A New Process for Green Extraction of Essential Oil from Eucalyptus Oleosa

Arabian Journal of Medicinal & Aromatic Plants Microwave-assisted hydro distillation of Eucalyptus oleosa

A new process for green extraction of essential oil from Eucalyptus

oleosa: Microwave-assisted hydro distillation

Saousan Chamali1,2,, Houcine Bendaoud1, Ezzeddine Saadaoui2, Walid Elfalleh1, Mehrez

Romdhane1*

1Laboratoire Energie, Eau, Environnement et Procèdes, (LEEEP) LR18ES35, Ecole Nationale d'Ingénieurs de Gabès, Université de Gabès, 6072 Gabès, Tunisia. 2Regional Station of Gabès Laboratory of Management and Valorization of Forest Resources, National research Institute of rural engineering, Water and Forests, Gabes, Tunisia

In this work, the potential of microwave-assisted Hydro-distillation (MAHD) process was evaluated for the extraction of Tunisian Eucalyptus oleosa essential oils and the results were compared with those of the conventional Hydro-distillation (HD) in terms of extraction time, essential oil yield, chemical composition, energy consumption and environmental impact. Results show that essential oil obtained by MAHD was quantitatively like those obtained by HD, but MAHD offered net advantages in terms of saving energy and extraction time (90 min against 25 min). In addition, the quality of the essential oil is improved thanks to a 50.42% increase in oxygenates compounds. Scanning electron microscopy (SEM) analyses of E. oleosa tissue structure before and after extraction were used as confirmation that microwave technique was faster than conventional HD. This study demonstrates the effectiveness of MAHD as a quick, green and efficient alternative for the extraction of essential oil from medicinal plants.

Keywords: Hydrodistillation, Microwave assisted hydrodistillation, Eucalyptus oleosa.

1. Introduction

As the knowledge about the long-term effects of excessive use of the synthetic molecules on human health increases, the demand to reduce the application of chemicals as antimicrobial

*Corresponding Author: Prof. Mehrez Romdhane Tel: +216-99617761; Fax: +216-75392190; E-mail: [email protected]

AJMAP V5N3, 2019 35 Arabian Journal of Medicinal & Aromatic Plants Microwave-assisted hydro distillation of Eucalyptus oleosa agents on the treatment of certain diseases increases dramatically. Simultaneously, there is an intense interest in the application of substances derived from plants as possible natural alternatives for conventional synthetic fungicides. Essential oils (EOs) are a complex mixture of volatile organic compounds produced as secondary metabolites whose functions are other than the nutrition. Well-known for their antiseptic and medicinal activities, they are also used in embalmment, and, due to their antimicrobial and antioxidant propriety, as natural food preservatives (Gil et al., 2010; Zhang et al., 2010). Eucalyptus is one of the diverse genus of flowering plants in the world. It belongs to the family Myrtaceae (subfamily Myrtoideae) and comprises about 747 species (Batish et al., 2008). Although most of the plants are native in Australia and Tasmania numerous species have been introduced in other parts of the world, including Tunisia. In 1957, Tunisia possessed 117 species of Eucalyptus, which have essentially been used as firewood, to produce mine wood and in the fight against erosion (Elaissi et al., 2011). Eucalyptus Eos rich in 1,8-cineole are among the 18 most commonly traded EOs in the world (Goldbeck et al., 2014). Today, they are widely used in food (Gilles et al., 2010; Vazquez et al., 2008), flavoring, perfumery (Muhlen et al., 2008), and in the pharmaceutical industries (Batish et al., 2008). They were traditionally extracted by hydrodistillation (HD) steam distillation and organic solvent extraction. However, these techniques present several disadvantages such as long extraction times, energy intensive technologies, and complex separation processes (Zhang et al., 2018). Hence, in order to find a suitable and cost-effective alternative to conventional methods, many researchers have been involved in replacing them with some new "green" methods. Recently, the use of microwave-assisted hydrodistillation (MAHD) for extraction of natural compounds from aromatic and medicinal plants has been increasingly developed. This is due to the advantages of the MAHD method: rapid heating, reduced hydrolytic effects, time saving and low energy and water consumption, all of which contribute to reducing environmental effect and costs (Al Mamoori and Al Janabi, 2018). Therefore, the objective of the current research was to make a comparative study of two processes of extraction of EOs of Tunisian Eucalyptus oleosa leaves: conventional hydrodistillation and microwave assisted hydrodistillation. This comparison is based on the level of the yield, the chemical composition, cost, energy consumption and safety environmental consideration. In addition, the conditions of microwave-assisted extraction have been optimized in order to improve the quality and the quantity of final product at lower cost.

