Hrev_master Antioxidation and anti-inflam- matory activities of blended essential oil Suriya Chaiwong, Kunthasaya Akkarasiritharattana, Khemjira Jarmkom, Punyanut Amorndoljai Faculty of Integrative Medicine, Rajamangala Univesity of Technology Thanyaburi Prathumthani, Thailand Abstract The mixtures of essential oils are increasing popularly in holistic medicine. The different ratios of oil components in the preparation can result in different health benefits. Therefore, this study aimed to develop a mixture of essential oil formula- tion having promising bioactivities for phar- maceutical and cosmetic applications. The five popular essential oils including vetiver oil, lavender oil, eucalyptus oil, lemongrass oil, and phlai oil were selected for blended formulation. The selected oils were mixed in a suitable ratio and their antioxidant and anti-inflammatory activities were evaluated using 2,2-Diphenyl-1-picrylhydrazyl radical scavenging and nitric oxide (NO) radical scavenging, respectively. The prepared blended essential oil formulation had clear yellow-pale color and good smell. The blended oil showed good antioxidative activity by acting as a hydrogen donator. However, the blended oil presents mild anti- inflammatory activity via NO quenching action. The results of this study exposed that the developed blended essential oil formula- tion has promising properties to be used as a natural antioxidant as well as an anti-inflam- matory agent in cosmetic and pharmaceuti- cal applications. Introduction Inflammation is an adaptatively physio- logical condition triggered by tissue injury, stress, or infection to recover tissue home- ostasis. In the inflammatory process, nitric oxide (NO) produced from activated macrophages plays the role of inflammatory mediator.1,2 NO can further react with super- oxide radicals to form reactive radical per- oxynitrite and directly damage the cellular components which can lead to cellular dys- function. Thus, the overproduction of NO causes cellular oxidative stress and con- ducts a higher severity level of inflamma- tion as well as may further induce carcino- genesis.3 Although inflammation is a recov- ery process of the body, self-damage caused by inflammation is unavoidable. Eventually, prolonged inflammation can lead to pathological conditions.2 Moreover, at the site of inflammation, an increase in cell and tissue oxidative stress was found. Reactive oxygen species (ROS) are well- known as molecular intermediates for oxidative stress. Moreover, ROS can also cause the severity of inflammation.4 Thus, the direct elimination of NO and ROS could be considered as potential targets for inflammation remedies. Essential oil is one of the interesting natural products that have been used for health promotion for a long time.5 Essential oils have earned great favor in the food, cosmetic, as well as pharmaceutical indus- tries owing to their biological activities such as analgesic, anti-inflammatory, antioxidant, antibacterial, antiviral, and antifungal.6,7 Recently, the use of essential oil in the form of a mixture is increasing particularly in holistic care because we can select the desired benefit of each essential oil into the mixture. The combination of essential oils may provide more efficacy than sole use. For example, the mixtures of essential oils produced various effects on antioxidant activity due to their free radical scavenging ability. This activity supports their use in food preservation and for the management of many ailments such as can- cers, and neurodegenerative, cardiovascu- lar, and immune system diseases.8 Based on holistic medicine principles, essential oils that have healthy balance property is often selected for blended essential oil prepara- tions.9 Popular essential oils that have been used in holistic care such as vetiver oil (Vetiveria zizanioides), lavender oil (Lavandula angustifolia), eucalyptus oil (Eucalyptus globulus), lemongrass oil (Cymbopogon citratus), and phlai oil (Zingiber montanum). These essential oils and their active constituents have previous- ly been reported as anti-inflammatory agents and antioxidants.2 However, the preparation of blended essential oil formu- lations by using these in different ratios can result in different health benefits. Therefore, this study aimed to develop a blended essential oil preparation for inflammation management. In addition, the bioactivities of prepared essential oil formulation includ- ing antioxidation and anti-inflammatory activities were also evaluated. The obtained formulation can be applied in cosmetic or pharmaceutical applications. Healthcare in Low-resource Settings 2023; volume 11:11548 Correspondence: Punyanut Amorndoljai, Faculty of Integrative Medicine, Rajamangala Univesity of Technology Thanyaburi Prathumthani, 12130 Thailand. Tel.: 0899864839 - Fax: 025921999. E-mail: punyanut_a@rmutt.ac.th Key words: antioxidant; anti-inflammation; essential oil; holistic