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Supercritical fluid extraction and characterisation of Moringa oleifera leaves oil
Separation and Purification Technology 118 (2013) 497–502
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Supercritical fluid extraction and characterisation of Moringa oleifera leaves oil Suwei Zhao, Dongke Zhang ⇑ Centre for Energy (M473), The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
a r t i c l e
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Article history: Received 24 April 2013 Received in revised form 18 June 2013 Accepted 27 July 2013 Available online 3 August 2013 Keywords: Carbon dioxide Essential oil Moringa oleifera Soxhlet extraction Supercritical fluid extraction
a b s t r a c t This work was aimed to extract and characterise the Moringa oleifera leaves oils using supercritical fluid extraction (SFE) and to compare the results against those obtained using the conventional Soxhlet extraction method. The oils extracted were analysed using GC–MS. The compounds were identified according to their retention indices and mass spectra (EI, 70 eV). The effects of pressure (30 MPa, 40 MPa, 50 MPa), temperature (40 °C, 60 °C, 80 °C) and extraction time (60 min, 90 min and 120 min) on the oil yield were investigated using a three-level orthogonal array design. The experimental results showed that while the oil yield increased with increasing any of the three parameters, temperature had the most significant effect. The highest yield of 6.34% was obtained under the condition of pressure 50 MPa, temperature 60 °C and an extraction time of 120 min. The Soxhlet extraction was performed at 78 °C for 8 h using hexane as the solvent. A total of 42 compounds were identified in the Soxhlet extracted oil while only 12 compounds were detected in the SFE extracted oil. Some compounds that were found in the Soxhlet extracts were not detected in the SFE samples. This is because the supercritical fluid extraction is more selective than the Soxhlet extraction method. The compositions of the oil extracted by SFE under the different conditions examined were mostly similar and the major compounds identified were 1,2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester, nonacosane, heptacosane, b-Amyrin, although their quantities differed. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Essential oils are complex mixtures of a few dozen to several hundred compounds [1], mainly formed by hydrocarbon and oxygenated terpenes and by hydrocarbon and oxygenated sesquiterpenes [2]. Essential oils have been traditionally used as food flavours, preservatives, pharmaceutical and cosmetic products [3]. Several other functions of the essential oils, including antibacterial, antifungal antioxidant and anti-inflammatory activities have also been recognised [4]. These pleasant pharmacological properties and chemical characteristics have been the focus of research and development interests from both the academia and pharmaceutical industry [5]. Essential oils can come from a large number of plant species that contain volatile chemical compounds such as orange, peppermint, lemon clove and spearmint. Steam distillation is a traditional technique for extracting essential oil as it is simple [6]. However, this technique suffers from some drawbacks in that thermal degradation, hydrolysis and solubilisation of some compounds in water that could alter the flavour and fragrance profile of many essential oils extracted using this technique. Moringa oleifera, commonly referred to as ‘‘Moringa’’, is one of the most widely cultivated tree species in the family Moringaceae. ⇑ Corresponding author. Tel.: +61 8 64887600; fax: +61 8 64887235. E-mail address: [email protected] (D. Zhang). 1383-5866/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.seppur.2013.07.046
This species originally comes from northern India but now distributes worldwide in the tropics and subtropics [7]. The trees range from 5 to 10 m in height and sometimes can be even 15 m. They can also grow in hot dry lands or destitute soils and are little affected by drought. M. oleifera is a traditionally important source of food as its leaves, flowers, pods, seeds and roots are locally used as vegetables. Moringa leaves have been taken to combat malnutrition, especially among infants and nursing mothers, because they contain more calcium than milk, more iron than spinach, more Vitamin C than oranges and more potassium than bananas [8]. Recent research has demonstrated that extracts of M. oleifera leaves possess several useful biological properties, such as antihypertensive [9], anti-fungal [10], antiulcer [11], antitumor and anticancer activities [12]. Aqueous leaves extracts can regulate thyroid hormone and can be used to treat hyperthyroidism and exhibit an antioxidant effect [13]. A recent report (Herpes simplex virus type 1 or HSV-1) also shows that Moringa leaves may be used as a prophylactic or therapeutic anti-HSV medicine and may be effective against the acyclovir-resistant variant [14]. These properties make M. oleifera extracts promising candidates for applications in the pharmaceutical industry. Most common methods for the extraction of important compounds from Moringa leaves are based on solvent extraction. However, this extraction has the major disadvantage of solvent residue being present in the extracts. Recently supercritical fluid extraction
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(SFE) has gained an increasing attention over the traditional techniques, like hydro-distillation and solvent extraction in the recovery of edible oils and essential oils. The commonly used fluid in SFE is CO2, which has several unique characteristics and physic-chemical properties. For example, CO2 is non-toxic, non-flammable, inexpensive, and odourless and has low critical pressure (7.38 MPa) and temperature (31.1 °C) and leaves no solvent residue in the products, thus potentially providing the oil of superior quality. SFE also has advantages over the traditional extraction techniques including operation at low temperatures thus enabling the preservation of the thermally labile compounds in the extracts. Moreover, the selectivity of carbon dioxide in relation to the oil can be easily adjusted by changing the temperature and pressure. SFE has been applied to the extraction of essential oils from different types of herbs such as Fennel seed [15], Origanum virens L. flower [16], lavender flowers [17] and ginger root [18]. To the best of the authors knowledge, no reports have been published on identifying essential oil compositions from M. oleifera leaf extracts obtained using supercritical CO2. Therefore, the main objective of this research was to extract and characterise the oils presented in the leaves of M. oleifera using SFE and to compare the results against those using the conventional Soxhlet extraction method. The effects of different parameters such as pressure, temperature and extraction time on the supercritical fluid extraction yield were also investigated.
