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Abstract Volatile components of Callistemon rigidus were analyzed by headspace solid phase microextraction-gas chromatography/mass spectrometry. The results showed that 34 volatile components, including alkenes, esters, alcohols, and a small amount of acids, heterocycles and alkanes, were identified from C. rigidus flowers. The main components were eucalyptol, aspidinol, terpilenol, vanillylacetone, α-phellandrene, 4-pyran-4-1,2,3-dihydro-3,5-dihydroxy-6-methyl, pinene, 3-methylcatechol, naphthalene, (+)-limonene, 7-methylxanthine and (+)-aromadendrene, the contents of which were 35.39%, 10.09%, 8.86%, 4.51%, 3.25%, 2.81%, 2.75%, 2.43%, 2.29%, 1.3%, 1.12% and 1.07%, respectively, all of which accounted for 75.69% of the total relative content. Monoterpenoids are the main components of various types of essential oils. This study provides a theoretical basis for the deep processing of this variety.
Key words Callistemon rigidus; Volatile chemical composition; GC-MS
Callistemon rigidus, also known as Hongpingshua and Jincengshu, is a woody plant in myrtaceae, native to Australia. The plants in this genus introduced into China include C. rigidus, Callistemon viminalis and Callistemon salignus［1］. C. rigidus leaves are lanceolate, and have oil glands. The flowers have no peduncles, often forming spikes at the top of branches, and the bloomed flowers look like a test-tube brush. Its flowering period lasts from March to July, and the flowers can be used as flower arrangement, or the plant can be trimmed into bonsai. Therefore, this plant has great ornamental value and is widely used in garden greening. In addition to an excellent garden ornamental plant, C. rigidus is also an aromatic plant. Its leaflets can be extracted for fragrant oil. Its essential oil is used for the preparation of cosmetics, soaps, daily necessities, detergent flavors, and also for medical and health care.
At present, Liu and Dong et al.［2］analyzed the volatile chemical constituents of C. rigidus branches and leaves, and obtained 44 volatile chemical constituents. Li［3］analyzed the volatile chemical constituents of C. viminalis leaves, and identified 13 kinds of volatile chemical constituents. Li and Hua et al.［4］identified two compounds from C. salignus leaves. Wu and Jiang et al.［5］identified 21 compounds from the volatile oil of C. rigidus leaves. There are no reports on the chemical components of C. rigidus flowers at home and abroad. In this study, C. rigidus flowers were extracted by steam distillation with ethyl acetate, and the volatile chemical constituents were identified by gas chromatography-mass spectrometry (GC-MS), obtaining the GC peak areas, which were processed by the normalization method to determine the relative content of each component. This study provides a scientific basis for further research and comprehensive utilization of C. rigidus plant resources. Materials and Methods
Instruments and Materials
Experiment instruments
QP2010 plus gas chromatograph-mass spectrometer (Shimadzu, Japan); P DMS-DVB solid phase micro-extraction instrument, 65 μm polydimethylsiloxane extraction head (Supelco, USA); HP-5M S quartz capillary column (30 m × 250 μm × 0.25 μm).
Plant material
C. rigidus flowers.
Experiment time and location
On June 12, 2018, C. rigidus flowers were collected from the campus of Kunming University in Yunnan, in the morning of a cloudy day. The C. rigidus flowers were in the flowering period in the local area. The experiment was conducted in the laboratory on the day of collection.
Experiment methods
Experiment design
Some of the newly flowered branches were randomly selected on the C. rigidus plants in the flowering stage, and the branches and leaves (small leaves on the C. rigidus branches) were removed. The flowers were sent to the laboratory for testing. The C. rigidus flowers were weighed into 400 g as 1 replicate, 3 replicates in total (1 200 g).
Extraction of volatile constituents
The C. rigidus flowers (400 g) were cut and extracted in 15 ml of methanol for 1.5 h. After passing through a 0.22 μm filter membrane, the filtrate was extracted with an appropriate amount of ethyl acetate in a condensation tube by steam distillation, obtaining the C. rigidus volatile constituents.