medicine. Contributions: SC, conceptualization; methodology; investigation; formal analysis; data curation; visualization; writing-original draft; writing-review and editing; final approval of the version to be published fund- ing acquisition; KA and KJ, conceptualiza- tion; final approval of the version to be pub- lished, funding acquisition. PA, conceptual- ization; methodology; investigation; formal analysis; data curation; writing-original draft; writing-review and editing; final approval of the version to be published; visualization; project administration; funding acquisition. Conflict of interest: the authors declare no potential conflict of interest, and all authors confirm accuracy. Ethics approval: there is no necessary ethical issue to be approved in this study. Informed consent: there are no patients partic- ipated in this study. Patient consent for publication: there are no patients participated in this study. Availability of data and materials: all data generated or analyzed during this study are included in this published article. Funding: this project was financial supported by the Program Management Unit for Area- based Development (PMU-A) (Fund no A1 3F650059). Acknowledgments: we are grateful to Assoc. Prof. Dr. Prasart Nuangchalerm for his sug- gestion on manuscript writing. Received for publication: 27 June 2023. Accepted for publication: 23 August 2023. Early access: 11 September 2023. This work is licensed under a Creative Commons Attribution 4.0 License (by-nc 4.0). ©Copyright: the Author(s), 2023 Licensee PAGEPress, Italy Healthcare in Low-resource Settings 2023; 11:11548 doi:10.4081/hls.2023.11548 Publisher's note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affili- ated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guar- anteed or endorsed by the publisher. [Healthcare in Low-resource Settings 2023; 11:11548] [page 77] Non -co mmerc ial us e o nly Materials and Methods Chemicals The essential oils including vetiver oil (Vetiveria zizanioides), lavender oil (Lavandula angustifolia), eucalyptus oil (Eucalyptus globulus), lemongrass oil (Cymbopogon citratus), and phlai oil (Zingiber montanum) were purchased from Royal Lotus. DPPH and gallic acid were obtained from Sigma Aldrich. Ascorbic acid was from Chem-supply. Sodium nitroprus- side and naphthyl ethylene diamine were bought from Himedia and Loba Chemie, respectively. Preparation of blended essential oil The blended essential oil formulations were prepared by using popular essential oils that have been used in holistic medicine. The selected essential oils were vetiver oil (Vetiveria zizanioides), lavender oil (Lavandula angustifolia), eucalyptus oil (Eucalyptus globulus), lemongrass oil (Cymbopogon citratus), and phlai oil (Zingiber montanum). The mixtures of selected oils in various ratios (by weight) were developed. Briefly, the largest amount of essential oil was pipetted into the vial first, followed by adding the minor compo- nents into the same vial. The blended oil was then homogenized and stored at 4°C prior to use. The physical characteristics of suitable obtained oil formulation such as color and odor were observed. Evaluation of bioactivities 2,2-Diphenyl-1-picrylhydrazyl radical scavenging assay The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical quenching ability of blend- ed essential oil was tested following a pre- vious report.10 The fresh DPPH solution was prepared by dissolving DPPH in methanol to obtain an absorbance unit of 1.1±0.02 at 515 nm. The sample was diluted with methanol into various concentrations (20-200 µg/mL). The sample solutions (80 µL) and DPPH solution (80 µL) were added to 96-well plates and incubated in the dark condition at room temperature for 30 min- utes. Then the absorbance of reactions was measured at 515 nm with a microplate read- er. Ascorbic acid was used as a positive con- trol. The experiment was done in triplicate. The % inhibition was achieved by using the following equation: %inhibition = [(ODcontrol-ODsample)/ODcontrol]×100. In vitro nitric oxide radical scavenging assay The NO radical scavenging test was employed to assess the anti-inflammatory activity of blended essential oil based on a previously reported method10. The 20 mM SNP solution was mixed with the blended essential oil solution (5-50 mg/mL) and incubated at ambient temperature for 180 min. Then the mixture was reacted with Griess reagent. The absorbance was read at 546 nm using a microplate reader. Gallic acid (1-20 mg/mL) was used as a positive control. The test was performed in tripli- cates. The % inhibition was achieved by using the following equation: %inhibition = [(ODcontrol-ODsample)/ODcontrol]×100. Statistical analysis Results were presented as mean±stan- dard deviation. The data were analyzed using the Microsoft Excel program. Results and Discussion Blended essential oil formulation The blended essential oil formulation was prepared by mixing five