J. Cent. South Univ. Technol. (2010) 17: 726-731
DOI: 10.1007/s11771-010-0547-8
Comparative analysis of volatile constituents between herbal pair flos lonicerae-caulis lonicerae and its single herbs
ZHAN Xue-hui(湛雪辉)1, XU Guang-wei(徐光伟)2, LI Fei(李飞)1, LI Xiao-ru (李晓如)2,
ZHOU Sui-an(周随安)1, CAO Fen(曹芬)1, LI Xia(李侠)1
1. School of Chemical and Biological Engineering, Changsha University of Science and Technology,
Changsha 410114, China;
2. School of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China
? Central South University Press and Springer-Verlag Berlin Heidelberg 2010
Abstract: Gas chromatography-mass spectrometry (GC-MS) and the chemometric resolution method (alternative moving window factor analysis, AMWFA) were used for comparative analysis of volatile constituents in herbal pair (HP) flos lonicerae-caulis lonicerae (FL-CL) and its single herbs. The temperature-programmed retention index (PTRI) was also employed for the identification of compounds. In total, 44, 39, and 50 volatile chemical components in volatile oil of FL, CL and HP FL-CL were separately determined qualitatively and quantitatively, accounting for 87.22%, 94.54% and 90.08% total contents of volatile oil of FL, CL and HP FL-CL, respectively. The results show that there are 32 common volatile constituents between HP FL-CL and single herb FL, 33 common volatile constituents between HP FL-CL and single herb CL, and 10 new constituents in the volatile oil of HP FL-CL.
Key words: herbal pair; flos lonicerae-caulis lonicerae; volatile oil; gas chromatography-mass spectrometry; alternative moving window factor analysis
1 Introduction
Herbal pair (HP) composed of two single herbal medicines is often used in the traditional Chinese medicine (TCM). The theory of compatibility is the core of the basic theories of TCM. Clarifying the physical changes and chemical reactions occurring during the decocting process of two single herbs is useful for elucidating the compatibility mechanism of TCM. Clinical pharmacodynamics of HP relies on its chemical components, so it is important to identify the components in HP and investigate the relationship between the chemical components in HP and its single herbs. The HP flos lonicerae-caulis lonicerae (FL-CL) is one of the commonly used HPs for clearing away heat and reducing fire, and is often used in the treatment of arthritis and sore throat [1]. FL has the function of clearing heat and detoxifying, and CL has the function of stimulating the menstrual flow and activating the collaterals [2]. Volatile oil is one kind of pharmacologically active constituents in herbal pairs [3], but the volatile chemical constituents of the HP FL-CL have not been reported yet. In this work, the volatile oil from FL, CL and HP FL-CL was extracted respectively, and then the combination of gas chromatography-mass spectrometry (GC-MS) and the chemometric resolution method (alternative moving window factor analysis, AMWFA) [4-5] were employed for the comparative analysis of the volatile constituents in HP FL-CL and its single herbs FL and CL. Furthermore, temperature-programmed retention index (PTRI) [6-9] was also employed for the identification of the compounds. Finally, the method of overall volume integration was used to the quantitative analysis.
2 Experimental
2.1 Instruments and medical materials
The GC-MS instrument was QP2010. Single herbal medicines FL and CL were purchased from and identified by Institute of Materia Medica, Hunan Academy of Traditional Chinese Medicine and Materia Medica (Changsha, China). n-alkane standard solutions of C8–C20 (mixture No. 04070) and C21–C40 (mixture No. 04071) were purchased from Fluka Chemika, Switzerland.
2.2 Extraction of volatile oil
2.2.1 Extraction of volatile oil of HP FL-CL
50 g of dried powders of each single herbal medicine, FL and CL, were exactly weighed and mixed and then processed according to the standard method described in Chinese Pharmacopoeia (2005 version) [10].
2.2.2 Extraction of volatile oil of single herbs
50 g of dried powders of each single herbal medicine FL and CL were exactly weighed respectively and then processed according to the same method as mentioned above.
