J. Cent. South Univ. Technol. (2008) 15: 791-795

DOI: 10.1007/s11771-008-0146-0

Analysis of volatile components in herbal pair

herba schizonepetae-ramulus cinnamomi

HU Chun-di(胡春弟)^{1, 2}, LI Xiao-ru(李晓如)¹, YU Lian-fang(余莲芳)¹,

XU Guang-wei(徐光伟)¹, LIU Shao-yin(刘少印)¹, LIANG Yi-zeng(梁逸曾)¹

 (1. School of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China;

2. School of Pharmacy, Xianning University, Xianning 437100, China)

Abstract: Analysis of volatile components in herbal pair (HP) herba schizonepetae-ramulus cinnamomi (HS-RC), single herb HS and RC was carried out by gas chromatography-mass spectrometry (GC-MS) data and chemometric resolution method (CRM). The two-dimensional data obtained from GC-MS instruments were resolved into a pure chromatogram and a mass spectrum of each chemical compound by CRM. In total, 47, 61 and 51 chemical components in volatile oil of HS, RC, and HP HS-RC were respectively determined qualitatively and quantitatively, accounting for 90.52%, 88.37%, and 88.72% total contents of volatile oil of HS, RC, and HP HS-RC, respectively. The number of the volatile components of HP HS-RC is almost the addition of that of two single herbs, but their relative contents are changed.

Key words: herbal pair; herba schizonepetae-ramulus cinnamomi; volatile component; gas chromatography-mass spectrometry; chemometric resolution method

1 Introduction

Herbal pair (HP), commonly used in traditional Chinese medicine (TCM) and consisted of two selected single herbs, is the basic unit of TCM compatibility^[1]. The chemistry of herbal pairs (HP chemistry) is the hard core of chemistry of the traditional Chinese medicinal prescriptions (TCMPs). Clarifying the chemical reactions and physical changes after the two selected single herbs are used together will be beneficial to revealing the principle of TCM compatibility and to analyze the recipes. Therefore, the study of HP chemistry will provide an effective method and a new model for the study of prescription chemistry in TCM, and also lay the foundation for the modernization of TCM.

HP herba schizonepetae (HS)-ramulus cinnamomi (RC) is one of the commonly used HPs for treating exterior syndromes^[1]. HS has the function of relieving exterior syndromes by dispelling pathogenic wind, promoting skin eruption by dispelling pathogens, and arresting bleeding, and RC dispelling  pathogenic factors from the exterior of the body by diaphoresis, warming the channels to relieve pain, and reinforcing “yang” to promote the flow of “qi”^[2]. The HP HS-RC is widely used in TCM clinic. For HPs of treating exterior syndromes, the essential oil is one group of pharmacologically active components^[3], but the volatile components in HP HS-RC have not yet been reported. In this work, the essential oil from HS, RC and HP HS-RC, was extracted respectively, and detected for the chemical components by gas chromatography-mass spectrometry (GC-MS), and the two-dimensional data were resolved and processed by the chemometric resolution method (CRM)^[4-6] to obtain pure chromatographic curves and MS profiles of two single herbs and the HP which was analyzed qualitatively and quantitatively for the resolved pure components by use of MS library and overall volume integration. Finally, the volatile chemical components of HP HS-RC were compared with those of two single herbs (HS and RC), and changes of their contents were discussed.

2.1 Instrumentals and reagents

The QP2010 gas chromatograph-mass spectrometer from Shimadzu, Japan, was used in this work. The single herbs HS and RC were purchased from Hunan Jiuzhitang Corporation, and identified by a researcher from Institute of Materia Medica, Hunan Academy of Traditional Chinese Medicine and Materia Medica, Changsha, Hunan, China.

2.2 Extraction of essential oil

2.2.1 Extraction of essential oil of HP HS-RC

Totally l00 g of HS and l00 g of RC crude herbs were mixed and soaked with 1 000 mL of distilled water in a standard extractor for extracting volatile oil, kept at room temperature for 30 min and then heated to boil at 100 ℃ in the closed system. Then, the essential oil was prepared with the standard method of steam distillation, according to Chinese Pharmacopeia (2000 version)^[7].

