Archives of Pharmacology and Pharmaceutical Sciences Research Article: RD-PHA-10003

The Chemical Compositions and Antibacterial Activity of Radiata Leaf Essential Oil

Asare Ebenezer Aquisman*,1,2 Droepenu Eric Kwabena1,2

1Graduate School of Nuclear and Allied Sciences, University of Ghana, Ghana

2Faculty of Resource Science and Technology, University Malaysia Sarawak, Malaysia

Abstract

The aim of this present study was to assess the yield, chemical constituent and antibacterial activity of Eucalyptus radiata leaf essential oil. Hydrodistillation method was used to obtain the E. radiata essential oil. Higher yields were obtained in mature leaf of E. radiata as compared to young leaf. Gas chromatography coupled with mass spectrometry (GC/SM) was used to analyse the chemical composition of the essential oil. The major compounds identified were, 1,8-cineole (i.e. 65.9% in young leaf oil sample and 62.8% in mature leaf oil sample), α-terpineol (10.3% in young leaf oil sample and 11.1% in mature leaf oil sample) and limonene (9.3% in young leaf oil sample and 10.9% in mature leaf oil sample). In terms of commercial feasibility, the E. radiata essential oil leaf collected in Odoben, Central part of Ghana satisfies the requirement. The E. radiata essential oil leaf was found to have potential antimicrobial activity against all tested bacterial {Staphylococcus aureus, (Gram +ve), Escherichia coli (Gram–ve), Acinetobacter baumanni (Gram –ve), Klebsielia pneumonia (Gram +ve) and Exiguobacterium aquaticum (Gram +ve)}. Keywords: Eucalyptus radiata, Antibacterial activity, Major compounds, Hydrodistillation, GC-MS