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2. Materials and methods 2.1. Raw material Leaves of Eucalyptus oleosa F. Muell were collected in south west of Tunisia (Gabes) in December 2015. As a first step, the leaves were cleaned and air-dried until a constant weight was achieved. The moisture of plant material was quantified by Karl Fischer method and was about 9.5 ± 0.04%. Leaves were then grinded and sieved and an average particle size of 1.5 mm was retained. Specimens were identified by the seed department of the National Institute of Research in Rural Engineering, Waters and Forests (Pr. Khouja Mohamed Larbi; INRGREF - Tunisia). They were harvested from Zerkine arboreta (Gabes- Tunisia), which was introduced from Australia in 1964-1965. 2.2. Isolation of essential oils 2.2.1. Conventional hydro-distillation

Dried leaves of Eucalyptus (50 g) were submitted to hydrodistillation with 600 mL of water for 90 min using a Clevenger-type apparatus according to the method recommended by the European Pharmacopoeia (European Pharmacopoeia, 1997). The volatile distillate was put into amber vials and stored at a temperature below 4°C prior to analysis. 2.2.2. Microwave assisted hydro-distillation Hydrodistillation assisted by microwaves was operated at atmospheric pressure using a household microwave oven, which was mechanically modified to perform the hydrodistillation (Stashenko et al., 2004). It is a 2.45 GHz multimode microwave reactor with an input and output powers of 1150W and maximum 800W, respectively. The dimensions of the oven cavity are 329 mm (H) x 557 mm (W) x 418 mm (D). 2.3. Chemicals All chemicals used were of analytical reagent grade. All reagents were purchased from Sigma-Aldrich-Fluka (Saint-Quentin, France). 2.4. Gas chromatography/mass spectrometry (GC-MS) Quantitative and qualitative analysis of the EOs were performed using gas chromatography- flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS) were used in quantitative and qualitative analysis of the essential oil. Chromatography analysis was done on a Varian Star 3400 (Les Ulis, France) Cx chromatograph; the system was equipped with a DB-5MS capillary column (30 m × 0.25 mm,

AJMAP V5N3, 2019 37 Arabian Journal of Medicinal & Aromatic Plants Microwave-assisted hydro distillation of Eucalyptus oleosa film thickness 0.25 μm). Injector and detector temperatures were fixed at 200 and 270°C, respectively. The column temperature gradually raised from 60 to 260°C at 5°C/min and held for 15 min. A second gradient of 40°C/min was used to attain 340°C. For analysis, the volatile oil was dissolved in petroleum ether. Samples were introduced in the split mode at a ratio of 1:10. Helium (purity 99.999%) was utilized as the carrier gas at 1 mL/min. The spectrometer detector (Varian Saturn GC–MS–MS 4D) was regulated for an electron multiplier voltage of 1500 V and emission current of 10 μ A. MS transfer line and injector temperatures were fixed at 300 and 200°C, respectively. The trap temperature was 250°C. Identification of the volatile constituents was carried out comparing their mass spectra with those reported in the literature and by computer matching with the Wiley 8 and Adams libraries and co-injection with authentic compounds whenever possible (Adams, 2007).

2.5. Scanning electronic microscopy Scanning electronic microscope (SEM) LEO 435 VP / EDX swiftEd3000 Oxford (Laboratoire de Génie Chimique Toulouse) were used to perform the microstructure of the dried leaves before and after ASE extraction. Single E. oleosa leaves were fixed on aluminum support and sputter coated with platinum and then observed by SEM, under standard vacuum conditions with the secondary electron detector. 2.6. Environmental impacts and energy consumption The energy consumption during the extraction of EOs was determined by a Wattmeter connected to the power supply circuit of the microwave oven. Energy consumption was directly read from the digital display in kWh. The electric consumption of each extraction process was calculated using the following equation: Electric consumption (kWh) = Power (kW) x Extraction time (h). Eq.1 The calculations of carbon dioxide rejected in the atmosphere were reported in previous studies (Levihn, 2014); 800 g of CO2 will be emitted into the atmosphere during the combustion of an amount of fuel needed to generate 1 kWh. In Clevenger type apparatus, cold water was used to condense the vapor mixture of water and oil. The calculations of wastewater have been made using the following equation:

