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Latin American applied research

versión On-line ISSN 1851-8796

Lat. Am. appl. res. vol.45 no.2 Bahía Blanca abr. 2015


Extraction of Mucuna aterrima seed oil using compressed propane and quantification of l-dopa in the defatted meal

C. Silva, D. Mantovani, V.A.S. Garcia§ and L. Cardozo-Filho*

Department of Technology, Maringá State University, 1800, Umuarama, Brazil.
Department of Food Engineering, University Federal of Parana, 1299, Curitiba,
§ Department of Food Engineering, University of São Paulo, 225, Pirassununga,
* Department of Chemical Engineering, Maringá State University, 5790, Maringá,

Abstract — The aim of this study was to extract the oil of Mucuna seeds using compressed propane as the solvent, in order to concentrate the L-Dopa content of the defatted Mucuna meal. The experiments were performed in a laboratory-scale unit with a constant propane flow rate of around 0.8 mL/min and temperature and pressure ranges of 30-60 °C and 80-120 bar, respectively. The results revealed that temperature is an important variable for the extraction yield obtained with propane. It was observed that the concentration of L-Dopa present in the defatted seed meal was greater than that in the seeds without the removal of oil. Quantitative analysis of the oil extracted under the different experimental conditions investigated showed no significant differences in terms of fatty acids (p>0.05), the major oil component being linoleic acid (omega-6), along with the presence of around 5% of linolenic acid (omega-3). The kinetics of the oil extraction with propane was also investigated for the operating conditions considered.

Keywords — Mucuna Aterrima; Propane; Supercritical Extraction.


Mucuna aterrima is a subtropical species of legumes, grown mainly in Asia, Africa and parts of the Americas (Vadivel and Janardhanan, 2000; Gurumoorthi et al., 2008). According to reports in the literature, L-Dopa, which is a non-protein amino acid used for the treatment of Parkinson's disease, is present in this species (Modi et al., 2008; Kasture et al., 2009; Bonis et al., 2010). In fact, Mucuna is considered to be the legume with the highest percentage of this important compound (Ingle, 2003). These seeds are also reported to be rich in antioxidants (Tripathi and Upadhyay, 2001; Bhat and Sridhar, 2008). Mucuna seeds have a high calorific value and are nutritionally comparable to some of the common edible beans and legumes (Bhat and Sridhar, 2008; Siddhuraju and Becker, 2005). Abud et al. (2009) reported that Mucuna seeds are also used to produce green manures, due to the high content of organic matter present in the plant.

Regarding its nutritional value, this legume can provide significant amounts of energy, vitamins and minerals. Other parts of the plant can also be used for medical purposes, for example, trichomes in the pods are used for de-worming, a root decoction is used for delirium, a root powder is used as a diuretic and anti-inflammatory agent and a paste of the fresh root is used in the treatment of lymphoedema (Kalidass and Mahapatra, 2014).

Mucuna seeds are rich in lipids (around 9.65 g/100g), proteins, carbohydrates and minerals. Mucuna oil is rich in essential fatty acids, predominantly linoleic acid (Ezeagu et al., 2005; Demune et al., 2003; Ajavi et al., 2006). The oil has a detrimental effect on the extraction of L-Dopa from Mucuna meal. As a result, the extraction of the oil from the seeds should contribute to increasing the yields of L-Dopa obtained from Mucuna (Garcia et al., 2012).

Supercritical fluid extraction (SFE) has been proposed as a procedure for the extraction of vegetable oils (Brunner, 2005; Illés et al., 2000; Freitas et al., 2008; Hamdan et al., 2008; Martinéz et al., 2011; Nimet et al., 2011; Carrara et al., 2012) because it has a high solvating power, leaves no toxic residues and involves low temperature (96.7 °C) and critical pressure (42.5 bar) conditions (Ndiaye et al., 2006). The solvent conditions lead to high permeation of the sample by the pressurized fluid, increasing the rate of extraction, due to the higher diffusivity, lower viscosity and lower surface tension compared to the liquid solvent. Thus, the pressurized fluid is considered most suitable for the extraction of vegetable oils (Pederssetti et al., 2011; Corso et al., 2010).

According to Hamdan et al. (2008), a higher quality vegetable oil is obtained when propane is used as a solvent in the extraction process. Illés et al. (2000) and Nimet et al. (2011) reported that the use of propane leads to higher efficiency in the extraction process, that is, a lower amount of solvent is required with respect to carbon dioxide and higher yields are obtained in a shorter time. Freitas et al. (2008) found that propane provided high yields and improved the extraction kinetics for oil extraction from grape seeds. This is due to the fact that propane exhibits better solubility in vegetable oils than carbon dioxide (Ndiaye et al., 2006). This effect was also observed for the extraction of oil from sesame seeds (Nimet et al., 2011), sunflower seeds (Pederssetti et al., 2011) and canola seeds (Corso et al., 2010).