Introduction etc. are few examples. Consequently, additional study of Research on essential oil is wide and ascending. Nevertheless, prospects of the essential oils of the species of among the countless essential oils studied, species affiliating family is necessitated.1,3,4 Especially; commercial, to the Myrtaceae family are most well received in terms of pharmaceutical purpose, and medicinal essential oils for characteristics. Essential oils from the family of Myrtaceae example Eucalyptus essential oil.2,5 The genus Eucalyptus are used in soaps, toothpastes and body creams according from the family of Myrtaceae has been planted for countless to Gillian DM & Luqman S, et al.1,2 The essential oil of of years as suggested by Luqman S & Ali B, et al.2,6 One tea tree (M. altrnifolia); nutmeg (M. fragrans); thyme (T. of the medicinal Eucalyptus essential oils understudied Vulgaris); clove (S. aromaticum); blue gum (E. globulus) is Eucalyptus rdiata. It is essential aromatic oil, used for different kinds of infectious conditions alone or in a mixture Citation: Asare Ebenezer Aquisman, Droepenu Eric Kwabena (2019) of other essential oils. It uses as anti-infective make it an The Chemical Compositions and Antibacterial Activity of Eucalyptus indicator of antimicrobial potential of the essential oil and Radiata Leaf Essential Oil, JArchives of Pharmacology and Pharmaceu- tical Sciences (APPS). RD-PHA-10003 its compounds. Nevertheless, the chemical composition and antimicrobial activities of Eucalyptus r. growing in Received Date: 28 June 2019; Accepted Date: 08 July 2019; Ghana is yet to be examined. A starting point of scientific Published Date: 11 July 2019 proof needs to be set out in order to better comprehend its *Corresponding author: Asare Ebenezer Aquisman, Graduate School therapeutic features, anti-infective ability and champion its of Nuclear and Allied Sciences, University of Ghana, Ghana, Faculty real use in medicinal practices. Therefore, this present study of Resource Science and Technology, University Malaysia Sarawak, was sketched to assess yield, chemical composition and Malaysia, Email [email protected] antimicrobial activity of Eucalyptus radiata leaf essential © 2019 - ReDelve. All Rights Reserved oil. Arch Pharmaco and Phar Sci(APPS)2019| volume 1 | issue 1 1 The Chemical Compositions and Antibacterial Activity of Eucalyptus Radiata Leaf Essential Oil Materials and methods auto – sampler at 24.79 psi and an inlet temperature of 250°C. Chromatographic separation was achieved by a BPX-5 capillary Plant collection column (29.5m×0.25mm i.d., 0.2μm film thickness) with a splitless The E. radiate fresh leaves were collected from a cultivated site injector and mass spectrometer detector. Oven temperature was -1 in Agona Odoben in the Central part of Ghana. Material samples programmed from 60°C to 2400C (5 min hold), at 6°C min , and -1 (Leaves) were identified and harvested under the assistance of local from 240°C to 300°C (15 min hold), at 6°C min rate. Helium was -1 farmer, Mr. Kojo Saah. To avoid loss of any volatile compounds used as the carrier gas (0.98ml min ). Spectra were achieved on all the plant material were processed within 32 hours. electron impact at 70eV, scanning from 35 to 550m/z. Each peak area of the gas chromatography components represented the % Preparation of Eucalptus radita leaf essential oil (percentage) composition of each individual component as a % (percentage) of all the peak zones (areas) obtained from electronic According to1,7 Eucalyptus is locally prepared in the form of poultices or infusions, which can be ingested as teas or used through integration computations using flame ionization detector (FID, inhalation. Infusions are usually for respiratory diseases whereas 250°C). 42 n-Alkanes were used as reference point in the poultices are for topical application. The medicinal application of measurement of relative retention indices (RRI). Eucalyptus essential oil is known for respiratory conditions. In Preparation of test samples this present study, the essential oil was obtained, as explained by Van Vuuren SF et al.8 using a Clevenger – type apparatus which Eucalyptus radiata essential oil extract was tested using disc was subject to hydrodistillation. Young and mature leaves were diffusion method on nutrient agar medium.9 This study was distilled independently throughout the extraction period. In short, performed by preparing serial diluted concentrations of 300mg/ a known amount of 500g of weighed fresh leaf plant material was mL, 150mg/mL, 75mg/mL, 35.5mg/mL, 17.75mg/mL and placed into 5000L round bottom flasks. About 850L of distilled 8.75mg/mL. was added into each round bottom flasks. Each round bottom flasks with a mixture of water and plant material was heated to Preparation of media (Bacteria broth) boiling point at a temperature of 100°C with the help of heating The bacteria of interest for the activity of the E. radiata essential mantle for 3 hours, 30 minutes. The heated mixture of plant and oils were obtained from the stock culture provided by Virology water produces water vapour and oil. The water helps in carrying Laboratory, UNIMAS (Universiti Malaysia Sarawak). Five out the volatile oils as well as prevention of overheating. The bacterial were cultured using Tryptone Soya broth (TSB) and water vapour and volatile oil mixture was condensed to liquid Tryptone Soya agar (TSA). The media (TSB and TSA) was form via the condensing tube. The water and essential oil mixture prepared based on the manufacturer’s instructions. A mass of run from the condenser and collected in the receiving tube of 2.50g of the dried broth was dissolved in 200.0mL distilled water, the Clevenger apparatus. Essential oil is immiscible in water, and then autoclaved at a temperature of 121°C for 18 minutes. following in a separation of the two liquids in the collecting tube. The bacterial was sub-cultured in a 10.0mL of broth, each in This permitted for easy segregation of the hydrosol and essential recommended glass vial bottle for 24.0 hours in an incubator with oil during collection. a shaker at a temperature of 37°C (normal body temperature). The essential oil yield was calculated using Equation 1, Turbidity (Optical Density/OD) of the bacterial broth after 24.0 hours was computed by UV Mini Spectrophotometer, model Percentage yield = Weight of essential oil/Weight of plant material * 100 1240 of Shimadzu brand at wavelength 575nm. The turbidity in Equation 1 the media was used to confirm the sterility of the media. It was compared to the nutrient broth standard until the desired Optical The assessed essential oil yield was recorded and stored in tightly Density of 0.6-0.9 was obtained. sealed amber bottles, away from light at 3°C for further analysis. Plate inoculation Chemical composition analysis The inoculation of the bacteria was performed by using Biohazard The essential oils were determined using Shimadzu Gas cabinent.9,10 1mL of the prepared broth was streaked over the entire Chromatography/Mass Spectrometer (GC/MS) QP 2010. A agar plate surface in four different directions using sterile cotton volume of 1µL was injected using a split ratio (200:1) with an bud. Before applying the test samples, the agar plate was then

