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       【摘要】 
       ObjectiveThe volatile components of Perilla frutescens were extracted by SPME and components separated were identified by gas chromatography (GC-MS) . MethodsFirst, the solid-phase microextraction(SPME) method was used to extract the volatile oils from Perillafrutescens, then the active ingredients of the volatile oil was determined by GC-MS .Results56 volatile compounds were identified, monoterpene , sesquiterpene, fatty acid and fatty acid esteer were predominant in the essential oil of P. frutescens. 2-Hexanoylfuran compound represented about 69.88% of the total essential, respectively, which was probably the main cause of the strong odor of P. frutescens.ConclusionSPME-GC-MS method is a simple, sensitive , and rapid method and suitable for the analysis of volatile constituents from P. frutescens.
       【关键词】  SPME; GC-MS; Volatile oil; Perilla frutescens
       DOI Logo：doi:10.3969/j.issn.10080805.2010.07.080
       Clc number:R284.2 Document code:A Article ID:10080805(2010)07173503
       Perilla frutescens is edible and medicinal. The leaves have a very pleasant sweet taste and are used as a spice, cooked as potherbs or fried. The volatile compounds of P. frutescens, which display a wide range of pharmacological activities, including antiasthmatic, antibacterial, antidote, antimicrobial, antipyretic, antiseptic, antispasmodic, antitussive, aromatic, carminative, diaphoretic, emollient, expectorant, pectoral, restorative, stomachic and tonic [1～6], so it has played an important role in clinical therapy. In addition, presents high pharmacological activity, low toxicity and rare complication , more and more interests have been attracted in recent years. The study sims to analyze the components in P. frutescens by SPME-GC-MS.
       1 Introduction
       Perilla frutescens is Annual herb of the mint family native to E. Asia, it is a traditional crop of China, India, Japan, Korea, Thailand, and other Asian countries. It"s deep purple stems and purple to red tinted leaves last all summer and fall. It is a very aromatic plant, with a strong minty smell.
       According to Makino, Furuta[7]， P. frutescens would be prescribed in Saiboku-to to exhibit other pharmacological effects than its anti-allergic activity, such as a sedative.The plant constituents confirm these uses in alternative medicine and ongoing studies have revealed that this plant is useful in curing many cancers as well as various other diseases and disorders. Further research has isolated such constituents as apigenin, ascorbic-acid, beta-carotene, caffeic-acid, citral, dillapiol, elemicin, limonene, luteolin, myristicin, perillaldehyde, protocatechuic-acid, quercetin, rosmarinic-acid, and more numerous to mention. Perilla seed oil has been used in paints, varnishes, linoleum, printing ink, lacquers, and for protective waterproof coatings on cloth. Volatile oils of the plant are also used in aroma therapy and for perfume.
       However, the volatile components of P. frutescens make the measurement of the volatile constituents as well as quality control of crude drugs and their medical preparations extremely difficult. Traditionally, the analysis of volatile compounds from traditional Chinese medicines (TCMs) is usually preceded by the extraction of essential oil by steam distillation,which often requires a large amount of sample and takes several hours to complete. The complex and time-consuming process for the preparation of samples sometimes further complicates the analytical results due to more influencing factors involved.Solid-phase microextraction (SPME) developed by Pawliszyn and coworkers in 1989 SPME proves to be a very sensitive and also low-cost,rapid method,which integrates sampling ,extraction and concentration of sample components,and sample introduction into a single solvent-free step and by using one device.SPME techniques offer a useful alternative to conventional techniques.So it is a solventless extraction technique widely used in application of extraction from plants, food, biological and environmental samples [8～13]. Gas chromatography is also a rapid way of analyzing volatile compounds in TCMs. The analytical process of GC chromatography involves the volatilization of the volatile components in the plant in a heater and the entrance into the GC column for the following chromatographic separation.With this method, the ground powder of a TCM is material extraction by SPME which can be directly applied for the analysis. Furthermore,the amount of sample used is greatly reduced and only a few grams of the ground powder of a TCM material are needed, which greatly simplify and shorten the analytical time for volatile compounds from TCMs.
