海南暗羅葉揮發(fā)油化學(xué)成分及其抗菌活性

李小寶，陳光英，宋小平*，韓長日，邵泰明

1海南師范大學(xué)省部共建-熱帶藥用植物化學(xué)教育部重點實驗室;2海南師范大學(xué)化學(xué)與化工學(xué)院，?？?71158

海南暗羅葉揮發(fā)油化學(xué)成分及其抗菌活性

李小寶1，2，陳光英1，2，宋小平1，2*，韓長日1，2，邵泰明1，2

1海南師范大學(xué)省部共建-熱帶藥用植物化學(xué)教育部重點實驗室;2海南師范大學(xué)化學(xué)與化工學(xué)院，?？?71158

本文采用水蒸氣蒸餾萃取法提取海南暗羅葉揮發(fā)油化學(xué)成分，利用氣相色譜-質(zhì)譜(GC-MS)從中分離鑒定出40種化學(xué)成分，占揮發(fā)油總量的67.03%。其中主要成分為1-甲基-5-亞甲基-8-(1-甲基乙基)-1，6-二烯環(huán)十烷(18.305%)，丁子香烯(6.256%)和γ-欖香烯(6.211%)。所得的揮發(fā)油抑菌試驗表明其對大腸桿菌和枯草芽孢桿菌表現(xiàn)出顯著的抑制活性。

海南暗羅;揮發(fā)油;氣相色譜-質(zhì)譜技術(shù);抗菌活性

Introduction

Genus Polyalthia(Annonaceae)comprises of about 70 species［1］，among which only 7 are indigenous in Hainan province.including P.cerasoides(Roxb.)Benth，P.consanguinea Merr，P.laui Merr，P.nemoralis A.DC，P.plagioneura Diels，P.rumphii(Bl.ex Hensch.) Merr and P.suberosa(Roxb.)Thw.Plants of the genus is known for its folkloric applications against a number of ailments such as hepatitis，lymphadenitis，pneumonia and so on［2］.Some plants of this genus are also used in folk medicines as abortifacien，febrifuge，antimalarial and anti-fungal agents［1-3］.In our previous study against human cancer cell lines of P.laui，its ethanol extract showed that it possessed some inhibitory activities towards four human tumor cells，including BEL-7402，K562，SPCA-1 and SGC-7409［4］.However，there was no report about chemical constituents and antimicrobial activity of the oil from P.laui.In this paper，we describe the chemical composition and antimicrobial activity of the volatile oils from the leaves of P.laui extracted by water-steam distillation to provide evidences for further development and application of the Chinese traditional herbal medicine.

Materials and Methods

Plant material

The leaves of P.laui were collected from Bawangling mountain in Hainan Province，P.R.China in May 2011，and identified as P.laui by vice-professor Qiongxin Zhong from the College of Life Science in Hainan Normal University.A voucher specimen has been pre-served in the Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education，Hainan Normal University.

Microbial strains and media

The essential oils from P.laui against 3 strains of bacteria(Escherichia coli，Bacillus subtilis，and Staphylococcus aureus).All microbial cultures were maintained on nutrient agar slants and were regular subculture.The purity of cultures was checked regularly by Gram-staining and standard microbiological methods.

Essential oil extraction

The fresh leaves(495 g)of P.laui were water distilled for 6 h using simultaneous distillation extraction.The extracts were dried with anhydrous sodium sulfate for two days then concentrated to dryness in a rotary evaporator，Essential oil was obtained by the standard extracting method described in the Chinese Pharmacopoeia and kept in vials at 4℃ until GC-MS analysis.

GC-MS analysis

The essential oils were analyzed by GC /MS on a HP7890A GC coupled to a 5975C MS(Agilent Technologies，USA)equipped with a HP-5MS silica capillary column(30 m ×0.25 mm I D，0.25 μm film thickness).Carrier gas was helium with a flow rate of 1 mL/min.The injector(the spilt flow ration was 50∶1) was set to 280℃.The GC temperature program was 30℃，10℃/min to 70℃，held for 2 min.The mass spectra in the electron impact mode were recorded at 70 eV with a scan range from 35-450 amu.The components in the essential oils of fresh materials were identified by comparison of their mass spectra with those of NIST08. L library data.

Antimicrobial activity assay

The antimicrobial activities of the essential oils from P.laui by the disc diffusion method［5］.In brief，a measured amount of the test samples was dissolved in definite volumes of solvent to give solutions of known concentrations(20，40，60 mg/mL).Then，sterile filter paper discs were impregnated with known amounts of the tested the essential oils and dried.These discs were placed on plates containing agar media seeded with the test organisms.The plates were incubated at 37 oC for 24 h and the antimicrobial activities were measured by the zones of inhibition expressed in mm.Test samples showing zones of inhibition with a diameter of 5.36 mm or more were considered active.

