Chemical Composition and Antibacterial Activity of Essential Oils from Different Parts of Leonurus japonicus Houtt.

The herb and fruits of Leonurus japonicus Houtt., named “Yimucao” and “Chongweizi”, respectively, in Chinese, have been widely used in China as gynecological medicines. The components of the essential oils obtained by hydrodistillation were investigated by GC-MS. The antibacterial activity of the essential oils was determined by micro-dilution assay. The results showed large variations in the chemical composition and antibacterial activity of the oils. The oil of “Yimucao” showed antibacterial activity against various Gram-positive bacteria and consisted mainly of sesquiterpenes and diterpenes, with phytone, phytol, caryophyllene oxide and β-caryophyllene being the most significant constituents, whereas the oil of “Chongweizi”, mainly made up of bornyl acetate and aliphatic hydrocarbons, was inactive in the antibacterial assay. Further study of the main compounds in “Yimucao oil” showed that β-caryophyllene had wide-spectrum activity against Gram-positive bacteria.

Thus, according to the GC and CG/MS analyses, the chemical components of "Yimucao oil" and "Chongweizi oil" were quite different. The "Yimucao oil" was mainly comprised sesquiterpenes and diterpenes, while "Chongweizi oil" was rich in aliphatic compounds, particularly aliphatic hydrocarbons. The result suggested that the significant difference in antibacterial activity of essential oils was relevant to the large variations in the composition.

Antibacterial Activity of the Main Compounds in "Yimucao Oil"
Further investigation on the chemical constituents of "Yimucao Oil" resulted in the isolation and identification of caryophyllene oxide (18) [11] and phytol (38) [12]. Their structures were elucidated by spectroscopic analyses, including HR-ESIMS and NMR techniques. Subsequent study on the antibacterial activity of the main compounds 17, 18 and 38 showed their MIC values against nine bacterial strains (Table 3).

Discussion
"Yimucao" and "Chongweizi", derived from different parts of L. japonicus, are two common drugs in Traditional Chinese Medicine. This work provided the first report of the comparison of the chemical composition and bioactivity between "Yimucao" and "Chongweizi". GC-MS analyses of the volatile constituents showed large variations between "Yimucao oil" and "Chongweizi oil". The most representative compounds in "Yimucao oil" were sesquiterpenes (45.37%) and diterpenes (32.77%), followed by monoterpenes (7.82%) and a small amount of aliphatic compounds (3.10%). However, "Chongweizi oil" is predominantly composed by aliphatic compounds (43.04%) and monoterpenes (15.11%), followed by sesquiterpenes (10.45%) and diterpenes (8.45%) represent the minor groups in this mixture. The results indicated that the variability in the composition of essential oils depend essentially upon the medicinal parts and the harvest season [13,14].
Then the essential oils also showed a very diverse antibacterial effect. The "Yimucao oil" exhibited antibacterial activity against several Gram-positive bacteria, while the "Chongweizi oil" was inactive in the assay. According to the above results, we could infer that the antibacterial activity of essential oil from L. japonicus may be due to the abundant sesquiterpenes and diterpenes rather than aliphatic compounds, especially due to caryophyllenes (accounting for 27.76% of the total oil). Although the "Chongweizi oil" showed no effect against the bacterial strains in our study, innovative researches about "Chongweizi" have led to the purification of two antimicrobial proteins [9,10]. These implied that "Chongweizi" is also an effective antibacterial drug, and the activity may be related to the rich proteins instead of essential oil.
Further investigation on the isolated compounds of "Yimucao oil" showed that β-caryophyllene had favorable antibacterial activity against Gram-positive bacteria, with MICs from 0.032 to 0.256 mg/mL and MBCs from 0.064 to 0.256 mg/mL. The results were consistent with the earlier reports [15][16][17]. In particular, β-caryophyllene was reported to show significant antibacterial activity against E. faecium and E. faecalis, the MICs (each 0.025 mg/mL) in the previous investigation were very similar to those in this study (each 0.032 mg/mL). In addition, the antibacterial assay displayed that β-caryophyllene was inactive against Gram-negative bacteria, including Enterobacter cloacae, Escherichia coli, Moraxella catarrhalis, Klebsiella pneumoniae and Pseudomonas maltophila, whereas some reports found β-caryophyllene to be active against other Gram-negative bacteria strains, such as Achromobacter xylosoxidans denitrificans, Escherichia coli, Chryseobacterium indologenes, Citrobacter freundii and Flavimonas oryzihabitans. However, the MICs against E. coli were obvious different among the reports (0.625-12.8 mg/mL) [15][16][17][18][19]. This may be the consequence of a problem with the solubility of β-caryophyllene, which we also experienced during our study.

Plant Material
The herb of L. japonicus ("Yimucao") was collected in May of 2012 from the field in Wenjiang District, Chengdu City, Sichuan Province, China. The ripe fruits of L. japonicus ("Chongweizi") were gathered in August of 2012 from the same field. Plant identity was verified by Prof. Min Li (Chengdu University of TCM, Sichuan, China). Voucher specimens were deposited at the School of Pharmacy, Chengdu University of TCM.

Volatile Oil Extraction
Air dried and powdered herb (5 kg) and fruits (5 kg) were separately subjected to hydrodistillation for 10 h using a modified Clevenger type apparatus with a water-cooled oil receiver to obtain the essential oils. The oils were dried over anhydrous sodium sulfate and kept in air tight glass bottles in a refrigerator for further experiments.

Antibacterial Activity Experiments
Three bacterial strains, S. aureus (ATCC25923), methicillin-resistant S. aureus (ATCC43300) and  Table 1). The in vitro antibacterial activity was determined by the standard agar dilution method, according to NCCLS (National Committee for Clinical Laboratory Standard) [20]. Volatile oil was dissolved in DMSO to a concentration of 3.2 mg/mL, and then distributed at various concentrations in triplicate with a volume of 100 μL in turbidity tubes. Bacterial suspension (5 μL) with a density of 1 × 10 6 CFU/mL in Mueller-Hinton (MH) agar was added to each tube. Positive control was a suspension of bacteria in 1 mL of MH agar, and negative control was medium without bacteria. The MIC values (minimum inhibitory concentration at which the microbes failed to grow into a visible spot) were determined after incubation at 37 °C for 24 h.

Analysis of Components
An Agilent 7890A/5975C Gas Chromatography-Mass Spectroscopy system equipped with GC-MSD was used for analysis with ionization achieved by electron impact at 70 eV. The HP-5 MS quartz capillary column (30 m × 0.25 mm, 0.25 μm film thickness) was used with helium (purity 99.999%) as carrier gas at a flow rate of 1.0 mL/min. Experimental conditions for GC analysis of volatile oil were: injection port temperature, 280 °C; column oven temperature, 100 °C for 5 min and programmed at 4 °C/min to 160 °C, kept constant at 160°C for 10 min, then programmed at 5 °C/min to 240 °C; 1 μL injection volume, and split ratio adjusted at 20:1. The mass spectrum of each peak was recorded in the total ion current mode of the mass spectrometer within a mass range of 20 to 800. Identification of oil constituents was achieved using NIST08 mass spectral database.

Conclusions
It can be concluded that β-caryophyllene is in fact the main antibacterial component in "Yimucao oil". However, it is very difficult to attribute the biological activities of a total essential oil to one or a few active principles, because the minor compounds also may be effective. In addition, it is known that the synergistic or antagonistic effect of a compound present in minor percentage in a mixture has to be considered as well [21]. Thus, further extensive studies on the antibacterial activity of "Yimucao oil" are necessary.