washed out with dichloromethane and then added to the extract collected in the amber bottle. The solution was bubbled using nitrogen gas to evaporate the dichloromethane. The mass of the extracted oil was then weighed to determine the extraction yield, expressed as the percent of the mass of the extracted oil to the dry mass of M. oleifera leaves loaded into the extraction vessel. 2.3. Experimental program design Several important parameters determine the efficacy of SFE including the pressure, temperature and extraction time. To study the effect of such multiple variables on the e oil extraction yield, an orthogonal array design was used to arrange the experiments and optimise the extraction process [19,20]. Orthogonal array design is a type of factorial design in which orthogonal array is used to assign factors to a series of experimental combinations. In this study, the effects of extraction pressure, temperature and time on the yield were investigated. An OA9 (33) orthogonal matrix with three factors, each factor containing three levels, was selected to arrange the experiments. Therefore, nine experiments were carried out at three levels of pressures (30 MPa, 40 MPa, 50 MPa), temperatures (40 °C, 60 °C, 80 °C), and extraction times of 60 min, 90 min, 120 min. All extraction experiments under the same conditions were performed in duplicate to ensure repeatability. 2.4. Soxhlet extraction
2. Experimental 2.1. Plant samples The M. oleifera leaves used in this work were provided by Department of Agriculture and Food of Western Australia (DAFWA). Prior to experimentation, the samples were air-dried at 40 °C for 8 h and the final water content of the leaves were determined to be 15.2% by drying a sample to 105 °C for 5 h. The airdried samples were then ground with a knife grinder (Model 3383-L30, Thomas Scientific, USA) and the fraction of particles under 250 lm was selected for all extraction experiments. The final sample was kept in a sealed plastic container and placed in a refrigerator before experimentation. 2.2. SFE The extraction experiments were carried out using an SFT Custom SCW-SFE system (Newark, DE, USA) as schematically shown in Fig. 1. Liquid CO2 was supplied from a CO2 cylinder with a siphon tube and introduced into the system through a dual piston pump. The pump was designed and operated for pressures up to 68 MPa. This system accommodated four parallel extraction vessels including two vessels for supercritical CO2 extraction and two vessels for supercritical water oxidation. The fluid was heated to a desired temperature through a preheater and a heating jacket around the vessel. A robust variable restrictor valve was used to control the overall flow rate and maintain the flow rate stable. In order to prevent sample plugging, the restrictor was warmed electrically. A flow meter was provided to indicate the flow rate of CO2. About 5 g of the air-dried M. oleifera leaves was weighed and loaded into the 50 ml supercritical CO2 vessel. Glass wool was packed at both ends of the extractor to stop entrainment of the sample. SFE started as soon as the desired pressure and temperature had been reached. The flow rate of expanded gas CO2 was maintained at approximately 2 L/min (NTP). The extract was collected in dichloromethane in an amber bottle. In order to improve the collection efficiency, the bottle was placed in an ice bath during the dynamic extraction stage. The precipitates in the tube lines was
The supercritical CO2 extraction experiments were benchmarked against the Soxhlet extraction method. In the Soxhlet extraction, approximately 5 g of ground M. oleifera leaves was placed in a cellulose thimble and transferred to a Soxhlet extractor. A round bottom extraction flask was filled with 150 ml n-hexane which was heated via a water bath set at 78 °C. The extraction process was continuously run for 8 h for each experimental run. After the extraction was completed, the solvent was removed at 50 °C at a reduced pressure using a rotary evaporator (model N-1000S-W, EYELA, Tokyo, Japan). After the evaporation of the solvent, the oil yield was determined as percent of the mass of extracted oil to the mass of dry M. oleifera leaves loaded in the extraction vessel. 2.5. GC–MS analysis The compositions of the M. oleifera oils extracted using the SFE and Soxhlet methods were determined using an Agilent 7890 N series gas chromatograph equipped with an Agilent 5975 mass selective detector and a HP-5MS column (30 0.25 mm (5%-Phenyl)-methylpolysiloxane column, film thickness 0.25 lm). Although Chuang et al. [10] have used GC–MS to analyse the M. oleifera leaves oil, the use of their method did not detect the very heavy molecular weight compounds. Thus, a new method was developed in this work to analyse all compositions in the oil. In brief, the oven temperature was initially set at 50 °C (held for 15 min), and then increased to 150 °C at a rate of 2 °C/min, held for 10 min at 150 °C, and finally increased to 280 °C at 2 °C/min, held for 20 min. The injector temperature was 250 °C. Helium was used as the carrier gas at a flow rate of 1.0 ml/min. The oil samples were diluted 10 times with dichloromethane and 0.2 ll of the diluted solution was injected into the GC in a split mode with a split ratio of 1/20. The temperatures of ion source and transfer line were 230 °C and 250 °C, respectively. The ionisation energy was 70 eV with a scan time of 1s and mass range of 20–550 amu. The percentages of compounds were calculated from the GC peak area without considering response factors. The compounds of the oil were identified by comparing of their mass spectra against the NIST (National Institute of Standards and Technologies) MS spectra library. The compounds were also confirmed by comparing
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Fig. 1. A schematic of the supercritical fluid extraction system: (1) CO2 cylinder; (2) CO2 and H2O pump; (3) co-solvent reservoir; (4) co-solvent pump; (5) preheater; (6) 100 ml jacketed H2O vessel; (7) 50 ml jacked H2O vessel; (8) 100 ml jacked CO2 vessel; (9) 50 ml jacked CO2 vessel; (10) pressure gauge; (11) condenser; (12) restrictor; (13) collection bottle and (14) flow meter.
Table 1 Three level orthogonal design and experimental results for extraction of Moringa oleifera leave oil with supercritical CO2 extractions. Run No.
Pressure (MPa)
Temperature (°C)
Dynamic time (min)
Yield (%)
Standard deviation
1 2 3 4 5 6 7 8 9
1(30) 1(30) 1(30) 2(40) 2(40) 2(40) 3(50) 3(50) 3(50)
1(40) 2(60) 3(80) 1(40) 2(60) 3(80) 1(40) 2(60) 3(80)
1(60) 2(90) 3(120) 3(120) 1(60) 2(90) 2(90) 3(120) 1(60)
4.00 5.18 4.87 4.23 4.72 5.55 4.37 6.34 5.99
0.16 0.24 0.15 0.12 0.15 0.11 0.11 0.10 0.17
14.06a 14.51 16.71 4.69b 4.84 5.57 0.88c
12.61 16.24 16.42 4.20 5.41 5.47 1.27
14.72 15.11 15.44 4.91 5.04 5.15 0.24 9.27
0.04
K values K1 K2 K3 k1 k2 k3 R Soxhlet extraction a b c
to 6.3%. A further orthogonal analysis was conducted; the K, k and R values were calculated and also listed in Table 1. The K value is the sum of the evaluation indices of all levels in each factor and k (the mean value of K) determines the optimal level and the optimal combination of factors. The optimal level for each factor could be obtained when k is the largest. The R value indicates the range between the maximum and minimum values of k and evaluates the importance of the factors. A larger R means a greater importance of the given factor [21]. According to the R values presented in Table 1, the most significant parameter affecting the extraction yield was temperature, followed by pressure and time. The highest yield of 6.3% was obtained under the condition of pressure 50 MPa, temperature 60 °C and time of 120 min. Note that the oil extracted using the Soxhlet was 9.27%, higher than those using the supercritical fluid extraction.
3.2. Effect of SFE conditions on the oil yield
P K Ai ¼ the extraction yield of oil at Ai . A ki ¼ K Ai =3. A A RAi ¼ maxfki g minfki g.
their retention indices with the data published in the literature [10].