This paper reports the results of an investigation on Mucuna aterrima oil extraction using propane as the solvent, aimed at the concentration of L-Dopa in the defatted meal. The experiments with compressed propane were carried in a laboratory-scale unit at different temperatures and pressures, but with a constant solvent flow rate of 0.8 mL/min. The characteristics of the extracted oil in terms of the fatty acids and the L-Dopa concentrations in the defatted meal are reported.


A. Materials
Black seeds of Mucuna (Mucuna aterrima) were obtained from Casa Agropecuaria with a moisture content of 9.3±0.2 %, determined by gravimetric analysis. The seeds were milled using an electric mill (IKA, model A11 B, Brazil) and a Tyler 60 sieve (Bertel, ASTM) was used to obtain particles with a mean diameter of 0.25 mm. For the extractions, propane obtained from Air Liquide (São Paulo, Brazil) with 99.9% purity was used. All solvents and reagents used in this study were of analytical grade. The standards of methyl heptadecanoate (>99 % purity), L-3,4-dihydro-xyphenylalanine (>99 % purity), derivatives of boron trifluoride-methanol and n-methyl-n-(trimethylsilyl) trifluoroacetamide were obtained from Sigma-Aldrich Chemical Co. Methanol, phosphoric acid and heptane (all HPLC grade), obtained from Tedia, were used as solvents to prepare the sample and the mobile phase for the HPCL analysis.

B. Extraction Apparatus and Procedure
The experiments were performed on laboratory-scale, as previously described by Garcia et al. (2012). Around 35 g of dried and finely comminuted seeds were placed into the extraction vessel. The solvent was pumped at a constant flow rate of 0.8 mL/min into the bed. The extract was then collected by opening the metering and needle valves, and the solvent mass flow was measured according to the pump recordings. The mass of extracted oil was weighed and the glass tube was reconnected to the equipment. This procedure was performed until no significant mass was extracted or, as in some cases, the extraction period exceeded a pre-established limit. The oil was collected initially at intervals of 3 min and after 15 min this was extended to 5 min. The experiments were conducted isothermally at constant pressure. The experimental temperature range investigated was 30 to 60 °C and the pressure range was 80 to 120 bar (Table 1). The extraction yield was calculated as the ratio between the mass of extracted oil and the raw material used.

Table 1. Experimental conditions and yield obtained in the extraction of Mucunaaterrima seed using pressurized propane. (T = Temperature; P = Pressure; Density = Solvent density; Yield = Extraction yield).

* Results in g/100 g of oil (100 times the mass of oil extracted by the mass of raw material fed).

C. Fatty Acids Analysis
The quantification of fatty acids in the Mucuna oil was carried out according to the AOAC standard method Ce 2-66 (Walker, 1990). In order to determine the total fatty acids content, derivatization of the oil with BF3/methanol was performed using methyl heptadecanoate (Sigma-Aldrich) as the internal standard. A Shimadzu gas chromatograph, fitted with a capillary column (ZBWAX, 30m x 0.25mm x 0.25 μm), was used and the column temperature was programmed to increase from 120 °C to 180 °C, at heating at a rate of 15 °C/min, and then to 240 °C, at a rate of 5 °C/min, holding this temperature for 5 min. The carrier gas flow rate was 1.5 mL/min (40 psi). The injector and detector temperature was 250 °C and the volume injected was 1.0 μL in the 1:50 split mode. The identification of the compounds was carried out through the injection of authentic standards of the fatty acids (Sigma-Aldrich).

D. L-Dopa Determination
The L-Dopa levels were quantified using high-performance liquid chromatography (Varian 920 LC) with a visible ultraviolet detector, equipped with a quaternary pump and autosampler. The analysis was performed according to Bezerra et al. (2003), using a Res Elut C18 column (Varian, 150 mm x 4.6 mm i. d.) at 30 °C. The mobile phase consisted of water, methanol and phosphoric acid (97.9:2:0.1 v/v/v), the flow rate was 0.5 mL/min, the detection was carried out at 280 nm and the injection volume was 20 µL. A chromatographic standard of L-3,4-dihydroxyphenylalanine was used to obtain the calibration curve (5 to 140 mg/L), which showed a regression coefficient (r) of 0.99. To quantify the samples, 0.1 g of defatted Mucuna meal was added to 5 mL of ultrapure Milli-Q water and placed in a sonication bath (Unique 1400A) for 5 min. The solution was then filtered (0.45 mm nylon membrane - Millipore) and 50 µL were transferred to a 1 mL volumetric flask and the volume was completed with the mobile phase (water, methanol and phosphoric acid). All analyses were carried out in duplicate.

