Study on extraction, chemical composition and antioxidant capacity of cinnamomum burmannii essential oil

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY VU THI THUY STUDY ON EXTRACTION, CHEMICAL COMPOSITION AND ANTIOXIDANT CAPACITY OF CINNAMOMUM BURMANNII ESSENTIAL OIL BACHELOR THESIS Study mode : Full time Major : Food technology Falcuty : International Program Office Batch : 2016 - 2020 THAI NGUYEN, 2022 ABTRACT Thai Nguyen University of Agriculture and Forestry Degree Program Bachelor of Food Technology Student name Vu Thi Thuy Student ID DTN1654190005 Thesis Title Supervisor(s) Study on extraction, chemical composition and antioxidant capacity of Cinnamomum burmannii essential oil MSc Dinh Thi Kim Hoa MSc Nguyen Thuong Tuan Supervisor’s Signature Abstract: The aims of this study was to develop a suitable technological process for extracting essential oil from Cinnamomum burmannii leaves by steam distillation method In addition, the quality, chemical compositions and antioxidant capacity of the essential oil also were evaluated The most suitable parameters for extraction process were the wilting time of 84 hours; the ratio between material and distill water of 5/50 (w/v); 50 minutes for extraction The amount of obtained essential oil was 0.6 - 0.7% The chemical ingredient of essential oil consisted of 23 compounds that were reported for chemical structures The antioxidant activity of the essential oil from Cinnamomum burmannii leaves was revealed with the IC50 of 41.10 (µg/ml) Keywords Extraction, Chemical composition, Antioxidant activity, Cinnamomum burmannii essential oil Number of pages 64 Date of Submission June 1st, 2022 ii ACKNOWLEDGEMENT During my studies and internship at Thai Nguyên University of agriculture and forestry, I have now completed my graduation thesis In order to complete this thesis, I have been guided by the devoted guidance of my supervisor, along with the help of Thai Nguyen University of Agriculture and Forestry, the Institute of Life Sciences, and the Advanced Program Office I also received enthusiastic cooperation from colleagues, help, and encouragement from my family members In response to that sentiment, though here I would like to express my deep gratitude and respect to all collectives and individuals who created conditions to help me throughout the internship process First of all, I would like to express my sincere thanks to the school management board, the Dean of the Faculty of Food Technology, and the collective of teachers in the Faculty of Food Technology, Thai Nguyen University of Agriculture and Forestry that taught and mentored me during these time, as well as my graduation internship In particular, I sincerely thank for the attention and guidance of the instructing supervisor MSc Dinh Thi Kim Hoa and MSc Nguyen Thuong Tuan, who directly guided me to implement this thesis successfully Through this, I would also like to express my gratitude to family, relatives, and friends for helping and encouraging me during my study and practice at school Finally, I would like to respectfully send my sincere thanks and best wishes to the teachers and teachers on the evaluation committee Thank you sincerely! Thai Nguyen, May 23, 2022 Student VU THI THUY iii TABLE OF CONTENT LIST OF FIGURES LIST OF TABLE ABBREVIATIONS PART I INTRODUCTION 1.1 Research rationale 1.2 Research objective 1.3 Detail goals 1.4 Limitations PART II LITERATURE REVIEW 2.1 Overview of Cinnamomum burmannii tree 2.1.1 Characteristic of Cinnamomum burmannii 2.1.2 Distribution, ecological characteristic and growth 2.1.3 Uses of Cinnamomum burmannii leaves 2.2 Overview of essential oil 10 2.2.1 Definition of essential oil 10 2.2.2 Physical and chemical properties of essential oil 11 2.2.3 The role of essential oils in plant life 12 2.2.4 Biological activities and uses of essential oils 14 