Eq.2

3. Results and discussion 3.1. Extraction technique

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There is a significant difference observed between the color intensities of the EOs extracted by HD and MAHD. Indeed, EOs extracted by HD were less colored than that one obtained by MAHD. Efficiency of microwave power were evaluated to determine the optimal conditions for rapidly extract the EO, in order to avoid the degradation of thermolabile molecules and to reduce the energy consumption. Figure 1 showed the total extraction time in relation with the microwave irradiation power for both methods. By increasing microwave power from 300 to 800 W, the total extraction period decreased. A microwave irradiation power of 600 W for 50 g of E. oleosa leaves was chosen as an optimum of power which permit to achieve extraction of the EO by MAHD in only 25 min. As shown in Figure 2, at 600 power settings, the extraction kinetics of EOs from E. oleosa leaves isolated by MAHD were compared with that of classical method of extraction HD. For both methods, the extraction temperature was equal to the boiling point of water (100◦C) at atmospheric pressure. With MAHD, the first EO droplets were observed after 10 min; while in the case of HD this was at 19 min. HD is an approved technique that is used as reference for the isolation of EOs (Stahl-Biskup and Saez, 2002).An extraction time of 25 min with MAHD provided yield comparable to those obtained after 90 min with HD. Consequently, the extraction time in MAHD was at least 3 times faster than that of the HD. Microwave extraction offers a fast delivery of energy to a total volume of water and also to the E.oleosa

Figure 1. Effect of extraction method and microwave power on total extraction time of EOs from E. oleosa leaves (mean values ± Standard deviations (SD); n = 3).

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Figure 2.Global yield profile of the E. oleosa EOs obtained by HD ( ) and MAHD (●) and as a function of the extraction time (mean values ± SD; n = 3). ⧍

2014). Other studies have cited that the yields of E. oleosa from the central Tunisia (Elaissi et al., 2007) and southern Tunisia (Ben Marzoug et al., 2010) were 2.6 and 4.8%, respectively. To our knowledge, no previous study reported the extraction of EOs isolated from our species of eucalyptus by MAHD. However, Gupta et al. (2013) were used a microwave power of 640 W to extract 1.2% of the EO from Indian Eucalyptus Citriodora leaves for 100 min by MAHD. Also, Moghaddam et al. (2013) were extracted 0.2% of EO from Iranian E. microtheca leaves by MAHD in 20 min at 700 W.

3.2. Chemical composition The results of the chemical identification of extracted E. oleosa EOs obtained by HD and MAHD at optimal conditions are summarized in Table 1. A total of 30 compounds were identified in HD EOs and 28 compounds in MAHD EOs, which accounted for 99.97%, 99.98% of the total oil composition, respectively. For the sake of simplicity, the discussion

AJMAP V5N3, 2019 40 Arabian Journal of Medicinal & Aromatic Plants Microwave-assisted hydro distillation of Eucalyptus oleosa has been limited to the major components detected for the both methods. These main volatile compounds are presented by 11 components detect at fraction higher than 2%.

Table 1. Chemical composition of Eucalyptus obtained from E.oleosaby conventional and microwave assisted hydrodistillation. E. oleosa N° KI Compound HD MAHD 1 936 α-pinene 12.28 2.53 2 955 camphene 0.24 0.25 3 959 2,4-thujadiene 0.40 3.16 4 982 β-pinene 11.36 0.09 5 1008 α-phellandrene 0.17 6 1028 m-cymene 17.02 8.50 7 1036 1,8-Cineole 15.31 25.65 8 1094 α-pinene epoxide 0.40 0.28 9 1135 3-terpinenol 1.60 1.61 10 1155 (E)-verbenol 8.86 16.00 11 1177 (E)-pinocamphone 0.69 0.48 12 1179 methyl phenylacetate 2.24 3.33 13 1184 (Z)-pinocarveol 0.99 0.44 14 1190 dihydrocarveol 0.90 0.98 15 1211 verbenone 13.70 20.98 16 1222 isodihydrocarveol 4.44 5.16 17 1267 (Z)-anethole 4.39 18 1349 (Z,E)-4,6,8-Megastigmatriene 0.86 0.46 19 1355 thymol acetate 0.34 0.53 20 1394 α-copaene 0.78 21 1467 β-caryophyllene 0.98 0.37 22 1493 valencene 0.20 0.33 23 1526 δ cadinene 0.20 24 1549 α-calacorene 0.38 0.39 25 1612 β-himachaleneoxide 2.05 1.40 26 1624 zingiberenol II 1.89 1.36 27 1635 γ-eudesmol 0.34 0.27 28 1647 himachalol 0.44 0.34 29 1661 7-epi-α-eudesmol 0.35 0.24 30 1669 bulnesol 0.22 0.14 31 1713 (Z,Z)-farnesol 0.34 0.32 Identified components 99.97 99.98 Monoterpene hydrocarbons 41.47 14.53 Monoterpenes oxygenated 46.89 75.97 Sesquiterpenes hydrocarbons 2.544 1.09 Sesquiterpenes oxygenated 6.01 4.46 Others 3.44 4.32 Total oxygenated compounds 56.34 84.75 Total no-oxygenated compounds 44.01 15.62 HD: hydrodistillation; MAHD: microwave assisted hydrodistillation.