essential oils. The mixing ratios are presented in Table 1. The physical characteristic of the obtained oil was the clear yellow-pale color. This ratio was the suitable smell from the proportions of the top note, middle note, and base note. 2,2-Diphenyl-1-picrylhydrazyl radi- cal scavenging activity The antioxidant potency of blended essential oil was evaluated using DPPH proton radical scavenging. The result found that tested oil and ascorbic acid exhibited DPPH radical scavenging activity in a dose- dependent manner. The highest tested con- centration of oil (300 mg/mL) and ascorbic acid (10 mg/mL) presented similar scaveng- ing activity up to 82.34±0.58 and 82.47±0.72 % DPPH radical scavenging (Figure 1). This suggested that blended essential oil carried a lower antioxidant potency of 30 times than ascorbic acid when compared with the highest tested con- centrations. The DPPH radical scavenging test was based on the reduction of DPPH, a hydrogen acceptor, in the presence of a hydrogen donator.11 Thus, the antioxidant activity of blended essential oil is due to its hydrogen-donating properties. Nitric oxide radical scavenging activity NO is an important inflammatory medi- ator. NO is over-released from activated Article Table 1. Mixing ratios of blended essential oil. Essential oil Ratio (by weight) Vetiver 2 Lavender 2 Eucalyptus 1.5 Lemongrass 1.5 Figure 1. 2,2-Diphenyl-1-picrylhydrazyl radical scavenging activity of blended essential oil and ascorbic acid. DPPH, 2,2-Diphenyl-1-picrylhydrazyl. Figure 2. Nitric oxide radical scavenging activity of blended essential oil and gallic acid. [page 78] [Healthcare in Low-resource Settings 2023; 11:11548] Non -co mmerc ial us e o nly [Healthcare in Low-resource Settings 2023; 11:11548] [page 79] macrophages during inflammation.12 Thus, NO scavenging is one of the mechanisms of anti-inflammatory agents. The anti-inflam- mation potency of blended essential oil was evaluated using inflammatory mediator NO radical scavenging. The result found that tested oil and gallic acid presented dose- dependent NO radical scavenging capacity (Figure 2). However, NO radical quenching at the highest tested concentration of essen- tial oil (27.41±0.87%) was approximately 2.1 folds lower than the scavenging efficacy of gallic acid (56.17±1.47%) at the highest tested dosage. This finding revealed that blended essential oil possessed mind anti- inflammatory activity. The increase in the used dosage may improve the anti-inflam- matory potency of this essential oil. Conclusions The present study has developed a blended essential oil formulation from the popular essential oils used in holistic medicine to use for pharmaceutical and cos- metic applications. The biological activities of prepared oil were evaluated and showed that the oil has a promising potential as an antioxidant by acting as a proton radical scavenger. In addition, the anti-inflammato- ry property of the oil was expanded via inflammatory mediator NO scavenging. References 1. Guzik T, Korbut R, Adamek-Guzik T. Nitric oxide and superoxide in inflam- mation. J Physiol Pharmacol 2003;54: 469-87. 2. Rawat A, Rawat M, Prakash OM, et al. Comparative study on eucalyptol and camphor rich essential oils from rhi- zomes of Hedychium spicatum Sm. and their pharmacological, antioxidant, and antifungal activities. Anais da Academia Brasileira de Ciências, 2022;94. 3. Knowles RG, Moncada S. Nitric oxide synthases in mammals. Biochem J 1994;298:249-58. 4. Mittal M, Siddiqui MR, Tran K, et al. Reactive oxygen species in inflamma- tion and tissue injury. Antiox Redox Signal 2014;20:1126-67. 5. Sadgrove NJ, Padilla-González GF, Phumthum M. Fundamental chemistry of essential oils and volatile organic compounds, methods of analysis and authentication. Plants 2022;11:789. 6. Ni ZJ, Wang X, Shen Y, et al. Recent updates on the chemistry, bioactivities, mode of action, and industrial applica- tions of plant essential oils. Trends Food Sci Technol 2021;110:78-89. 7. Turek C, Stintzing FC. Stability of essential oil: a review. Compr Rev Food Sci Food Saf 2013;12:40-53. 8. Horvathova E, Navarova J, Galova E, et al. Assessment of antioxidative, chelat- ing, and DNA-protective effects of selected essential oil components (eugenol, carvacrol, thymol, borneol, eucalyptol) of plants and intact Rosmarinus officinalis oil. J Agric Food Chem 2014;62;6632-9. 9. Halm MA. Essential oils for manage- ment of symptoms in critically ill patients. Ame J Crit Care 2008;17:160- 3. 10. Chaiwong S, Puttarak P, Sretrirutchai S, Kaewsuwan S. In Vitro Anti-inflamma- tory and Antioxidative Activities of Isolated Interruptins from Cyclosorus terminans. Lat Ame J Pharma 2019;38: 1677-82. 11. Kadhum AAH, Al-Amiery AA, Musa AY, Mohamad AB. The antioxidant activity of new coumarin derivatives. Int J Mol Sci 2011;12:5747-61. 12. Muntané J, De la Mata M. Nitric oxide and cancer. World J Hepatol 2010;2: 337-44. Article Non -co mmerc ial us e o nly