Arch Pharmaco and Phar Sci(APPS)2019| volume 1 | issue 1 The Chemical Compositions and Antibacterial Activity of Eucalyptus Radiata Leaf Essential Oil left for 5-10 minutes. A 10μl volume of test extract (Eucalyyptus Compound Young Mature r. essential oil) of concentrations 300mg/mL, 150mg/mL, 75mg/ α – Pinene 0.9±0.02 1.4±0.01 mL, 35.5mg/mL , 17.75mg/mL and 8.75mg/mL were each pupated onto the discs (6.0mm diameter) and gently pressed α – Thujene 2.4±0.01 1.9±0.02 onto the agar plate using sterile forceps for 12.0 minutes at 25°C β – Pinene 0.8±0.01 1.1±0.02 (room temperature). A disc pupated with methanol and 30.0μg of Sabinene 1.2±0.01 1.1±0.03 tetracycline as standard antibacterial agent were used as negative Mycrene 1.2±0.02 1.4±0.01 and positive controls respectively. Each of the test samples were tested in triplicate for the bacterium used. The plate samples were α – Terpinene 1.3±0.01 1.0±0.01 then incubated at a normal body temperature (37°C) for 24 hours Limonene* 9.3±0.02 10.9±0.07 before the inhibition zone of each sample disc investigated. The 1,8 – Cineole* 65.9±0.04 62.8±0.09 inhibition zone was measured in diameter (mm) to exhibit the γ – Terpinene Tr 0.1±0.01 presence of antibacterial activity for all the samples compared to the positive control. (E) - β – Ocimene Tr Tr ρ – Cymene 0.6±0.01 1.0±0.01 Statistical analysis Terpinolene 1.0±0.01 0.9±0.01 The measurements of all the assessments were replicated 3 (three) z - 3 – Hex-en-1-ol 0.9±0.02 0.6±0.02 times for each assay. All the data received are presented as mean ± Linalool 0.7±0.01 0.4±0.02 SD. The analysis of data was performed by using windows SPSS version 17.0. To statistically evaluate the results, Kendall’s tau-b Trans-ρ-menth-2-en-1-ol 0.3±0.01 0.5±0.02 correlation analysis (level of significant is 0.05) was employed Terpinene-4-ol 0.3±0.01 0.1±0.01 to comprehend the relationship of the oil yield and the other γ-Terpinol 0.2±0.03 0.4±0.04 parameters. Neral 0.6±0.02 0.3±0.01 Results α-Terpineol* 10.3±0.06 11.1±0.02 The E. radiata essential oil young and mature leaves were extracted Geranial 0.3±0.01 0.5±0.01 independently by hydrodistillation method and each of the γ-Elemene 0.2±0.01 0.1±0.03 percentage yields was calculated. Table 1.0 show the percentage yield produced from both young and mature leaf of Eucalyptus r. Geraniol 0.4±0.03 0.3±0.01 . Spathulemol Tr 0.1±0.01

Table 1 E.Radiataessential oil yield (level of significant is 0.05) γ-Eudesmol 0.2±0.01 0.4±0.02 α-Eudesmol 0.1±0.02 0.1±0.02 Plant extract Yield (%; w/w) β-Eudesmol 0.2±0.03 0.1±0.01 Young leaf 2.23±0.1 Total % area 98.9±0.43 98.6±0.54 Mature leaf 3.51±0.1 *(bold) = Major compounds, tr = trace amount (less than 0.1% compo- sition)

Chemical composition of E. radiata Number of replicate = 3

The major components of the E. radiata matured and young leaf Antibacterial efficacy essential oil identified by Gas Chromatography/Mass Spectroscopy The effect of E. radiata essential oil leaf extracts on are shown in Table 2 based on their percentage composition. Staphylococcus aureus, (Gram +ve), Escherichia coli (Gram– Table 2Chemical composition of Eucalyptus radiataleaf essential oil ve), Acinetobacter baumanni (Gram –ve), Klebsielia pneumonia (level of significant is 0.05) (Gram +ve) and Exiguobacterium aquaticum (Gram +ve) are shown in Table 3.