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Both HD oil and MAHD extract contained the same main compounds vary more or less according to the extraction technique used such as 1,8-cineole (15.31 and 25.65%, respectively), m-cymene (17.0 and 28.50%, respectively), α-pinene (12.28 and 2.53%, respectively), β-pinene (11.36 and 0.09%, respectively), (E)-verbenol (8.86 and 16.00%, respectively), verbenone (13.70 and 20.98%, respectively), isodihydrocarveol (4.44 and 5.16%, respectively), methyl phenylacetate (2.24 and 3.33%, respectively), and β- himachalene oxide (2.05 and 1.40%, respectively). Great differences concerning the quantity of some compounds were specially found with respect to the type of extraction method: such as α-pinene present six times in MAHD (12.28 %) than in HD (2.53%) and (E)-verbenol detect two times in MAHD (16.00 %) than in HD (8.86%),a markedly lower quantity of β-pinene was found in MAHD (0.09%) compared to HD (11.36%), and the absence of myrtenal in (Z)-anethole compared to MAHD method (4.39%). By referring to the literature, the investigated E. oleosa HD essential oil showed a marked difference in composition, by comparison to EOs from the same species collected in the southern Tunisia (Ben Marzoug et al., 2010). This essential oil was characterized by higher content of m-cymene and verbenone and lower content of α-pinene, γ-eudesmol, limonene and 1,8-cineole. Moreover, some of the compounds, namely β-himachaleneoxid, zingiberenol II and isodihydrocarveol found exclusively in our EOs were never found in others reports. A higher amount of oxygenated compounds was observed in the E. oleosa EOs obtained by MAHD process. Indeed, this difference in the relative amount between the different constituents was due to the difference in the duration of the extraction by the different processes. Therefore, it was inferred that the MAHD extracted better quality EOs than those isolated by HD because they were richer in the main component having commercial value such as 1,8-cineole and m-cymene. 3.3. Energy and environmental impact: The cost saving of EOs extraction from E. oleosa is clearly advantageous for the MAHD method in terms of time, energy consumption and environmental effect (Table2). MAHD procedure showed a significant saving in the electric consumption was clearly. Indeed, the energy requirement needed for EOs to be extracted thoroughly from plant matrix, based on the power consumptions of the microwave stove (in MAHD)and the heating mantle (in HD), were 1,2 kWh by HD against only 0.43kWh by MAHD. Concerning environmental effects, MAHD offered a lower quantity of carbon dioxide rejected in the atmosphere

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(346.7±12.22CO2/g EO) compared to HD method (1013 ±122.20 g CO2/g EO). Wastewater used in MAHD (198±7.64 L/kg EO) was inferior to those in HD (634±4.04 L/kg EO).

Table 2. Energy and environmental impact of conventional and microwave-assisted hydrodistillation. Extraction method HD MAHD Power (W) 800 800 Extraction time (min) 109 30 Electric consumption (kWh) 1.27±0.15 0.43±0.02

CO2 rejected (g CO2/g of EO) 1013±122.20 346.7±12.22 Waste water rejected (L/kg of EO) 634±4.04 198±7.64 HD: hydrodistillation; MAHD: microwave assisted hydrodistillation. Values are given as mean ± SD (n = 3); Standard deviations (SD) did not exceed 5%.

