Stability, Bio-functional Nanoemulsion Derived from Thai Herbal for Enhanced Topical Efficacy

Stability, Bio-functional Nanoemulsion Derived from Thai Herbal for Enhanced Topical Efficacy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Stability, Bio-functional Nanoemulsion Derived from Thai Herbal for Enhanced Topical Efficacy Chutikan Sakphisutthikul This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8788292/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The objective of this study was to develop and evaluate a stable nanoemulsion for the treatment of dermatitis using the traditional Thai recipe of Khun Nithets Sukkij, consisting of turmeric, cannabidiol (CBD), and cottonseed oil. Using the cold process cream method, two emulsifiers, Viscolam and Aquamax, were compared using four formulations (Dev#1-Dev#4). The study employed several evaluation methods including accelerated stability testing (Freeze-Thaw cycles), analysis of nanoparticles with a Zeta sizer, chemical integrity with Fourier transform infrared (FTIR) spectroscopy, antimicrobial activity with the agar well diffusion method, antioxidant capacity with the DPPH assay, and in vitro safety with the MTT assay. The experiment resulted in the successful stabilization of a nanoemulsion with an optimal particle size of 162.67 nm, which was highly colloidally stable, as indicated by a zeta potential of -72 to -96 mV. The formulations showed good antimicrobial activity, with maximum inhibition zones against Staphylococcus aureus (16.30 ± 2.73 mm) and Candida albicans (19.17 ± 0.55 mm), but no inhibitory activity against Escherichia coli . The research found that the formulated nanoemulsion, namely, formulation Dev#2, is safe (cell viability>80%), stable, and cost-effective topical formulation that effectively integrates traditional Thai herbal knowledge with modern dermatological applications. Integrative & Complementary Medicine Stability Bio-functional Nanoemulsion Thai herbal Efficacy Figures Figure 1 Figure 2 Figure 3 Highlights • Successful transformation of the unstable traditional “Khun Nithets Sukkij” recipe into a high-stability nanoemulsion. • Cold-process technique effectively preserved the chemical integrity of heat-sensitive CBD and Curcumin. • Exceptional colloidal stability achieved with a zeta potential of -76.20 to -92.23 mV and particle sizes under 200 nm. • Potent antimicrobial activity demonstrated against S. aureus and C. albicans , common skin pathogens. • Cost-effective formulation (Dev#2) identified using Aquamax, reducing production costs by 50% without compromising quality. Introduction Dermatitis is a significant public health concern in Thailand as the tropical climate contributes substantially to the emergence of various skin-related problems and diseases affecting the skin and subcutaneous tissues. [ 1 ] According to statistical data published in 2023 among outpatient visits, skin-related ailments were listed among the top ten causes of morbidity in the country.[ 2 ] Despite the fact that topical corticosteroids are still considered the standard of care for inflammatory dermatoses, their extended use is often linked with adverse effects such as skin atrophy and discoloration. [ 3 ] In addition, the economic cost is significant; Thailand has imported dermatological products worth approximately 2.4 billion baht in 2022, which highlights the urgent need for safe, efficient, and locally produced alternative treatments. [ 4 ] In a bid to address these issues, the Thai Ministry of Public Health has invested heavily in reviving the traditional Thai medicine. One of the most notable examples of this effort has been the traditional recipe of Khun Nithets Sukkij which is now included in the Thai National List of Essential Herbal Medicines for the treatment of psoriasis and chronic skin diseases. This synergistic three-component formula contains 3 major bioactive ingredients, which included turmeric ( Curcuma longa L.), cannabis ( Cannabis sativa L.), and cottonseed oil ( Gossypium herbaceum L.). The main polyphenol of turmeric curcumin and cannabidiol (CBD) from cannabis exhibit strong anti-inflammatory, antioxidant, and antimicrobial properties. Cottonseed oil, rich in Vitamin E and unsaturated fatty acids also aids in skin barrier repair and possesses natural antioxidant properties. [ 5 ] The traditional preparation, although therapeutically promising, is severely limited in practical application. The initial formulation is an oil-based unstable blend that is prone to phase separation resulting in low percutaneous uptake and insufficient patient compliance.[ 6 ] To overcome these limitations, nanotechnology offers an advanced drug delivery approach. As droplets with diameter sizes between 20 and 500 nm, nanoemulsions have become essential in improving solubility and bioavailability of lipophilic active compounds, including CBD and curcumin. These systems enhance penetration via the intercellular route of the stratum corneum. Moreover, the use of a non-thermal cold-process method is essential to stabilize the chemical composition of these heat-sensitive herbal extracts during formulation. [ 7 – 8 ] The proposed study aims to develop and comprehensively characterize a modernized nanoemulsion-based topical delivery system based on the Khun Nithets Sukkij formula. The study aims to assess the physicochemical properties, including physical stability during Freeze-Thaw cycles, nanoparticle size distribution, and chemical integrity using Fourier Transform Infrared (FTIR) spectroscopy. In addition, antimicrobial activity against Staphylococcus aureus and Candida albicans and antioxidant capacity was evaluated using the DPPH assay. Lastly, an in vitro cytotoxicity test in RAW 264.7 macrophage cells was conducted to determine the safety profile. This systematic approach aims to demonstrate a stable and cost-effective topical formulation that incorporates not only traditional Thai medical knowledge but also the latest pharmaceutical nanotechnology. Materials and methods Bioactive Compounds and Herbal Materials The bioactive components used in the nanoemulsion formulation were selected to modernize the traditional Thai dermatological recipe of Khun Nithets Sukkij. Curcumin extract derived from Curcuma longa L. was obtained from Bangkok Chemical