2.2.5 Cinnamomum burmannii essential oil 15 2.3 Methods extraction of essential oil 17 2.3.1 Hydrodistillation 18 2.3.2 Solvent extraction 19 2.3.3 Enfleurage 21 2.3.4 Maceration 21 2.3.5 Cold pressing extraction 21 PART III METHODOLOGY 23 3.1 Material and research scope 23 3.1.1 Material 23 3.1.2 Research scope 23 3.2 Work place and time to proceed 23 3.3 Equipment and chemicals 24 3.4 Research content 25 iv 3.4.1 The research on factors affecting the content of Cinnamomum burmannii essential oil 25 3.4.2 Determination of the chemical composition of Cinnamomum burmannii essential oil by GC-MS method 25 3.4.3 Investigation of antioxidant capacity of essential oils 26 3.5 Research methods 26 3.5.1 The process of extracting essential oil from Cinnamomum burmannii leaves 26 3.5.2 Experimental design method 27 3.5.3 Determination of the chemical composition of Cinnamomum burmannii essential oil by GC-MS method 29 3.5.4 Investigation of antioxidant capacity of essential oil 29 3.5.5 Determination of moisture and ash content in Cinnamomum burmannii leaves 31 3.5.6 Determination of some physicochemical parameters of Cinnamomum burmannii essential oil 33 3.6 Data satistical analysis methods 36 PART IV RESULT AND DICUSSION 37 4.1 Moisture and ash content in Cinnamomum burmannii leaves 37 4.2 The result of studying factors affecting the distillation process of essential oils 37 4.2.1 Effect of extraction time 37 4.2.2 Effect of the material/distilled water ratio on essential oil content 38 4.2.3 Effect of wilting time of raw material on essential oil content 39 4.4 Result for determining the chemical composition of Cinnamomum burmannii essential oil by GC-MS method 41 4.5 Result for determining the antioxidant capacity of Cinnamomum burmannii essential oil 42 4.6 Result for determining some physicochemical of essential oils 44 4.7 Completing the extraction process of Cinnamomum burmannii essential oils 46 PART V CONCLUSION 48 5.1 Conclusion 48 5.2 Recommendation 48 REFERENCES 50 APPENDIXES 55 v LIST OF FIGURES Figure 2.1 Cinnamomum burmannii Tree Figure 2.2 Main molecules of essential oils 10 Figure 3.1 Cinnamomum burmannii leaves 23 Figure 3.2 The process of extracting from Cinnamomum burmannii leaves 26 Figure 3.3 Reaction mechanism of 2,2-diphenyl-1-picrylhydrazyl (DPPH) with antioxidant 30 Figure 4.1 Graph showing the effect of time on essential oil content 38 Figure 4.2 The graph shows the effect of the amount of distilled water on the essential oil content 39 Figure 4.3 Graph showing the effect of wilting time on essential oil content 40 Figure 4.4 Correlation between free radical inhibitory activity and concentration of Cinnamomum burmannii essential oil 43 Figure 4.5 Correlation between free radical inhibitory activity and concentration of Ascorbic acid 43 Figure 4.6 Cinnamomum burmannii essential oil 45 Figure 4.7 The extraction process of Cinnamomum burmannii essential oils 46 LIST OF TABLE Table 3.1 Experiment Chemicals 24 Table 3.2 Experiment Equipment 24 Table 3.3 Laboratory instruments 25 Table 3.4 The experimental design to determine the effect of extraction time on essential oils 27 Table 3.5 Experimental design to determine the effect of (material/distilled water) ratio on essential oils 28 Table 3.6 The experimental design to determine the effect of wilting time of raw material on essential oils 28 Table 4.1 The moisture and ash content of Cinnamomum burmannii leaves 37 Table 4.2 Results of survey on extraction time of essential oils 37 Table 4.3 Result of effecting the material/distilled water ratio on essential oil content 38 Table 4.4 Results affect the wilting time of raw material on essential oil content 40 Table 4.5 Chemical composition of Cinnamomum burmannii essential oil 41 Table 4.7 Antioxidant activity of Ascorbic acid with DPPH 43 Table 4.8 Some physicochemical of Cinnamomum