E. Statistical analysis
The experimental results were analyzed using the software program SAS (Statistic Analysis System), version 9.3 (2010). The difference between the means was determined applying Tukey's test (95% confidence interval).


A. Extraction Yields
The experimental conditions and extraction yields obtained from the extraction of Mucuna seed oil using pressurized propane as the solvent are shown in Table 1. The total oil content in the Mucuna seeds was determined by the conventional method of Soxhlet extraction and the value obtained was 5.73±0.2% (w/w). The extraction yield was defined in this study as 100 times the mass of oil extracted divided by the mass of raw Mucuna seeds. In order to allow a direct comparison between the results obtained under different experimental conditions, all of the values for yield shown in Table 1 were obtained after 25 min of extraction. The total extraction time varied according to the experimental conditions, aiming to achieve exhaustive extraction with the compressed solvent.

As can be seen in Table 1, the effect of temperature is more pronounced than that of pressure since, under the experimental conditions investigated, propane is a compressed liquid and changes in its density with variations in the pressure are small. The highest extraction yield of 2.24 % was obtained in run 4, at a pressure of 120 bar and temperature of 60 °C. The positive effect of temperature on the supercritical extraction with propane of oils from sunflower seeds (Nimet et al., 2011), canola seeds (Pedersseti et al., 2011) and sesame seeds (Corso et al., 2010) has been previously reported. Mesomo et al. (2012) studied the extraction of ginger oil using propane as the solvent and reported that pressure was the most important variable in relation to the extraction yield, although the temperature also positively influenced the extraction process, the highest oil yields being obtained under higher temperature and pressure conditions, as observed in this study.

The kinectics curves for the extraction of Mucuna seed oil with compressed propane as the solvent can be seen in Fig. 1. It can be observed that an increase in the temperature favors the initial rates of extraction. This figure shows a comparison of the results obtained in this study with data reported by Garcia et al. (2012) using carbon dioxide as the solvent at 150 bar and 40 °C. It can also be noted that the extraction process with propane is much faster than that with carbon dioxide. In the case of extraction with supercritical carbon dioxide around 0.48 % of the final yield was obtained within 40 min and the yield increased by 2.42 % with the use of propane at 120 bar and 60 °C. This result is due to the fact that propane is a better solvent for vegetable oils than carbon dioxide (Freitas et al., 2008; Ndiaye et al., 2006; Corso et al., 2010). Hamdan et al. (2008) noted that propane is a suitable solvent for the rapid and efficient extraction of oleoresin from cardamom seeds, since the process is highly efficient in terms of cost and can be carried out in a short period of time.

Figure 1. Kinetic curves for Mucuna aterrima seed oil extraction using pressurized propane. Experimental conditions are labeled according to Table 1.

B. Composition of Mucuna aterrima oil
The results for the quantification of fatty acids in the Mucuna oil are shown in Table 2. It can be observed that the chemical profiles for the extracted Mucuna oils are similar and consistent with results presented in the literature (Garcia et al., 2012). Ezeagu et al. (2005) and Ajayi et al. (2006) determined the fatty acids compositions of oils extracted by petroleum ether (boiling point range 40-60 °C) from Mucuna seeds and the results are also in agreement with those obtained in this study. An analysis of the chemical distribution of the fatty acids of the oils extracted at the different temperatures and pressures investigated in this study indicated no significant differences between the results considering a significance level of 5% (p>0.05) in the ANOVA test. In the literature, several authors have reported the same finding for the extraction of oil from sunflower seeds, canola seeds, sesame seeds, cardamom seeds and rice bran (Nimet et al., 2011; Pederssetti et al., 2011; Corso et al., 2010; Hamdan et al., 2008; Sparks et al., 2006).

Table 2. Quantification of fatty acids in the Mucuna aterrima oils extracted with pressurized propane.