Co., Ltd. (Thailand), with quality verified by a Certificate of Analysis (COA). High-purity cannabidiol (CBD) extract from Cannabis sativa L. was supplied by WP Science Co., Ltd. (Thailand). The oil phase consisted of refined cottonseed oil (low gossypol content) derived from Gossypium herbaceum L. and sourced from Statfold Seed Oil Ltd. (Netherlands). These active ingredients were maintained at a constant weight ratio of 1:1:2 (curcumin: cottonseed oil) to preserve the pharmacological synergy of the original traditional formulation. Formulation Excipients and Reagents To facilitate cold-process emulsification, two primary polymeric emulsifiers were evaluated: Viscolam (Bangkok Chemical, Thailand) and Aquamax (Chemipan, Thailand). Isopropyl Myristate (IPM) was employed as an emollient to enhance skin penetration. Additional pharmaceutical-grade excipients included liquid glycerin (humectant), ethylenediaminetetraacetic acid (EDTA) (chelating agent), and phenoxyethanol (preservative). Deionized (DI) water served as the continuous phase for all oil-in-water (O/W) nanoemulsion systems. For antioxidant assays, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were used as the free radical and standard reference, respectively. Microbial Strains and Cell Culture Reagents The antimicrobial efficacy was evaluated against clinically relevant pathogens: Staphylococcus aureus (DMST: 5815), Escherichia coli (DMST: 12743), and the fungal strain Candida albicans (DMST: 8840). Culture media included Brain Heart Infusion (BHI) broth/agar for bacterial strains and Sabouraud Dextrose (SD) broth/agar for the fungal strain, all sourced from Himedia (India). For safety evaluations, RAW 264.7 macrophage cells were utilized. Cell culture reagents, including Dulbecco’s Modified Eagle’s Medium (DMEM), Fetal Bovine Serum (FBS), Penicillin-Streptomycin, and MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), were of analytical grade. Diclofenac was used as a comparative control in the cytotoxicity assays. Formulation Development The nanoemulsions were prepared using the cold process cream method. This technique was selected for its ability to produce a stable emulsion at ambient temperature, thereby preserving the chemical integrity of heat-sensitive herbal compounds during emulsification. The core active ingredients—curcumin extract, CBD extract, and cottonseed oil—were maintained in a 1:1:2 ratio, consistent with the traditional Khun Nithets Sukkij formulation. Four development formulations (Dev#1–Dev#4) were created, differing by emulsifier (Viscolam or Aquamax). The detailed compositions are presented in Table 1 . Table 1 Composition of Experimental Nanoemulsion Formulations Component Function Dev#1 (%) Dev#2 (%) Dev#3 (%) Dev#4 (%) Part A Viscolam Emulsifier 3.00 - 3.00 - Aquamax Emulsifier - 3.00 - 3.00 Isopropyl Myristate Emollient 3.00 3.00 3.00 3.00 Part B Liquid Glycerin Humectant 2.00 2.00 2.00 2.00 DI Water Solution 87.30 87.30 86.30 86.30 EDTA Chelating Agent 0.20 0.20 0.20 0.20 Part C (Active) Curcumin Extract Active 1.00 1.00 2.00 2.00 CBD Extract Active 1.00 1.00 1.00 1.00 Cottonseed Oil Active 2.00 2.00 2.00 2.00 Part D Phenoxyethanol Preservative 0.50 0.50 0.50 0.50 Dev#1 : treatment 1 (Viscolam3%+Curcumin1%) Dev#2 : treatment 2 (Aquamax3%+Curcumin1%) Dev#3 : treatment 3 (Viscolam3%+Curcumin2%) Dev#4 : treatment 4 (Aquamax3%+Curcumin2%) Physicochemical Characterization Stability Testing : Accelerated stability assessment was performed using a Freeze-Thaw protocol. The formulations were subjected to 6 cycles of alternating temperatures: 45°C for 24 hours, followed by -20°C for 24 hours. Following the cycles, samples were visually inspected for any signs of phase separation, creaming, or changes in color and odor. Particle Size and Zeta Potential A Zeta sizer instrument was utilized to measure the average droplet size (nm) and zeta potential (mV) of the nanoemulsion. These parameters are critical for assessing the system's physical properties and predicting its long-term colloidal stability. pH Measurement The pH of each formulation was measured using a calibrated pH meter to ensure compatibility with the natural pH of human skin. Chemical Integrity (FTIR) Fourier Transform Infrared (FTIR) spectroscopy was used to analyze the chemical composition of the final nanoemulsion in comparison with the original formula. This analysis aimed to confirm that the key functional groups from the active ingredients were retained after the formulation process. Bio-functional Evaluation Antimicrobial Activity : The antimicrobial efficacy of the formulations was assessed using the agar well diffusion method. This assay was performed against microorganisms relevant to skin infections: Staphylococcus aureus (Gram-positive bacterium), Escherichia coli (Gram-negative bacterium), and Candida albicans (fungus). Efficacy was quantified by measuring the diameter of the inhibition zone in millimeters (mm). Antioxidant Activity The antioxidant potential of the nanoemulsion was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. In Vitro Safety (Cytotoxicity ) : An MTT assay was conducted to evaluate the potential cytotoxicity of the selected nanoemulsion on RAW 264.7 macrophage cells. The objective was to determine the concentration range at which the formulation maintained high cell viability, indicating its safety for topical use. Statistical Analysis All experiments were conducted in triplicate (n = 3), and the results are presented as mean ± standard deviation (SD). Statistical significance was determined using one-way ANOVA and paired t-tests; p-values < 0.05 were considered significant. Results and discussion Physical Characterization and Accelerated Stability The production of the Thai traditional dermatological nanoemulsion was effective in converting an unstable combination of herbs into a robust topical delivery system and displayed high physical integrity in comparison with the initial formula. The accelerated stability test using six Freeze-Thaw cycles showed that all the formulations developed (Dev#1-Dev#4) were homogeneous and no phase separation was observed, the color was uniform, and the odor remained stable (Fig. 1 -A). Conversely, the initial formula was found to exhibit immediate phase separation between the oil and the herbal powder