GC-MS analysis method
The HP-5M S quartz capillary column (30 m × 250 μm × 0.25 μm) was used with the column temperature of -60 -325 ℃ (350 ℃). The temperature program for the column oven was started with the initial temperature at 80 ℃, which was held for 0.5 min and raised at 5 ℃/min to 100 ℃, which was held for 2 min and raised at 5 ℃/min to 150 ℃, which was also held for 2 min and then increased at 5 ℃/min to 200 ℃, which was maintained for 5 min and finally raised at 5 ℃/min to 250 ℃ which was maintained for 10 min. The carrier gas was He (99.99%), and unsplit stream sampling was adopted with an inlet temperature at 230 ℃. EI ionization was carried out with an ion source temperature at 230 ℃. The MS detection was performed with the MS quadrupole temperature at 150 ℃ under the mass scanning range m/z: 30-550 u, the injection volume of 1 μ and the solvent delay of 3 min.
Statistic analysis
The gas chromatography-mass spectrometry-computer combined instrument was employed to perform analysis and identification. The total ion chromatogram (GC/MS) of the volatile constituents from C. rigidus flowers was obtained, and the chemical information represented by each peak in the chromatogram was analyzed using the software TurboMass Ver (5.4.2 version). The NIST/WIELY standard spectrum library was searched by computer for the mass spectrometry data of each component, and the volatile components of the flowers were qualitatively analyzed. The GC peak areas were subjected to normalization to obtain the relative content of each component. Results and Analysis
Main aroma constituents of C. rigidus flowers
The volatiles were analyzed according to the above GC-MS conditions to obtain the total ion chromatogram of the volatile chemical constituents of C. rigidus flowers (Fig. 1). Through mass spectrometry database retrieval, combined with relevant literatures, a total of 34 components in C. rigidus flowers were identified from the base peak, relative abundance and other aspects. According to the peak area normalization method, the relative content of each component was determined, and the results are shown in Table 1.
It can be seen from the analysis in Table 1 that 34 volatile components were identified from C. rigidus flowers. The main components were eucalyptol, aspidinol, terpilenol, vanillylacetone, α-phellandrene, 4-pyran-4-1,2,3-dihydro-3,5-dihydroxy- 6-methyl, pinene, 3-methylcatechol, naphthalene, (+)-limonene, 7-methylxanthine and (+)-aromadendrene, the contents of which were 35.39%, 10.09%, 8.86%, 4.51%, 3.25%, 2.81%, 2.75%, 2.43%, 2.29%, 1.3%, 1.12% and 1.07%, respectively, all of which accounted for 75.69% of the total relative content. Among the main volatile components, alcohols were the main volatile components in C. rigidus flowers, and had the highest relative content of 44.86%. It indicated that the components playing the main role in the aroma of C. rigidus flowers were olefins, followed by esters and alcohols［6-9］, which can be concluded from the total ion chromatogram (Fig. 1). According to the research data, monoterpenoids accounted for a larger mass fraction in this study. The volatile substances of flowers are composed of a series of low molecular volatile compounds, mainly fatty acid derivatives, benzene rings, terpenoids, nitrogenous substances and other fragrant substances［10-12］. The results of this analysis were compared with the chemical constituents of C. rigidus branches and leaves analyzed by Liu and Dong. The flowers, branches and leaves all contain pinene, α-phellandrene, eucalyptol, methyl palmitate and other compounds, but there are greater differences. Moreover, different varieties in Callistemon differ obviously in chemical components from different parts of the plants, and the growth conditions have a great influence on the plant metabolites.
Effects of aroma constituents
According to related references, α-phellandrene is an important raw material for synthesizing terpene synthetic perfumes such as geraniol and linalool. α-Pinene and β-pinene are the main chemical components of turpentine［13-14］. α-Pinene has the smell of pine and is the main substance for synthesizing sandalwood perfumes. It is mainly used to synthesize perfumes. β-Pinene is a raw material for the synthesis of various terpene perfumes. α-Terpinene has a citrus flavor and can be used to blend perfumes and essential oils such as lemon and mint［15］. γ-Terpinene can be used to make various plant perfumes［16］. Conclusions
The unique aroma of C. rigidus flower makes it stand out among the varieties. Volatile components are the material basis of the aroma of food, and multiple volatile components determine the aroma characteristics of food together. Volatile substances are an important factor affecting the flavor quality of food.