3.4. Effect of extraction method on the plant pores morphology The SEM micrographs of the E. oleosa leaf before and after different extraction methods were presented in Figure 3. Figure 3A is a micrograph of the untreated leaf. Images from the treated E. oleosa leaves by HD (Figure 3B) and by MAHD (Figure 3C) are also presented for comparison. As shown in Figure 3A, the glandular trichomes containing the EOs are distributed on the external surface of the E. oleosa leaf. After HD extraction, these secretory structures were empty, but still intact (Figure 3B), while after MAHD, an important modifications were clearly seen as a huge destruction and large perforation on the plant epidermises ensuring the loss of bioactive compounds from the leaf sample (Figure 3C). Such observations can be attributed to the microwave irradiation mechanism. In fact, the higher dielectric constant of the water permitted more absorption of the microwave energy in the raw material. Thus, this absorbed irradiation caused a fast increase in localized temperature inside the plant tissue, which exceeded the capacity of the glands for expansion and consequently a faster rupture of the glandular walls was observed.

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Figure 3.SEM images of untreated E.oleosa leaf (A), after HD extraction (B) and after MAHD extraction (C) at 600 W.

4. Conclusion This work confirms that MAHD method offers important advantages over HD, i.e., shorter extraction duration, better chemical composition, reduced cost, less energy consumption and lighter environmental impact. SEM images of E. oleosa leaf after MAHD and HD indicated that microwaves cause a rapid rupture of the glandular walls resulting in higher isolation efficiency at a shorter time. GC–MS results indicated the better quality of MAHD essential

AJMAP V5N3, 2019 44 Arabian Journal of Medicinal & Aromatic Plants Microwave-assisted hydro distillation of Eucalyptus oleosa oil with higher amount of oxygenated compounds. Significantly lower energy consumption with MAHD renders this method being more ecofriendly than HD. Finally, MAHD was presented as a rapid, green, economical and efficient process suitable for the extraction of the high quality of EOs from the medicinal plants especially Eucalyptus leaves.

Acknowledgment The authors wish to express their gratitude to the Ministry of Higher Education and Scientific Research for providing financial assistance Conflicts of Interest We declare that we have no conflict of interest. References Adams, R. P. (2007). Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy, (4th ed). Allured publishing corporation .Carol Stream, Illinois. Al Mamoori, F., & Al Janabi, R. (2018). Recent advances in microwave-assisted extraction (MAE) of medicinal plants: a review. International Research Journal of Pharmacy, 9, 22– 29.https://dx.doi.org /10.7897/2230-8407.09684. Batish, D.R., Singh, H.P., Kohli, R.K., &Kaur, S. (2008). Eucalyptus essential oil as a natural pesticide. Forest Ecology and Management, 256, 2166–2174. https://dx.doi.org /10.1016/j.foreco.2008.08.008. Ben Marzoug, H.N., Bouajila, J., Ennajar, M., Lebrihi, A., Mathieu, F., Couderc, F., Abderraba, M., &Romdhane, M. (2010). Eucalyptus (gracilis, oleosa, salubris, andsalmonophloia) Essential Oils: Their Chemical Composition, Antioxidant, and Antimicrobial Activities. Journal of Medicinal Food, 13,1–8. https:// dx.doi.org /10.1089/jmf.2009.0153 Elaissi, A., Chraif, I., Bannour, F., Farhat, F., Salah, M.B., Chemli, R., &Khouja, M.L. (2007). Contribution to the Qualitative and Quantitative Study of Seven Eucalyptus Species Essential Oil Harvested of Hajeb’sLayoun Arboreta (Tunisia). Journal of Essential Oil Bearing Plants, 10, 15-25. http://dx.doi.org/10.1080/0972060X.2007.10643513. Elaissi, A., Medini, H. M., Simmonds, F., Lynenc, F.F., Chemli, R., Skhiri, F. H., &Khouja, M.L. (2011).Variation in volatile leaf oils of seven Eucalyptus species harvested from Zerniza arboreta (Tunisia).Chemistry & Biodiversity,8, 362-372.https:// dx.doi.org /10.1002/cbdv.201000103. European Pharmacopoeia. (1997). 3rd ed. Council of Europe,Strasbourg, p. 121. Gil, L., Tadesse, W., Tolosana, E.,&López, R. (2010). Eucalyptusspecies management, history, status and trends in Ethiopia.Ethiopian Institute of Agricultural Research, Addis Ababa,62-68.

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