at Cycle 0. The pH of the nanoemulsions were maintained between 5.5 and 6.0 which is within the physiological pH range of the human skin (4.06.0) therefore, minimizing the risk of skin irritation. This increased stability is explained by the fact that polymeric emulsifiers (Viscolam and Aquamax) were used, which offer steric stability due to the creation of a strong interfacial film on the droplets of oil, preventing coalescence. These observations are in line with the findings of Xiao, et al. (2025), who observed that the physical stability and droplet properties of nanoemulsions intended for topical delivery are directly determined by the choice of the oil phase and emulsifier composition. [ 9 ] Nanoparticle Properties A Zeta sizer was used to analyze the nanoparticles and confirmed that Dev#1, Dev#2 and Dev#3 had mean droplet sizes within the optimal nanoemulsion range of 61.28-260.33 nm. (Fig. 1 -B and C). Conversely, the formulations had highly negative zeta potentials. In particular, the values of zeta potential were in the range of -76.20 to -92.23 mV (Fig. 1 -D) indicating excellent colloidal stability due to high electrostatic repulsions that effectively prevent the aggregation of the particles. Droplet sizes smaller than 200 nm are essential for topical delivery systems because they greatly multiply the specific surface area, thereby enhancing the deeper delivery of active constituents such as Curcumin and CBD through the stratum corneum using intercellular routes or hair follicles. Based on the colloidal science principles, zeta potentials of over ± 30 mV are considered indicative of high stability, and the values observed in this study far exceeded this threshold providing a strong scientific explanation for the long term stability of these herbal nanoemulsions.[ 10 – 11 ] Chemical Integrity Analysis with FTIR The chemical integrity of the herbal actives was confirmed to be preserved by FTIR analysis during the cold-process formulation. The nanoemulsion produced was found to retain the primary characteristic functional groups of the active ingredients, as indicated in the spectra of the final product. The major peaks included the C-O of phenolic and ether groups (~ 1160 cm − 1 ) of Curcumin, C-H of the alkyl groups (~ 2925 cm − 1 ) of CBD and the C = O ester (~ 1740 cm − 1 ) and the methylene groups (~ 2856 cm − 1 ) of the fatty acid in cottonseed oil were the major peaks. This ascertains that the active functional groups remained undegraded during the emulsification process and therefore are retained in their active form in the finished product. The preservation of these functional groups of typical chemicals proves that the “Cold Process” technique applied in this work guarantees that heat-sensitive active ingredients, namely cannabinoids and curcuminoids, remain intact and are not destroyed during their production through the conventional hot-processing method of cream manufacturing. This chemical integrity, allows the modernized nanoemulsion to retain the therapeutic qualities of the original Thai traditional wisdom.[ 12 ] Bio-functional Efficacy The antimicrobial effect of the nanoemulsion was tested against pathogens known to cause skin infections. Quantitative data obtained by the agar well diffusion technique were as follows: The nanoemulsion showed good activity against Gram-positive bacterium Staphylococcus aureus and its maximum inhibition zone is 16.30 ± 2.73 mm. It also showed good antifungal activity against Candida albicans , with a maximum inhibition zone of 19.17 ± 0.55 mm. Conversely, the formulation had no inhibitory effect against Gram-negative bacteria Escherichia coli . The findings are clinically relevant because S. aureus and C. albicans are common pathogens involved in skin infections. The selective nature of the given activity is significant. It is assumed that the selectivity is due to inherent differences in the structure of the cell wall of the microbes; the lipophilic herbal compounds may be more easily embedded in the peptidoglycan layer of Gram-positive bacteria and the chitin-based wall of fungi, whereas the complex outer membrane of Gram-negative bacteria, including E. coli , may act as a significant permeability barrier. [ 13 ] Table 2 and Fig. 3 illustrate these results. Table 2 The antimicrobial efficacy of nanoemulsion. Formulation ID Antimicrobial Activity ( S. aureus Inhibition Zone mm) Antimicrobial Activity ( C. albicans Inhibition Zone mm) Dev#1 16.30 ± 2.73 17.72 ± 0.65 Dev#2 14.62 ± 1.72 18.59 ± 0.83 Dev#3 12.47 ± 2.72 19.17 ± 0.55 Dev#4 9.54 ± 1.68 18.52 ± 1.37 Optimal Formulation Selection and Safety Assessment Dev#2, which employed the emulsifier Aquamax, was selected as the optimal formulation based on its superior physicochemical performance and greater cost-effectiveness. This selection was justified by two main factors: 1) Cost-Effectiveness: Aquamax was identified as a more economical emulsifier which is a crucial aspect of scalable production. 2) Performance: Dev#2 exhibited the highest level of physical stability and favorable nanoscale characteristics (droplet size: 162.67 nm; zeta potential: -85.77 mV) compared with the other best-performing formulations. The in vitro safety profile of optimal Dev − 2 formulation was then evaluated. The MTT assay performed on RAW 264.7 cells demonstrated that the nanoemulsion maintained cell viability of more than 80% across concentration ranging from 125 to 2,000 µg/mL. This finding indicates high biocompatibility and low cytotoxicity of the formulation implying its suitability for tropical application. The selected formulation is thus stable, efficient, safe, and cost-effective, making it a strong candidate for further development.