J. Chil. Chem. Soc, 52, N° 1 (2007)

ESSENTIAL OIL FROM MARCHANTIA CONVOLUTA: EXTRACTION AND COMPONENTS

 

HUI CAO, MEI JUAN JI, HONG XIAN WANG*

College of Chemistry and Chemical Engineering, Nantong University, Nantong 226000, PR China, e-mail: jianbo_xiai@yahoo.com.cn

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ABSTRACT

The essential oil of Marchantia convoluta was obtained by supercritical fluid extraction (SFE) using methanol as a modifier. The effects of different parameters, such as pressure, temperature, modifier volume and extraction time, on the extraction of essential oil from M. convoluta were investigated. Maximum global yields were obtained using the following conditions: extraction temperature, 40 ºC; dynamic time, 40 min; pressure, 15 Mpa and modifier volume, 35 ml. The essential oil extracts were analyzed by capillary gas chromatography with mass spectrometric detector (GC-MS). The obtained results were compared with references.

Key words: Marchantia convoluta; essential oil; supercritical fluid extraction; GC-MS

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1. INTRODUCTION

Marchantiaceae plants are well-known traditional Chinese medicinal herbs. They were extensively used as antipyretic in countryside and used to treat tumefaction of skins, protect liver and treat hepatitis [1-3]. A great number of Marchantiaceae plants, such as Marchantia polymorpha, M. convoluta and M. paleacea are found and identified in Guangxi Zhuang Autonomous District in China, and the former was only found in China [4]. These species live in together and are difficult to distinguish one from the others because of their genetic similarity.

Compared with M. polymorpha, M. convoluta was neglected many years ago because it is quite rare. The extracts from M. convoluta can strongly inhibit tumors in human liver and lung cancer cell lines [1-2]. The dried leaves of M. convoluta are used in China to protect livers and to treat tumefaction of skins. The major constituents identified in M. convoluta were flavonoids, triterpenoids, and steroids [5-9]. The flavonoids mainly consist of quercetin, luteolin, apigenin and their O- and C-glycosides [5-6]. A high dosage of flavonoids from M. convoluta (20 and 40 µg/mL) can significantly reduce the activity of Alanine aminotranferease (ALT) and Alanine aminotranferease (AST) in the serum of mice with acute hepatic injury caused by CCl₄ and increase the contents of Total protein (TP) and Alkaline phosphatase (ALP). Moreover, flavonoids from Marchantia convoluta can inhibit obviously bacteria and the auricle tympanites of mice incurred by dimethylbenzene [2]. It possessed distinct effect of antibiosis, anti-inflammation and diuresis in mice, and anti-hepatitis B Virus activity as well [10].

As an alternative for conventional processes, such as organic solvent extraction and steam distillation, the supercritical fluid extraction (SFE) of essential oils has received great attentions in the field of food, pharmaceutical and cosmetic industries in the past several years [11-20]. SFE allows a continuous modification of solvent power and selectivity by means of changing the solvent density. This technique features a fluid with liquid density that solubilizes solids like a liquid solvent and diffusion power that permeates through solid materials very easily likes a gas. The solubilization power increases with the density of the fluid and the higher density of a supercritical fluid may be available at high pressures, which allow it to dissolve large quantities of organic compounds. The dissolved compounds can be recovered from the fluid by reduction of its density via decreasing the pressure or increasing the temperature. This separation process prevents the degradation of the compounds in the extract due to the low temperature. Compared with the organic solvent extraction, the SCF extraction technique has also many other advantages, such as low operating temperature, shorter extraction time, high selectivity and lower solvent residue.

The intrinsic drawback of CO₂ in the SFE extraction is its low polarity, which limits the dissolution of polar analytes and makes the extraction of these compounds difficult. Nevertheless, this limitation may be overcame by adding small amounts of polar modifiers, such as methanol or ethanol. Moreover, SFE appears to be a cost-effective technique at laboratory scale though some more experiments are still need to be carried out for the large scale unit operation.

We herein investigate the effects of various parameters on the supercritical fluid extraction of M. convoluta, such as pressure, temperature, modifier volume and dynamic extraction time. To the best of our knowledge, no report has yet appeared on the supercritical fluid extraction of such plant species.