burmannii essential oils 45 ABBREVIATIONS ABTS 2,2'-azino-bis (3-ethylbenzothiazoline-6sulfonic acid) DPPH 2,2-diphenyl-1-picrylhydrazy GC-MS Gas Chromatography-Mass Spectrometry HPLC High-Performance Liquid Chromatography IA Acid index IC50 Half Maximal Inhibitory Concentration IE Saponification index IS Ester index SD Standard Deviation SDE Simultaneous Distillation Extraction SFE Supercritical Fluid Extraction SPME Solid-phase Micro-extraction W/V Weight/Volume PART I INTRODUCTION 1.1 Research rationale Essential oils are natural ingredients Currently, in the world, people are increasingly interested in medical products derived from essential oils to ensure health and have no negative effects As a result, both domestic and international research on essential oils is rising in number and quality Vietnam is one of the nations in the tropical monsoon area that has a large and diversified collection of essential oils and aromatic plants, with over 300 essential oil species collected such as Peppermint (1.3%-2%), Gratissimum (0.9%-1.67%), Lemongrass (0.7%-1.5%), Rosemary (0.3%-0.5%), Cinnamon (1%-2.5%), They are used quite commonly in the market due to they mostly contain the main components such as cinnamic aldehyde, geraniol, menthol, borneol, citral, citronellal, menthol, … that pleasant aroma, anti-cold effect, antiinflammatoryand stress reduction Some essential oils can cure cancer such as Frankincense, Lemongrass, and Orange Cinnamomum burmanniiis a woody plant in Bao Lac district, Cao Bang province Cinnamomum burmanniileaves are often used by people as raw materials for food processing In addition, people often boil the leaves for a bath or sauna to prevent and treat diseases such as malaria, and bacterial infections, to control blood pressure and headaches Based on the above applications, we can see that Cinnamomum burmannii leaves are a useful ingredient for health, not only as a food additive but also as a common healing method for people However, these effects are insufficient to explain the phenomenon clearly from a scientific standpoint, and up to this point, there have been no in-depth studies on this leaf Therefore, I conducted the topic: “ Study on extraction, chemical composition and antioxidant capacity of Cinnamomum burmannii essential oil.” Thereby contributing to creating more directions and to clarify for the application of Cinnamomum burmanniiessential oil in the fields of medicine, pharmaceuticals, and cosmetics Simultaneously, maximizing the potential and improving the value of Cinnamomum burmannii essential oil on the market 1.2 Research objective Develop extraction procedure, investigate the chemical composition, and test the biological activity of the obtained Cinnamomum burmannii essential oil 1.3 Detail goals - Conduct an experiment to extract Cinnamomum burmannii essential oil by direct steam distillation method - Determination of some physicochemical indexes and chemical compositions of essential oils - Dertermination of antioxidant properties of Cinnamomum burmannii essential oil 1.4 Limitations Limitations that are expected to be encountered throughout the study: - Raw materials: Because Cinnamomum burmannii leaves are available in Bao Lac town, Cao Bang province, it is difficult to collect and transport raw materials - There is currently no study on the plant's composition and activities of Cinnamomum burmannii REFERENCES English document Al-Dhubiab, B E (2012) Pharmaceutical applications and phytochemical profile of Cinnamomum Burmannii Pharmacognosy reviews, 6(12), 125 Abid Shah SF and Bashir S (2019) Comparative Proximate Analysis of Cinnamon And Lemongrass for its Nutritive Value JSM Human Nutri Food Sci 5: 3 Atti-Santos, A C., Rossato, M., Serafini, L A., Cassel, E., & Moyna, P (2005) Extraction of essential oils from lime (Citrus latifolia Tanaka) by