In this study, the main volatile components of C. rigidus were determined by solid phase microextraction-gas chromatography/mass spectrometry, and 34 volatile substances were identified, including alkenes, esters, alcohols and a small amount of acids, heterocycles and alkanes, the combination of which constitutes the rich aroma of C. rigidus. Its volatile components were mainly monoterpenoids, the relative content of which accounts for 84.99% of the total. The main components all have characteristic odor, which plays an important role in the normal growth and development of plants, and monoterpenoids among them can be used in food, essential oil, industry and so on［17］. Monoterpenoids are mostly volatile oily liquids with a special odor. They are widely found in plants and are the main components of perfumes, resins, pigments and the like of certain plants. For instance, rose oil, eucalyptus oil and turpentine contain a variety of terpenoids. Terpenoids also have anti-cough, wind dispelling, deworming, analgesic and other effects, and are indispensable raw materials for food and cosmetic industries.
Yanrong BAI et al. Analysis of Volatile Chemical Constituents in Callistemon rigidus Flowers by GC-MS
References
［1］SHI FP. Garden ornamental aromatic plants［J］. Southwest Horticulture, 2005(1): 33-33.
［2］LIU BM, DONG XM, LIN X, et al. Chemical components of the essential oil from Callistemon rigidus R. Br.［J］. Journal of Tsinghua University: Science and Technology, 2010, 50(9): 29-41.
［3］LI C, TAN HB, QIU SX, et al. Chemical constituents from the leaves of Callistemon viminalis［J］. Natural Product Research and Development, 2017(29): 954-958.
［4］lIU YQ. Analysis of the volatile oils from the flowers of Magnolia liliflora Desr. by GC-MS［J］. Lishizhen Medicine and Materia Medica Research, 2008. 19(8): 1911-1912.
［5］CHEN HG, YANG ZN, ZHAO C, et al. Analysis of volatile oil from the flower of Magnolia grandiflora by SPME/GC/MS［J］. Chinese Journal of Spectroscopy Laboratory, 2010, 27(4): 1440-1442.
［6］SHI L, WANG JM, KANG WY. Analysis of volatile constituents in two species of genus Magnoloia by HS-SPME-GC-MS［J］．China Journal of Chinese Materia Medica, 2008, 33(12): 1429-1433 ［7］WU FH. Research progress on new techniques of solid phase extraction［J］. Analysis and Testing Technology and Instruments, 2012, 18(2): 114-120.
［8］WANG X, TANG XW, ZHOU D, et al. Analysis of volatile compounds of lily (Lilium spp.) by SPME-GC-MS［J］. Modern Instruments, 2011, 17(5): 47-49.
［9］DAI Y, XU HR. Analysis on aromatic components of Longjing tea using SPME-GC/MS methods［J］. Tea, 2008, 34(2): 85-88.
［10］BAI SS. Applications of solid phase microextraction combined with gas chromatography technology for the determination of several organic pollutants［D］. Baoding: Hebei Agricultural University, 2013.
［11］BI SF, CHENG XX, CHEN JW, et al. Analysis of volatile components of Chrysanthemum indieum L. flowers from Huangshan by head space-solid phase micro-extraction combined with GC-MS［J］. Journal of Beijing Union University: Natural Science, 2016, 30(2): 88-92.
［12］RAN H, FENG LL, MAO YZ, et al. Identification and analysis of volatile components in essential oil from four Lauraceae wild species leaves in Chongqing by GC-MS［J］. Scientia Silvae Sinicae, 2018, 54(7): 91-103.
［13］LI L. Application of α-pinene chemical properties［J］. Guangxi Chemical Industry, 2000, 29(1): 36-38, 48.
［14］LIAO SL. Synthesis, biological activity and structure-activity relationship of novel (-)-β-pinene derivatives［D］. Beijing: Chinese Academy of Forestry, 2016.