2.2.2 Extraction of essential oil of single herb

The essential oil of single herb HS (l00 g) and RC (l00 g) was respectively extracted in the same way as mentioned above.

2.3 Detection conditions of volatile oil

Chromatogram conditions were as follows. A 30 m×0.25 mm (I.D.) capillary column was used. Column temperature was held initially at 40 ℃, and next heated to 120 ℃ at a rate of first 2 ℃/min, then 10 ℃/min to 230 ℃ and maintained for 20 min. Helium carrier gas was employed at a constant flow-rate of 1.0 mL/min. The inlet temperature was kept at 250 ℃ and interface temperature was kept at 280 ℃.

The mass spectrometer conditions were as follows. The spectrometers were operated in an electron-impact (EI) mode, the ionization energy was 70 eV, the ionization source temperature was 230 ℃, the scan range of atomic mass unit was 20-600, the scan rate was 3.8 s^-1, the multiplier voltage was 1.28 kV and the solvent delay was 2 min.

2.4 Experimental data analysis

Experimental data analysis was performed on a Pentium Ⅲ 850 (Intel) personal computer. All programs were coded in Matlab 6.5 for windows. Resolved spectra were identified by matching against the standard mass spectral database of National Institute of Standards and Technology (NIST), which contains about  compounds.

3 Results and discussion

3.1 Qualitative analysis of essential oil

Generally, the GC-MS method is employed to analyze the volatile chemical constituents of TCMs. TCMs, however, usually contain too many nature organic compounds, and so the constituents in TCMs are very complicated, the retention time of similar constituents is very close. The hyphenated chromatography instruments combined with the chemometric resolution methods (CRMs) provide powerful tools for the resolution of such complex systems. CRM is an efficient method on two-dimensional data of chromatograms and mass spectra. The principle and resolution method could be found in Refs.[4-6]. This method has been successfully employed to analyze the volatile constituents in TCM^[8-16].

The total ionic current (TIC) chromatogram profiles of the volatile oil from the single herb HS, RC, and HP HS-RC are shown in Fig.1. There are a lot of peaks and their contents vary greatly in these TIC profiles. Furthermore, many chromatographic peaks overlap with one another, even some peaks that look like the pure peaks are also overlapped by several components. If direct search in the NIST mass spectral database without further data processing, incorrect identification of compounds may be obtained. However, if the overlapped peaks and the components with low content were

Fig.1 TIC profiles of volatile oils from HS (a), RC (b) and HP HS-RC (c)

In Fig.2, the amplified profile of peak cluster A shows the TIC curve of peak cluster A, which is so smooth that it looks like a pure chromatographic peak with only one component. Direct search in the NIST mass database can get methoxyphenyl oxime for the middle of the peak cluster A with the similarity index up to 94%. However, the former and the latter of the same peak cluster could not get the similar results, and the similarity index is low. So a conclusion can be drawn that peak cluster A is an overlapping peak. Obviously, such a search will definitely lead to lower reliability and lower accuracy for qualitative identification and it is also difficult to carry out quantitative analysis because of overlapping peaks.

Fig.2 Amplified TIC curve of peak cluster A

When CRM is adopted to resolution of peak cluster A, the result shows that peak cluster A is a system with three components (Fig.3), that is, 3-ethyl-2, 4-dimethyl- resolved into pure spectra and chromatograms with CRM, the qualitative analysis of components would be improved to a reliable extent. Now peak cluster A (the retention time between 13.82 and 13.98 min) in Fig.1(b) is taken as an example to show the analytical proceeding with CRM.

Fig.3 Resolved chromatograms of peak cluster A: 1—3-ethyl-2, 4-dimethyl-pentane; 2—1-ethyl-4-methyl-benzene; 3—4- methyl-nonane

pentane, 1-ethyl-4-methyl-benzene and 4-methyl-nonane with similarity index 96.82%, 99.27% and 97.14%, respectively, and relative content 0.06%, 0.41% and 0.10% respectively. The reliability and the accuracy of qualitative analysis of each component, therefore, have been improved greatly because the results are obtained from its pure mass spectrum.