3. Results and discussion 3.1. M. oleifera leaves oil yields The oil yields at the different sets of pressure, temperature and time were studied under an OA9 (33) design. The results presented in Table 1 are the average of the two measurements under the same conditions, along with the standard deviation of the measurements. The results shown in Table 1 indicate that there were substantial differences in the oil yields obtained under different SFE conditions. The oil yields of Run 1 to Run 9 ranged from 4.0%
To evaluate the influence of the level of each of the influencing factors, namely, pressure, temperature and extraction time, on the extraction yield, the variation in the oil yield as a function of the changes in the different levels of the factors was depicted in Fig. 2. As expected, the extraction yield was enhanced significantly with increasing pressure. This is because raising the extraction pressure at a constant temperature led to a higher fluid density thus increased the solubility of the oil. Temperature affects the density of the fluid, the volatility of the extract components and desorption of the extracts from the matrix. It can be seen from Fig. 2b that increasing temperature from 40 °C to 60 °C resulted in a significant increase in the oil extraction yield, while further increasing temperature from 60 °C to 80 °C resulted in only a small increase in the yield. This can be explained as that at a higher temperature, although the fluid density decreases, the solubility of the oil still increases. Therefore, the extraction yield under different temperatures depends on a complex balance between the supercritical CO2 density and the volatility of the extracted components under a given condition. The extraction time had a positive influence on the extraction yield as shown in Fig. 2c. The extraction yield gradually increased with increasing time, and reached a maximum at 2 h within the tested range.
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3.3. Oil composition Table 2 compares the compositions of essential oils extracted with the supercritical CO2 extraction and the Soxhlet extraction. It can be seen that 42 compounds were identified in the M. oleifera leaves oil obtained by the Soxhlet extraction. In general, Benzene,1-ethyl-3-methyl- (5.95%), Benzene,1,2,4-trimethyl-(16.96%), Heptacosane (7.45%) and Nonacosane (18.65%) were the major components in the M. oleifera leaves oil. These compounds are different from those found by Chuang et al. [10] whose leave samples were from Taichung, Taiwan. In their study, it was revealed that pentacosane (17.41%), hexacosane (11.20%), (E)-phytol (7.66%)
Fig. 2. Effect of pressure (a), temperature (b) and extraction time and (c) on the yield of extracted oil from Moringa oleifera leaves.
and 1-[2,3,6-trimethyl-phenyl]-2-butanone (3.44%) were the major components. The presence of c-Tocopherol and dl-a-Tocopherol were foundfor all extracts, accounting for a high percentage of the total extracts, ranging from 0.97% to 6.54%. These two chemicals have been recognised with medicinal properties and used against approximately 80 diseases, such as cancer, cystic fibrosis cardiovascular diseases, cell membrane, DNA damage by free radicals, oxidation of low density lipoproteins, disorders of the skin, eye, lungs, and other lipid body constituents [22]. The results of tocopherol in this study are consistent with the study of Sánchez-Machado et al. [23] who has also detected these two components from M. oleifera cultivated in the Northwest Mexico. From Tables 2 and 3, it is clear that there were significant differences in the essential oil compositions isolated by the SFE and Soxhlet extractions. There were a number of constituents indentified in the oil obtained by the Soxhlet extraction that were not detected in any extracts from the supercritical CO2 extraction. Cao et al. [24] found that there were less compounds separated by GC analyses from SFE extracts than Soxhlet extracts when they compared the volatile components of Marchantia convolute obtained by supercritical carbon dioxide extraction and Soxhlet extraction with petrol ether. They explained that the results were different from each other because of the different methods in dealing with the extracts. Other authors have attempted to compare the hydro-distillation method with SFE, they have obtained similar findings that there was asmaller number of constituents in the supercritical fluid extraction [25]. Probably all these missing compounds were still present in the SFE extracts but at lower contents than those obtained in the hydro-distillation. This may be a consequence of the smaller quantities of plant material used in the SFE extraction and the correspondingly low amounts of extracts. However, Asghar et al. [26] believed that this might be due to the difference in the two methods for trapping the volatile oil: in the supercritical CO2 extraction, a part of the extracted components escaped along with CO2 from the vessel containing dichloromethane, but in hydro-distillation trapping the oil was simultaneously performed along with the condensing steam in the pipe. From the present experimental observations, it is believed that as the supercritical fluid extraction is amore selective technique than the Soxhlet extraction, some of the components that were not detected in the SFE samples might have not been extracted in SFE. Table 2 shows that there were 12 compounds detected in the SFE extracts and the extracts obtained by SFE under different conditions had similar compositions. However, the quantitative compositions of the products were quite different, especially for the SFE run 8 under conditions of 50 MPa, 60 °C and 120 min. Under this condition, the main compounds indentified in the extracted oil were 1,2-Benzenedicarboxylic acid,mono (2-ethylhexyl) ester (76.23%), followed by Nonacosane (13.37%), heptacosane (4.98%) and b-Amyrin (1.50%). However, in the other SFE experiments, the major constituent in the extracted oil was Nonacosane, accounting for nearly 50%. Other main compounds indentified in those samples were Pentacosane, Heptacosane, Dimethoate, nhexadecanoic acid, c-Tocopherol, Sulfurous acid, hexyl pentadecyl ester, dl-a-Tocopherol, 1,30-Triacontanediol,c-Sitosetrol and bAmyrin. It is interesting to note that the extractions of cuticular waxes, represented by the long chain alkanes, were verified in all the experiments. The most abundant alkanes were Nonacosane (13.37–60.06%), Pentacosane (1.00–6.32%), and Heptacosane (4.98–22.66%). This can be explained by the fact that cuticular waxes are located on the surfaces of the leaves and are readily available for extraction with little mass transfer resistance, white the oil compounds may be in the internal structure of the leaves and the mass transfer mechanism is related to multiple diffusion stages [25]. However, no general tendency was observed regarding the effect of extraction conditions on the percentage of these
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S. Zhao, D. Zhang / Separation and Purification Technology 118 (2013) 497–502 Table 2 Composition (W%) of light compounds from Moringa oleifera leaves oil obtained by the SFE and Soxhlet extractions. No.
