CONSTITUENTS AND BACTERIOSTATIC ACTIVITY OF VOLATILE MATTER FROM FOUR FLOWER PLANT SPECIES

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1 Indian J. Agric. Res., 44 (3) : , 2010 AGRICULTURAL RESEARCH COMMUNICATION CENTRE / indianjournals.com CONSTITUENTS AND BACTERIOSTATIC ACTIVITY OF VOLATILE MATTER FROM FOUR FLOWER PLANT SPECIES Xue Meng, Zhiying Wang and Hui Lv Northeast Forestry University, Harbin , China ABSTRACT This study was conducted to identify major constituents in the volatile matter from four indoor flowering plants and to evaluate them for bacteriostatic activity of the most abundant compounds. Volatile matter was collected by a dynamic collection method from Gardenia jasminoides, Ceropegia woodii, Pilea nummulariifolli, and Rhoeo purpurea. GC-MS analyses identified that the most abundant terpene compounds were á-pinene and ocimene (14.19% of total volatile matter) in G. jasminoides, á-pinene, camphene, ocimene and eucalyptole (43.91% of total volatile matter) in C. woodii, á-pinene and camphene (45.52% of total volatile matter) in P. nummulariifollia, and á-pinene, camphene and eucalyptole (46.42% of total volatile matter) in R. purpurea. These four terpene monomers have demonstrated strong bacteriostatic activity against six bacterial cultures. The most effective was á-pinene (5μl/ml) which had 100% bacteriostatic rate on all the bacterial cultures. The least effective (34% bacteriostatic rate) was eucalyptole (10μl/ml) against Saprophytic staphylococcus. Key words: Flowering plant, Terpene, Bacterium, Bacteriostatic ability, GC-MS. INTRODUCTION Indoor flowering plants are used as an adornment all over the world, and are endowed with various cultural connotations in different regions. Besides, they are also beneficial to many human activities (Jia,1999; Wu CC and Wu WZ,2006). Bacterial contamination of indoor air is harmful for human health. During the lengthy winter in Northeast China, the poor air ventilation condition inside the houses provides ideal environment for bacteria growth (Huang et al,2007).. However, flowers maybe helpful for us to solve this annoying problem, for recent studies showed that many volatile compounds emitted by flowering plants are known to control bacterial growth(hu et al,2007). Therefore, understanding the relationship between flower volatile matters and their antibacterial activities will help to fully appreciate the contribution of ornamentals to indoor environment and human health. Volatile matter should be collected under natural condition in order to minimize interferences from organic solvents and the resultant loss of natural constituents, or addition of impurities. The dynamic top collection method followed in this study is simple, fast and reliable. The sample represents both the complete spectrum as well as the exact content of natural volatile organic compounds (VOCs) (Ki and Takeda,1999; Finch,1982; Santos et al,1990). This collection method is the most widely applied in contemporary researches, such as the study of four landscape trees. The volatile matter was analyzed by gas chromatography-mass spectrometry (GC-MS) and evaluated for bacteriostatic activity (Guo,2007). * Corresponding author: Z.Y. Wang zyw0451@sohu.com.