Other peaks in chromatographic profile of RC and all peaks in two chromatographic profiles of HS and HP HS-RC were also resolved in the same way as peaks cluster A. Therefore, 47 components in HS, 61 components in RC, and 51 components in HP HS-RC were identified.

3.2 Quantitative analysis of volatile components

The overall volume integration method was employed in this work on all the resolved chromatogram peaks in order to obtain the quantitative results of each component. The qualitative constituents of single herbs HS, RC and HP HS-RC account for 90.52%, 88.37% and 88.72% of the total contents of essential oil, respectively. The major chemical compounds of volatile oils from HS, RC and HP HS-RC are shown in Table 1.

Table 1 Major chemical components of volatile oils from HS, RC and HP HS-RC

3.3 Comparison of volatile components in HP HS-RC with HS and RC

From Table 1 one can see that most of the volatile chemical components of HP HS-RC are almost from addition of two single herbs (HS and RC). The major constituents in the HP HS-RC are either from single herb HS, such as 5-methyl-2-(1-methylethylidene)-cyclohex- anone, 5-methyl-2-(1-methylethyl)-cyclohexanone, (2R- trans)-5-methyl-2-(1-methylethyl)-cyclohexanone, or from single herb RC, such as 3-(2-methoxyphenyl)- 2-propenal, benzaldehyde, or from the common constituents in two single herbs, such as D-limonene, benzylidenemalonaldehyde, α-bisabolol, tetradecanal. But the relative contents of these components in the HP are different from those in the single herbs. The experimental results also indicate that some new constituents which were not found in the two single herbs appeared in the HP, such as (E)-3,7-dimethyl-2,6- octadien-1-ol acetate, (S)-1-methyl-4-(5-methyl-1- methylene-4-hexenyl)-cyclohexene, octanal, 3-phenyl-2- propen-1-ol acetate, and the relative contents of these constituents are all very low. The physical changes, such as solubilizing effect and co-dissolving effect, and chemical reactions occurring during the decocting process are probably responsible for the appearance of these new constituents.

The chemical compounds of HP HS-RC are from two single herbs HS and RC, but not the simple addition of the two single herbs because of the chemical changes and physical processes in the process of decocting two single-herbs. As we all know, volatile oil is pharmacologically active. However, these active volatile components do not come directly from single herbs or simple addition of volatile components in single herbs. They usually form a new group of volatile components after a HP is decocted. This means that chemical components especially pharmacologically active compounds in the recipe might be different from those of single herbs in TCM. For this reason, much attention should be paid to chemical components study of HP.

1) Totally, 47, 61, and 51 volatile chemical components in HS, RC, and HP HS-RC are respectively determined qualitatively and quantitatively, accounting for 90.52%, 88.37%, and 88.72% total contents of volatile oil of HS, RC, and HP HS-RC, respectively.

2) The number of volatile components of HP HS-RC is almost addition of that of two single herbs, but some relative contents of them are changed.

3) Because of chemical reactions and physical changes during the course of decocation, some new components, such as (E)-3,7-dimethyl-2,6-octadien-1-  olacetate,(S)-1-methyl-4-(5-methyl-1-methylene-4-hexenyl)-cyclohexene, octanal, 3-phenyl-2-propen-1-ol acetate, are found in HP HS-RC.

[1] XU Qing-hua, LIU Li-yun, ZHAO Rui-hua. Collection of drug pairs in traditional Chinese medicine [M]. Beijing: Publishing House of Traditional Chinese Medicine, 1996: 360. (in Chinese)

[2] TIAN Dai-hua. Practical dictionary of traditional Chinese drugs [M]. Beijing: Publishing House of People’s Healthy, 2000: 165-167, 984-985 (in Chinese)

[3] SHEN Ying-jun. Study on exterior-releasing drugs and prescriptions in traditional Chinese medicine [M]. Beijing: Publishing House of Traditional Chinese Medicine, 2005: 198-199. (in Chinese)

[4] KVALHEIM O M, LIANG Y Z. Heuristic evolving latent projections-resolving 2-way multicomponent data (I): Selectivity, latent-projective graph, datascope, local rank and unique resolution [J]. Anal Chem, 1992, 64(8): 936-945.