Results and Discussion

Chemical constituents of the essential oils

Essential oils from the fresh leaves(495 g)of P.laui with water distilled were analyzed by GC/MS.The identified constituents and the irrelative percentages were shown in Table 2 and the chromatogram were showed in Fig1.

Fig.1 The total ion chromatogram of essential l oil from P.laui

Table 1 Analysis of the Chemical Composition of Essential Oil from P.laui by GC-MS Method

7 22.720［1S-(1α，3aα，3bβ，6aβ，6bα)］-decahydro-3a-methyl-6-methylene-1-(1-methylethyl)-Cyclobuta［1，2，3，4］dicyclopentene C15H24 204.188 0.443 95 8 22.901 1-ethenyl-1-methyl-2，4-bis(1-methylethenyl)-CyclohexaneC15H24 204.188 1.364 91 9 23.371 ［1aR-(1aα，4α，4aβ，7bα)］-1a，2，3，4，4a，5，6，7boctahydro-1，1，4，7-tetramethyl-1H-Cycloprop［e］azulene C15H24 204.188 0.937 99 10 23.666 Caryophyllene C15H24 204.188 6.256 99 11 23.863［3aS-(3aα，3bβ，4β，7α，7aS*)］-octahydro-7-methyl-3-methylene-4-(1-methylethyl)-1-H-Cyclopenta［1，3］cyclopropa1，2］benzene C15H24 204.188 0.323 96 12 24.000 2，6-dimethyl-6-(4-methyl-3-pentenyl)-Bicyclo［3.1.1］hept-2-ene C15H24 204.188 0.807 98 13 24.510 Z，Z，Z-1，4，7，-Cycloundecatriene，1，5，9，9-tetramethyl C15H24 204.188 2.562 98 14 24.677［1aR-(1aα，4aβ，7α，7aβ，7bα)］-decahydro-1，1，7-trimethyl-4-methylene-1-H-Cycloprop［e］azulene C15H24 204.188 0.305 99 15 24.812 2-isopropyl-5-methyl-9-methylene-Bicyclo［4.4.0］dec-1-eneC15H24 204.188 0.195 93 16 25.270 ［s-(E，E)］-1-methyl-5-methylene-8-(1-methylethyl)-1，6-Cyclodecadiene C15H24 204.188 18.305 97 17 25.794 6-ethenyl-6-methyl-1-(1-methylethyl)-3-(1-methylethylidene)-(S)-Cyclohexene C15H24 204.188 1.082 93 18 25.993 1，2，4a，5，6，8a-hexahydro-4，7-dimethyl-1-(1-methylethyl)-Naphthalene C15H24 204.188 1.001 97 19 26.203 1，2，3，5，6，8a-hexahydro-4，7-dimethyl-1-(1-methylethyl)-(1S-cis)-Naphthalene C15H24 204.188 2.600 98 20 26.363 ［1S-(1α，7α，8aα)］-1，2，3，5，6，7，8，8a-octahydro-1，8a-dimethyl-7-(1-methylethenyl)-Naphthalene C15H24 204.188 0.243 93 21 26.553 ［1S-(1α，4aβ，8aα)］-1，2，4a，5，6，8a-hexahydro-4，7-dimethyl-1-(1-methylethyl)-Naphthalene C15H24 204.188 0.374 94 22 26.839 Cyclohexanemethanol C7H14O 222.198 0.769 91 23 27.047 Cyclohexane C15H24 204.188 3.842 91 24 27.571 ［1aR-(1aα，4aα，7β，7aβ，7bα)］-decahydro-1，1，7-trimethyl-4-methylene-1H-Cycloprop［e］azulen-7-ol C15H24O 220.183 4.282 96 25 27.689 Caryophyllene oxide C15H24O 220.183 3.319 93 26 28.096 Naphthalene C15H24 204.188 0.968 92 27 28.482 Ledol C15H26O 222.198 1.490 90 28 29.001 tau-Muurolol C15H26O 222.198 1.194 96 29 29.097 1，2，3，4，4a，7，8，8a-octahydro-1，6-dimethyl-(1-methylethyl)-，［1S-(1α，4α，4aβ，8aβ)］-1-NaphthalenolC15H26O 222.198 1.300 96 30 29.294 α-Cadinol C15H26O 222.198 3.246 99 31 29.562［1S-(1α，7α，8aα)］-1，2，3，5，6，7，8，8a-octahydro-1，8adimethyl-7-(1-methylethenyl)-Naphthalene C15H24 204.188 0.830 92 32 30.215［2R-(2α，4aα.，8aβ)］-1，2，3，4，4a，5，6，8a-octahydro-4a，8-dimethyl-2-(1-methylethenyl)-Naphthalene C15H24 204.188 0.145 93 33 30.453 Isocaryophillene C15H24 204.188 0.230 90 34 30.694 7R，8R-8-Hydroxy-4-isopropylidene-7-methylbicyclo［5.3.1］undec-1-ene C15H24O 220.183 0.560 90 35 31.968 1，4-dimethyl-7-(1-methylethyl)-Azulene C15H18 198.141 0.266 99 36 32.210 1，2，3，4，5，6-Hexahydro-1，1，5，5-tetramethyl-2，4a-methanonaphthalen-7(4a-H)-one C15H22O 218.167 0.207 90 37 34.196 cis，cis，cis-7，10，13-Hexadecatrienal C16H26O 234.198 0.079 96 38 35.235 n-Hexadecanoic acid C16H32O2 256.24 0.131 99