hydrodistillation and supercritical carbon dioxide Brazilian Archives of Biology and Technology, 48(1), 155-160 Azmir, J., Zaidul, I S M., Rahman, M M., Sharif, K M., Mohamed, A., Sahena, F., & Omar, A K M (2013) Techniques for extraction of bioactive compounds from plant materials: A review Journal of food engineering, 117(4), 426-436 Baig S (2021) Analysis of cinnamon (proximate analysis) J Nutraceuticals Food Sci Vol.6 No.1:2 Berry, Paul E Sampson, F Bruce "Laurales" Encyclopedia Britannica , ngày 28 tháng năm 2019, https://www.britannica.com/plant/Laurales Bottu, H M., Mero, A., Husanu, E., Tavernier, S., Pomelli, C S., Dewaele, A., & Brennan, L (2022) The ability of deep eutectic solvent systems to extract bioactive compounds from apple pomace Food Chemistry, 386, 132717 Cardoso-Ugarte, G A., López-Malo, A., & Sosa-Morales, M E (2016) Cinnamon (Cinnamomum zeylanicum) essential oils In Essential oils in food preservation, flavor and safety (pp 339-347) Academic Press Cardoso-Ugarte, G A., López-Malo, A., & Sosa-Morales, M E (2016) Cinnamon (Cinnamomum zeylanicum) essential oils In Essential oils in food preservation, flavor and safety (pp 339-347) Academic Press 10 Chandurkar, P., Tripathi, N., Choudhary, A., & Murab, T (2014) Antibacterial properties of cinnamon stick oil with special reference to 50 Streptococcus pyogenes and Pseudomonas aeruginosa Int J Curr Microbiol App Sci, 3(2), 177-178 11 Cheng B, Xu Y, Zeng F, Yu X, Ding J, Wu Y, et al Chemical constituents of essential oil of Cinnamomum burmanniif heyneanum Yunnan Zhiwu Yanjiu 1992;14:105–10 12 Christenhusz, M J M.; Byng, J W (2016) "The number of known plants species in the world and its annual increase" Phytotaxa 261 (3): 201–217 13 Deng CC, Huo LN, Li PY, et al Chemical constituents and antioxidant activity of essential oils from leaves of Cinnamomum burmanniiin Guangxi Zhongguo Shiyan Fangjixue Zazhi 2010;16:105–9 14 El-Baroty, G S., Abd El-Baky, H H., Farag, R S., & Saleh, M A (2010) Characterization of antioxidant and antimicrobial compounds of cinnamon and ginger essential oils African journal of biochemistry research, 4(6), 167-174 15 Ghosh, M.N.(1998) Fundamentals of Experimental Pharmacology, 2nd Edn., Scientific Book Agency, Calcutta, 174-179 16 Goldschmidt, S (1920) Über zweiwertigen Stickstoff: Das Triphenyl‐ hydrazyl.(II Mitteilung über Amin‐Oxydation) Berichte der deutschen chemischen Gesellschaft (A and B Series), 53(1), 44-62 17 Goldschmidt, S., & Renn, K (1922) Zweiwertiger Stickstoff: Über das α, α‐ Diphenyl‐β‐trinitrophenyl‐hydrazyl.(IV.MitteilungüberAmin‐ Oxydation) Berichte der deutschen chemischen Gesellschaft (A and B Series), 55(3), 628-643 18 Guenther, Ernest, and Darrell Althausen The essential oils Vol New York: van Nostrand, 1948 19 Handa, SS, Vasisht, K, et.al (2006) Compendium of Medicinal and Aromatic Plants-Asia, II, ICS-UNIDO, AREA Science Park, Padriciano, Trieste, Italy, 79-83 20 Harborne, J.B (1998) Phytochemical methods- A guide to modern techniques of plant analysis, 3rd Edn, Springer (India) Pvt Ltd, New delhi, 5-32 21 Johner, J C., Hatami, T., & Meireles, M A A (2018) Developing a supercritical fluid extraction method assisted by cold pressing for extraction of pequi (Caryocar brasiliense) The journal of supercritical fluids, 137, 34-39 51 22 Kuspradini, Harlinda, et al "Bioactivity of essential oils from leaves of Dryobalanops lanceolata, Cinnamomum Burmannii, Cananga odorata, and Scorodocarpus borneensis." Agriculture and Agricultural Science Procedia (2016): 411-418 23 Lin, J., & Rouseff, R L (2001) Characterization of aroma‐impact compounds in cold‐pressed grapefruit oil using time–intensity GC– olfactometry and GC–MS Flavour and Fragrance Journal, 16(6), 457-463 24 Liu F, Li P, Wang Y The chemical constituents of essential oils from different organs in Cinnamomum burmanni in Yuebei