［15］XIE ZJ, HOU YQ, WANG W, et al. The allelopathic functions of terpenoids and its application prospects［J］. Chinese Agricultural Science Bulletin, 2010, 26(24): 233-237.
［16］GU WX, DUAN SS, LUO SM. Ecological characteristic of terpenoids and their allelopathic effects to plants［J］. Journal of South China Agricultural University, 1998, 19(4): 5.
［17］LIU BM, PENG W. Component analysis of essential oil from melaleuca Leucadendron L.［J］. Journal of Instrumental Analysis, 1999, 18(6): 70-72.
Key words Callistemon rigidus; Volatile chemical composition; GC-MS
Callistemon rigidus, also known as Hongpingshua and Jincengshu, is a woody plant in myrtaceae, native to Australia. The plants in this genus introduced into China include C. rigidus, Callistemon viminalis and Callistemon salignus［1］. C. rigidus leaves are lanceolate, and have oil glands. The flowers have no peduncles, often forming spikes at the top of branches, and the bloomed flowers look like a test-tube brush. Its flowering period lasts from March to July, and the flowers can be used as flower arrangement, or the plant can be trimmed into bonsai. Therefore, this plant has great ornamental value and is widely used in garden greening. In addition to an excellent garden ornamental plant, C. rigidus is also an aromatic plant. Its leaflets can be extracted for fragrant oil. Its essential oil is used for the preparation of cosmetics, soaps, daily necessities, detergent flavors, and also for medical and health care.
At present, Liu and Dong et al.［2］analyzed the volatile chemical constituents of C. rigidus branches and leaves, and obtained 44 volatile chemical constituents. Li［3］analyzed the volatile chemical constituents of C. viminalis leaves, and identified 13 kinds of volatile chemical constituents. Li and Hua et al.［4］identified two compounds from C. salignus leaves. Wu and Jiang et al.［5］identified 21 compounds from the volatile oil of C. rigidus leaves. There are no reports on the chemical components of C. rigidus flowers at home and abroad. In this study, C. rigidus flowers were extracted by steam distillation with ethyl acetate, and the volatile chemical constituents were identified by gas chromatography-mass spectrometry (GC-MS), obtaining the GC peak areas, which were processed by the normalization method to determine the relative content of each component. This study provides a scientific basis for further research and comprehensive utilization of C. rigidus plant resources. Materials and Methods
Instruments and Materials
Experiment instruments
QP2010 plus gas chromatograph-mass spectrometer (Shimadzu, Japan); P DMS-DVB solid phase micro-extraction instrument, 65 μm polydimethylsiloxane extraction head (Supelco, USA); HP-5M S quartz capillary column (30 m × 250 μm × 0.25 μm).
Plant material
C. rigidus flowers.
Experiment time and location
On June 12, 2018, C. rigidus flowers were collected from the campus of Kunming University in Yunnan, in the morning of a cloudy day. The C. rigidus flowers were in the flowering period in the local area. The experiment was conducted in the laboratory on the day of collection.
Experiment methods
Experiment design
Some of the newly flowered branches were randomly selected on the C. rigidus plants in the flowering stage, and the branches and leaves (small leaves on the C. rigidus branches) were removed. The flowers were sent to the laboratory for testing. The C. rigidus flowers were weighed into 400 g as 1 replicate, 3 replicates in total (1 200 g).
Extraction of volatile constituents
The C. rigidus flowers (400 g) were cut and extracted in 15 ml of methanol for 1.5 h. After passing through a 0.22 μm filter membrane, the filtrate was extracted with an appropriate amount of ethyl acetate in a condensation tube by steam distillation, obtaining the C. rigidus volatile constituents.
GC-MS analysis method
The HP-5M S quartz capillary column (30 m × 250 μm × 0.25 μm) was used with the column temperature of -60 -325 ℃ (350 ℃). The temperature program for the column oven was started with the initial temperature at 80 ℃, which was held for 0.5 min and raised at 5 ℃/min to 100 ℃, which was held for 2 min and raised at 5 ℃/min to 150 ℃, which was also held for 2 min and then increased at 5 ℃/min to 200 ℃, which was maintained for 5 min and finally raised at 5 ℃/min to 250 ℃ which was maintained for 10 min. The carrier gas was He (99.99%), and unsplit stream sampling was adopted with an inlet temperature at 230 ℃. EI ionization was carried out with an ion source temperature at 230 ℃. The MS detection was performed with the MS quadrupole temperature at 150 ℃ under the mass scanning range m/z: 30-550 u, the injection volume of 1 μ and the solvent delay of 3 min.