2 158 INDIAN JOURNAL OF AGRICULTURAL RESEARCH So far, very little is known about the bacteriostatic activity of volatile matter from indoor flowering plants (Zhang,2007; Jiang et al,2004; Liu et al,2006). In this study, four popular indoor flowering plants of Northeast China were selected to collect volatile matter, and the most abundant constituents were evaluated for bacteriostatic activity. Findings from this research can be used for selection of indoor flowering plants, utilization of plant volatile matter, and synthesis of biological bactericide. MATERIAL AND METHODS Materials Experimental plants Potted plants of Gardenia jasminoides Ellis, Ceropegia woodii Schlechte Lindl, Urticaceae Pilea Lindl and Commelinaceae Commelina Lindl, were purchased from a flower market in Haerbin, Heilong Jiang Province, China. Bacterial cultures The bacterial cultures were Bacillus subtilis, Micrococcus roseus, Pseudomonas aeruginosa, Bacillus anthracis, Staphylococcus saprophyticus, and Micrococcus luteus. Bacterial stocks were provided by the Key Laboratory of Forestry Pest Biology of China National Ministry of Forestry, Northeast Forestry University, China. Monomer compounds for bacteriostatic activity assay α-pinene was manufactured by Shanghai Chemical Reagent Factory (Shanghai, China). Ocimene (SAFC Biosciences Brand), eucalyptol and camphene were purchased from Sigma-Aldrich, USA. Experimental Methodology Collection of volatile matter and constituent analysis Samples of volatile matter were collected in June, 2008, on sunny days when the air temperature was C and humidity at 70-80%. To avoid daily environmental variation interferences, all the samples were collected between 12:00-13:00 hours. Healthy plants free of disease symptoms and injury were selected. After placing plants inside the polythene bags, the bag openings were sealed to allow the volatile matter to enrich for 15 min. The volatile matter inside the bag was then evaporated into a stainless steel tank. The control was the blank air collected from empty bags. Temperature, humidity, and light conditions were recorded at the time when the samples were collected (Zheng et al,2002). The volatile matter was then conducted into an ENTECH7100 precondensor for condensation. The constituents of the volatile matter were analyzed by GC-MS on a Shimadzu GCMS-QP5050 mass spectrometer coupled with a Shimadzu GC-17G gas chromatograph. The sample load was 400ml. The gas chromatograph was fitted with a Shimadzu Cp2Sil chromatographic column: CB (30m 0.25mm 0.25μm). Helium was used as a carrier gas at a pressure of 6kPa. The injection port was maintained at 250 C, the interface temperature 230 C; and the split ratio was Oven temperature was programmed as 0 C for 2 min, followed by increment of 5 C /min to reach 100 C (hold for 0.50 min), then increment of 20 C /min until reaching 250 C, and it was kept at 250 C for 8 min. Ionization mode was electron impact ionization (El) with electron energy 70 ev; ion source temperature 200 C. The scanning range was from 10 amu to 400amu. Mass spectra were obtained at 0.14 s. The spectra of the compounds were matched with National Institute of Standards and Technology (NIST) library. The identity of the compound was determined by the percent similarity of the spectra and also by comparing with the calibration standards. Relative content (%) of each constituent was estimated based on the retention time of the respective peak (CONG,1987; Yang et al,2006). Preparation of bacterial cultures Bacterial cultures were revived on beef cream-peptone culture agar culture medium. The cultures were diluted to 10 6 ~10 7 cfu/ml suspension with salt water (Guo, 2007).

