Abstract
Cymbopogon flexuosus is widely cultivated for essential oil which is having various health benefits apart from perfumery uses. Microwave radiation combined with hydrodistillation was investigated for efficient and rapid extraction of essential oil from lemongrass. Yield and citral content were dependent on various parameters like weight of raw material, volume of water, rehydration time, extraction time and applied power. Based on these parameters, optimized conditions were found at 100 g of raw material, 500 ml of water, 3 h of rehydration time, 45 min of extraction time and 850 W applied power. Essential oil was found to contain higher percentage of oxygenated compounds, mainly citral (> 72%). Microscopic analysis provided the basis to understand the extraction mechanism. The outcome of this study recommends that hydrodistillation using microwave may further be utilized for extraction of from various medicinal plants and scale up.
Introduction
Cymbopogon flexuosus (lemongrass), commonly known as lemongrass, is a tall, coarse perennial grass containing 1-2% essential oil on a dry basis extracted from the leaves (Husain et al., 1988, Nikos). Citral, mixture of neral and geranial isomers (Figure 1) (Rauber, Guterres & Schapoval 2005), is the major compound found in the lemongrass oil(Schaneberg and Khan, 2002) and used in synthesis of ionone, vitamin A and beta-carotene [2]. Several studies reported that lemongrass oil possess antimicrobial (), antifungal and antibacterial activity against a diverse range of organisms. Also, the essential oil was proved to be superior fungicide compared to synthetic one (Nikos) and found to be non-phytotoxic in nature (Naik). It is possible to treat muscle aches, acne, flatulence, athlete's foot, excessive perspiration, scabies and oily skin using lemongrass oil [4]. Also, the oil is useful as anxiolytic, sedative or anticonvulsive agent (Blanco, Costa, Freire, Santos Jr. & Costa, 2009). Lemongrass oil was found to be useful in inhibiting the activity of β-glucuronidase and some tested human, plant and animal pathogens (Saleem et al., 2003). Apart from various medicinal applications, essential oil is useful for perfumery because of easy solubility with other essential oils and blending agents [3, 4].
Driven by the potential applications of essential oil extracted from lemongrass, oil was extracted using conventional method like solvent extraction (Sargenti & Lancas, 1997), steam distillation (Marongiu, Piras, Porcedda & Tuveri, 2006; Sargenti & Lancas, 1997; Silou, Malanda & Loubaki, 2004), hydrodistillation (Marongiu, Piras, Porcedda & Tuveri, 2006, Parikh and Desai, 2011) and novel technique like supercritical fluid extraction (Marongiu, Piras, Porcedda & Tuveri, 2006; Sargenti & Lancas, 1997). Though the conventional techniques are well established, they have energy and environmental issues. The elevated temperature for longer extraction time can cause chemical modiï¬cation of the oil components and a loss of the most volatile molecules in case of steam distillation and hydrodistillation. For solvent extraction, it is vey difficult to have solvent free product and this process, also, leads to loss of the most volatile compounds. Although supercritical fluid extraction is known to be a clean technology giving acceptable yields and purity, its major disadvantage lies in its high operating pressure and cost. These drawbacks have led to search for 'green' extraction technique in order to have not only environment friendly but also energy efficient method.
In recent years, microwave extraction is used for extraction of various natural products from plant matrices because of large reduction in solvent consumption [7] and extraction time [7] with better efficiencies compared to conventional techniques. Also, fewer chemical changes of original plant components were observed in case of microwave extraction compared to hydrodistillation [19, 20]. Essential oil obtained by microwave irradiation has increased antimicrobial as well as antioxidant activities compared to oil obtained by hydrodistillation [23]. Major advantages of microwave extraction lie in its ability for volumetric heating [7] and maximum penetration of electromagnetic energy in biological tissues [8]. A detailed review of microwave extraction for natural products and its comparison with various conventional as well as novel techniques were given by Desai and Parikh (2010). The comparison showed that microwave yields better extraction in less time. Also, novel techniques supercritical fluid extraction and pressurized fluid extraction involve high operating pressure creating safety concern and need higher investment compared to the moderate of microwave extraction.
Extraction of essential oil from Cymbopogon flexuosus (lemongrass) using microwave extraction has not been reported yet. In the present study, microwave irradiation is combined with hydrodistillation to accelerate the extraction of essential oil from lemongrass. The aim of this study is to examine the feasibility of microwave extraction as an efficient technique for the extraction of essential oil. The parameters affecting the yield of essential oil (weight of plant materials, volume of water, rehydration time and irradiation power) are studied in order to optimize the experimental conditions.