[ 14 ] Conclusions This study successfully transformed the ancient and time-tested Khun Nithets Sukkij herbal formula into a stable and bio-functional nanoemulsion (Dev#2), thereby integrating traditional Thai medical knowledge with modern pharmaceutical nanotechnology. The formulation overcame the limitations of the traditional oil-based remedy by providing a robust nanoscale delivery system with high colloidal stability (zeta potential 80%). Overall, the study offers a scientific justification for the use of this novel formulation as a safe and cost-effective alternative for dermatitis treatment. Furthermore, it contributes to the valorization of Thai herbal resources and supports the advancement of the local pharmaceutical industry. Declarations CRediT author statement Jurarat Sirisukheepradit : Conceptualization; Methodology; Data curation; Writing - Original draft preparation. Roshan Kumar Mahato : Visualization; Writing - Reviewing and Editing. Chutikan Sakphisutthikul : Visualization; Investigation; Supervision; Writing - Reviewing and Editing. References C Pichanee ,K Silada Kanokrungsee, V Noldtawat, Suthasanee Prasertsook, L Surachanee and B Waranya. Sensitive Skin in Thais: Prevalence, Clinical Characteristics, and Diagnostic Cutoff Scores. Journal of Cosmetic Dermatology , 2025. https://doi.org/10.1111/jocd.70181. E.A Belachew and A.K Sendekie. Health-related quality of life and its determinants in patients with different dermatological disorders at the University of Gondar Comprehensive Specialized Hospital. BMC Res Notes . 2023. https://doi.org/10.1186/s13104-023-06442-8. D DiRuggiero and M DiRuggiero. Beyond Skin Deep: The Systemic Impact of Topical Corticosteroids in Dermatology. J Clin Aesthet Dermatol . 2025;18(1-2 Suppl 1):S16-S20. H Parima , P Unchalee , N Sureerat , C Niphon and S Jiruth. Budget Impact Analysis of Botulinum Toxin Type A for Patients with Severe Blepharospasm in Thailand. Clinicoecon Outcomes Res . 2025. http//doi:10.2147/CEOR.S540982 H.K Cho, C.G Park and H.B Lim. Construction of a Synergy Combination Model for Turmeric ( Curcuma longa L.) and Black Pepper ( Piper nigrum L.) Extracts: Enhanced Anticancer Activity against A549 and NCI-H292 Human Lung Cancer Cells. Current Issues in Molecular Biology . 2024. https://doi.org/10.3390/cimb46060332. H Schoenfelder, Y Wiedemann and D.J Lunter. Development and characterization of topical formulation for maintenance therapy containing sorbitan monostearate with and without PEG-100-stearate. Int J Cosmet Sci . 2025. https://doi:10.1111/ics.13023 G Lucia, D.D Giulia, V Giulia, C.B Maria , T Lihua, L Cristina and B.R Anna Development, characterization and in vitro assessment of a novel self-microemulsifying drug delivery system to increase cannabidiol intestinal bioaccessibility. International Journal of Pharmaceutics . 2025. https://doi.org/10.1016/j.ijpharm.2025.126284. H.D Lee, L Sunyoung Lim, K.S Sung and K Joohee. Advancements in Skin-Mediated Drug Delivery: Mechanisms, Techniques, and Applications. Advanced Healthcare Materials . 2024. https://doi.org/10.1002/adhm.202302375. T Xiao, X Ma and H Hu. Advances in emulsion stability: A review on mechanisms, role of emulsifiers, and applications in food. Food Chem X . 2025. http://doi:10.1016/j.fochx.2025.102792. H Xiang , S Xu , J Li , S Pan and X Miao . Particle Size Effect of Curcumin Nanocrystals on Transdermal and Transfollicular Penetration by Hyaluronic Acid-Dissolving Microneedle Delivery. Pharmaceuticals (Basel) . 2022. http://doi:10.3390/ph15020206. Z Németh , I Csóka and R J Semnani. Quality by Design-Driven Zeta Potential Optimisation Study of Liposomes with Charge Imparting Membrane Additives. Pharmaceutics . 2022. http://doi:10.3390/pharmaceutics14091798. N Choudhary and B Sekhon. An overview of advances in the standardization of herbal drugs. Journal of Pharmaceutical Education and Research . 2011(2), 55-70. S Bose , D.V Singh , T.K Adhya , N Acharya . Escherichia coli, but Not Staphylococcus aureus, Functions as a Chelating Agent That Exhibits Antifungal Activity against the Pathogenic Yeast Candida albicans . J Fungi (Basel) . 2023.http://doi:10.3390/jof9030286. B.A Witika , P.A Makoni and S.K Matafwali. Biocompatibility of Biomaterials for Nanoencapsulation: Current Approaches. Nanomaterials (Basel) . 2020. http://doi:10.3390/nano10091649. Additional Declarations The authors declare no competing interests. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8788292","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":585820038,"identity":"57ea5fdb-0ab8-450e-b42f-53c7808e6fda","order_by":0,"name":"Chutikan Sakphisutthikul","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtElEQVRIiWNgGAWjYDACCRBxwMZAAspPIFZLGkyLAdFaDpOghX92j/GHH2fOG0vOSGD88IPhTx5hS+6cMZPsuXHbTFoigVmyh8GgmKAWA4kcMwaeD7dt5CQSGKSB/MQGIrQYf/zz4RxIC/NvYrUYSPPcOAByGBtxtkjcSCuTljmTbCzZ87DNssfAmLAW/hnJmz++OWZnOON48uEbPyrkCGtBAoxAxQYkqB8Fo2AUjIJRgBsAAN5mOcHaB9UeAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-3557-1204","institution":"Khon Kaen University","correspondingAuthor":true,"prefix":"","firstName":"Chutikan","middleName":"","lastName":"Sakphisutthikul","suffix":""}],"badges":[],"createdAt":"2026-02-04 15:33:05","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-8788292/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8788292/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101924982,"identity":"5f7dcb81-07d4-47f5-9ece-4d779e070be4","added_by":"auto","created_at":"2026-02-05 06:04:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":839889,"visible":true,"origin":"","legend":"\u003cp\u003eA: Stability testing , B-D: Nanoparticle analysis\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8788292/v1/0514728153bc3eec3b5c8301.png"},{"id":101924981,"identity":"afc33ccf-3e98-4795-b5a0-022fb9563964","added_by":"auto","created_at":"2026-02-05 06:04:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":213604,"visible":true,"origin":"","legend":"\u003cp\u003eChemical Analysis with FTIR\u003cstrong\u003e \u003c/strong\u003eA: Traditional formula, B: Developed formula (Dev#2)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8788292/v1/4ea38d7c80b03e19677ce83d.png"},{"id":101943525,"identity":"14d03443-c24f-4579-b7ac-1cd1e75623a7","added_by":"auto","created_at":"2026-02-05 09:42:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":546044,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition Zone of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e. and \u003cem\u003eCandida albicans\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8788292/v1/1a9fedcec02e3a9bceaa1fd3.png"},{"id":101944659,"identity":"c1f4f557-ddfc-490d-9929-b34fbd26b415","added_by":"auto","created_at":"2026-02-05 09:53:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2538650,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8788292/v1/7743d5f0-588d-4d8f-9d5c-a515e4107ced.pdf"},{"id":101924985,"identity":"6c627bb1-f2de-4fac-8341-c74ceb7f7d94","added_by":"auto","created_at":"2026-02-05 06:04:52","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1055872,"visible":true,"origin":"","legend":"\u003cp\u003eAbstract infographic\u003c/p\u003e","description":"","filename":"Abstractinfographic.png","url":"https://assets-eu.researchsquare.com/files/rs-8788292/v1/0605e58261da1d634da910e7.