3 Assay of bacteriostatic activity The chemicals were diluted into 8 concentrations with sterile water of containing 5% Tween 80 before being tested for bacteriostatic activity on 6 bacterial cultures. The concentrations were as following: 1.25μl/ml, 2.5μl/ml, 5μl/ml, 10μl/ ml, 50μl/ml, 100μl/ml, 200μl/ml and 500μl/ml. After mixing 1ml of the diluted solution with 9 ml beef cream-peptone agar culture medium (55 C), the medium was poured into each Petri dish to make solid agar plate. The final volatile concentration in the plate was 0.125μl/ml, 0.25μl/ml, 0.5μl/ml, 1μl/ ml, 5μl/ml, 10μl/ml, 20μl/ml and 50μl/ml ( v: v; volatile matter: medium). For bacteriostatic activity assay, an aliquot of 10μl bacterial suspension was spread evenly onto the surface of the agar medium. Three replicates were maintained for each concentration of every volatile monomer. The control was the inoculated plate containing no volatiles. The blank was the plate containing volatiles without bacterial inoculation. The cultures were incubated at 37 C for 24 hours. The antibacterial rate was estimated using the following equation: Vol. 44, No. 3, evaluate concentration effect of the four terpene monomers (P<0.05). RESULTS AND DISCUSSION Constituents of the volatile matter GC/MS analysis produced the total ion current chromatogram of VOCs for volatile samples emitted from the four flowering plants (Fig. 1). The molecular structure of each constituent was determined by matching the spectra to the database, followed by calibration to the respective standards. Each peak area was normalized to the total ion current profile to estimate content percentage of each constituent (Table 1-4). Reason peaks whose content was below 0.1%, and contained Si, Br impurities were not identification, this test got string of discontinuous peak numbers. From C. woodii, 41 peaks were isolated, of which 24 compounds were identified. Of the total volatile matter, 70.73% was terpenes (13 compounds), 5.82% was taxanes (5 compounds), and 1.52% was ketones (2 compounds). From U. Pilea, 43 peaks were isolated and 31 compounds were identified. Of the total volatile No. of bacterial colonies in control-no. of colonies in treated plate Antibacterial rate % = 100% No. of bacterial colonies in control Data analysis The peaks in the volatile matter total ion current chromatogram (profile) were identified by matching with the NIST 2.0 spectra library and following the method for structural identification (Zeng et al,2003; Zhao and Sun,2003; Ma,2002). The content percentage of each peak was estimated by area-normalization method. Peaks whose content was below 0.1%, and contained Si, Br and other impurities were excluded from further identification. The probability unit regression method in the SPSS10.0 program (Jing et al, 2008) was used to determine the median effect concentrations (EC 50 ) value of the four terpene monomers on the 6 bacterial cultures. The chi-square test was conducted to matter, 62.36% was terpenes (15 compounds), 3.34% was taxanes (8 compounds), 0.7% was ketones (3 compounds), and 0.91% was lipids (3 compounds). From C. Commelina, 39 peaks were isolated, and 26 compounds were identified. Of the total volatile matter, 64.69% was terpenes(14 compounds), 3.2% was taxanes (4 compounds), and 0.45% was aldehydes (3 compounds). From G. jasminoides, 38 peaks were isolated and 23 compounds were identified. Of the total volatile matter, 34.36% was terpenes (4 compounds), 4.36% was taxanes (7 compounds), 2.53% was lipids (5 compounds), and 0.69% was alcohols (2 compounds).

4 160 INDIAN JOURNAL OF AGRICULTURAL RESEARCH Table 1: Volatile constituents of C. woodii and their relative qualities as identified through GC-MS analysis PeakNo. Molecular formula Compounds Relative content(%) Retentiontime(min) Percentsimilarity 1 C 4 (E)-2-butylene C 8 Pentane C 6 N 2 Tetramethylethylenediamine C 7 -heptanone C 6 3-methyl pentane C 13 O 4 Di Ethyl phenylmalonate CCL 4 Carbon tetrachloride C 7 1,3,5-cycloheptatriene C 8 H 12 Cyclopentane C 7 O 5-methyl-3-hexanone C 10 Tricyclene C 10 Thujene C 10 á-pinene C 10 Camphene C 10 Sabinene C 10 â-pinene C 10 â-pinene C 10 Terpinolene C 10 O-cymene C 10 O Eeucalyptol C 10 Ocimene C 10 4-cyclohexadiene C 11 Undecane C 10 O L-Camphor The percentage of each constituent in total volatile matter was contained in Table 1to 4. In G. jasminoides, the relative content of terpenes including á-pinene and ocimene was 14.19%. In C. woodii, the relative content of terpenes including á-pinene, camphene, ocimene and eucalyptol was 43.91%, of which eucalyptol was the highest at 33.8%. In U. Pilea, the relative content of terpenes including á- pinene and camphene was 44.52%, of which 29.51% was á-pinene. In C. Commelina, the relative content of terpenes including á-pinene, camphene and eucalyptol was 46.42%, of which 31.06% was á-pinene. The most abundant terpene was á-pinene in C. Commelina, ocimene in G. jasminoides, camphene in U. Pilea, and eucalyptol in C. woodii. From the above analysis, it can be concluded that terpenes represent a large portion of the total volatiles in flowering plants. Next, the four most abundant terpene monomers were tested for bacteriostatic activity. The bacteriostatic activity of the four terpene monomers The four terpene monomers were diluted into 8 concentrations with sterile water of containing 5% Tween 80 before being tested. The data in Table 5 indicate that á-pinene had very strong bacteriostatic activity against B. subtilis, S. saprophyticus, and M. luteu. When á-pinene was applied at 1μl/ml concentration, the