       Gas chromatography hyphenated with mass spectrometry (GC/MS) is a commonly used method for characterization and identification of volatile organic compounds in complex mixtures. Some publications are available concerning the essential oil composition of Perilla by GC-MS [14～17], in which the essential oils were extracted by steam distillation and identified by GC-MS.However, little has been reported concerning the volatile compounds from Perilla after extraction by solid-phase microextraction. SPME-GC-MS is of great significance for the rapid identification and quality control of TCMs. The simplicity and the high sensitivity of the procedures render them the desirable techniques for the analysis of volatile compounds.
       2 Experiment
       2.1 MaterialSamples of wild P. frutescens were obtained commercially from Jiang Province,China in August 2007. Samples were air dried,ground in a high-speed rotary cutting pulverizer, and then screened to give fractions 75 ?m in size.
       2.2 Extraction of volatile constituentsThe manual SPME holder was used with a 100m polydimethylsiloxane fibre assembly (Supelco,Bellefonte,USA).Before use, the fibre was conditioned as recommended by the manufacturer. Prior to extraction,the fiber was activated for 30 min in the GC QP2010(SHIMAZHU, Japan) with an injector temperature of 250℃. The sample (0.5 g) of 40 mparticle size was hermetically sealed in an 5 ml vial, then the SPME fibre was explosed in the headspace of sealed for 30 min at 80℃ to absorb the analytes. Only that part of the vial with the solid matrix was submerged, to keep the SPME fibre as cool as possible to improve the vapour phase/absorbent fibre coating partition coefficient.After that ,the fiber was withdrawn and directly inserted into the GC-MS inlet for desorption of the volatiles for 3 min.
       GC-MS analysis was performed on a Shimadzu GC-2010 gas chromatography instrument coupled to a Shimadzu QP2010 mass spectrometer (Compaq-Pro Linear data system, class5k software), equipped with a HP-5 capillary column (30 m×0.25 mm I.D., film thickness 0.25 mm).The column was maintained at 40°C for 3 min, programmed to 100°C at a rate of 5°C/min, held for 1 min, then programmed to 160°C at a rate of 3°C/min, then held for 1min, and programmed to 220°C at a rate of 6°C/min, held for 16 min. The temperature of the injection port and interface was set at 260°C. Helium was used as the carrier gas with a flow rate of 0.76 ml/min. One microlitre of the samples was injected in the 1:10 split mode. The mass spectrometer was operated under electron impact (EI) mode at ionization energy of 70 eV and the scan rate was 0.5 scan/s. The mass spectrometer was operated with a scan mass range of 33 to 450 atomic mass units. The ionization source temperature was 260°C. The volation constituents (tabe 1) were identified using the NIST Mass Spectral Database and comparison of their linear retention indices (relative to C6-C24 alkanes on the HP-5 capillary column). The percentage composition of the volatile was computed from the GC peak area normalization without any corrections.
       3 RESULT and DISCUSSION
       3.1 Chemical Composition of the Essential OilThe analysis was performed under the analytical conditions indicated above directly. Fig. 1 illustrates GC-MS chromatograms resulting from the SPME sampling.