Arch Pharmaco and Phar Sci(APPS)2019| volume 1 | issue 1 3 The Chemical Compositions and Antibacterial Activity of Eucalyptus Radiata Leaf Essential Oil Table 3 Effect ofEucalyptus radiata essential oil on selected bacteria Essential oil Concentration Plant Staphylococcus aureus Escherichia Coli Acinetobacter baumanni Klebsielia pneumonia Exiguobacteriu maquaticum (mg/mL) Part (Gram + ve), (Gram -ve) (Gram –ve) (Gram+ve) (Gram +ve)

Control 3.07±0.01mm 3.04±0.02mm 3.04±0.01mm 3.05±0.01mm 3.06mm±0.01 300 Young 1.63±0.02xmm 1.77±0.03ymm 0.97±0.03mm 1.69±0.04mm 0.89±0.02mm

Mature 1.99±0.02ymm 1.61±0.02mm 0.98±0.01ymm 2.01±0.02xymm 1.05±0.04xmm

150 Young 1.01±0.01xmm 0.92±0.03mm 0.77±0.02mm 0.98±0.01mm 0.83±0.01mm

Mature 1.41±0.02mm 1.42±0.02mm 0.91±0.01mm 1.69±0.02xmm 0.97±0.03mm

75 Young 0.64±0.01mm 0.79±0.01mm 0.84±0.03xmm 0.81±0.04 mm 0.61±0.04mm

Mature 1.02±0.02mm 0.89±0.02mm 1.77±0.04xmm 1.07±0.02 mm 0.97±0.02mm

35.5 Young 0.51±0.02mm 0.63±0.02xmm 0.52±0.01mm 0.46± 0.01mm 0.57±0.01mm

Mature 0.81±0.01xmm 0.61±0.02mm 0.64±0.04mm 0.72±0.02mm 0.64± 0.03mm

17.5 Young 0.47±0.03mm 0.055±0.01xmm 0.48 ±0.01mm 0.41±0.02mm 0.46±0.01mm

Mature 0.51±0.01mm 0.62±0.01mm 0.58±0.03mm 0.52±0.01mm 0.59±0.02xmm

8.75 Young 0.41±0.01mm 0.46±0.03mm 0.32±0.02mm 0.41±0.02mm 0.48±0.03xmm

Mature 0.46±0.02mm 0.59±0.04xmm 0.49±0.04mm 0.52± 0.01mm 0.55±0.02mm

Discussion species grown in Zambia accounted 1,8-cineole, α-terpineol The results obtained in the chemical analysis of young and limonene as the major compounds of E. radiata 12 13 and mature Eucalyptus r. essential oil samples are shown essential oil sample. Furthermore, Bendaoud et al. in in Table 2.0. In all, twenty-six (26) compounds were 2009 in its study on GC/MS analysis and antimicrobial and recognized within six (6) samples constituting for 91.5– antioxidant activities of essential oil of Eucalyptus radiate 98.5% with the average standard deviation of ± 0.01 of the reported 69.5% of 1,8-cineole, 11.9% of α-pinene and 4.8% oil composition. The major compound found in all the six of trans-pinocarveol as the major compounds obtained from 13 14 oil samples irrespective of plant age was 1,8-cineole (i.e. oil sample of Tunisia. In addition, Mulyaningsih, et al. in 65.9 % ±0.04 in young leaf oil sample and 62.8% ±0.09 in 2011 in its study on antibacterial activity of essential oils from mature leaf oil sample). Also, α-terpineol (10.3% ±0.06 in Eucalyptus and of selected components against multidrug- young leaf oil sample and 11.1%±0.02 in mature leaf oil resistant bacterial pathogens noted 82.70% of 1,8-cineole, sample) and limonene (9.3%±0.02 in young leaf oil sample 7.00% of α-terpineol and 3.7% of α-pinene as the major 14 and 10.9%±0.07 in mature leaf oil sample) were other major compounds of Eucalyptus r. oil sample from Germany. 15 compounds. E. radiata was proven to produce medicinal type According to, Luis, et al. in 2015 in its study on chemical of essential oil because of rich in 1,8-cineole. The chemical composition, antioxidant, antibacterial and antiquorum compositions of Eucalyptus radiata leaf essential oil from sensing activities and essential oils reported 68.51%, 8.60% different parts of the globe have been formerly accounted. and 6.07% of limonene, α-terpineol and α-terpinyl acetate Singh in 1994 in its study on chemical composition of the leaf were the major compounds obtained respectively from the 15 oil of Eucalyptus radiata reported 1,8-cineole, α-terpineol oil sample collected from Portugal. Gillian in 2016 in and limonene as the major compounds (i.e. 74.25%, 11.60% its study on antimicrobial activity and chemical analysis and 4.59% respectively).11 Chisowa in 1997 in its study on of Eucalyptus radiata leaf essential oil noted 65.7% of chemical composition of essential oils of Three Eucalyptus 1,8-cineole, 12.8% of limonene and 6.5% α-terpineol as the