Retention time
Retention index
Component
Formula
Soxhlet
1 2 3 4 5
3.149 3.46 3.532 3.608 3.734
772.5 780.5 782.3 784.2 787.3
C5H5N C8H18 C7H8 C8H18 C8H16
0.78 0.68 1.27 0.50 0.78
6 7
4.181 4.335
798.7 802.6
C8H18 C8H16
1.75 0.14
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
4.973 5.138 6.162 6.488 7.592 8.042 9.504 11.722 12.462 12.568 13.094 14.225 15.673 18.678 19.9 22.636
818.7 822.8 848.6 856.8 884.9 896.1 914 937.9 945.7 947 952.5 964.5 980.1 1011.8 1024.2 1052
C9H20 C8H16 C8H10 C8H10 C8H10 C9H20 C9H12 C9H12 C9H12 C9H12 C9H12 C9H12 C9H12 C9H12 C9H10 C10H14
0.17 0.43 0.83 4.63 2.82 0.14 0.16 1.04 5.95 2.39 4.31 2.55 16.96 4.14 0.39 0.64
24
25.045
1076.6
C10H14
0.99
25 26 27 28 29 30 31 32 33 34
27.702 28.066 30.681 32.903 67.973 82.66 86.93 89.137 112.38 114.0047
1104.2 1108.9 1138.4 1163 1708 1907 1966 1997.4 2510 2558
C10H14 C10H14 C10H14 C10H8 C5H12NO3PS2 C19H40 C16H32O2 C20H42 C25H52 C16H22O4
1.59 2.12 0.58 1.21 0.52 0.22 0.15 0.31 2.14
35 36 37 38
119.844 126.724 131.169 133.089
2725.9 2924.3 3052.3 3108
39 40 41 42
133.73 137.421 138.639 139.232
3126.7 3232.8 3267.8 3283.9
Pyridine Hepane,2-methylTobuene Heptane,3-methylCyclohexane,1,3-dimethyl,cisOctane Cyclohexane,1,4-dimethyl,cisOctane,2-methylCyclohexane,ethylEthylbenzene Benzene,1,3-dimethylo-Xylene Nonane Benzene,(1-methylethyl)Benzene,propylBenzene,1-ethyl-3-methylBenzene,1-ethyl-4-methylBenzene,1,3,5-trimethylBenzene,1-ethyl-2-methylBenzene,1,2,4-trimethylBenzene,1,2,3-trimethylIndane Benzene,1-ethyl-2,4dimethylBenzene,1-ethyl-2,3dimethylBenzene,1,2,3,5-tetramethylBenzene,1,2,4,5-tetramethylBenzene,1,2,3,4-tetramethylNphthalene Dimethoate Nonadecane n-Hexadecanoic acid Eicosane Pentacosane 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl)ester Heptacosane Nonacosane c-Tocopherol Sulfurous acid, hexyl pentadecyl ester dl-a-Tocopherol 1,30-Triacontanediol c-Sitosetrol b-Amyrin
SFE1
1.48
SFE2
SFE3
0.66
0.90
SFE4
1.63
SFE5
1.03
SFE 6
0.94
0.31
SFE7
SFE8
SFE9
1.77
0.24
1.55
0.62
0.11
4.73
5.09 0.51
3.88
4.28 0.78
5.53
3.57
4.32
6.32
1.00 76.23
C27H56 C29H60 C28h48O2 C21H44O3S
7.45 18.65 0.75 0.34
20.00 54.98 1.34 1.44
21.35 60.06 1.55 0.95
20.86 54.80 2.16 0.84
21.86 51.99 1.87 1.40
21.80 55.51 2.35 0.93
19.72 49.74 3.38 1.18
22.66 49.71 1.84 0.79
4.98 13.37 0.41 0.24
20.52 48.97 3.54 0.96
C29H50O2 C30H62O2 C29H50O C30H50O
1.05 3.06 0.86 4.60
2.67 2.82 1.54 8.14
1.42 4.34 0.97 4.83
1.83 6.03 1.70 5.51
2.97 3.52 2.77 6.47
1.89 6.14 1.31 5.48
2.40 10.05 2.74 5.53
3.52 3.20 2.85 6.72
0.57 0.86 0.47 1.50
3.00 7.61 2.95 6.18
branch alkanes, except in the case of experimental 8. This was probably because the mass-transfer mechanisms of the cuticular waxes and the oil components were quite different. Under the SFE run 8 condition, the fluidwith a very high pressure and a mild temperature, the extraction time was very long, so the dissolution power of supercritical CO2 under this condition was very high and more readily able to overcome the larger mass transfer resistances of the components in the internal structures of the leaves, therefore, it increased the extraction of the oil components [27]. As discussed before, the extraction under this condition produced the highest yield of M. oleifera leaves oil.
4. Conclusions Supercritical fluid extraction has been applied to the extraction of essential oil from M. oleifera leaves and the effects of pressure, temperature and extraction time on the extraction yield have been investigated. The results indicated that temperature had the most significant effect on the oil yield followed by pressure and
extraction time. The oil yield increased with an increase in any of these three factors. The yield and composition of the oils obtained by the SFE and Soxhlet extractions were also compared. Although the yield of the SFE process (the highest yield was 6.3%) was lower than that of the Soxhlet extraction (9.3%), the SFE offered the advantage of shorter extraction times, being 2 h rather than 8 h for the Soxhlet extraction, and lower temperatures. Besides, there were substantial differences in the oil compositions obtained using the SFE technique and Soxhlet extraction method, as some compounds identified in the Soxhlet extraction samples were not extracted by SFE method. Chemical analysis revealed that although the compounds in all SFE extracts were roughly the same, their quantities differed. Therefore, it is possible to manipulate the composition of the oil extracted by changing the operating parameters of the SFE extraction. Acknowledgments The authors gratefully acknowledge the financial and other support provided by the Australian Research Council under the ARC
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Linkage Projects Scheme (ARC Linkage Project LP100200135), BHP Billiton Iron Ore Pty Ltd, ENN Group, and ANSAC Pty Ltd. Dr Henry Brockman of Department of Agriculture and Food of Western Australia (DAFWA) provided the Moringa oleifera leaf samples and many invaluable insights into the growth and nature of Moringa oleifera.