39 37.196 Phytol C20H40O 296.308 0.044 91 40 37.680 mono(2-ethylhexyl)ester1-2-Benzenedicarboxylic acid C16H22O4 278.152 0.143 91

The oil constituents are classified into sesquiterpene hydrocarbons(44.2%)，oxygenated sesquite-rpenes (8.2%)and others.Among 40 identified constituents，representing 67.03%of the oil，1-methyl-5-methylene-8-(1-methylethyl)-1，6-cyclodecadiene(18.305%)，caryophyllene(6.256%)，γ-elemene(6.211%)were the most abundant.The oil was characterized by the high percentage of sesquiterpene hydrocarbons and oxygenated sesquiterpenes.The antimicrobial activity of the essential oil suggested this plant had the potential use as an important antimicrobial source.

Antimicrobial activity

All experiments were carried out in triplicate.The antibacterial activity was assessed by evaluating the presence of inhibition zone values.Results of inhibition zone(Table 1)showed that the essential oil of P.laui had different antibacterial activities against the three bacteria tested.The bacteriostatic test showed that the essential oil had remarkable inhibitory efffects on E.coli and B.subtilis.

Table 1 The bacterial inhibition zone of different concentrations(±s，mm，n=3)

Table 1 The bacterial inhibition zone of different concentrations(±s，mm，n=3)

Diameter(mm) Bacteria 20 mg/mL 40 mg/mL 60 mg/mL Benzylpenicillin sodium(1 mg/mL)Ethanol E.coli 15.06±1.12 17.44±1.54 22.24±2.07 14.32±0.70－B.subtilis 17.02±1.47 17.84±1.59 26.18±2.07 12.91±0.65 －S.aureus 13.56±1.16 14.72±1.23 18.13±1.98 40.29±0.50－

1 Padmaa MP，Khosa RL.Phytoconstituents from the genus Polyalthia-a review.J Pharm Res，2009，2:594-605.

2 Yao JZ，Liang HQ，Liao SX.Active constituents of Polyalthia nemoralis A.DC.Acta Pharm Sin，1994，29:845-850.

3 Han GY，Shen QH，Jiang W，et al.Study on biological activity from the three kinds of Annonaceae.Nat Prod Res Dev(天然產(chǎn)物研究與開發(fā))，1998，11:33-35.

4 Yuan Y，Huang GJ，Wang TS，et al.In vitro screening of five Hainan plants of Polyalthia(Annonaceae)against human cancer cell lines with MTT assay.Phytother Res，2011，5: 837-841.

5 Bauer AW，Kirby WM，Sherris JC，et al.Antibiotic susceptibility testing by a standardized single disc method.Am J Clin Pathol，1966，45:493-496.

October 24，2011 Accepted March 5，2012

The National Natural Science Foundation of China (81160391 and 20862005);The Natural Science Foundation of Hainan Province(211015).

Composition and Antimicrobial Activities of Essential Oil of Leaves of Polyalthia laui Merr.

LI Xiao-bao1，2，CHEN Guang-ying1，2，SONG Xiao-ping1，2*，HAN Chang-ri1，2，SHAO Tai-ming1，21Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education;Hainan Normal University;2College of chemistry and chemical engineering，Hainan Normal University;Hainan 571158，China

The essential oil obtained by water-steam distillation extraction of the leaves of Polyalthia laui was analyzed by gas chromatography-mass spectrometry(GC-MS).Forty compounds were characterized accounting for 67.03%in the oil.The major components were 1-methyl-5-methylene-8-(1-methylethyl)-1，6-Cyclodecadiene(18.305%)，Caryophyllene(6.256%)and γ-Elemene(6.211%).The bacteriostatic test showed that the essential oil had remarkable inhibitory effects for Escherichia coli and Bacillus subtilis.

Polyalthia laui Merr;essential oil;GC-MS;antimicrobial activity

1001-6880(2012)05-0590-04

*Corresponding author Tel:86-898-65889422;E-mail:sxp628@126.com

R284.2

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