5 Vol. 44, No. 3, Table 2: Volatile constituents of U. Pilea and their relative qualities as identified through GC-MS analysis PeakNo. Molecular formula Compounds Relative content(%) Retentiontime(min) Percentsimilarity 1 C 4 (E)-2-butylene C 8 Pentane C 4 H 3 CL Hexachlorobuta-1,3-diene C 8 Cis-3-Hexenyl acetate C 5 H 9 Cl Butyl-chloroformate C 8 1,1,3,3-tetramethyl-2,4-cyclobutanediol C 4 Ethyl acetate C 3 H 2 CL 4 O 1,1,3-trichloro-2-propanone C 2 H 4 CL 2 Dichloroethane C 6 H 6 2ÿ4ÿ6-cycloheptatrieN-1-one C 7 Cycloheptatriene C 10 H 20 Tert-Butylcyclohexane C 10 Tricyclene C 10 á-pinene C 10 Camphene C 10 Sabinene C 10 â-pinene C 10 Myrcene C 10 H 22 Decane C 6 H 4 CL 2 1,3-Dichlorobenzene C 10 1-Isopropyl-2-methylbenzene C 10 O Cineole C 10 1-Methyl-4-(1-methylethylidene)-Cyclohexene C 10 H 12 2ÿ4-dimethylstyrene C 10 Undecane C 9 O n-nonaldehyde C 18 Naphthalene C 12 n-dodecane C 12 Bihexyl C 15 (+)-á-longipinene C 14 H 30 Tetradecane antibacterial rate was 100%, and all the other concentration treatments over 5μl/ml produced the same result. Eucalyptol and ocimene had the best bacteriostatic effect on M. luteus. The antibacterial rate was 100% when 1μl/ml of the chemicals was supplemented in the culture medium. Eucalyptol was the least effective on S. saprophyticus, because the antibacterial rate was only 14% in the 1μl/ml eucalyptol concentration treatment. Camphene had the best bacteriostatic effect on S. saprophyticus and M. luteus. The antibacterial rate was 100% at 1μl/ml camphene concentration treatment. However, bacteriostatic activity of camphene was not as good on P. aeruginosa when the antibacterial rate was only 28% in the 1μl/ml concentration treatment.

6 162 INDIAN JOURNAL OF AGRICULTURAL RESEARCH Table 3: Volatile constituents of C. Commelina and their relative qualities as identified through GC-MS analysis PeakNo. Molecular formula Compounds Relative content(%) Retentiontime(min) Percentsimilarity 1 C 4 (E)-2-butylene C 8 Pentane C 6 H 13 N Tert-Octylamine C 8 Butyl methacrylate C 7 Cycloheptatriene C 5 H 10 4-hydroxy-3- methoxy- butyraldehyde C 10 Tricyclene C 10 á-pinene C 10 Camphene C 10 Sabinene C 10 â-pinene C 10 Myrcene C 10 H 22 Decane C 8 O n-capryl(ic) aldehyde C 10 1-Isopropyl-2-methylbenzene C 10 O Eucalyptol C 10 H 12 2,4-dimethylstyrene C 11 Undecane C 10 H 20 O n-decanal, C 10H8 Azulene C 12 Bihexyl C 12 Bihexyl C 15 (+)-Alpha-longipinene C 15 Germacrene C 12 Bihexyl C 15 Isolongifolene C 15 Longifolene C 15 Cedrene Camphene was also very effective in suppressing growth of B. subtilis and M. Roseus. Among the four monomers, á-pinene showed the highest bacteriostatic activity against S. saprophyticus, M. Roseus, B. anthracis, P. aeruginosa, and M. luteus.the antibacterial rate from the eight concentration treatments of each terpene monomer was contained in Table 6. The nonparametric test demonstrated significant differences among the 8 concentration treatments for each monomer against the six bacterial cultures (P<0.05). The median effect concentrations of the four terpene monomers(ec 50 ) Probability unit regression calculation was performed to estimate the EC 50. The EC 50 values of the four terpene monomers on the 6 bacterial cultures were shown in Table 7. Based on GC-MS analysis and bacteriostatic activity assay, volatile matter differ both qualitatively and quantitatively among the four flowering plants. C. Commelina contained the highest level of á- pinene, whereas it was ocimene in G. jasminoides,