1 Fatty acids - Results in g/100 g of oil
2 SFA - saturated fatty acid
3 MUFA - monounsaturated fatty acids
4 PUFA - polyunsaturated fatty acid

The total amount of polyunsaturated fatty acids (PUFAs) in Mucuna seed oil was around 56%, while the relative contents of monounsaturated fatty acids (MUFAs) and saturated fatty acids (SFAs) were approximately 12% and 33%, respectively. The results also show that the major polyunsaturated fatty acid in the Mucunaaterrima oils extracted under different experimental conditions was linoleic acid (omega-6) and the linolenic acid (omega-3) content was around 5%. Garcia et al. (2012) reported similar results for the quantification of fatty acids in M. aterrima oil obtained by extraction with carbon dioxide as the solvent. These results indicate that the nature of the solvent employed in supercritical extraction does not influence the fatty acids composition of the oils extracted.

Kalidass and Mahapatra (2014) compared the composition of two different oils extracted, using chloroform and methanol as solvents, from the seeds of Mucuna var. utilis (Wall ex. Wight). They reported a composition of more than 56% unsaturated fatty acids, comprised of mono unsaturated fatty acids (oleic acid) and poly unsaturated fatty acids (linoleic acid and linolenic acid), with linoleic acid being the major component.

C. Quantification of L-Dopa in Defatted Meal
Table 3 shows the results for the oil yield and the L-Dopa content of the defatted meal, for all experimental conditions, applying a total extraction time of 60 min. As can be seen in Table 3, the different extraction conditions influence significantly (p<0.05) the percentages of L-Dopa in the defatted meal obtained and 5.08 % of L-Dopa was obtained under the best experimental conditions in terms of oil yields. On comparing the percentages of L-Dopa in the raw seeds (without oil removal) and in the defatted meal, it can be noted that oil extraction by SFE increases the concentration of L-Dopa (Table 3). The results for the concentration of L-Dopa found in the defatted meal in this study (3.79-5.08 %) compared with that in Mucuna pruriens seeds without oil removal reported in the literature (2.4 %) (Chikagwa-Malunga et al., 2009) verify the advantage of the use of oil extraction with supercritical carbon dioxide with regard to the subsequent L-Dopa concentration in the Mucuna meal. Garcia et al. (2012) evaluated the process of removing the oil from the seeds of M. aterrima with carbon dioxide under supercritical conditions to concentrate L-Dopa and reported a value of around 4.62 % in the defatted meal, this concentration, in all cases, being higher than that obtained for the seeds without oil extraction.

Table 3. Process conditions, yield obtained and L-dopa percentage in the extraction of oil from Mucuna aterrima seeds using pressurized propane.

* Mean followed by same lowercase did not differ statistically (p > 0.05) in relation to the raw seeds and mean followed by same uppercase letters did not differ statistically (p > 0.05) from each other.

Adebowale et al. (2005) evaluated the physico-chemical, nutritional and anti-nutritional properties of six Mucuna varieties and reported that M. pruriens and Mucuna deeringiana contained around 4.99% and 3.87% of L-dopa, respectively. Bhat et al. (2008) reported 5.15% of L-dopa in seeds of M. pruriens. Diallo and Berhe (2003) evaluated the processing of M. pruriens for use in human food products and reported 4.93% of L-dopa in the seeds. Kalidass and Mahapatra (2014) reported 3.24 % and 3.64 % of L-dopa in seeds of Mucuna var. utilis (Wall. ex Wight), respectively. The environmental conditions, such as latitude, light intensity and nitrogen source, are directly related to levels of L-Dopa present in the seeds of Mucuna (Bhat et al., 2008). This would explain the higher L-Dopa values in seeds reported in other studies. Teixeira et al. (2003) reported the effect of particle diameter on the extraction of L-dopa obtained from M.pruriens. For the intact seed residue the L-Dopa content in the Mucuna bean extracted at 20 °C and pH 7.0, with an extraction time of 1 h, was 4.22%, while for a particle size of 1 mm this content decreased to 1.54%.


In the extraction process carried out with propane an increase in the temperature had a positive effect on the extraction yield and the kinetic curves for the extraction in the range investigated. The results verified a yield of 2.43 % at 60 °C and 120 bar with an extraction time of 60 min. The chemical profiles for the oils extracted with propane under different experimental conditions were similar. L-Dopa contents of >3.8 % were observed for the defatted meal obtained under the conditions of maximum oil yield and for all extraction conditions studied the concentration of L-Dopa was greater than that obtain for the seeds without oil extraction.

The authors are grateful to CNPq, Pro-Engenharias - CAPES and Maringá State University (UEM) for scholarships.

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Received: September 11, 2013.
Accepted: April 22, 2014.
Recommended by Subject Editor: Mariano Martín Martin