Shengwuxue Zazhi 2007;24:25–7 25 Mitra, Somenath, ed (2003) Sample Preparation Techniques in Analytical Chemistry Wiley-Interscience p 113 26 Nanasombat, S., & Wimuttigosol, P (2011) Antimicrobial and antioxidant activity of spice essential oils Food Science and Biotechnology, 20(1), 45-53 27 Preuss, H.G., Echard, B., Polansky, M.M., Anderson, R., 2006 Whole cinnamon and aqueous extracts ameliorate sucrose-induced blood pressure elevations in spontaneously hypertensive rats J Am Coll Nutr 25 (2), 144–150 28 Pensuk, W., Padumanonda, T., & Pichaensoonthon, C (2007) Comparison of the chemical constituents in Michelia alba flower oil extracted by steam distillation, hexane extraction and enfleurage method Journal of Thai Traditional & Alternative Medicine, 5(1), 30-39 29 Rasul, M G (2018) Conventional extraction methods use in medicinal plants, their advantages and disadvantages Int J Basic Sci Appl Comput, 2, 10-14 30 Rowaan PA The essential oil from the leaf of the Padang-cinnamon tree Chem Weekbl 1936;33:698–9 31 Salleh WMNHW, Ahmad F, Yen KH, Zulkifli RM A review on chemical constituents and biological activities of the genus Beilschmiedia (Lauraceae) Trop J Pharm Res 2015; 14: 2139-2150 32 Shan B, Cai YZ, Brooks JD, Corke H The in vitro antibacterial activity of dietary spice and medicinal herb extracts Int J Food Microbiol 2007;117:112–9 52 33 Shan, B., Cai, Y Z., Brooks, J D., & Corke, H (2007) Antibacterial properties and major bioactive components of cinnamon stick (Cinnamomum Burmannii): activity against foodborne pathogenic bacteria Journal of agricultural and food chemistry, 55(14), 5484-5490 34 Spietelun, Agata; Pilarczyk, Michał; Kloskowski, Adam; Namieśnik, Jacek (2010) "Current trends in solid-phase microextraction (SPME) fibre coatings" Chemical Society Reviews 39 (11): 4524–37 35 Su J, Chen L, Li B, Li L Extraction and composition analysis of volatile components in leaves of Cinnamomum burmanniiB1 Shipin Kexue 2010;31:399-401 36 Soares, M D O., Vinha, A F., Sousa, C., Castro, A., & Pires, P C (2020) Food preservative potential of lemongrass (Cymbopogon citratus) essential oil Prime Archives in Agricultural Research, 1-20 37 Tan, H.T., 2005 Herbs and spices of Thailand Times Publishing Ltd., Singapore 38 Tran, T H., Nguyen, D C., Phu, T N N., Bach, L G., & Nguyen, T D (2019) Research on lemongrass oil extraction technology (hydrodistillation, microwaveassisted hydrodistillation) Indonesian Journal of Chemistry, 19(4), 1000-1007 39 Uysal, B., Sozmen, F., Aktas, O., Oksal, B S., & Kose, E O (2011) Essential oil composition and antibacterial activity of the grapefruit (Citrus Paradisi L) peel essential oils obtained by solvent‐free microwave extraction: comparison with hydrodistillation International journal of food science & technology, 46(7), 1455-1461 40 Wagner, H, Bladet, S, et.al (1996) Plant Drug Analysis-A TLC Atlas, 1st Edn, Springer verlag Berlin, Heidel berg, New York, 195-214 41 Wang, R., Wang, R., & Yang, B (2009) Extraction of essential oils from five cinnamon leaves and identification of their volatile compound compositions Innovative Food Science & Emerging Technologies, 10(2), 289-292 42 Zhang J, Wen S, Fulin L, Huang S, Diao S, Zhu Y, et al Effects of temperature, light and pH on DPPH radical scavenging activity of anthocyanin extracted from fruit of Cinnamomum Burmannii J Food Sci 2009;30:120–3 43 Ziegenfuss, T.N., Hofheins, J.E., Mendel, R.W., Landis, J., Anderson, R.A., 2006 Effects of a water-soluble cinnamon extract on body composition and 53 features of the metabolic syndrome in pre-diabetic men and women J Int Soc Sports Nutr 3, 45–53 Vietnam document 44 Đỗ Tất Lợi (1985), Tinh dầu Việt Nam, Nhà xuất Y học TP Hồ Chí Minh 45 Nam, Hương Liệu–Mỹ Phẩm Việt Chính Sách Đầu Tư Và Cơng Tác Quản Lý Ngành Tinh Dầu 46 Nguyễn Ngọc Yến, Bùi Nguyễn Anh Thư Nguyễn Minh Kha, 