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eStability, Bio-functional Nanoemulsion Derived from Thai Herbal for Enhanced Topical Efficacy\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Highlights","content":"\u003cp\u003e\u0026bull; \u003cstrong\u003eSuccessful transformation\u003c/strong\u003e of the unstable traditional \u0026ldquo;Khun Nithets Sukkij\u0026rdquo; recipe into a high-stability nanoemulsion.\u003c/p\u003e\n\u003cp\u003e\u0026bull; \u003cstrong\u003eCold-process technique\u003c/strong\u003e effectively preserved the chemical integrity of heat-sensitive CBD and Curcumin.\u003c/p\u003e\n\u003cp\u003e\u0026bull; \u003cstrong\u003eExceptional colloidal stability\u003c/strong\u003e achieved with a zeta potential of -76.20 to -92.23 mV and particle sizes under 200 nm.\u003c/p\u003e\n\u003cp\u003e\u0026bull; \u003cstrong\u003ePotent antimicrobial activity\u003c/strong\u003e demonstrated against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e, common skin pathogens.\u003c/p\u003e\n\u003cp\u003e\u0026bull; \u003cstrong\u003eCost-effective formulation (Dev#2)\u003c/strong\u003e identified using Aquamax, reducing production costs by 50% without compromising quality.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eDermatitis is a significant public health concern in Thailand as the tropical climate contributes substantially to the emergence of various skin-related problems and diseases affecting the skin and subcutaneous tissues. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] According to statistical data published in 2023 among outpatient visits, skin-related ailments were listed among the top ten causes of morbidity in the country.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] Despite the fact that topical corticosteroids are still considered the standard of care for inflammatory dermatoses, their extended use is often linked with adverse effects such as skin atrophy and discoloration. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] In addition, the economic cost is significant; Thailand has imported dermatological products worth approximately 2.4\u0026nbsp;billion baht in 2022, which highlights the urgent need for safe, efficient, and locally produced alternative treatments. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIn a bid to address these issues, the Thai Ministry of Public Health has invested heavily in reviving the traditional Thai medicine. One of the most notable examples of this effort has been the traditional recipe of Khun Nithets Sukkij which is now included in the Thai National List of Essential Herbal Medicines for the treatment of psoriasis and chronic skin diseases. This synergistic three-component formula contains 3 major bioactive ingredients, which included turmeric (\u003cem\u003eCurcuma longa\u003c/em\u003e L.), cannabis (\u003cem\u003eCannabis sativa\u003c/em\u003e L.), and cottonseed oil (\u003cem\u003eGossypium herbaceum\u003c/em\u003e L.). The main polyphenol of turmeric curcumin and cannabidiol (CBD) from cannabis exhibit strong anti-inflammatory, antioxidant, and antimicrobial properties. Cottonseed oil, rich in Vitamin E and unsaturated fatty acids also aids in skin barrier repair and possesses natural antioxidant properties. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe traditional preparation, although therapeutically promising, is severely limited in practical application. The initial formulation is an oil-based unstable blend that is prone to phase separation resulting in low percutaneous uptake and insufficient patient compliance.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] To overcome these limitations, nanotechnology offers an advanced drug delivery approach. As droplets with diameter sizes between 20 and 500 nm, nanoemulsions have become essential in improving solubility and bioavailability of lipophilic active compounds, including CBD and curcumin. These systems enhance penetration via the intercellular route of the stratum corneum. Moreover, the use of a non-thermal cold-process method is essential to stabilize the chemical composition of these heat-sensitive herbal extracts during formulation. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe proposed study aims to develop and comprehensively characterize a modernized nanoemulsion-based topical delivery system based on the Khun Nithets Sukkij formula. The study aims to assess the physicochemical properties, including physical stability during Freeze-Thaw cycles, nanoparticle size distribution, and chemical integrity using Fourier Transform Infrared (FTIR) spectroscopy. In addition, antimicrobial activity against Staphylococcus aureus and Candida albicans and antioxidant capacity was evaluated using the DPPH assay. Lastly, an in vitro cytotoxicity test in RAW 264.7 macrophage cells was conducted to determine the safety profile. This systematic approach aims to demonstrate a stable and cost-effective topical formulation that incorporates not only traditional Thai medical knowledge but also the latest pharmaceutical nanotechnology.