Materials and Methods
Materials
Leaves of Cymbopogon flexuosus (lemongrass) were collected from Navsari Agriculture University, Navsari in the month of May, 2010. The leaves were dried at room temperature under shed for 2 days and stored in moisture free environment at room temperature.
Methods
Microwave Extraction
Microwave extraction (ME) was performed in a cavity of 50 l supplied by M/s Falcon Microwave Technology, Mumbai, India. The unit is equipped with two magnetrons, 850 W each. The time, temperature and power were controlled by Indusoft Software package. The rehydrated plant material was kept in a flask of 2 l capacity along with water. The absorbed water molecules residing in the glands of leaves get vaporized because of microwave irradiation which in turn causes rapture of the cell walls. During this process, essential oil gets vaporized and carried along with water vapor towards the condenser, kept above the microwave cavity. The condensed essential oil and water are then decanted and separated. The oil, thus, collected is dehydrated and stored at 2 oC. The maximum temperature of the system was boiling point of water and system was operated at atmospheric pressure.
Various parameters like weight of raw material, volume of water, rehydration of raw material, size of raw material and power may affect the extraction process and, hence, yield. In hydrodistillation study, it was observed that grinding had an adverse effect on the yield of the essential oil, hence, leaves of size 2.5 cm have been used for the study (Communicated paper).
Analysis by GC-MS
The essential oils obtained by microwave extraction were analyzed by gas chromatography-mass spectrometry (Clarus 600 GC-MS, Perkin Elmer) using Rxi-5Sil-MS column having arylene stabilized equivalent of 5% diphenyl (30 m x 0.25 mm x 0.25 µm film thickness). The conditions maintained for obtaining GC -MS spectra were: Carrier gas He with a flow rate of 0.7 ml/min; split 1:50; injection volume 0.1 µl; injection temperature 250 oC; oven temperature progress from 80 to 200 oC at 5 oC/min and from 200 to 290 oC at 20 oC/min; the ionization mode used was electronic impact at 70 eV.
Identification of components was carried out by comparison of the mass spectral fragmentation patterns with those stored in MS database (National Institute of Standards and Technology).
Scanning Electron Microscope (SEM)
Surface morphology of specimen was studied using SEM (ESEM EDAX XL-30, Philips). The examination was carried under vacuum (0.8 torr) and accelerating voltage of 30 V.
Result and Discussion
Effect of Weight of Raw Material
Raw materials of different weights were studied to optimize the solid loading (Figure 5.2). For lower solid loading, the yield was found to be constant. However, citral content increased which might be due to either decrease in loss of volatiles or less extraction of higher molecular weight compounds. The decrease in yield as well as citral content was observed after 100g. With increase in weight of raw material, distribution of microwave radiation per particle decreases at a fixed applied power which in turn results in low dielectric heating and, thus, a reduced effect of microwave radiation. Also, the material near the surface of vessel will have higher absorption of microwaves compared the material residing in the interior part of the vessel [26]. This may provide non uniform heating of the material. Hence longer extraction time may require for achieving same level of extraction for increased weight of raw material. Similar results wert obtained for extraction of curcuminoids [27], piperine and RRP when solid loading was increased.
Figure 5.2: Effect of weight of raw material (Extraction conditions: Volume of water: 500 ml; Rehydration time: 1 hr; Power applied: 850 W; Extraction time: 45 min)
Effect of Volume of Water
Volume of water was varied from 0 - 1000 ml. As shown in Figure 5.3, with increase in volume, decrease in yield as well as citral was observed after 500 ml. Initially, water content was less, resulted in instantaneous heating, which may lead to loss of volatile compounds or extraction of compounds which are otherwise difficult to get extracted. Water, having polar nature, absorbs significant portion of microwaves which decreases the penetration depth for microwave radiation. For this reason, higher water amount can lead to incomplete extraction of essential oil and lesser amount of citral since more amount of heat is required to reach the boiling point of the water. This was, also, observed in the extraction of triterpenoid saponins from Ganoderma atrum [28].
Figure 5.3: Effect of volume of water (Extraction conditions: Weight of raw material: 100 ml; Rehydration time: 1 hr; Power applied: 850 W; Extraction time: 45 min)
Effect of Rehydration Time
Since microwaves are absorbed the most by polar compound, experiments were carried out with water soaked leaves for different rehydration time. The water soaked raw material was kept for few minutes to drain excess water. The amount of water uptake for 50 g raw material at different rehydration time is given in Table 5.3. Water uptake was almost constant after 3 h.