2019 Khảo sát thành phần hóa học hoạt tính kháng oxy hóa tinh dầu hương thảo (Rosmarinus Officinalis L.) Tạp chí Nghiên cứu khoa học Phát triển kinh tế Trường Đại học Tây Đô 06: 190-201 47 Nguyễn Thị Thu Thảo, Phùng Thị Lan Hương, Hoàng Thị Lý (2021) Nghiên cứu thành phần hóa học ứng dụng sản xuất dầu cao xoa tinh dầu quế trồng tỉnh Phú Thọ Study Aids & Test Prep Documents 48 Phát, Đ T., Quyên, N T C., Ngân, T T K., Nhựt, P T., Hiền, T T., Việt, N T., & Cường, L X (2019) Nghiên cứu mơ hình động học trình chiết xuất thành phần hóa học tinh dầu vỏ Cam (Citrus sinensis) Journal of Science and Technology, 2(4), 19-25 49 Phúc, N Đ., Danh, P H., Anh, T T., & Phương, T H Y (2020) Chiết xuất tinh dầu Hương thảo phương pháp chưng cất nước Journal of Science and Technology, 3(2), 21-25 Internet document 50 https://duoclieuvietnam.org/tinh-dau-thuc-vat-va-nhung-ung-dung-trong-doisong-trang-thong-tin-duoc-lieu-cay-thuoc-vi-thuoc-viet-nam/ 51 The Seasoning and Spice Association, 2010 Culinary Herbs and Spices Available from:http://www.seasoningandspice.org.uk/ssa/background_culinary-herbsspices.aspx 52 https://caythuoc.org/que-ranh-tren-tren-dieu-tri-phong-thap-dau-nhucxuong-khop-va-chan-an.html 54 APPENDIXES Table A.1: Tables of data processed by SPSS software of results of survey on extraction time of essential oils ANOVA Essentialoilcontent Sum of Squares df Mean Square Between Groups 653 163 Within Groups 000 10 000 Total 653 14 F Sig 11124178.682 000 Essentialoilcontent Duncan Extractiontime N Subset for alpha = 0.05 1.0000 2.0000 3.0000 5.0000 4.0000 3 121433 219573 508600 618633 644233 Sig 1.000 1.000 1.000 1.000 1.000 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000 Table A.2: Tables of data processed by SPSS software of the effect of distilled water volume on essential oil content ANOVA Essentialoilcontent Sum of Squares df Mean Square Between Groups 049 012 Within Groups 000 10 000 Total 049 14 F 1219847.233 Sig .000 55 Essentialoilcontent Duncan Volumeofdistillwater N Subset for alpha = 0.05 1.0000 2.0000 5.0000 3.0000 4.0000 3 439301 520067 549053 560737 612702 Sig 1.000 1.000 1.000 1.000 1.000 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000 Table A.3: Tables of data processed by SPSS software of effect of sample wilting time on essential oil content ANOVA Essentialoilcontent Sum of Squares df Mean Square Between Groups 069 017 Within Groups 000 10 000 Total 069 14 F Sig 2865178.722 000 Essentialoilcontent Duncan wiltingtime N Subset for alpha = 0.05 5.0000 1.0000 2.0000 3.0000 4.0000 Sig 537534 577983 606900 682010 722533 1.000 1.000 1.000 1.000 1.000 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000 56 Table A.4: Tables of data processed by SPSS software of Antioxidant activity of Cinnamomum burmanniiwith DPPH ANOVA Dpph Sum of Squares Between Groups Mean Square 4143.900 1035.975 588 10 059 4144.487 14 Within Groups Total df F Sig 17630.615 000 Dpph Duncan factor N Subset for alpha = 0.05 1.00 2.00 3.00 4.00 5.00 3 32.5521 45.6011 63.9890 76.4201 89.2322 Sig 1.000 1.000 1.000 1.000 1.000 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000 Table A.5: Tables of data processed by SPSS software of antioxidant activity of Ascorbic acid with DPPH ANOVA Ascorbic acid Sum of Squares Between Groups Mean Square 4011.432 1002.858 007 10 001 4011.439 14 Within Groups Total df F 1519481.944 Sig .000 Ascorbic acid Duncan factor N Subset for alpha = 0.05 1.00 2.00 3.00 4.00 5.00 Sig 39.8153 49.0517 59.0333 74.8700 84.4801 1.000 1.000 1.000 1.000 1.000 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000 57 Figure A.1: Some pictures of the research process Essential oil distillation set Pipe cooler 58 Separation of water and essential oils Cinnamomum burmanniiessential oil 59 Investigation of the antioxidant activity of essential oils Wilting process of leaves 60 61 62 63 64