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[13] P. Tahiliani, A. Kar, Role of Moringa oleifera leaf extract in the regulation of thyroid hormone status in adult male and female rats, Pharmacol. Res. 41 (3) (2000) 319–323. [14] V. Lipipun, M. Kurokawa, R. Suttisri, P. Taweechotipatr, P. Pramyothin, M. Hattori, K. Shiraki, Efficacy of Thai medicinal plant extracts against herpes simplex virus type 1 infection in vitro and in vivo, Antiviral Res. 60 (3) (2003) 175–180. [15] E. Reverchon, J. Daghero, C. Marrone, M. Mattea, M. Poletto, Supercritical fractional extraction of fennel seed oil and essential oil: experiments and mathematical modeling, Ind. Eng. Chem. Res. 38 (8) (1999) 3069–3075. [16] F. Gaspar, Extraction of essential oils and cuticular waxes with compressed CO2: effect of extraction pressure and temperature, Ind. Eng. Chem. Res. 41 (10) (2002) 2497–2503. [17] M. Akgün, N.A. Akgün, S. Dinçer, Extraction and modeling of lavender flower essential oil using supercritical carbon dioxide, Ind. Eng. Chem. Res. 39 (2) (2000) 473–477. [18] B.C. Roy, M. Goto, T. Hirose, Extraction of ginger oil with supercritical carbon dioxide: experiments and modeling, Ind. Eng. Chem. Res. 35 (2) (1996) 607– 612. [19] W. Liu, Y.-J. Fu, Y.-G. Zu, M.-H. Tong, N. Wu, X.-L. Liu, S. Zhang, Supercritical carbon dioxide extraction of seed oil from Opuntia dillenii Haw. and its antioxidant activity, Food Chem. 114 (1) (2009) 334–339. [20] M. Khajeh, Y. Yamini, F. Sefidkon, N. Bahramifar, Comparison of essential oil composition of Carum copticum obtained by supercritical carbon dioxide extraction and hydrodistillation methods, Food Chem. 86 (4) (2004) 587–591. [21] X. Wu, D.Y.C. Leung, Optimization of biodiesel production from camelina oil using orthogonal experiment, Appl. Energy 88 (11) (2011) 3615–3624. [22] R.A. Jacques, J.G. Santos, C. Dariva, J.V. Oliveira, E.B. Caramão, GC/MS characterization of mate tea leaves extracts obtained from high-pressure CO2 extraction, J. Supercrit. Fluids 40 (3) (2007) 354–359. [23] D.I. Sánchez-Machado, J. López-Cervantes, N.J. Ríos Vázquez, Highperformance liquid chromatography method to measure [alpha]- and [gamma]-tocopherol in leaves, flowers and fresh beans from Moringa oleifera, J. Chromatogr. A 1105 (1–2) (2006) 111–114. [24] H. Cao, J.B. Xiao, M. Xu, Comparison of volatile components of Marchantia convoluta obtained by supercritical carbon dioxide extraction and petrol ether extraction, J. Food. Comp. Anal. 20 (1) (2007) 45–51. [25] N. Aghel, Y. Yamini, A. Hadjiakhoondi, S.M. Pourmortazavi, Supercritical carbon dioxide extraction of Mentha pulegium L. essential oil, Talanta 62 (2) (2004) 407–411. [26] A. Safaralie, S. Fatemi, F. Sefidkon, Essential oil composition of Valeriana officinalis L. roots cultivated in Iran: comparative analysis between supercritical CO2 extraction and hydrodistillation, J. Chromatogr. A 1180 (1– 2) (2008) 159–164. [27] L.H.C. Carlson, R.A.F. Machado, C.B. Spricigo, L.K. Pereira, A. Bolzan, Extraction of lemongrass essential oil with dense carbon dioxide, J. Supercrit. Fluids 21 (1) (2001) 33–39.
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Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Supercritical fluid extraction and characterisation of Moringa oleifera leaves oil Suwei Zhao, Dongke Zhang ⇑ Centre for Energy (M473), The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
a r t i c l e
i n f o
Article history: Received 24 April 2013 Received in revised form 18 June 2013 Accepted 27 July 2013 Available online 3 August 2013 Keywords: Carbon dioxide Essential oil Moringa oleifera Soxhlet extraction Supercritical fluid extraction
a b s t r a c t This work was aimed to extract and characterise the Moringa oleifera leaves oils using supercritical fluid extraction (SFE) and to compare the results against those obtained using the conventional Soxhlet extraction method. The oils extracted were analysed using GC–MS. The compounds were identified according to their retention indices and mass spectra (EI, 70 eV). The effects of pressure (30 MPa, 40 MPa, 50 MPa), temperature (40 °C, 60 °C, 80 °C) and extraction time (60 min, 90 min and 120 min) on the oil yield were investigated using a three-level orthogonal array design. The experimental results showed that while the oil yield increased with increasing any of the three parameters, temperature had the most significant effect. The highest yield of 6.34% was obtained under the condition of pressure 50 MPa, temperature 60 °C and an extraction time of 120 min. The Soxhlet extraction was performed at 78 °C for 8 h using hexane as the solvent. A total of 42 compounds were identified in the Soxhlet extracted oil while only 12 compounds were detected in the SFE extracted oil. Some compounds that were found in the Soxhlet extracts were not detected in the SFE samples. This is because the supercritical fluid extraction is more selective than the Soxhlet extraction method. The compositions of the oil extracted by SFE under the different conditions examined were mostly similar and the major compounds identified were 1,2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester, nonacosane, heptacosane, b-Amyrin, although their quantities differed. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Essential oils are complex mixtures of a few dozen to several hundred compounds [1], mainly formed by hydrocarbon and oxygenated terpenes and by hydrocarbon and oxygenated sesquiterpenes [2]. Essential oils have been traditionally used as food flavours, preservatives, pharmaceutical and cosmetic products [3]. Several other functions of the essential oils, including antibacterial, antifungal antioxidant and anti-inflammatory activities have also been recognised [4]. These pleasant pharmacological properties and chemical characteristics have been the focus of research and development interests from both the academia and pharmaceutical industry [5]. Essential oils can come from a large number of plant species that contain volatile chemical compounds such as orange, peppermint, lemon clove and spearmint. Steam distillation is a traditional technique for extracting essential oil as it is simple [6]. However, this technique suffers from some drawbacks in that thermal degradation, hydrolysis and solubilisation of some compounds in water that could alter the flavour and fragrance profile of many essential oils extracted using this technique. Moringa oleifera, commonly referred to as ‘‘Moringa’’, is one of the most widely cultivated tree species in the family Moringaceae. ⇑ Corresponding author. Tel.: +61 8 64887600; fax: +61 8 64887235. E-mail address: [email protected] (D. Zhang). 1383-5866/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.seppur.2013.07.046
This species originally comes from northern India but now distributes worldwide in the tropics and subtropics [7]. The trees range from 5 to 10 m in height and sometimes can be even 15 m. They can also grow in hot dry lands or destitute soils and are little affected by drought. M. oleifera is a traditionally important source of food as its leaves, flowers, pods, seeds and roots are locally used as vegetables. Moringa leaves have been taken to combat malnutrition, especially among infants and nursing mothers, because they contain more calcium than milk, more iron than spinach, more Vitamin C than oranges and more potassium than bananas [8]. Recent research has demonstrated that extracts of M. oleifera leaves possess several useful biological properties, such as antihypertensive [9], anti-fungal [10], antiulcer [11], antitumor and anticancer activities [12]. Aqueous leaves extracts can regulate thyroid hormone and can be used to treat hyperthyroidism and exhibit an antioxidant effect [13]. A recent report (Herpes simplex virus type 1 or HSV-1) also shows that Moringa leaves may be used as a prophylactic or therapeutic anti-HSV medicine and may be effective against the acyclovir-resistant variant [14]. These properties make M. oleifera extracts promising candidates for applications in the pharmaceutical industry. Most common methods for the extraction of important compounds from Moringa leaves are based on solvent extraction. However, this extraction has the major disadvantage of solvent residue being present in the extracts. Recently supercritical fluid extraction
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(SFE) has gained an increasing attention over the traditional techniques, like hydro-distillation and solvent extraction in the recovery of edible oils and essential oils. The commonly used fluid in SFE is CO2, which has several unique characteristics and physic-chemical properties. For example, CO2 is non-toxic, non-flammable, inexpensive, and odourless and has low critical pressure (7.38 MPa) and temperature (31.1 °C) and leaves no solvent residue in the products, thus potentially providing the oil of superior quality. SFE also has advantages over the traditional extraction techniques including operation at low temperatures thus enabling the preservation of the thermally labile compounds in the extracts. Moreover, the selectivity of carbon dioxide in relation to the oil can be easily adjusted by changing the temperature and pressure. SFE has been applied to the extraction of essential oils from different types of herbs such as Fennel seed [15], Origanum virens L. flower [16], lavender flowers [17] and ginger root [18]. To the best of the authors knowledge, no reports have been published on identifying essential oil compositions from M. oleifera leaf extracts obtained using supercritical CO2. Therefore, the main objective of this research was to extract and characterise the oils presented in the leaves of M. oleifera using SFE and to compare the results against those using the conventional Soxhlet extraction method. The effects of different parameters such as pressure, temperature and extraction time on the supercritical fluid extraction yield were also investigated.