       Table 1 percentage Composition of Essential Oils of P. frutescens
       NOCompoundsRT%peack area1Butanal, 3-methyl-3.0930.112Butanal, 2-methyl-3.2250.0232,4-Hexadienal, (E,E)-3.7580.074Hydrogen azide4.2740.0151-Butanol, 3-methyl-4.6040.016Acetic acid, butoxyhydroxy-, butyl ester5.4110.007Hexanal6.2780.0582-Propanol, 1-(1-methylethoxy)-6.5810.0892-Hexenal7.9710.06101-Hexanol8.4790.01111,2,4,5-Tetrazine, 3,6-dipropyl-9.5350.0012alpha.-Pinene10.5400.1013Camphene11.1140.0414Pentane, 2,4-dimethyl-2-nitro-11.4230.0115Benzaldehyde11.6030.1216Bicyclo[3.1.0]hexane, 4-methylene-1-(1-methylethyl)-11.8800.0617beta.-Pinene12.0540.20181-Octen-3-ol12.1390.15195-Hepten-2-one, 6-methyl-12.3050.0920beta.-Myrcene12.4120.13213-Octanol12.7010.1122Cyclohexene, 1-methyl-4-(1-methylethenyl)-, (S)-13.7610.0823Benzeneacetaldehyde14.3230.0424trans-Linaloloxide15.1350.02251,6-Octadien-3-ol, 3,7-dimethyl-16.0937.2226Nonanal16.2480.0527Pivalic acid vinyl ester16.7460.01284-Octanone17.7490.0229Docosanoic acid 1-methyl-butyl ester18.5600.03303-Cyclohexen-1-ol, 4-methyl-1-(1-methylethyl)-, (R)-18.9920.0231Methyl Salicylate $$ Benzoic acid, 2-hydroxy-, methyl ester19.4360.0532p-menth-1-en-8-ol19.5350.03332,2-Dimethyl-3-heptanone19.9291.0334erythro-9,10-Dibromopentacosane20.4760.05354,7-Methanobenzofuran,2,2"-oxybis[octahydro-7,8,8-trimethyl-,20.6411.01362-Hexanoylfuran21.73069.8837erythro-9,10-Dibromopentacosane22.8930.08382-Undecanone23.1320.30394-Chloro-3-(2-furoyloxy)phenyl 2-furoate23.1970.42404-(2-Methylcyclohex-1-enyl)-but-2-enal23.5405.84412,6-Octadienoic acid, 3,7-dimethyl-, methyl ester24.2290.1642Phenol, 2-methoxy-4-(1-propenyl)-25.5220.02432,6-Octadien-1-ol, 3,7-dimethyl-, acetate26.4370.0244Cyclobuta[1,2:3,4]dicyclopentene, decahydro-3a-methyl-6-methylene-1-(1-methylethyl)-, [1S-(1.alpha.,3a.alpha.,3b.beta.,6a.beta.,6b.alpha.)]-26.7720.02451,7-Octadiene, 2-methyl-6-methylene-26.9560.02
       Table 1
       NOCompoundsRT%peack area46Bicyclo[7.2.0]undec-4-ene, 4,11,11-trimethyl-8-methylene-28.2134.49473,5-Dinitrobenzoic acid, 3,7,11-trimethyldodeca-2,6,10-trienyl ester29.5980.51481,6-Cyclodecadiene, 1-methyl-5-methylene-8-(1-methylethyl)-, [s-(E,E)]-30.5890.2249trans-.alpha.-Bergamotene30.8512.19501,3,6-Heptatriene, 2,5,6-trimethyl-31.1580.0951alpha.-Farnesene31.3790.13521,6,10-Dodecatrien-3-ol, 3,7,11-trimethyl-, [S-(Z)]-33.5620.4053Caryophyllene oxide34.5091.0154Hexadecanoic acid, methyl ester44.1360.34559,12-Octadecadienoic acid, methyl ester 47.3770.62569-Octadecenoic acid, methyl ester, (E)-47.5212.15
       Fig.1 Typical chromatograms of volatile components of P. frutescens
       The result obtained from qualitative analysis of the essential oils are shown in Table 1.The chromatographic profile of P. frutescens reveal that it contained 56 volatile compounds. The monoterpene , sesquiterpene, fatty acid and fatty acid esteer predominant in the essential oil of P. frutescens. The main components were 1,6-Octadien-3-ol, 3,7-dimethyl(7.22%), 2-Hexanoylfuran (69.88%), 4-(2-Methylcyclohex-1-enyl)-but-2-enal (5.84%), Bicyclo[7.2.0] undec-4-ene, 4,11,11-trimethyl-8-methylene- (4.49%), trans-.alpha.-Bergamotene (2.91%), Caryophyllene oxide (1.10%), 9-Octadecenoic acid, methyl ester, (E)- (2.15%). By comparision of our result with the available literature regarding the volatile of P. frutescens.There was singnicant difference. It was estimated that the geographical environment, climate and different methods for extraction were leading affection factor.Such as,the 2-Hexanoylfuran compound represented about 69.88% of the total essential, which was probably the main cause of the strong odor of perilla frutescensby.
       4 CONCLUSION
       In summary,this paper describes the volatile components of P. frutescens are extracted by SPME ,which include many advantages such as simple, sensitive ,mild conditions and rapid method suitable for the analysis of volatile constituents from P. frutescens.We are promising for its application to other traditional Chinese medicines.
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