2. Materials and methods

2.1. Plant materials

M. convoluta were collected in Shangling City of Guangxi Zhuang Autonomous District in August 2004. The specimen was identified by Zhou Zi-jing, at Biology Department of Guangxi Chinese Medical University. The dried leaves were stored in a dark place at 4 ºC for 20 days. Immediately prior to the extraction process, the leaves were ground in a blender to produce a powder with an approximate size of 0.4 mm.

2.2. Reagents and instruments.

HPLC grade methanol and analytical grade petroleum ether were purchased from Shanghai Analytical Reagent Co., Ltd. Carbon dioxide (99.99% purity), was obtained from Nantong Jetair Gas Co., Ltd. (Nantong, China). HA121-50-01-C supercritical CO₂ extraction system was provided by Nantong Huaan Supercritical Extraction Co., Ltd. (Nantong, China). GC/MS analyses were performed on a Agilent 5975 gas chromatograph equipped with a mass spectrometric detector (Agilent, USA).

2.3. Supercritical fluid extraction (SFE)

A 10-ml stainless steel vessel was used as the extraction vessel. Supercritical fluid extractions were performed at certain pressures and temperatures for duration of 20 min, static, followed by extraction. In order to prevent sample plugging, the restrict point was warmed electrically. The supercritical CO₂ flow rate through the restrictor was approximately 0.3–0.4 ml/min (compressed). Plant powder (5.0 g) was well mixed with 2 mm diameter glass beads, and was then charged into the 10-ml extraction vessel. The essential oil was extracted from the plant using supercritical CO₂ under various conditions according to the Taguchi method. Table 1 showed the experimental conditions for each of the SFE runs. The extracted analytes were collected with dichloromethane in a 5.0-ml volumetric flask. The final volume of the extract was adjusted to 5.0 ml with dichloromethane. In order to improve the efficiency, a 5.0-mL volumetric flask was placed in an ice bath during the dynamic extraction stage. For all the modifier studies, methanol was spiked directly into the extraction vessel with charged sample prior to the extraction.

Four millilitres of solution were poured into a 20 ml beaker. Bubbling of the solution was done by using argon gas in order to evaporate the solution. Then the weight of essential oil was measured. Finally, the extraction yield was calculated.

2.4. Gas chromatography-mass spectrometry (GC-MS)

GC/MS analyses were carried out on a HP-5 fused silica column (60 m × 0.25 mm, 5% phenyl- methylpolysiloxane stationary phase, film thickness of 0.25 µm). The oven temperature was programmed 60 ºC for 4 min, and then increased to 250 ºC at a rate of 6 ºC /min. The injector and detector temperatures were set at 250 and 260 ºC, respectively. The carrier gas (helium) was adjusted to a linear velocity of 30 cm/s. The SFE extracts (1.0 µl) were injected into GC/MS (without any further dilution) using the split mode with a split ratio of 1/60. The ionization energy was 70 eV with a scan time of 1 s and mass range of 40–540 amu. The percentages of compounds were calculated by the area normalization method without considering response factors. The components in the essential oil were identified by comparison of mass spectra with those of library or authentic compounds. Data obtained were conformed by comparison of their retention indices, either with those of authentic compounds or with data published in the literature [20].

3. RESULTS AND DISCUSSION

3.1. Optimization of the experimental conditions

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, amount of the modifier and dynamic extraction time are generally considered as the most important factors. Table 1 showed different conditions of SFE according to the Taguchi experimental design. All the selected factors were examined by a four-level orthogonal array design. A full evaluation of the effect of four factors from four levels on the yield needs 256 (4⁴) experiments. In order to reduce the number of experiments, a L₄ (4⁴) orthogonal design was performed (Table 1). By this way, only sixteen experiments were carried out. The yields obtained under orthogonal experiments were also shown in Table 1. The yields of essential oil were 0.87– 4.57 % (w/w).

The mean values of the extraction yields for the corresponding factors at each level were calculated according to the assignment of the experiment (Fig. 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. Fig. 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 10, 20, 30 and 40 min of dynamic extractions. Results showed that increasing dynamic extraction time to 40 min enhanced the extraction of most components.