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBioactive Compounds and Herbal Materials\u003c/h2\u003e \u003cp\u003eThe bioactive components used in the nanoemulsion formulation were selected to modernize the traditional Thai dermatological recipe of Khun Nithets Sukkij. Curcumin extract derived from \u003cem\u003eCurcuma longa\u003c/em\u003e L. was obtained from Bangkok Chemical Co., Ltd. (Thailand), with quality verified by a Certificate of Analysis (COA). High-purity cannabidiol (CBD) extract from \u003cem\u003eCannabis sativa\u003c/em\u003e L. was supplied by WP Science Co., Ltd. (Thailand). The oil phase consisted of refined cottonseed oil (low gossypol content) derived from \u003cem\u003eGossypium herbaceum\u003c/em\u003e L. and sourced from Statfold Seed Oil Ltd. (Netherlands). These active ingredients were maintained at a constant weight ratio of 1:1:2 (curcumin: cottonseed oil) to preserve the pharmacological synergy of the original traditional formulation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFormulation Excipients and Reagents\u003c/h3\u003e\n\u003cp\u003eTo facilitate cold-process emulsification, two primary polymeric emulsifiers were evaluated: Viscolam (Bangkok Chemical, Thailand) and Aquamax (Chemipan, Thailand). Isopropyl Myristate (IPM) was employed as an emollient to enhance skin penetration. Additional pharmaceutical-grade excipients included liquid glycerin (humectant), ethylenediaminetetraacetic acid (EDTA) (chelating agent), and phenoxyethanol (preservative). Deionized (DI) water served as the continuous phase for all oil-in-water (O/W) nanoemulsion systems. For antioxidant assays, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were used as the free radical and standard reference, respectively.\u003c/p\u003e\n\u003ch3\u003eMicrobial Strains and Cell Culture Reagents\u003c/h3\u003e\n\u003cp\u003eThe antimicrobial efficacy was evaluated against clinically relevant pathogens: Staphylococcus aureus (DMST: 5815), Escherichia coli (DMST: 12743), and the fungal strain Candida albicans (DMST: 8840). Culture media included Brain Heart Infusion (BHI) broth/agar for bacterial strains and Sabouraud Dextrose (SD) broth/agar for the fungal strain, all sourced from Himedia (India). For safety evaluations, RAW 264.7 macrophage cells were utilized. Cell culture reagents, including Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM), Fetal Bovine Serum (FBS), Penicillin-Streptomycin, and MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), were of analytical grade. Diclofenac was used as a comparative control in the cytotoxicity assays.\u003c/p\u003e\n\u003ch3\u003eFormulation Development\u003c/h3\u003e\n\u003cp\u003eThe nanoemulsions were prepared using the cold process cream method. This technique was selected for its ability to produce a stable emulsion at ambient temperature, thereby preserving the chemical integrity of heat-sensitive herbal compounds during emulsification. The core active ingredients\u0026mdash;curcumin extract, CBD extract, and cottonseed oil\u0026mdash;were maintained in a 1:1:2 ratio, consistent with the traditional Khun Nithets Sukkij formulation. Four development formulations (Dev#1\u0026ndash;Dev#4) were created, differing by emulsifier (Viscolam or Aquamax). The detailed compositions are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComposition of Experimental Nanoemulsion Formulations\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eComponent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFunction\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDev#1 (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDev#2 (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDev#3 (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDev#4 (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePart A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eViscolam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEmulsifier\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAquamax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEmulsifier\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsopropyl Myristate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEmollient\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePart B\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiquid Glycerin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHumectant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDI Water\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSolution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e87.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e86.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e86.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEDTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChelating Agent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePart C (Active)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCurcumin Extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eActive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCBD Extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eActive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCottonseed Oil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eActive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePart D\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenoxyethanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePreservative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003cp\u003eDev#1 : treatment 1 (Viscolam3%+Curcumin1%)\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003cp\u003eDev#2 : treatment 2 (Aquamax3%+Curcumin1%)\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003cp\u003eDev#3 : treatment 3 (Viscolam3%+Curcumin2%)\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section4\"\u003e \u003cp\u003eDev#4 : treatment 4 (Aquamax3%+Curcumin2%)\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhysicochemical Characterization\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eStability Testing\u003c/span\u003e: Accelerated stability assessment was performed using a Freeze-Thaw protocol. The formulations were subjected to 6 cycles of alternating temperatures: 45\u0026deg;C for 24 hours, followed by -20\u0026deg;C for 24 hours. Following the cycles, samples were visually inspected for any signs of phase separation, creaming, or changes in color and odor.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eParticle Size and Zeta Potential\u003c/strong\u003e \u003cp\u003eA Zeta sizer instrument was utilized to measure the average droplet size (nm) and zeta potential (mV) of the nanoemulsion. These parameters are critical for assessing the system's physical properties and predicting its long-term colloidal stability.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003epH Measurement\u003c/strong\u003e \u003cp\u003eThe pH of each formulation was measured using a calibrated pH meter to ensure compatibility with the natural pH of human skin.