2.3 Analytical conditions of volatile oil
2.3.1 Chromatography conditions
DB-1 capillary column (30 m×0.25 mm (length×inner diameter)) was used. The column temperature was set at 50 ℃ (maintained for 3 min) initially, and then increased to 250 ℃ (maintained for 10 min) at a rate of 5 ℃/min. The carrier gas was helium with a constant flow rate of 1.0 mL/min. The inlet temperature and interface temperature were kept at 250 ℃.
2.3.2 Mass spectrometer conditions
Electron impact (EI+) mass spectra were recorded at 70 eV. The scan was carried out in the range of 35-500 amu with 5 scan/s. The ionization source temperature was set at 200 ℃. Solvent cut time was 3.5 min.
2.4 Temperature-programmed retention indices
VANDENDOOL and KRATZ [11] proposed a quasilinear equation for temperature-programmed retention index as follows:
(1)
where Ix is the temperature-programmed retention index of the compound studied; tn, tn+1 and tx are the retention time of the two standard n-alkanes containing n and n+1 carbons and the compound studied, respectively. Eq.(1) was used to calculate retention index in this work.
2.5 Data analysis
Data analysis and all the calculations were performed on an HP V3911 personal computer, and all programs of chemometric resolution methods were coded in MATLAB 6.5 for windows. Resolved spectra were identified by matching with the standard mass spectral database of the National Institute of Standards and Technology (NIST).
3 Results and discussion
Because volatile oil of TCMs is a very complex system, the GC-MS data from volatile oil often involve a great number of overlapped peaks and even embedded ones. AMWFA is an efficient method for resolving the overlapped peaks and embedded ones, and is an extensive and conjoint version of multicomponent spectral correlative chromatography (MSCC) [12] and subwindow factor analysis (SFA) [13]. This method can utilize the cross-information hidden in two systems to determine the number of common components in different samples, and then to identify their corresponding spectra of common components automatically. The principle and resolution of AMWFA method can be found in Refs.[4-5]. AMWFA was successfully employed to comparatively analyze the volatile components in recipe jingfangsan and their single herbs [14], different species of Clematis [15], Pericarpium Citri Reticulatae Viride and Pericarpium Citri Reticulatae [16].
3.1 Resolution of overlapped peaks by AMWFA
The total ion chromatograms (TICs) of herbal medicines FL, CL and HP FL-CL are shown in Fig.1. To illustrate the extraction of the pure mass spectra efficiently, peak clusters marked by X (retention time: 36.109-36.305 min) and Y (retention time: 36.133- 36.323 min) were picked out as examples and then processed by AMWFA (Fig.2).
Multicomponent spectral correlative chromatography (MSCC) and inverse projection-MSCC (IP-MSCC) were used to investigate the relationship of components between peak clusters X and Y. The results of MSCC and IP-MSCC are shown in Fig.3, which indicates that the mass spectrum feature of the compounds in peak cluster X is closely correlated with that in peak cluster Y, and the two peak clusters contain common components. The results obtained by common rank analysis (Fig.4) show that the number of common components in the two peak clusters is 2, because the first two values (No.1 and No.2) in Y-axis are almost equal to zero.
Peak cluster X was taken as the base matrix, and peak cluster Y as the target matrix. The moving window searching was conducted on peak cluster Y with fixed window size 3, and then the common rank map (Fig.5(a)) was obtained by plotting the number of common ranks versus the number of scan points. During the process of moving window scanning, the mass spectra and the similarity between the two close mass spectra were obtained by calculating with formula. The spectral auto-correlative curve (Fig.5(b)) was also obtained by plotting similarity versus the number of scan points. In Fig.5(a), there are two regions of scan points (5-22, 38-53), in which the number of common components is close to 1 in the common rank map, and two flat regions (indicated by R1 and R2 in Fig.5(b)) with similarity being close to 1 appearing in the corresponding spectral autocorrelative curve. If the number of common components is equal to 1, a pure spectrum can be acquired from the corresponding region with correlation coefficient close to 1 in the spectral auto-correlative curve. In Fig.5(b), two flat parts indicated by R1 and R2 show the regions where the two identified mass spectra are picked out. By matching search from NIST107 standard mass spectral database, the resolution result by AMWFA shows that the two common components in peak clusters X and Y are [Z, Z]-9,12-octadecadienoic acid methyl ester (R1) and [Z, Z, Z]-9,12,15-octadecatrienoic acid methyl ester (R2), with the match similarities of 98.764% and 97.798%, respectively. The pure chromatograms of peak clusters X and Y are shown in Fig.6, which indicates that peak clusters X and Y are overlapped ones of two components.