7 Vol. 44, No. 3, Table 4: Volatile constituents of G. jasminoides a nd their relative qualities as identified through GC-MS analysis PeakNo. Molecular formula Compounds Relative content(%) Retentiontime(min) Percentsimilarity 1 C 4 (E)-2-butene C 4 H 11 N Tert-butylamine C 4 H 3 Cl 1-chloro-1- butyl-3- alkyne C 8 Acrylic acid methylbutyl ester C 8 2,2,4,4-tetramethyl-1,3-cyclobutanediol C 6 H 10 4-methyl-1,3-pentadiene C 6 H 6 Benzene C 5 H 10 Isobutyric acid methyl ester C 6 H 12 Methyl 2-methylbutyrate C 6 H 10 2-butenoic acid, ethyl ester C 10 á-pinene C 10 H 22 Decane C 10 Trans-1,2-Bis-(1-methylethenyl)-cyclobutane C 10 á-pinene C 10 3,7-dimethyl-1,3,7-octatriene C 8 Methyl benzoate C 11 Undecane C 9 O (E)-2-nonen-1-ol C 10 Naphthalene C 12 Bihexyl, C 16 H 34 Cetane; Hexadecane C 14 H 30 Tetradecane C 17 H 34 5-heptadecene camphene in U. Pilea, and eucalyptol in C. woodii. The levels of bacteriostatic activity varied significantly among the four terpene monomers (Chen et al,1998; Ma,1985; Xie et al,1999; QI et al,2000). S. saprophyticus and M. luteu were the most susceptible bacterial cultures to á-pinene, B. subtilis for camphene, M. luteus for ocimene, and M. luteus for eucalyptol. Overall M. luteus is more sensitive to the four terpene compounds. This study evaluated the four terpene monomers for bacteriostatic activity against six common bacterial cultures. More studies are needed to validate similar function on other organisms such as fungi, actinomycetes and virus. It is important to understand the associated mechanism in order to fully utilize plant volatile compounds as biological agents. This study used GC-MS analysis to identify constituents of the volatile matter. The identified molecules were analogs to the known molecules. Commercial chemicals, á-pinene, camphene, ocimene and eucalyptol, were used in bacteriostatic activity assay. Whether these molecules have exactly the same function as the counterparts in natural plant volatiles also needs to be clarified. Moreover, only the bacteriostatic activity of the four most abundant constituents were evaluated in this study. The whole spectrum of volatile matter may have additional function, which will be investigated in future study.

8 164 INDIAN JOURNAL OF AGRICULTURAL RESEARCH Table 5: The bacteriostatic activity of four terpene monomers on six bacterial cultures Terpene Concentration(μl/ml) Colony inhibition (%) monomers B. subtilis M. Roseus P.aeruginosa B. anthracis S.saprophyticus M. luteus α-pinene Eucalyptole Ocimene Camphene CONCLUSIONS In the volatile matter from the four flowering plants, terpenes were the most abundant compounds. Terpene content was 18.54% in G. jasminoides, 70.73% in C. woodii, 62.36% in U. Pilea, and 63.46% in C. Commelina. Content of á- pinene was 0.31%, 5.89%, 29.51% and 31.06% in G. jasminoides, C. woodii, U. Pilea, C. Commelina, respectively. Ocimene was only detected in G. jasminoides and C. woodii, while camphene in C. woodii, U. Pilea and C. Commelina, eucalyptol in C. woodii and C. Commelina.