washed out with dichloromethane and then added to the extract collected in the amber bottle. The solution was bubbled using nitrogen gas to evaporate the dichloromethane. The mass of the extracted oil was then weighed to determine the extraction yield, expressed as the percent of the mass of the extracted oil to the dry mass of M. oleifera leaves loaded into the extraction vessel. 2.3. Experimental program design Several important parameters determine the efficacy of SFE including the pressure, temperature and extraction time. To study the effect of such multiple variables on the e oil extraction yield, an orthogonal array design was used to arrange the experiments and optimise the extraction process [19,20]. Orthogonal array design is a type of factorial design in which orthogonal array is used to assign factors to a series of experimental combinations. In this study, the effects of extraction pressure, temperature and time on the yield were investigated. An OA9 (33) orthogonal matrix with three factors, each factor containing three levels, was selected to arrange the experiments. Therefore, nine experiments were carried out at three levels of pressures (30 MPa, 40 MPa, 50 MPa), temperatures (40 °C, 60 °C, 80 °C), and extraction times of 60 min, 90 min, 120 min. All extraction experiments under the same conditions were performed in duplicate to ensure repeatability. 2.4. Soxhlet extraction
2. Experimental 2.1. Plant samples The M. oleifera leaves used in this work were provided by Department of Agriculture and Food of Western Australia (DAFWA). Prior to experimentation, the samples were air-dried at 40 °C for 8 h and the final water content of the leaves were determined to be 15.2% by drying a sample to 105 °C for 5 h. The airdried samples were then ground with a knife grinder (Model 3383-L30, Thomas Scientific, USA) and the fraction of particles under 250 lm was selected for all extraction experiments. The final sample was kept in a sealed plastic container and placed in a refrigerator before experimentation. 2.2. SFE The extraction experiments were carried out using an SFT Custom SCW-SFE system (Newark, DE, USA) as schematically shown in Fig. 1. Liquid CO2 was supplied from a CO2 cylinder with a siphon tube and introduced into the system through a dual piston pump. The pump was designed and operated for pressures up to 68 MPa. This system accommodated four parallel extraction vessels including two vessels for supercritical CO2 extraction and two vessels for supercritical water oxidation. The fluid was heated to a desired temperature through a preheater and a heating jacket around the vessel. A robust variable restrictor valve was used to control the overall flow rate and maintain the flow rate stable. In order to prevent sample plugging, the restrictor was warmed electrically. A flow meter was provided to indicate the flow rate of CO2. About 5 g of the air-dried M. oleifera leaves was weighed and loaded into the 50 ml supercritical CO2 vessel. Glass wool was packed at both ends of the extractor to stop entrainment of the sample. SFE started as soon as the desired pressure and temperature had been reached. The flow rate of expanded gas CO2 was maintained at approximately 2 L/min (NTP). The extract was collected in dichloromethane in an amber bottle. In order to improve the collection efficiency, the bottle was placed in an ice bath during the dynamic extraction stage. The precipitates in the tube lines was
The supercritical CO2 extraction experiments were benchmarked against the Soxhlet extraction method. In the Soxhlet extraction, approximately 5 g of ground M. oleifera leaves was placed in a cellulose thimble and transferred to a Soxhlet extractor. A round bottom extraction flask was filled with 150 ml n-hexane which was heated via a water bath set at 78 °C. The extraction process was continuously run for 8 h for each experimental run. After the extraction was completed, the solvent was removed at 50 °C at a reduced pressure using a rotary evaporator (model N-1000S-W, EYELA, Tokyo, Japan). After the evaporation of the solvent, the oil yield was determined as percent of the mass of extracted oil to the mass of dry M. oleifera leaves loaded in the extraction vessel. 2.5. GC–MS analysis The compositions of the M. oleifera oils extracted using the SFE and Soxhlet methods were determined using an Agilent 7890 N series gas chromatograph equipped with an Agilent 5975 mass selective detector and a HP-5MS column (30 0.25 mm (5%-Phenyl)-methylpolysiloxane column, film thickness 0.25 lm). Although Chuang et al. [10] have used GC–MS to analyse the M. oleifera leaves oil, the use of their method did not detect the very heavy molecular weight compounds. Thus, a new method was developed in this work to analyse all compositions in the oil. In brief, the oven temperature was initially set at 50 °C (held for 15 min), and then increased to 150 °C at a rate of 2 °C/min, held for 10 min at 150 °C, and finally increased to 280 °C at 2 °C/min, held for 20 min. The injector temperature was 250 °C. Helium was used as the carrier gas at a flow rate of 1.0 ml/min. The oil samples were diluted 10 times with dichloromethane and 0.2 ll of the diluted solution was injected into the GC in a split mode with a split ratio of 1/20. The temperatures of ion source and transfer line were 230 °C and 250 °C, respectively. The ionisation energy was 70 eV with a scan time of 1s and mass range of 20–550 amu. The percentages of compounds were calculated from the GC peak area without considering response factors. The compounds of the oil were identified by comparing of their mass spectra against the NIST (National Institute of Standards and Technologies) MS spectra library. The compounds were also confirmed by comparing
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Fig. 1. A schematic of the supercritical fluid extraction system: (1) CO2 cylinder; (2) CO2 and H2O pump; (3) co-solvent reservoir; (4) co-solvent pump; (5) preheater; (6) 100 ml jacketed H2O vessel; (7) 50 ml jacked H2O vessel; (8) 100 ml jacked CO2 vessel; (9) 50 ml jacked CO2 vessel; (10) pressure gauge; (11) condenser; (12) restrictor; (13) collection bottle and (14) flow meter.
Table 1 Three level orthogonal design and experimental results for extraction of Moringa oleifera leave oil with supercritical CO2 extractions. Run No.