Statistic analysis
The gas chromatography-mass spectrometry-computer combined instrument was employed to perform analysis and identification. The total ion chromatogram (GC/MS) of the volatile constituents from C. rigidus flowers was obtained, and the chemical information represented by each peak in the chromatogram was analyzed using the software TurboMass Ver (5.4.2 version). The NIST/WIELY standard spectrum library was searched by computer for the mass spectrometry data of each component, and the volatile components of the flowers were qualitatively analyzed. The GC peak areas were subjected to normalization to obtain the relative content of each component. Results and Analysis
Main aroma constituents of C. rigidus flowers
The volatiles were analyzed according to the above GC-MS conditions to obtain the total ion chromatogram of the volatile chemical constituents of C. rigidus flowers (Fig. 1). Through mass spectrometry database retrieval, combined with relevant literatures, a total of 34 components in C. rigidus flowers were identified from the base peak, relative abundance and other aspects. According to the peak area normalization method, the relative content of each component was determined, and the results are shown in Table 1.
It can be seen from the analysis in Table 1 that 34 volatile components were identified from C. rigidus flowers. The main components were eucalyptol, aspidinol, terpilenol, vanillylacetone, α-phellandrene, 4-pyran-4-1,2,3-dihydro-3,5-dihydroxy- 6-methyl, pinene, 3-methylcatechol, naphthalene, (+)-limonene, 7-methylxanthine and (+)-aromadendrene, the contents of which were 35.39%, 10.09%, 8.86%, 4.51%, 3.25%, 2.81%, 2.75%, 2.43%, 2.29%, 1.3%, 1.12% and 1.07%, respectively, all of which accounted for 75.69% of the total relative content. Among the main volatile components, alcohols were the main volatile components in C. rigidus flowers, and had the highest relative content of 44.86%. It indicated that the components playing the main role in the aroma of C. rigidus flowers were olefins, followed by esters and alcohols［6-9］, which can be concluded from the total ion chromatogram (Fig. 1). According to the research data, monoterpenoids accounted for a larger mass fraction in this study. The volatile substances of flowers are composed of a series of low molecular volatile compounds, mainly fatty acid derivatives, benzene rings, terpenoids, nitrogenous substances and other fragrant substances［10-12］. The results of this analysis were compared with the chemical constituents of C. rigidus branches and leaves analyzed by Liu and Dong. The flowers, branches and leaves all contain pinene, α-phellandrene, eucalyptol, methyl palmitate and other compounds, but there are greater differences. Moreover, different varieties in Callistemon differ obviously in chemical components from different parts of the plants, and the growth conditions have a great influence on the plant metabolites.
Effects of aroma constituents
According to related references, α-phellandrene is an important raw material for synthesizing terpene synthetic perfumes such as geraniol and linalool. α-Pinene and β-pinene are the main chemical components of turpentine［13-14］. α-Pinene has the smell of pine and is the main substance for synthesizing sandalwood perfumes. It is mainly used to synthesize perfumes. β-Pinene is a raw material for the synthesis of various terpene perfumes. α-Terpinene has a citrus flavor and can be used to blend perfumes and essential oils such as lemon and mint［15］. γ-Terpinene can be used to make various plant perfumes［16］. Conclusions
The unique aroma of C. rigidus flower makes it stand out among the varieties. Volatile components are the material basis of the aroma of food, and multiple volatile components determine the aroma characteristics of food together. Volatile substances are an important factor affecting the flavor quality of food.