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eChemical Integrity (FTIR)\u003c/strong\u003e \u003cp\u003eFourier Transform Infrared (FTIR) spectroscopy was used to analyze the chemical composition of the final nanoemulsion in comparison with the original formula. This analysis aimed to confirm that the key functional groups from the active ingredients were retained after the formulation process.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBio-functional Evaluation\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eAntimicrobial Activity\u003c/span\u003e: The antimicrobial efficacy of the formulations was assessed using the agar well diffusion method. This assay was performed against microorganisms relevant to skin infections: \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (Gram-positive bacterium), \u003cem\u003eEscherichia coli\u003c/em\u003e (Gram-negative bacterium), and \u003cem\u003eCandida albicans\u003c/em\u003e (fungus). Efficacy was quantified by measuring the diameter of the inhibition zone in millimeters (mm).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAntioxidant Activity\u003c/strong\u003e \u003cp\u003eThe antioxidant potential of the nanoemulsion was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eIn Vitro Safety (Cytotoxicity\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e: An MTT assay was conducted to evaluate the potential cytotoxicity of the selected nanoemulsion on RAW 264.7 macrophage cells. The objective was to determine the concentration range at which the formulation maintained high cell viability, indicating its safety for topical use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll experiments were conducted in triplicate (n\u0026thinsp;=\u0026thinsp;3), and the results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical significance was determined using one-way ANOVA and paired t-tests; p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePhysical Characterization and Accelerated Stability\u003c/h2\u003e \u003cp\u003eThe production of the Thai traditional dermatological nanoemulsion was effective in converting an unstable combination of herbs into a robust topical delivery system and displayed high physical integrity in comparison with the initial formula. The accelerated stability test using six Freeze-Thaw cycles showed that all the formulations developed (Dev#1-Dev#4) were homogeneous and no phase separation was observed, the color was uniform, and the odor remained stable (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e-A). Conversely, the initial formula was found to exhibit immediate phase separation between the oil and the herbal powder at Cycle 0. The pH of the nanoemulsions were maintained between 5.5 and 6.0 which is within the physiological pH range of the human skin (4.06.0) therefore, minimizing the risk of skin irritation. This increased stability is explained by the fact that polymeric emulsifiers (Viscolam and Aquamax) were used, which offer steric stability due to the creation of a strong interfacial film on the droplets of oil, preventing coalescence. These observations are in line with the findings of Xiao, et al. (2025), who observed that the physical stability and droplet properties of nanoemulsions intended for topical delivery are directly determined by the choice of the oil phase and emulsifier composition. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eNanoparticle Properties\u003c/h2\u003e \u003cp\u003eA Zeta sizer was used to analyze the nanoparticles and confirmed that Dev#1, Dev#2 and Dev#3 had mean droplet sizes within the optimal nanoemulsion range of 61.28-260.33 nm. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e-B and C). Conversely, the formulations had highly negative zeta potentials. In particular, the values of zeta potential were in the range of -76.20 to -92.23 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e-D) indicating excellent colloidal stability due to high electrostatic repulsions that effectively prevent the aggregation of the particles. Droplet sizes smaller than 200 nm are essential for topical delivery systems because they greatly multiply the specific surface area, thereby enhancing the deeper delivery of active constituents such as Curcumin and CBD through the stratum corneum using intercellular routes or hair follicles. Based on the colloidal science principles, zeta potentials of over \u0026plusmn;\u0026thinsp;30 mV are considered indicative of high stability, and the values observed in this study far exceeded this threshold providing a strong scientific explanation for the long term stability of these herbal nanoemulsions.[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eChemical Integrity Analysis with FTIR\u003c/h2\u003e \u003cp\u003eThe chemical integrity of the herbal actives was confirmed to be preserved by FTIR analysis during the cold-process formulation. The nanoemulsion produced was found to retain the primary characteristic functional groups of the active ingredients, as indicated in the spectra of the final product. The major peaks included the C-O of phenolic and ether groups (~\u0026thinsp;1160 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of Curcumin, C-H of the alkyl groups (~\u0026thinsp;2925 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of CBD and the C\u0026thinsp;=\u0026thinsp;O ester (~\u0026thinsp;1740 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and the methylene groups (~\u0026thinsp;2856 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of the fatty acid in cottonseed oil were the major peaks. This ascertains that the active functional groups remained undegraded during the emulsification process and therefore are retained in their active form in the finished product. The preservation of these functional groups of typical chemicals proves that the \u0026ldquo;Cold Process\u0026rdquo; technique applied in this work guarantees that heat-sensitive active ingredients, namely cannabinoids and curcuminoids, remain intact and are not destroyed during their production through the conventional hot-processing method of cream manufacturing. This chemical integrity, allows the modernized nanoemulsion to retain the therapeutic qualities of the original Thai traditional wisdom.