GC-MS Analysis of Chemical Constituents of Volatile Oils of the Yao Medicine Thunbergia grandiflora from Different Habitats in Guangxi

Shuang LIANG Xun ZHANG Jianjun HUANG Qifa HE Shuang QIN Huqiang YANG Yihang ZHOU

Abstract [Objectives] This study was conducted to compare and analyze the chemical constituents of the volatile oils of the Yao medicine Thunbergia grandiflora from different habitats.

[Methods] A quartz capillary column DB-1MS， an EI ion source and a quadrupole mass analyzer were used for analysis. The chromatographic and mass spectrum information obtained was automatically retrieved and analyzed by data processing system and its memory spectrum library （Nist.08）. The relative content of each chemical component in the volatile oil was determined by the peak area normalization method.

[Results] Forty nine chromatographic peaks were isolated from the sample produced in Shitun， Bailongtan Town， Mashan County， and 24 chemical constituents were identified， accounting for 88.78% of the total volatile oil. Forty nine chromatographic peaks were isolated from the sample produced in Hongdu Village， Chengjiang Town， Duan County， and 30 chemical constituents were identified， accounting for 88.38% of the total volatile oil. Forty eight chromatographic peaks were isolated from the sample produced in Longwan Township， Duan County， and 25 chemical constituents were identified， accounting for 80.01% of the total volatile oil. Nine chemical constituents were common to the volatile oils of the samples from the three habitats.

[Conclusions] It could be seen that the main components of volatile oils from T. grandiflora produced in different areas are different.

Key words Thunbergia grandiflora; Volatile oil; GC-MS

Laoyazui refers to the whole plant of Thunbergia grandiflora （Roxb. ex Willd.） Roxb. in Acanthaceae， which has rich plant resources. T. grandiflora tastes bitter and astringent， and is neutral in nature. It has the functions of relaxing muscles and collaterals， opening orifice， dissipating stasis and relieving pain， diminishing swelling and stopping bleeding. In the area where Guangxi Yao people gather， it is mainly used for the treatment of nephritis and edema， trauma injury， rheumatic arthralgia， low back pain， lumbar muscle strain， dysmenorrhea， carbuncles and fracture. In this study， the chemical composition of the volatile oil in T. grandiflora materials from different habitats in Guangxi was investigated by gas chromatography-mass spectrometry （GC-MS）， and their common components were analyzed and compared， aiming to provide a theoretical basis for the clinical application， quality control and resource exploitation and utilization of T. grandiflora.

Materials and Methods

Experimental materials

The materials were collected from ① Dahua County， ② Shitun， Bailongtan Town， Mashan County， ③ Hongdu Village， Chengjiang Town， Duan County， and ④ the area near Longwan Township， Duan County. The samples used in the experiment were all identified by Professor Teng of Guangxi University of Chinese Medicine as the whole plant of T. grandiflora （Roxb. ex Willd.） Roxb. in Acanthaceae.

Instruments and reagents

Agilent 7890A-5975C gas chromatograph-mass spectrometer （Agilent Technologies）; quartz capillary column （DB-1MS， 30 m×0.25 mm×0.25 μm）; volatile oil extractor; ML3002 electronic analytical balance[Mettler-Toledo Instruments （Shanghai） Co.， Ltd.].

Anhydrous sodium sulfate （analytically pure， Guangdong Guanghua Sci-Tech Co.， Ltd.）; ethyl acetate （chromatographically pure， Merck Limited Partnership）; pure water.

Experimental methods

Extraction of volatile oil

A certain amount of T. grandiflora （150 g） was pulverized to 20 mesh， and added in a 2 000 ml round bottom flask， which was added with distilled water 10 times the amount of the sample. The materials were soaked for 1 h， and then the flask was connected to a volatile oil extractor and a reflux condenser. The sample was subjected to steam distillation according to the appendix method （without the addition of xylene） in the 2015 edition of Chinese Pharmacopoeia until the distillate contained no oil droplets （about 6 h）， obtaining a light yellow oil with special flavor. Finally， the collected volatile soil was dissolved and collected with ethyl acetate， dried with anhydrous sodium sulfate and kept in a refrigerator in a sealed state.