Fig.1 TICs of volatile oils from FL (a), CL (b) and HP FL-CL (c)
Fig.2 TIC curves for peak clusters X (a) and Y (b)
Fig.3 Results obtained by MSCC (a) and IP-MSCC (b) analysis
Fig.4 Results of common rank analysis
Fig.5 Common rank map (a) and spectral auto-correlative curve (b)
Fig.6 Resolved chromatograms for peak clusters X (a) and Y (b)
3.2 Quantitative analysis of volatile components
The overall volume integration method was used for all the chromatogram peaks in order to obtain the quantitative result of each component. In total, 44, 39, and 50 volatile components in volatile oils of FL, CL, and HP FL-CL were respectively determined qualitatively and quantitatively, accounting for 87.22%, 94.54% and 90.08% total contents of volatile oil of FL, CL, and HP FL-CL, respectively. The main volatile chemical components are given in Table 1.
3.3 Comparison of volatile components between HP FL-CL and single herbs FL and CL
From Table 1, the main volatile chemical components in FL are n-hexadecanoic acid (30.91%), 9,12-octadecadienoic acid ethyl ester (26.42%), hexadecanoic acid methyl ester (5.17%), [Z, Z, Z]- 9,12,15-octadecatrienoic acid methyl ester (4.97%) and tetradecanoic acid (3.36%); the main volatile chemical components in CL are n-hexadecanoic acid (45.86%), 9,12-octadecadienoic acid ethyl ester (39.02%), tetradecanoic acid (1.82%) and pentadecanoic acid (1.07%); the main volatile chemical components in HP FL-CL are n-hexadecanoic acid (44.73%), 9,12- octadecadienoic acid ethyl ester (26.59%), tetradecanoic acid (3.07%), hexadecanoic acid methyl ester (2.13%), [Z, Z, Z] 9,12,15-octadecatrienoic acid methyl ester (1.95%) and 6,10,14-trimethyl-2-pentadecanone (1.57%).
Table 1 Relative contents and PTRIs of main chemical components of volatile oils from FL, CL and HP FL-CL
The comparison of the volatile oil components between HP FL-CL and single herbs FL, CL shows that there are 32 common volatile chemical components between HP FL-CL and single herb FL, and 33 common volatile chemical components between HP FL-CL and single herb CL. The result also indicates that 10 new chemical components, such as 1-octen-3-one, appear in the volatile oil of HP FL-CL, and the relative contents of these components are all very low. This information is useful and necessary to the further pharmacological study of HP FL-CL. The physical changes and chemical reactions in the process of decoction are probably responsible for the appearance of these new components [17-18], such as oxidation, reduction, solubilizing effect and co-dissolving effects.
4 Conclusions
(1) The main volatile components in HP FL-CL are n-hexadecanoic acid (44.73%), 9,12-octadecadienoic acid ethyl ester (26.59%), tetradecanoic acid (3.07%), hexadecanoic acid methyl ester (2.13%), [Z, Z, Z]- 9,12,15-octadecatrienoic acid methyl ester (1.95%), and 6,10,14-trimethyl-2-pentadecanone (1.57%).
(2) There are 32 common volatile chemical components between HP FL-CL and single herb FL, and 33 common volatile chemical components between HP FL-CL and single herb CL.
(3) Because of physical changes and chemical reactions in the process of decoction, 10 new components, such as 1-octen-3-one, are found in HP FL-CL.
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Foundation item: Project(20976017) supported by the National Natural Science Foundation of China
Received date: 2009-10-09; Accepted date: 2010-01-28
Corresponding author: LI Xiao-ru, Professor; Tel: +86-731-88836376; E-mail: xrli@mail.csu.edu.cn
(Edited by CHEN Wei-ping)