In this study, the main volatile components of C. rigidus were determined by solid phase microextraction-gas chromatography/mass spectrometry, and 34 volatile substances were identified, including alkenes, esters, alcohols and a small amount of acids, heterocycles and alkanes, the combination of which constitutes the rich aroma of C. rigidus. Its volatile components were mainly monoterpenoids, the relative content of which accounts for 84.99% of the total. The main components all have characteristic odor, which plays an important role in the normal growth and development of plants, and monoterpenoids among them can be used in food, essential oil, industry and so on［17］. Monoterpenoids are mostly volatile oily liquids with a special odor. They are widely found in plants and are the main components of perfumes, resins, pigments and the like of certain plants. For instance, rose oil, eucalyptus oil and turpentine contain a variety of terpenoids. Terpenoids also have anti-cough, wind dispelling, deworming, analgesic and other effects, and are indispensable raw materials for food and cosmetic industries.
Yanrong BAI et al. Analysis of Volatile Chemical Constituents in Callistemon rigidus Flowers by GC-MS
References
［1］SHI FP. Garden ornamental aromatic plants［J］. Southwest Horticulture, 2005(1): 33-33.
［2］LIU BM, DONG XM, LIN X, et al. Chemical components of the essential oil from Callistemon rigidus R. Br.［J］. Journal of Tsinghua University: Science and Technology, 2010, 50(9): 29-41.
［3］LI C, TAN HB, QIU SX, et al. Chemical constituents from the leaves of Callistemon viminalis［J］. Natural Product Research and Development, 2017(29): 954-958.
［4］lIU YQ. Analysis of the volatile oils from the flowers of Magnolia liliflora Desr. by GC-MS［J］. Lishizhen Medicine and Materia Medica Research, 2008. 19(8): 1911-1912.
［5］CHEN HG, YANG ZN, ZHAO C, et al. Analysis of volatile oil from the flower of Magnolia grandiflora by SPME/GC/MS［J］. Chinese Journal of Spectroscopy Laboratory, 2010, 27(4): 1440-1442.
［6］SHI L, WANG JM, KANG WY. Analysis of volatile constituents in two species of genus Magnoloia by HS-SPME-GC-MS［J］．China Journal of Chinese Materia Medica, 2008, 33(12): 1429-1433 ［7］WU FH. Research progress on new techniques of solid phase extraction［J］. Analysis and Testing Technology and Instruments, 2012, 18(2): 114-120.
［8］WANG X, TANG XW, ZHOU D, et al. Analysis of volatile compounds of lily (Lilium spp.) by SPME-GC-MS［J］. Modern Instruments, 2011, 17(5): 47-49.
［9］DAI Y, XU HR. Analysis on aromatic components of Longjing tea using SPME-GC/MS methods［J］. Tea, 2008, 34(2): 85-88.
［10］BAI SS. Applications of solid phase microextraction combined with gas chromatography technology for the determination of several organic pollutants［D］. Baoding: Hebei Agricultural University, 2013.
［11］BI SF, CHENG XX, CHEN JW, et al. Analysis of volatile components of Chrysanthemum indieum L. flowers from Huangshan by head space-solid phase micro-extraction combined with GC-MS［J］. Journal of Beijing Union University: Natural Science, 2016, 30(2): 88-92.
［12］RAN H, FENG LL, MAO YZ, et al. Identification and analysis of volatile components in essential oil from four Lauraceae wild species leaves in Chongqing by GC-MS［J］. Scientia Silvae Sinicae, 2018, 54(7): 91-103.
［13］LI L. Application of α-pinene chemical properties［J］. Guangxi Chemical Industry, 2000, 29(1): 36-38, 48.
［14］LIAO SL. Synthesis, biological activity and structure-activity relationship of novel (-)-β-pinene derivatives［D］. Beijing: Chinese Academy of Forestry, 2016.
［15］XIE ZJ, HOU YQ, WANG W, et al. The allelopathic functions of terpenoids and its application prospects［J］. Chinese Agricultural Science Bulletin, 2010, 26(24): 233-237.
［16］GU WX, DUAN SS, LUO SM. Ecological characteristic of terpenoids and their allelopathic effects to plants［J］. Journal of South China Agricultural University, 1998, 19(4): 5.
［17］LIU BM, PENG W. Component analysis of essential oil from melaleuca Leucadendron L.［J］. Journal of Instrumental Analysis, 1999, 18(6): 70-72.