoxyphenol (5), that is, δ 6.25 (dt, J= 9.5, 1.5 Hz), and 5.97 (dt, J= 9.5, 4 Hz) and 5.20 (t, J= 8 Hz) assigned to those of the olefinic methine groups of CH-7,6 of cyclohexene and the olefinic proton of CH-8 on the butylidene side chain of (Z)-ligustilide (1), δ 6.21 (d, J= 9.3 Hz) and δ 5.91 (m) could be recognized as the signals of H-7 and H-6 of senkyunolide A (2), and δ 6.93 (s), 6.88 (d, J= 8.1 Hz), 6.85 (d, J= 8.1 Hz), 6.62 (d, J= 16.2 Hz) to H-2, H-6, H-5 and H-7 of compound 5.

A few weak signals at δ 7.33–7.90 were not assigned because the separation of the corresponding compounds failed, and compounds 1, 2, and 5 predominated to the extent that the other compound's signals were suppressed among the chemical constituents of CSPD A of the rhizomes of L. chuanxiong. By comparison of the spectral data with published values, the unknown peaks were preliminarily ascribed to those of butylphthalide and butylidene phthalide, which has been isolated and identified from the rhizomes of L. chuanxiong previously (Wang and Gao 1985).

Although the relative area of each peak in the HPLC profiles of CSPD A of the rhizomes of L. chuanxiong showed distinct differences among seven samples (Table 2), the 15 peaks mentioned in the section of results were determined to be common in CSPD A of the rhizomes of L. chuanxiong. On the basis of careful comparison of the HPLC profiles of the seven samples, as well as the assignment of the major peaks with comparison of control substances, a conclusion could be obtained that the HPLC profiles could also be used easily for original identification of L. chuanxiong. The differences from relative content of the major similar constituents of their CSPDs explained the differences in the relative signal intensity of the corresponding ¹H NMR spectroscopy, and this property belongs to the natural character of the specific metabolites L. chuanxiong.

Materials and Methods