Pressure (MPa)
Temperature (°C)
Dynamic time (min)
Yield (%)
Standard deviation
1 2 3 4 5 6 7 8 9
1(30) 1(30) 1(30) 2(40) 2(40) 2(40) 3(50) 3(50) 3(50)
1(40) 2(60) 3(80) 1(40) 2(60) 3(80) 1(40) 2(60) 3(80)
1(60) 2(90) 3(120) 3(120) 1(60) 2(90) 2(90) 3(120) 1(60)
4.00 5.18 4.87 4.23 4.72 5.55 4.37 6.34 5.99
0.16 0.24 0.15 0.12 0.15 0.11 0.11 0.10 0.17
14.06a 14.51 16.71 4.69b 4.84 5.57 0.88c
12.61 16.24 16.42 4.20 5.41 5.47 1.27
14.72 15.11 15.44 4.91 5.04 5.15 0.24 9.27
0.04
K values K1 K2 K3 k1 k2 k3 R Soxhlet extraction a b c
to 6.3%. A further orthogonal analysis was conducted; the K, k and R values were calculated and also listed in Table 1. The K value is the sum of the evaluation indices of all levels in each factor and k (the mean value of K) determines the optimal level and the optimal combination of factors. The optimal level for each factor could be obtained when k is the largest. The R value indicates the range between the maximum and minimum values of k and evaluates the importance of the factors. A larger R means a greater importance of the given factor [21]. According to the R values presented in Table 1, the most significant parameter affecting the extraction yield was temperature, followed by pressure and time. The highest yield of 6.3% was obtained under the condition of pressure 50 MPa, temperature 60 °C and time of 120 min. Note that the oil extracted using the Soxhlet was 9.27%, higher than those using the supercritical fluid extraction.
3.2. Effect of SFE conditions on the oil yield
P K Ai ¼ the extraction yield of oil at Ai . A ki ¼ K Ai =3. A A RAi ¼ maxfki g minfki g.
their retention indices with the data published in the literature [10].
3. Results and discussion 3.1. M. oleifera leaves oil yields The oil yields at the different sets of pressure, temperature and time were studied under an OA9 (33) design. The results presented in Table 1 are the average of the two measurements under the same conditions, along with the standard deviation of the measurements. The results shown in Table 1 indicate that there were substantial differences in the oil yields obtained under different SFE conditions. The oil yields of Run 1 to Run 9 ranged from 4.0%
To evaluate the influence of the level of each of the influencing factors, namely, pressure, temperature and extraction time, on the extraction yield, the variation in the oil yield as a function of the changes in the different levels of the factors was depicted in Fig. 2. As expected, the extraction yield was enhanced significantly with increasing pressure. This is because raising the extraction pressure at a constant temperature led to a higher fluid density thus increased the solubility of the oil. Temperature affects the density of the fluid, the volatility of the extract components and desorption of the extracts from the matrix. It can be seen from Fig. 2b that increasing temperature from 40 °C to 60 °C resulted in a significant increase in the oil extraction yield, while further increasing temperature from 60 °C to 80 °C resulted in only a small increase in the yield. This can be explained as that at a higher temperature, although the fluid density decreases, the solubility of the oil still increases. Therefore, the extraction yield under different temperatures depends on a complex balance between the supercritical CO2 density and the volatility of the extracted components under a given condition. The extraction time had a positive influence on the extraction yield as shown in Fig. 2c. The extraction yield gradually increased with increasing time, and reached a maximum at 2 h within the tested range.
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3.3. Oil composition Table 2 compares the compositions of essential oils extracted with the supercritical CO2 extraction and the Soxhlet extraction. It can be seen that 42 compounds were identified in the M. oleifera leaves oil obtained by the Soxhlet extraction. In general, Benzene,1-ethyl-3-methyl- (5.95%), Benzene,1,2,4-trimethyl-(16.96%), Heptacosane (7.45%) and Nonacosane (18.65%) were the major components in the M. oleifera leaves oil. These compounds are different from those found by Chuang et al. [10] whose leave samples were from Taichung, Taiwan. In their study, it was revealed that pentacosane (17.41%), hexacosane (11.20%), (E)-phytol (7.66%)
Fig. 2. Effect of pressure (a), temperature (b) and extraction time and (c) on the yield of extracted oil from Moringa oleifera leaves.
and 1-[2,3,6-trimethyl-phenyl]-2-butanone (3.44%) were the major components. The presence of c-Tocopherol and dl-a-Tocopherol were foundfor all extracts, accounting for a high percentage of the total extracts, ranging from 0.97% to 6.54%. These two chemicals have been recognised with medicinal properties and used against approximately 80 diseases, such as cancer, cystic fibrosis cardiovascular diseases, cell membrane, DNA damage by free radicals, oxidation of low density lipoproteins, disorders of the skin, eye, lungs, and other lipid body constituents [22]. The results of tocopherol in this study are consistent with the study of Sánchez-Machado et al. [23] who has also detected these two components from M. oleifera cultivated in the Northwest Mexico. From Tables 2 and 3, it is clear that there were significant differences in the essential oil compositions isolated by the SFE and Soxhlet extractions. There were a number of constituents indentified in the oil obtained by the Soxhlet extraction that were not detected in any extracts from the supercritical CO2 extraction. Cao et al. [24] found that there were less compounds separated by GC analyses from SFE extracts than Soxhlet extracts when they compared the volatile components of Marchantia convolute obtained by supercritical carbon dioxide extraction and Soxhlet extraction with petrol ether. They explained that the results were different from each other because of the different methods in dealing with the extracts. Other authors have attempted to compare the hydro-distillation method with SFE, they have obtained similar findings that there was asmaller number of constituents in the supercritical fluid extraction [25]. Probably all these missing compounds were still present in the SFE extracts but at lower contents than those obtained in the hydro-distillation. This may be a consequence of the smaller quantities of plant material used in the SFE extraction and the correspondingly low amounts of extracts. However, Asghar et al. [26] believed that this might be due to the difference in the two methods for trapping the volatile oil: in the supercritical CO2 extraction, a part of the extracted components escaped along with CO2 from the vessel containing dichloromethane, but in hydro-distillation trapping the oil was simultaneously performed along with the condensing steam in the pipe. From the present experimental observations, it is believed that as the supercritical fluid extraction is amore selective technique than the Soxhlet extraction, some of the components that were not detected in the SFE samples might have not been extracted in SFE. Table 2 shows that there were 12 compounds detected in the SFE extracts and the extracts obtained by SFE under different conditions had similar compositions. However, the quantitative compositions of the products were quite different, especially for the SFE run 8 under conditions of 50 MPa, 60 °C and 120 min. Under this condition, the main compounds indentified in the extracted oil were 1,2-Benzenedicarboxylic acid,mono (2-ethylhexyl) ester (76.23%), followed by Nonacosane (13.37%), heptacosane (4.98%) and b-Amyrin (1.50%). However, in the other SFE experiments, the major constituent in the extracted oil was Nonacosane, accounting for nearly 50%. Other main compounds indentified in those samples were Pentacosane, Heptacosane, Dimethoate, nhexadecanoic acid, c-Tocopherol, Sulfurous acid, hexyl pentadecyl ester, dl-a-Tocopherol, 1,30-Triacontanediol,c-Sitosetrol and bAmyrin. It is interesting to note that the extractions of cuticular waxes, represented by the long chain alkanes, were verified in all the experiments. The most abundant alkanes were Nonacosane (13.37–60.06%), Pentacosane (1.00–6.32%), and Heptacosane (4.98–22.66%). This can be explained by the fact that cuticular waxes are located on the surfaces of the leaves and are readily available for extraction with little mass transfer resistance, white the oil compounds may be in the internal structure of the leaves and the mass transfer mechanism is related to multiple diffusion stages [25]. However, no general tendency was observed regarding the effect of extraction conditions on the percentage of these
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S. Zhao, D. Zhang / Separation and Purification Technology 118 (2013) 497–502 Table 2 Composition (W%) of light compounds from Moringa oleifera leaves oil obtained by the SFE and Soxhlet extractions. No.