[5] LIANG Y Z, KVALHEIM O M, KELLER H R, MASSART D L, KIECHLE P, ERNI F. Heuristic evolving latent projections- resolving 2-way multicomponent data (II): Detection and resolution of minor constituents [J]. Anal Chem, 1992, 64(8): 946-953.

[6] LIANG Y Z, KVALHEIM O M, RAHMANI A. Resolution of strongly overlapping two-way multicomponent data by means of Heuristic Evolving Latent Projections [J]. J Chemom, 1993, 7(1): 15-43.

[7] Chinese Pharmacopoeia Committee. Chinese pharmacopoeia [M]. Beijing: Publishing House of Chemical Industry, 2000, Appendix 64. (in Chinese)

[8] GONG F, LIANG Y Z, CUI H, CHAU F T, CHAN B T P. Determination of volatile components in peptic power by gas chromatography-mass spectrometry and chemometric resolution [J]. J Chromatogr A, 2001, 909: 237-247.

[9] GONG F, LIANG Y Z, XU Q S, CHAU F T. Gas chromatography-mass spectrometry and chemometric resolution applied to the determination of essential oils in Cortex Cinnamomi [J]. J Chromatogr A, 2001, 905: 193-205.

[10] GONG Fan, LIANG Yi-zeng, FUNG Ying-sing. Analysis of volatile components from Cortex cinnamomi with hyphenated chromatography and chemometric resolution [J]. J Pharma Biomed Anal, 2004, 34(5): 1029-1047.

[11] LI Xiao-ru, LAN Zheng-gang, LIANG Yi-zeng. Analysis of the volatile chemical constituents of Radix Paeoniae Rubra by GC-MS and chemometric resolution [J]. J Cent South Univ Technol, 2007, 14(1): 57-61.

[12] LI Xiao-ru, LIANG Yi-zeng, GUO Fan-qiu. Analysis of volatile oil in rhizoma ligustici chuanxiong–radix paeoniae rubra by gas chromatography–mass spectrometry and chemometric resolution [J]. Acta Pharmacologica Sinica, 2006, 27(4): 491-498.

[13] WU Ming-jian, SUN Xian-jun, DAI Yuan-hui, HUANG Lan-fang. Determination of constituents of essential oil from Angelica sinensis by gas chromatography-mass spectrometry [J]. J Cent South Univ Technol, 2005, 12(4): 430-436.

[14] CHEN Yong, LI Xiao-ru, ZHAO Jun, ZHOU Tao, ZOU Qiao. Chemical components analysis of volatile oil in drug pair Herba Ephedrae-Ramulus Cinnamomi by GC-MS and CRM [J]. J Cent South Univ Technol, 2007, 14(4): 509-513.

[15] LI Xiao-ru, LIANG Yi-zeng, GUO Fan-qiu, LI Xiao-ning, ZENG Zhong-da. Analysis of volatile oil in Semen Persicae-Flos Carthami by GC-MS and CRM [J]. Chinese J Anal Chem, 2007, 35(4): 532-536. (in Chinese)

[16] LI Guo-hui, LI Xiao-ru, ZOU Qiao, TAN Bin-bin. Determination of volatile components in Herba Ephedrae-Rhizoma seu Radix Notopeterygii by GC-MS and chemometric resolution method [J]. J Cent South Univ, 2007, 38(5): 888-892. (in Chinese)

Foundation item: Project(20235020) supported by the National Natural Science Foundation of China

Received date: 2008-05-19; Accepted date: 2008-07-18

Corresponding author: LI Xiao-ru, Professor; Tel: +86-731-8836376; E-mail: xrli@mail.csu.edu.cn

(Edited by YANG Hua)