Sample treatments

A certain amount of the ethyl acetate solution of volatile oil （1 ml） was transferred in a centrifuge tube and added with an appropriate amount of anhydrous sodium sulfate to dehydrate it. The sample was then centrifuged at 1 000 r/min in a centrifuge for 20 min， obtaining a supernatant as the test solution.

GC-MS conditions

The GC was equipped with quartz capillary column DB-1MS capillary column （30 m×0.25 mm×0.25 μm）. The temperature program was started with an initial temperature of 50 ℃， which was held for 1 min， and then increased at 5 ℃/min to 250 ℃， which was held for 5 min. The carrier gas was He （≥99.99%）. The GC separation was performed with a column flow rate of 1.0 ml/min， an inlet temperature of 220 ℃ and a split ratio of 20∶1， the injection volume was 1 μl， and the solvent was delayed by 3.0 min.

The MS conditions were as follows： EI source， electron energy 70 eV， ion source temperature 230 ℃， quadrupole temperature 150 ℃， MS interface temperature 280 ℃， full scan （Scan） acquisition mode， mass range m/z 50-500 amu， and scanning interval 0.2 s.

Determination methods

The sample prepared under "Extraction of volatile oil" was diluted with ethyl acetate， and water contained in it was removed with anhydrous sodium sulfate. Filtration was performed with a 0.45 μm microporous filter membrane， obtaining a filtrate， which was determined according to the chromatographic conditions under "GC-MS conditions" for a total ion current chromatogram of the volatile oil of T. grandiflora. The chemical composition represented by each chromatographic peak was searched through the Nist.08 mass spectrometry data system.

GC-MS analysis

The GC and MS information obtained was automatically searched and analyzed by the data processing system and the internal memory library （Nist.08）， and the relative percentage content of each chemical component in the volatile oil was determined by the peak area normalization method.

Results and Analysis

GC-MS results

The GC-MS analysis of the extracted volatile oils from T. grandiflora produced in the three areas was conducted according to the above test conditions. After the total ion current chromatogram was determined， it was searched in combination with the mass spectrometry database （Nist 02 and Wiley 275）， and the peak area normalization method was used to determine the relative content of each component in the sample. The total ion current chromatograms of the volatile oils are shown in Fig. 1， Fig. 2， and Fig. 3， respectively， and the analysis results of their components are shown in Table 1， Table 2， and Table 3， respectively.

Component analysis

There were 49 chromatographic peaks isolated from T. grandiflora produced in Shitun， Bailongtan Town， Mashan County， and 24 chemical components were identified， accounting for 88.78% of the total volatile oil. The main components were palmitic acid 22.11%， 3-methyl-3-ethyl-pentanol 21.62%， linoleic acid 6.49%， etc. Forty nine chromatographic peaks were isolated from T. grandiflora produced in Hongdu Village， Chengjiang Town， Duan County， and 30 chemical components were identified， accounting for 88.38% of the total volatile oil. The main components were D-limonene 11.84%， palmitic acid 16.17%， linoleic acid 5.63%， 3-methyl-4-isopropylphenol 5.46%， etc. There were 48 chromatographic peaks separated from T. grandiflora produced in Longwan Township， Duan County， and 25 chemical components were identified， accounting for 80.01% of the total volatile oil. The main components were 4-methoxysalicylaldehyde 13.5%， 2-pentylfuran 5.57%， palmitic acid 27.22%， linoleic acid 5.41%， etc.[1-2]. Among the chemical constituents of volatile oil from T. grandiflora in the three producing areas， there were 9 components common to samples from the three habitats， as shown in Table 4.

Conclusions

The results of this study reflected the differences in the types and contents of the chemical constituents of the volatile oil of T. grandiflora from different producing areas， which might be caused by environmental differences. The differences in the types and contents of the chemical components of T. grandiflora and the characteristic components may affect the pharmacology and efficacy. The research results of this study provide a scientific basis for further development and utilization of the Yao medicine T. grandiflora[3].

References

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[2] DU CZ， WANG H， FENG X， et al. GC-MS analysis of volatile oil in Agrimonia pilosa Ledeb. from different regions[J]. Jiangsu Agricultural Sciences， 2014， 42（4）： 253-255. （in Chinese）

[3] DU CZ， QIN JP， CHEN YP， et al. GC-MS analysis of volatile oil components in Schizonepeta tenuifolia Briq. from various habitats[J]. Hubei Agricultural Sciences， 2014， 53（1）： 187-190. （in Chinese）