Table 5.3: Amount of water uptake at different rehydration time
Rehydration time, h
60
120
180
240
24
Amount of water uptake, ml
74
103
124
125
128
Figure 5.4 shows the effect of different rehydration time on the yield of essential oil. The yield was found to be constant. However, the extraction time was reduced greatly from 60 min to 35 min with increase in rehydration time. Extraction time was found to be constant for rehydration time of 3 h and onwards. The absorbed water molecule gets heated very rapidly on exposure to microwave radiation which in turn vaporizes and ruptures the cell. With increase in rehydration time, amount of water molecules absorbed also increased leading to generation of more vapor, thus, imparting more pressure on the cell wall and, finally, rupture of cell wall in lesser time. Citral content was found to be almost constant over entire range of study except 24 h. The rehydration caused improvement in extraction efficiency of various natural products [12, 27].
(a: Extraction condition: Weight of raw material: 100 ml; Volume of water: 500 ml; Power applied: 850 W)
Effect of Extraction Time
The extraction time was varied to observe the effect of radiation on essential oil yield and citral content. As shown in Figure 5.4, the yield increased with increase in extraction time. The rate of extraction was very fast upto 35 min and then it decreased for the rest of extraction. However, yield was found to be constant after 45 min of extraction indicating end of extraction. Citral content was found to increase with increase in extraction time and then a fall was observed which may be because of dilution effect i.e. extraction of compounds which require more time to get vaporized. Similar trend was observed for extraction of triterpenoid saponins and gly acid where no further extraction was achieved after some time.
Figure 5.5: Effect of extraction time (Extraction conditions: Weight of raw material: 100ml; Volume of water: 500 ml; Rehydration time: 1 hr; Power applied: 850 W)
Effect of Power Applied
With increase in power level, microwave absorption by polar compounds becomes very rapid which enhances heating rate and, ultimately, rate of extraction. The extraction was carried out at 850 W and 1700 W. In both the cases, the yield and the citral content were found to be almost constant and the extraction time reduced from 45 min to 35 min by increasing power from 850 W to 1700 W. However, the reduction in extraction time was only 10 min indicating more energy consumption at higher power. In case of curucminoids and piperine extraction, higher yield was achieved with decreased purity may be due to extraction of other impurities at higher power.
Composition of Essential Oil
The total ion chromatogram of essential oil is given in Figure 6. Compositions of essential oil obtained for different experimental condition are given in Table 2. Figure 7 shows the variation of hydrocarbons and oxygenated compounds with respect to different experimental conditions. As seen from the table and figure, oxygenated compounds dominated the composition contributing above 90 % of total composition. Oxygenated compounds are found to be odoriferous and responsible for the characteristic aroma of essential oil (Ferhat, 2006). Among oxygenated compounds, citral was the major compound present, ranging from 73 - 88% of total oxygenated compounds. Citral is the mixture of neral and geranial. Percentage contribution of geranial (52 - 60%) was found to be higher compared to that of neral (40 - 48%) in citral.
It is difficult to say that a clear trend showing the variation of hydrocarbons and oxygenated compounds with change in experimental conditions was observed. However, presence of caryophyllene oxide has affected the percentage of citral content by inducing dilution effect. At lower amount of raw material, lower volume of water, higher rehydration time, higher extraction time and higher power, percentage of caryophyllene oxide increased may be because of extreme operating conditions.
Surface morphology of leaves
Adaxial side of lemongrass leaves was examined by SEM. The comparison of micrographs of untreated (Figure ) and microwave treated (Figure ) leaves shows that the microwave radiation caused physical changes in the structure of leaves. The cells containing essential oil were found to be present adjacent to non photosynthetic tissues (Lewinsohn). After microwave extraction, the cells were empty while the non photosynthetic tissues were only dried. Since the polar compounds have absorbed the microwave radiation efficiently, instantaneous energy transfer and vaporization of the compounds occurred which lead to rupture of cell walls. This phenomenon caused the release of volatiles from the cell wall, thus, emptying the cells. Similar morphological changes were observed by (Ferhat and Reila)
Conclusions
Microwave extraction was employed to extract essential oil from Cymbopogon flexuosus for the first time using open vessel apparatus. By studying the effect of various parameters on microwave extraction, optimal conditions for extraction of essential oil from lemongrass were: weight of raw material 100 g, volume of water 500 ml, rehydration time 3 h, extraction time: 45 min and power applied: 850 W. Essential oil was found to be rich with oxygenated compounds (> 90%), mainly citral. The citral content varied from 72 - 80% with change in operating conditions. Surface morphology of lemongrass leaves revealed that microwave radiation caused rupture of cell wall because of localized heating. The method utilized was found to be rapid and efficient and provided green approach for extraction of essential oil from lemongrass. The results suggest that the combination of microwave with hydrodistillation can further be utilized for extraction of volatile compounds from plant materials and scale up.
