Marchantiaceae plants are well-known traditional Chinese medicinal herbs and extensively used to treat tumefaction of skins, protect liver and treat hepatitis and used as antipyretic in countryside (Chen and Xiao, 2005; Xiao et al. 2005a; Xiao et al. 2005b; Zhu et al. 2005). There are a large number of Marchantiaceae plants in Guangxi Zhuang Autonomous District such as Marchantia polymorpha, M. convoluta and M. paleacea. These species live in together and it is difficult to distinguish one from the others because of their genetic similarity.

Compared to M. polymorpha, M. convoluta is quite rare and was thought of negligible by people many years ago. The major identified constituents in M. convoluta were flavonols, triterpenoids, and steroids (Zhu et al. 2003; Cao et al. 2005; Chen and Xiao, 2005; Xiao et al. 2005a; Xiao et al. 2005b; Zhu et al. 2005; Chen and Xiao, 2006; Xiao et al. 2006a). The flavonoids of M. convoluta mainly consist of quercetin, luteolin, apigenin and their O- and C-glycosides (Chen and Xiao, 2005; Xiao et al. 2005a; Xiao et al. 2005b; Zhu et al. 2005; Chen and Xiao, 2006; Xiao et al. 2006b). Dried leaves are used in China to protect livers and to treat tumefaction of skins. A high dosage of flavonoids from M. convoluta (20 and 40 µg/ml) could significantly reduce the activity of ALT (Alanine aminotranferease) and AST (Alanine aminotranferease) in the serum of mice with acute hepatic injury caused by CCl4 and increase the contents of TP (Total protein) and ALP (Alkaline phosphatase), as well as inhibit the auricle tympanites of mice caused by dimethylbenzene. Flavonoids from M. convoluta strongly inhibited colibacillus, tyhoid bacillus, Staphylococcus aureus, bacillus enteritidis, hemolytic streptococci type B and Diplococcus pneumoniae and possess distinct effect of antibiosis, anti-inflammation and diuresis in mice (Xiao et al. 2005a), as well as flavonoids from M. convoluta has anti-hepatitis B Virus activity (Xiao et al. 2005b). Extracts from M. convoluta stronglyinhibit tumors in human liver and lung cancer cell lines (Chen and Xiao, 2006; Xiao et al. 2006a).

A Suprex MPS/225 system (Pittsburgh, PA) in the SFE mode was used for all the extractions. The extraction vessel was a 10 ml stainless steel vessel. Supercritical fluid extractions were conducted at pressures of 5, 10, 15 and 20 MPa and temperatures of 35, 45, 55 and 65ºC for a duration of 20 min, static, followed by 15, 25, 35, or 45 min, dynamic. A Durafow manual variable restrictor (Suprex) was used in the SFE system to collect the extracted analytes. The essential oil was extracted from the plant using supercritical CO₂ under various conditions according to the Taguchi method. Since various parameters potentially affect the extraction process, the optimization of the experimental conditions represents a critical step in the development of a SFE method. In fact, pressure and temperature of the fluid, percentage of the modifier and the extraction times are generally considered as the most important factors. The optimization of the method can be carried out step-by-step or by using an experimental design. The result shows different conditions of experiments carried out with SFE for extractions of M. convolutaaccording to the Taguchi experimental design. All the selected factors were examined using a four-level orthogonal array design with an L₄16 (4⁴) matrix. In general, a full evaluation of the effect of four factors from three levels on the yield needs 256 (4⁴) experiments. In order to reduce the number of experiments, a L₄ (4⁴) orthogonal design graph was used, reducing the number of experiments to 16. The extraction yields were 0.87-4.69%.

The mean values of the extraction yields for the corresponding factors at each level were calculated according to the assignment of the experiment. For example, the extraction yields of the four trials at 15 MPa were evaluated as mean values of the corresponding four runs. The mean values of the four levels of each factor (e.g., pressure) reveal how the extraction yield changes when the level of that factor is changed. For the complete recovery of the main components of the plant, higher pressures are necessary. This is because raising the extraction pressure, at constant temperature, leads to higher fluid density, which increases the solubility of the analytes. To obtain quantitative recovery of analytes, they must be efficiently partitioned from the sample matrix into the supercritical fluid. The influence of temperature on the composition of the extracts was studied. Higher temperature resulted in lower extraction yield. Higher temperature can decrease fluid density and thus reduce extraction efficiency. For all the analytes, the volume of the modifier was found not to be a significant parameter. The influence of the dynamic extraction time on the composition of the extracts was studied. Extraction was performed with supercritical carbon dioxide at the static extraction step of 20 min, followed by 15, 25, 35 and 45 min of dynamic extractions. Results showed that increasing dynamic extraction time to 35 min enhanced the extraction of most components. Thus, the best conditions, obtained by preliminary test, for the extraction of oil were: extraction temperature: 35ºC; dynamic time: 35 min;pressure: 15 Mpa and modifier volume, 40 ml.

The compounds from the oil produced by SFE were identified and quantified by GC-MS. GC separation gave 50 peaks, from which 46 were identified by MS library matching. The major compounds identified in SFE extract were:benzothiazole (11.82%), 2-ethylhexanoic acid (9.82%), ethylphenoxybenzene (8.99%), acetic acid octadecyl ester (8.82%), 4-cyanothiophenol (5.49%), cedrol (4.60%), 9,12-octadecadienoic acid ethyl ester (3.25%), 2(3H)-benzothiazolone (2.79%), octadecanoic acid ethyl ester (2.39%), n-hexadecanoic acid (2.08%), 1,1'-(3-methyl-1-propene-1,3-diyl) bis-Benzene (2.07%). The total content of organic acids and esters was 32.19%.

Different methods of natural products extraction yield different efficiencies Several reports about compositions of the extract from M. convoluta were reported. Chen and Xiao (2005) separated and determined flavonoids of M. convoluta by RP-HPLC. Cao et al (2005) extracted bioactive components from M. convoluta with 80% ethanol. The extract was suspended in water and extracted with petroleum ether, EtOAc and n-BuOH successively. The petroleum ether extract and EtOAc extract were analyzed by capillary gas chromatography with mass spectrometric detector. The results were different from each other because of different methods dealing with the extract.As shown in the table 2 and discussed by Cao et al. (2005), the composition of the SFE products and the extracts extracted by petrol ether and ethyl acetate are different. Higher levels of ester (accounting for 57.21%) were found in the extracts extracted by petrol etherwhile higher levels of terpenes and derivatives were found in the SFE product. The benzothiazole content in the SFE extract is considerable (11.82%) andthe organic acids and esters accounted for 32.19 %.This is similar to report by Cao et al. (2005). On the other hand,Cao et al reported that higher benzothiazolecontent(14.97%) in the ethyl acetate extract whileorganic acids and esters accounted for 36.01% in the petrol ether extract extracted. Cao et al also reported that a phytol content of 6.32% in the petrol ether extract, whereas it was not found in the SFE products.

The authors wish to thank Jiangsu Provincial Key Laboratory of Coastal Wetland Bio-resources and Environmental Protection, Yancheng Normal College.

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