9 Vol. 44, No. 3, Table 6: The nonparametric tests of bacteriostatic rate of four terpene monomersmonomers on six bacterial cultures-chi-square tests) Terpene monomers Chi-square B. subtilis M.Roseus P. aeruginosa B. anthracis S. saprophyticus M. luteus α-pinene x 2 = x 2 = x 2 = x 2 = x 2 = x 2 = P<0.05 P<0.05 P<0.05 P<0.05 P<0.05 P<0.05 Eucalyptol x 2 = x 2 = x 2 = x 2 = x 2 = x 2 = P<0.05 P<0.05 P<0.05 P<0.05 P<0.05 P<0.05 Camphene x 2 = x 2 = x 2 = x 2 = x 2 = x 2 = P<0.05 P<0.05 P<0.05 P<0.05 P<0.05 P<0.05 Ocimene x 2 = x 2 = x 2 = x 2 = x 2 = x 2 = P<0.05 P<0.05 P<0.05 P<0.05 P<0.05 P<0.05 Table 7: The median effect concentrations (EC 50, μl/ml) of the four terpene monomers monomers on 6 bacterial cultures Terpene B. subtilis M.Roseus P. aeruginosa B. anthracis S. saprophyticus M. luteus 95%CI 95%CI 95%CI 95%CI 95%CI 95%CI Camphe Ocimen Eucalypto Alph-Apinen * 95% CI stands for 95% confidence interval.

10 166 INDIAN JOURNAL OF AGRICULTURAL RESEARCH Fig.1: Total ion current chromatogram (GC-MS) of the volatile matter from 4 flowering plants The bacteriostatic activity of á-pinene, camphene, ocimene and eucalyptol on the six bacterial cultures M. luteus is the most sensitive bacterium to terpenes; the antibacterial ratio was 100% when treated by the four terpene monomers at 1μl/ml concentration. S. saprophyticus showed the strongest resistance to eucalyptol, the antibacterial ratio was 34% at 10μl/ml concentration. á-pinene had the strongest bacteriostatic activity, 100% antibacterial ratio of the six bacterial cultures was achieved at the 5μl/ml concentration. REFERENCE Chen Z.X. et al. (1998) Chin. L.A. 14(56):51-54 CONG P. Z.( 1987) In: Science Press, Beijing,

11 Vol. 44, No. 3, Finch S.(1982) In: Springer Verlag,New York Inc Guo A.J.(2003). Nor Hor. 6:36-37 Guo A.J.(2007) Ph.D. Thesis, Northeast Forestry University, Heilongjiang. Hu R.H. etal.(2007) A H Ag Sci. 35: ÿ9131 Huang L.et al. (2007) TJU Med Sci. 28: Jia Y.Y.( 1999) In :Chinese Friendship Publication Company,Beijing, 258 Jiang J.H. et al. (2004) J S Fores Sci & Technol. 31:7-12 Jing T Z. etal.(2008) In: Northeast Forestry University Publishing House, Heilongjiang, Ki K and Takeda. (1999) En Technol. 30: Liu Q. et al.(2006) J S Fores Sci & Technol. 3:2-3 Ma X.J. (1985) En Sci. 6: Ma L.D.(2002)In: Fudan University Press, Shanghai, QI J.Z. etal. (2000) Ur En Ur Ecol.13:36-38 Santos F.D. et al. (1990) Rev. Cien.Farm.12:39-46 Wu Cc. and Wu WZ.(2006) In: Chinese Forestry Publishing houseÿbeijingÿ2-4 Xie H.L. et al. (1999) H N Ar UN. 33: Yang B. et al.(2006) Chin Mat Med. 24: Zeng Z.etal. (2003) An Chem. 31: Zhang W.(2007) Ph.D. Thesis, Hunan University, Hunan,134 Zhao Y. X. and Sun X. Y.(2003) In: Science Press, Beijing, Zheng H.etal. (2002) PR FORES TECHNOL. 5:30