Supercritical fluid CO2 extraction of essential oil from Marchantia convoluta: global yields and extract chemical composition
Keywords: GC-MS, Marchantia convoluta, supercritical fluid extraction, yields.
The essential oil of Marchantia
convoluta was obtained by supercritical (carbon dioxide) extraction
using methanol as a modifier. Global yields were determined according
to the orthogonal design. The effects of different parameters, such
as pressure, temperature, modifier volume and extraction time, on
the supercritical fluid extraction (SFE) of essential oil from M.
convoluta were investigated. Maximum global yields were obtained
using the following conditions: extraction temperature,
plants are well-known traditional Chinese medicinal herbs and extensively
used to treat tumefaction of skins, protect liver, treat hepatitis
and used as antipyretic in countryside (Chen and Xiao,
2005; Xiao et al. 2005a; 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 be distinguished one from the others because
of their genetic similarity. M. convoluta was only found in
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 (Cao et al. 2005; Chen
and Xiao, 2005; Chen and Xiao, 2006; Xiao
et al. 2005a; Xiao et al. 2005b; Xiao
et al. 2006a; Zhu et al. 2005; Zhu
et al. 2003). 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
The extraction of essential oil components using solvent at high pressure, or supercritical fluids (SCF), has received much attention in the past several years, especially in food, pharmaceutical and cosmetic industries, because it presents an alternative for conventional processes such as organic solvent extraction and steam distillation (Fekete et al. 1996; Assis and Lanças, 1999; Doraiswamy et al. 1999; Eikani et al. 1999). Supercritical fluid extraction allows a continuous modification of dissolution power and selectivity by changing the solvent density. It has the density of a liquid and solubilizes solids like a liquid solvent, but has a diffusion power similar to a gas and permeates through solid materials very easily. The power of solubilization increases with the density of the fluid; high densities of a supercritical fluid are possible at high pressures and allow it to dissolve large quantities of organic compounds. The dissolved compounds can be recovered from the fluid by reduction of its density, by means of decreasing the pressure or increasing the temperature. This low temperature separation process prevents the degradation of the chemical compounds of the extract due to heat, as in steam distillation (Anitescu and Doneanu, 1998; Lanças and Sargenti, 1998; Gamiz-Gracia and Luque, 2000; Michielin et al. 2005; Raeissi and Peters, 2005; Sovová, 2005).
An essential drawback in the use of supercritical CO2 is its low polarity, making the extraction of polar analytes difficult. Nevertheless, this limitation may be overcome by adding small amounts of polar modifiers, such as methanol or ethanol to the supercritical CO2, in order to increase its solution power. In the present work, the modifier methanol enhanced the solubility of solutes in supercritical CO2 and thus the efficiency of extraction increased.
SFE appears to be a cost-effective technique in laboratory scale, but an accurate economic evaluation for large-scale units requires supplementary experiments. The advantages of SFE-CO2 extraction over the petrol ether extraction include: low operating temperature, hence no thermal degradation of most of the labile compounds; shorter extraction period; high selectivity in the extraction of compounds; no solvent residue with negative effects on the oils quality. The essential oils of plants have usually been isolated by either hydrodistillation or solvent extraction. The disadvantages of all these techniques are: low yield, loss of volatile compounds, long extraction time, toxic solvent residues and degradation of unsaturated compounds, giving undesirable off-flavour compounds, due to heat.
The aim of the present work is to investigate the effects of different parameters, such as pressure, temperature, modifier volume and dynamic extraction time, on the supercritical fluid carbon dioxide extraction of M. convoluta. To the best of our knowledge, no report has yet appeared on the SFE of the plant species.
whole plants of Marchantia convoluta were collected in Shangling
City of Guangxi Zhuang Autonomous District in August, 2003. The specimen
(No 20041364) was identified by Zhou Zi-jing, at Biology Department
of Guangxi Chinese Medical University. The dried leaves were stored
in dark at
grade methanol and analytical grade petroleum ether were purchased
from Hanbon Company Limited. Carbon dioxide (99.99% purity) contained
in a cylinder with an eductor tube, was obtained from CSU Co. (
Suprex MPS/225 system (
Four millilitres of solution were poured into a 20 mL beaker. The solvent was evaporated by bubbling argon gas through the solution. Then the weight of essential oil was measured and the extraction yield was calculated.
analyses were performed using a Shimadzu GC-9A gas chromatograph
equipped with a FID and a HP-5 fused silica column (
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. Table 1 shows different conditions of experiments carried out with SFE for extractions of M. convoluta according 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 (Table 1), reducing the number of experiments to 16. The yields obtained under orthogonal conditions are also shown in Table 1. The extraction yields were 0.87% - 4.69%.
In this study, interactions among variables were not incorporated in the matrix and focus was placed on the main effects of the four most important factors. The results of the SFE experiments, based on extraction yields, are given in Table 1.
mean values of the extraction yields for the corresponding factors
at each level were calculated according to the assignment of the experiment
(Figure 1). 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. Figure
1 shows the variations in extraction yield as a function of change
in different levels of the factors studied. 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:
The compounds from the oil produced by SFE using no. 9 orthogonal test conditions were identified and quantified by GC-MS (Table 2). The total ion chromatograph of SFE using Expt no. 9 orthogonal test condition was shown in Figure 2. GC separation gave 50 peaks, among which 46 were identified by MS library matching. The peak area of compounds identified accounted for 84.16% of total peak area.
The major compounds identified in SFE extract no. 9 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%.
Extraction of natural products by different methods may yield different chemical components (Stashenko et al. 1996; Kohler et al. 1997; Vinatoru, 2001; Kim and Lee, 2002; Pourmortazavi et al. 2003; Lucchesi et al. 2004; Menaker et al. 2004; Seger et al. 2004; Braga et al. 2005; Fulzele et al. 2005; Michielin et al. 2005; Sporring et al. 2005). Several studies on compositions of the extract from M. convoluta were reported (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). Zhu et al. (2003) separated β-sitosterol and stigmasterol from the methanol extract. 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 (GC-MS) (Cao et al. 2005). 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 ether while higher levels of terpenes and derivatives were found in the SFE product. The benzothiazole content in the SFE extract is considerable (11.82%) and the organic acids and esters accounted for 32.19%. This is similar to the report by Cao et al. (2005). On the other hand, Cao et al. (2005) reported that higher benzothiazole content (14.97%) in the ethyl acetate extract while organic acids and esters accounted for 36.01% in the petrol ether extract. Cao et al. (2005) also reported that a phytol content of 6.32% in the petrol ether extract, whereas it was not found in the SFE products.
authors wish to thank Jiangsu Provincial Key Laboratory of Coastal
Wetland Bio-resources and Environmental Protection,
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