Thus, the best conditions, obtained by preliminary test, for the extraction of oil were: extraction temperature, 40 ºC; dynamic time, 40 min; pressure, 15 Mpa and modifier volume, 35 ml.

3.2 GC-MS analysis

The GC-MS profiles of SFE extract was shown in Fig. 2. The analytical results of GC-MS were shown in Table 2. Twenty-nine compounds were separated by gas chromatographic analyses from SFE extract and eleven compounds were identified by reference standards. The peak area of compounds identified accounted for 73.62% of total peak area. The major compounds of Marchantia convoluta SFE extract (NO.10) were 22, 23-dihydro-stigmasterol (31.26%), n-hexadecanoic acid (20.35%), stigmasterol (4.55%) and octadecanoic acid (5.75%).

Compared with the conferences [1, 7, 8], the SFE extract of M. convoluta showed a relatively simple GC–MS chromatographic pattern. Detailed identification of the compounds found in Marchantia convoluta oil, produced by SFE under NO. 10 orthogonal test conditions, were shown in Table 2. The results obtained from reference [8] were also shown in Table 2, for comparison.

M. convoluta was only found in China. There are few reports about the chemical components of the essential oil. Only two plant sterols, namely 22, 23-dihydro-stigmasterol and stigmasterol were isolated from Marchantia convoluta by Zhu et al [7]. There are may be some novel essential oil of M. convoluta. However, it was not able to identify all compounds in the essential oil of M. convoluta only through GC-MS. Isolation and identification of chemical components from Marchantia convoluta need further work.

GC analyses were performed on a HP-5 fused silica column (60 m×0.25 mm, 5% phenyl-methylpolysiloxane stationary phase, film thickness of 0.25µm). The oven temperature was programmed 60 ºC for 4 min, and then increased to 250 ºC at a rate of 6 ºC /min. The injector and detector temperatures were set at 250 and 260 ºC, respectively. The carrier gas (helium) was adjusted to a linear velocity of 30 cm/s.

3.3 Compared with references

Different extraction compositions could be obtained by different extraction methods applied to natural products [21-24]. Zhu et al separated β-sitosterol and stigmasterol from the methanol extract [7]. Xiao et al separated and determined flavonoids from M. convoluta by RP-HPLC [6]. Cao et al 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 [8]. 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 [8], 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 report by Cao et al [8]. On the other hand, Cao et al 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 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.

22,23-dihydro-stigmasterol and stigmasterol were plant sterols. Phytosterol is one kind of active constituents, and can be obtained from some sorts of vegetable oil [25]. Phytosterol and its derivatives are widely applied in pharmaceutical, food, and cosmetic industry due to their special biological-activity, physical, and chemical properties [26]. Phytosterol obtained from vegetable oil usually consists of stigmasterol, β-sitosterol, campesterol, and brassicasterol.

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 efficiency of extraction increased. The higher polar components such as n-hexadecanoic acid (20.35%) and octadecanoic acid (5.75%) were largely found in the SFE extract. Because it is lower polarity for petrol ether, the lower polar compounds were main components in petrol ether extraction.

SFE appears to be a cost-effective technique at laboratory scale, but an accurate economic evaluation for large-scale units requires supplementary experiments. The advantages of SFE over the solvent 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, losses of volatile compounds, long extraction times, toxic solvent residues and degradation of unsaturated compounds, giving undesirable off-flavor compounds, due to heat.

4. CONCLUSIONS

The supercritical fluid extraction of essential oil in Marchantia convoluta has been studied. The results showed that plant sterols were main components found in the SFE products. The oil obtained by SFE of Marchantia convoluta showed some differences in composition compared with the oil obtained by solvent extraction. The SFE method offers obvious advantages over petrol ether extraction, namely: shorter extraction time; lower cost (energy cost is fairly higher for performing solvent extraction than that required for reaching supercritical conditions) and cleaner features (as no great volume of organic solvent is involved). The method contributes to the automation of pharmaceutical industry.

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(Received: November 16, 2006 - Accepted: December 11,2006),