Retention time
Retention index
Component
Formula
Soxhlet
1 2 3 4 5
3.149 3.46 3.532 3.608 3.734
772.5 780.5 782.3 784.2 787.3
C5H5N C8H18 C7H8 C8H18 C8H16
0.78 0.68 1.27 0.50 0.78
6 7
4.181 4.335
798.7 802.6
C8H18 C8H16
1.75 0.14
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
4.973 5.138 6.162 6.488 7.592 8.042 9.504 11.722 12.462 12.568 13.094 14.225 15.673 18.678 19.9 22.636
818.7 822.8 848.6 856.8 884.9 896.1 914 937.9 945.7 947 952.5 964.5 980.1 1011.8 1024.2 1052
C9H20 C8H16 C8H10 C8H10 C8H10 C9H20 C9H12 C9H12 C9H12 C9H12 C9H12 C9H12 C9H12 C9H12 C9H10 C10H14
0.17 0.43 0.83 4.63 2.82 0.14 0.16 1.04 5.95 2.39 4.31 2.55 16.96 4.14 0.39 0.64
24
25.045
1076.6
C10H14
0.99
25 26 27 28 29 30 31 32 33 34
27.702 28.066 30.681 32.903 67.973 82.66 86.93 89.137 112.38 114.0047
1104.2 1108.9 1138.4 1163 1708 1907 1966 1997.4 2510 2558
C10H14 C10H14 C10H14 C10H8 C5H12NO3PS2 C19H40 C16H32O2 C20H42 C25H52 C16H22O4
1.59 2.12 0.58 1.21 0.52 0.22 0.15 0.31 2.14
35 36 37 38
119.844 126.724 131.169 133.089
2725.9 2924.3 3052.3 3108
39 40 41 42
133.73 137.421 138.639 139.232
3126.7 3232.8 3267.8 3283.9
Pyridine Hepane,2-methylTobuene Heptane,3-methylCyclohexane,1,3-dimethyl,cisOctane Cyclohexane,1,4-dimethyl,cisOctane,2-methylCyclohexane,ethylEthylbenzene Benzene,1,3-dimethylo-Xylene Nonane Benzene,(1-methylethyl)Benzene,propylBenzene,1-ethyl-3-methylBenzene,1-ethyl-4-methylBenzene,1,3,5-trimethylBenzene,1-ethyl-2-methylBenzene,1,2,4-trimethylBenzene,1,2,3-trimethylIndane Benzene,1-ethyl-2,4dimethylBenzene,1-ethyl-2,3dimethylBenzene,1,2,3,5-tetramethylBenzene,1,2,4,5-tetramethylBenzene,1,2,3,4-tetramethylNphthalene Dimethoate Nonadecane n-Hexadecanoic acid Eicosane Pentacosane 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl)ester Heptacosane Nonacosane c-Tocopherol Sulfurous acid, hexyl pentadecyl ester dl-a-Tocopherol 1,30-Triacontanediol c-Sitosetrol b-Amyrin
SFE1
1.48
SFE2
SFE3
0.66
0.90
SFE4
1.63
SFE5
1.03
SFE 6
0.94
0.31
SFE7
SFE8
SFE9
1.77
0.24
1.55
0.62
0.11
4.73
5.09 0.51
3.88
4.28 0.78
5.53
3.57
4.32
6.32
1.00 76.23
C27H56 C29H60 C28h48O2 C21H44O3S
7.45 18.65 0.75 0.34
20.00 54.98 1.34 1.44
21.35 60.06 1.55 0.95
20.86 54.80 2.16 0.84
21.86 51.99 1.87 1.40
21.80 55.51 2.35 0.93
19.72 49.74 3.38 1.18
22.66 49.71 1.84 0.79
4.98 13.37 0.41 0.24
20.52 48.97 3.54 0.96
C29H50O2 C30H62O2 C29H50O C30H50O
1.05 3.06 0.86 4.60
2.67 2.82 1.54 8.14
1.42 4.34 0.97 4.83
1.83 6.03 1.70 5.51
2.97 3.52 2.77 6.47
1.89 6.14 1.31 5.48
2.40 10.05 2.74 5.53
3.52 3.20 2.85 6.72
0.57 0.86 0.47 1.50
3.00 7.61 2.95 6.18
branch alkanes, except in the case of experimental 8. This was probably because the mass-transfer mechanisms of the cuticular waxes and the oil components were quite different. Under the SFE run 8 condition, the fluidwith a very high pressure and a mild temperature, the extraction time was very long, so the dissolution power of supercritical CO2 under this condition was very high and more readily able to overcome the larger mass transfer resistances of the components in the internal structures of the leaves, therefore, it increased the extraction of the oil components [27]. As discussed before, the extraction under this condition produced the highest yield of M. oleifera leaves oil.
4. Conclusions Supercritical fluid extraction has been applied to the extraction of essential oil from M. oleifera leaves and the effects of pressure, temperature and extraction time on the extraction yield have been investigated. The results indicated that temperature had the most significant effect on the oil yield followed by pressure and
extraction time. The oil yield increased with an increase in any of these three factors. The yield and composition of the oils obtained by the SFE and Soxhlet extractions were also compared. Although the yield of the SFE process (the highest yield was 6.3%) was lower than that of the Soxhlet extraction (9.3%), the SFE offered the advantage of shorter extraction times, being 2 h rather than 8 h for the Soxhlet extraction, and lower temperatures. Besides, there were substantial differences in the oil compositions obtained using the SFE technique and Soxhlet extraction method, as some compounds identified in the Soxhlet extraction samples were not extracted by SFE method. Chemical analysis revealed that although the compounds in all SFE extracts were roughly the same, their quantities differed. Therefore, it is possible to manipulate the composition of the oil extracted by changing the operating parameters of the SFE extraction. Acknowledgments The authors gratefully acknowledge the financial and other support provided by the Australian Research Council under the ARC
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Linkage Projects Scheme (ARC Linkage Project LP100200135), BHP Billiton Iron Ore Pty Ltd, ENN Group, and ANSAC Pty Ltd. Dr Henry Brockman of Department of Agriculture and Food of Western Australia (DAFWA) provided the Moringa oleifera leaf samples and many invaluable insights into the growth and nature of Moringa oleifera.
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