Comparative studies of essential oils of Clausena anisata ( Hook ) using solvent-free microwave extraction and hydrodistillation methods

The use of plants for medicinal purposes dates back to early man. Medicinal plants have played a pivotal role in both ancient and modern cultures, with a large human population in developing countries greatly depending on plant resources for health care because they are cheaper and safer and have little or no side effects when compared to modern synthetic drugs (Agbor & Ngogang 2005). It is believed that plants not only possess nutritional properties but also medicinal values (Kala 2005). Pharmaceutical, food and cosmetic industries extensively use plant extracts. Also, it is important to study medicinal plants in order to document their active chemical compounds and various functions in curing different kinds of ailments (Nostro et al. 2000).


Introduction
The use of plants for medicinal purposes dates back to early man.Medicinal plants have played a pivotal role in both ancient and modern cultures, with a large human population in developing countries greatly depending on plant resources for health care because they are cheaper and safer and have little or no side effects when compared to modern synthetic drugs (Agbor & Ngogang 2005).It is believed that plants not only possess nutritional properties but also medicinal values (Kala 2005).Pharmaceutical, food and cosmetic industries extensively use plant extracts.Also, it is important to study medicinal plants in order to document their active chemical compounds and various functions in curing different kinds of ailments (Nostro et al. 2000).
Herbs play a vital role in maintaining human health.Volatile oils or essential oils have been used for medical purposes because of their medicinal properties and are sourced from natural products.They assume great importance because of their antioxidant capacities (Rohloff 2003).Essential oils are secondary plant metabolites known for their fragrance and food flavour properties.They consist of complex chemical compounds such as monoterpenes and sesquiterpenes, phenyl propanoids and oxygenated compounds (Nostro et al. 2000).Essential oils offer promising potential for future applications in the fields of agriculture, medicine, pharmaceuticals and biotechnology.This may be as a result of their function as signalling compounds produced between different types of organisms and diverse biological systems coupled with their general antimicrobial effects, antioxidative effects and medicinal properties.
Essential oils can be extracted from various plant parts through different methods, of which hydrodistillation (HD), steam distillation and steam or water distillation are the most common (Bowles 2003;Surburg & Panten 2006).Other methods include solvent extraction, aqueous infusion, cold or hot pressing, effleurage, supercritical fluid extraction and phytonic process

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Scan this QR code with your smart phone or mobile device to read online.(Da Porto, Decorti & Kikic 2009;Hunter 2009;Martínez 2008;Pourmortazavi & Hajimirsadeghi 2007;Surburg & Panten 2006).The discovery and identification of biologically active secondary metabolites such as essential oils from new, promising drug species is testimony to the fact that the study of medicinal plants has clearly evolved.
Clausena, owing to its medical properties, has provided numerous natural products of interest to researchers in the field of pharmacology.Clausena is a genus of about 23 species of evergreen shrubs, occurring mostly in Asia (Burkill 1997).Its widespread availability in different parts of the world, including India, Tropical Asia and South Africa, is a great advantage.They are very easy to grow, are immune to pests and diseases and can withstand heavy pruning (Swarbrick 1997).It is therefore essential to compare the chemical components of essential oils extracted from C. anisata using solvent-free microwave extraction (SFME) and HD methods.

Extraction of essential oil using solvent-free microwave extraction
SFME was performed using the Milestone Dry DIST (2004) apparatus in accordance with the operational parameters, as described by Okoh, Sadimenko and Afolayan (2011).The multimode reactor comes with a twin magnetron (2800 W, 2450 MHz each) which delivers a maximum power of 500 W in 5 W increments.The rotating microwave diffusers in the unit ensure homogeneous microwave distribution throughout the plasma-coated polytetrafluoroethylene (PTFE) cavity.The attached infra-red sensor was used to regulate the temperature.
The reflux of condensed water by a circulating cooling system at 5 °C was used to keep the conditions of the temperature and water constant.Clausena anisata leaves (250 g) were placed into the reactor without the addition of water or any solvent, with exhaustive extraction of the essential oil accomplished in 30 min.

Extraction of essential oil using hydrodistillation
Clausena anisata leaves (250 g) were hydrodistilled for 3 h in an all-glass Clevenger apparatus in accordance with the description of the British Pharmacopoeia (1980), as similarly used by Okoh et al. (2011) with slight modifications, where the heat was supplied to the heating mantle (100°C) and the essential oil was extracted with 51 mL of distilled water for 3 h.The essential oil was collected and analysed without delay.

Gas chromatography-mass spectroscopy analysis
The chemical profiling of essential oil extracted through HD and SFME was performed using gas chromatography-mass spectroscopy (GC-MS).This was carried out using an Agilent (6890N) GC with CTC Combi Pal Autosampler and Agilent (5975B) MS.Identification of the chemical components of the essential oils was accomplished by correlating their mass spectra and retention indices with those of the Wiley 275 library (Okoh et al. 2011;Omoruyi, Afolayan & Bradley 2014).
The column type was ZB 274305, semivolatiles (30 m, 0.25 mm ID, 0.25 µm film thickness).The spectrogram of each identified compound was determined by the integration of the peak areas.

Results
The essential oils obtained from the leaves of C. anisata through SFME and HD are pale yellow and colourless, respectively.Higher oil yield of 5.2 mL/100 g was obtained through SFME compared to 3.7 mL/100 g obtained through HD.The GC-MS spectral data for the essential oil obtained by HD and SFME are shown in Figures 1 and 2, respectively.

Discussion
The essential oils of C. anisata contain numerous monoterpenes and sesquiterpenes, with α-Pinene, α.      predominant compounds.Monoterpenes are used for an array of therapeutic purposes such as anti-bacterial and anti-fungal treatment (Marchese et al. 2017).Sesquiterpenes are thought to be very effective in fighting cancer.The findings of this study showed that the amount of essential oils obtained through the SFME was more than that extracted through the HD extraction method from a qualitative point of view, which was still in accordance with Lucchesi, Chemat and Smadja (2004), who reported that extraction of essential oil from three aromatic herbs (basil, garden mint and thyme) using SFME method resulted in higher amounts of essential oils from both qualitative and quantitative points of view; thus, the method used for essential oil extraction will determine the type of chemical compounds found.

Conclusion
Essential oils are very important as a good source of healing to mankind.The results obtained from this study probably justify their usage in local communities for healing purposes.
The SFME and HD methods of extraction gave distinctive compounds that are imperative in pharmaceuticals and cosmeceuticals.SFME yielded more essential oils than the HD method.

TABLE 2 :
Kovat's index of chemical compounds and structural formula for the solvent-free microwave extraction.

TABLE 2 (
Continues): Kovat's index of chemical compounds and structural formula for the solvent-free extraction method.