Arch Pharmaco and Phar Sci(APPS)2019| volume 1 | issue 1 The Chemical Compositions and Antibacterial Activity of Eucalyptus Radiata Leaf Essential Oil major compounds obtained from the oil sample collected compared with the inhibition rate of young leaf. from South Africa.1 Five (5) out of six of these previous studies 1,8-cineole is the major compound found which is In addition, Acinetobacter baumanni against young leaf and in confirmation with the findings of this study. The only mature leaf of Eucalyptus radiata exhibited inhibition within deviation was with the Eucalyptus radiata oil sample noted the range of 0.32±0.02mm to 0.97±0.03mm and 0.49±0.04mm by Luis et al.15 in 2015 in its study on chemical composition, to 0.98±0.01mm respectively. The inhibition rate at 8.75mg/ antioxidant, antibacterial and antiquorum sensing activities mL–300mg/mL was considerably high in mature leaf oil and essential oils.15 This sample was not only lacking of the sample of E. radiata against Acinetobacter baumanni as major compound 1,8-cineole, but have limonene compound compared with young leaf oil sample of E. radiata. as the major. The variations can be attributed to the variations in environmental and genetic factors owing to differences of The interactions of young and mature leaf essential essential oil seeds and location.1,13,14,16 oil samples against Klebsielia pneumonia at various concentrations showed an active inhibition within the range In this present study, Eucalyptus radiate leaf essential oil of 0.41±0.02mm to 1.69±0.04mm and 0.52±0.01mm to test samples were lacking of sixty-four (64) undesirable 2.01±0.02mm respectively. The inhibition rate of mature leaf compounds (isovaleradehyde and phellandrene), making the essential oil extract at concentration of 300mg/mL was found E. radiata essential oil desirable for medicinal applications. to have a significant antibacterial activity against Kliebsiela pneumonia as compared to the same concentration of young In relation to commercial practicality i.e. 60.0% to 65.0% leaf. 1,8-cineole content, about 5/6 of the tested samples satisfy this requirement. The inhibition in terms of young and mature oil samples against Exiguobacterium aquaticum at 8.75mg/mL–300mg/ At the serial dilution concentration of 8.75mg/mL–300mg/ mL were within the range of 0.48±0.03mm to 0.89±0.02mm mL in Table 3, the inhibition activity of essential oil and 0.55±0.02mm to 1.05±0.044mm respectively. From samples in E. radiate young leaf and mature leaf on the Table 3, it was observed that there is high inhibition rate of bacteria Staphylococcus aureus, ranges from 0.41±0.01mm oil extract of mature leaf against Exiguobacterium aquaticum to 1.63±0.02mm and 0.46±0.02mm to 1.99±0.02mm than young leaf type. respectively. High inhibition rate was observed from all the serial concentrations (8.75mg/mL–300mg/mL) in essential Furthermore, it was observed that the E. radiata essential oil leaf was found to have the highest potential antimicrobial oil derived from both mature and young leaf E. radiata. activity against all tested bacterial. Nevertheless, Kliebsiela There is a higher inhibition rate in mature leaf essential oil pneumonia, staphylococcus aureus, Escherichia coli and against Staphylococcus aureus than young leaf oil sample. Acinetobacter baumanni were more sensitive to E. radiata The result obtained proves that the E. radiata mature leaf essential oil leaf. There is also significant antibacterial essential oil sample at 300mg/mL has a strong antibacterial activity against Exiguobacterium aquaticum. It can be activity against Staphylococcus aureus with fairly strong explained that the antimicrobial properties of Eucalyptus antibacterial activity against Staphylococcus aureus of leaf radiata was found to be dose dependent. Hence, the locally extract. use of Eucalyptus radiata for the treatment of infectious diseases such as respiratory ailments prescribed by some E. coli (Escherichia coli) was inhibited by the young native doctors and herbalists is a vital attempt because of leaf and mature leaf essential oil samples in all the serial presence of some bioactive components (1,8-cineole) which concentration as shown in Table 3, within the range of has a medicinal purposes. 0.46±0.03mm to 1.77±0.03mm and 0.59±0.04mm to 1.61±0.02mm respectively. At 17.5mg/mL, 35.5mg/mL and Limitations 300mg/mL the young leaf oil sample of Eucalyptus radiata gave strong inhibition against Escherichia coli as compared Due to lack of support, the toxicity study to assess the safety with mature leaf oil sample of Eucalyptus radiata. There of E. radiata essential was ignored. Therefore, future toxicity was higher inhibition rate of mature leaf essential oil sample studies could be conducted to determine the toxicity profile at 8.75mg/mL, 75mg/mL and 150mg/mL against E coli as of the essential oil.

Arch Pharmaco and Phar Sci(APPS)2019| volume 1 | issue 1 The Chemical Compositions and Antibacterial Activity of Eucalyptus Radiata Leaf Essential Oil

Conclusion 6. Anna C, Claire K, William H, et al. Rodale’s Illustrated Encyclope- dia of Herbs. 5th ed. Rodale Press: USA; 1998. The study of Eucalyptus radiata essential oil leaf has exhibited for the first time, that the oil sourced from 7. Van Vuuren SF, Docrat Y, Kamatou GPP, et al. Essential oil com- Odoben in the Central part of Ghana has a desirable profile, position and antimicrobial interactions of understudied tea tree i.e. (62.8%–65.9% 1,8-cineole). Also have antibacterial species. South African Journal of Botany. 2014;92:7–14. potentials for medicinal purpose. 8. Isaac JU, Fasihuddin AB, Hauwa AU. Phytochemical Screening of Acknowledgments Essential Oils and Antibacterial Activity and Antioxidant Proper- ties of Barringtoniaasiatica(L) Leaf Extract.Biochemistry Research The authors acknowledge the contribution of colleagues International. 2019;1–6. from Analytical Laboratory and Bioanalytical Laboratory 9. Asare EA. Quality assessment of some selected brands of anti–ma- of Faculty of Resource Science and Technology (FRST), larial drugs used in Ghana: A Case Study of Agona West Munici- Universiti Malaysia Sarawak. pality.International Nuclear Information System. 2016;48(23):76. Conflicts of interest 10. Singh AK. Chemical composition of the leaf oil of Eucalyptus radiata Sieb, ex DC subsp. Robertsonii (Blakely) L. Johnson et D. The authors declare that they have no conflicts of interest. Blaxell: A rich source of of Pharmacopoeia grade. Journal of Essential Oil Research. 1994;6:657–659. Referrences 11. Chisowa EH. Chemical composition of essential oils of Three Eucalyptus species grown in Zambia.Journal of Essential Oil Re- 1. Gillian DM. Antimicrobial activity and Chemical analysis of search.1997;9:653–655. Eucalyptus radiata leaf essential oil.South Africa: University of the Witwatersrand; 2016:1–156. 12. BendaoudH, Bouajila J, Rhouma A, et al. GC/MS analysis and antimicrobial and antioxidant activities of essential oil of Eu- 2. RautJS,Karuppayil SM. A status review on the medicinal proper- calyptus radiate.Journal of the Science of Food and Agricul- ties of essential oils. Industrial Crops and Products. 2014;62:250– 264. ture.2009;89:1292–1297. 3. Mohd SA, Birhamu D,Tanweer A. Antimicrobial activity of es- 13. Mulyaningsih S, SporerF, Reichling J, et al. Antibacterial activi- sential oils extracted from medicinal plants against the pathogenic ty of essential oils from Eucalyptus and of selected components microorganisms: A review. Biological Sciences and Pharmaceutical against multidrug-resistant bacterial pathogens.Pharm Biol. Research.2014;2(1):1–7. 2011;49:893–899. 4. Van VuurenS, ViljoenA. Plant-based Antimicrobial studies - 14. Luis A, Duarte A, Gominho J, et al. Chemical composition, an- methods and approaches to study the interaction between natural tioxidant, antibacterial and antiquorum sensing activities of and products. Planta Medica.2011;77:1168–1182. essential oils. Industrial Crops and Products. 2015;79:274–282. 5. Ali B, Al-Wabel NA, Shams S, et al. Essential oils used in aro- matherapy: A systemic review. Asian Pacific Journal of Tropical 15. CoppenJJW, Hone GA. Eucalyptus oils: a review on production Biomedicine. 2015;5:601–611. and markets (NRI Bulletin 56). 1992:01–53.

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