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBio-functional Efficacy\u003c/h2\u003e \u003cp\u003eThe antimicrobial effect of the nanoemulsion was tested against pathogens known to cause skin infections. Quantitative data obtained by the agar well diffusion technique were as follows: The nanoemulsion showed good activity against Gram-positive bacterium \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and its maximum inhibition zone is 16.30\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73 mm. It also showed good antifungal activity against \u003cem\u003eCandida albicans\u003c/em\u003e, with a maximum inhibition zone of 19.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55 mm. Conversely, the formulation had no inhibitory effect against Gram-negative bacteria \u003cem\u003eEscherichia coli\u003c/em\u003e. The findings are clinically relevant because \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e are common pathogens involved in skin infections. The selective nature of the given activity is significant. It is assumed that the selectivity is due to inherent differences in the structure of the cell wall of the microbes; the lipophilic herbal compounds may be more easily embedded in the peptidoglycan layer of Gram-positive bacteria and the chitin-based wall of fungi, whereas the complex outer membrane of Gram-negative bacteria, including \u003cem\u003eE. coli\u003c/em\u003e, may act as a significant permeability barrier. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrate these results.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe antimicrobial efficacy of nanoemulsion.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFormulation ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntimicrobial Activity\u003c/p\u003e \u003cp\u003e(\u003cem\u003eS. aureus\u003c/em\u003e Inhibition Zone mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAntimicrobial Activity\u003c/p\u003e \u003cp\u003e(\u003cem\u003eC. albicans\u003c/em\u003e Inhibition Zone mm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDev#1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e16.30\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e17.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDev#2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e14.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e18.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDev#3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e12.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e19.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDev#4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e18.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eOptimal Formulation Selection and Safety Assessment\u003c/h2\u003e \u003cp\u003eDev#2, which employed the emulsifier Aquamax, was selected as the optimal formulation based on its superior physicochemical performance and greater cost-effectiveness. This selection was justified by two main factors: 1) Cost-Effectiveness: Aquamax was identified as a more economical emulsifier which is a crucial aspect of scalable production. 2) Performance: Dev#2 exhibited the highest level of physical stability and favorable nanoscale characteristics (droplet size: 162.67 nm; zeta potential: -85.77 mV) compared with the other best-performing formulations.\u003c/p\u003e \u003cp\u003eThe in vitro safety profile of optimal Dev\u0026thinsp;\u0026minus;\u0026thinsp;2 formulation was then evaluated. The MTT assay performed on RAW 264.7 cells demonstrated that the nanoemulsion maintained cell viability of more than 80% across concentration ranging from 125 to 2,000 \u0026micro;g/mL. This finding indicates high biocompatibility and low cytotoxicity of the formulation implying its suitability for tropical application. The selected formulation is thus stable, efficient, safe, and cost-effective, making it a strong candidate for further development.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study successfully transformed the ancient and time-tested Khun Nithets Sukkij herbal formula into a stable and bio-functional nanoemulsion (Dev#2), thereby integrating traditional Thai medical knowledge with modern pharmaceutical nanotechnology. The formulation overcame the limitations of the traditional oil-based remedy by providing a robust nanoscale delivery system with high colloidal stability (zeta potential \u0026lt; -70 mV). The nanoemulsion exhibited notable antimicrobial efficacy against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e; and demonstrated a high safety profile in skin-related cells (cell viability \u0026gt; 80%). Overall, the study offers a scientific justification for the use of this novel formulation as a safe and cost-effective alternative for dermatitis treatment. Furthermore, it contributes to the valorization of Thai herbal resources and supports the advancement of the local pharmaceutical industry.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eCRediT author statement\u003c/h2\u003e\u003cp\u003e \u003cb\u003eJurarat Sirisukheepradit\u003c/b\u003e: Conceptualization; Methodology; Data curation; Writing - Original draft preparation. \u003cb\u003eRoshan Kumar Mahato\u003c/b\u003e: Visualization; Writing - Reviewing and Editing. \u003cb\u003eChutikan Sakphisutthikul\u003c/b\u003e: Visualization; Investigation; Supervision; Writing - Reviewing and Editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eC Pichanee ,K Silada Kanokrungsee, V Noldtawat, Suthasanee Prasertsook, L Surachanee and B Waranya. Sensitive Skin in Thais: Prevalence, Clinical Characteristics, and Diagnostic Cutoff Scores. \u003cem\u003eJournal of Cosmetic Dermatology\u003c/em\u003e, 2025. https://doi.org/10.1111/jocd.70181.\u003c/li\u003e\n\u003cli\u003eE.A Belachew and A.K Sendekie. Health-related quality of life and its determinants in patients with different dermatological disorders at the University of Gondar Comprehensive Specialized Hospital. \u003cem\u003eBMC Res Notes\u003c/em\u003e. 2023. https://doi.org/10.1186/s13104-023-06442-8.\u003c/li\u003e\n\u003cli\u003eD DiRuggiero and M DiRuggiero. Beyond Skin Deep: The Systemic Impact of Topical Corticosteroids in Dermatology. \u003cem\u003eJ Clin Aesthet Dermatol\u003c/em\u003e. 2025;18(1-2 Suppl 1):S16-S20. \u003c/li\u003e\n\u003cli\u003eH Parima , P Unchalee , N Sureerat , C Niphon and S Jiruth. Budget Impact Analysis of Botulinum Toxin Type A for Patients with Severe Blepharospasm in Thailand. \u003cem\u003eClinicoecon Outcomes Res\u003c/em\u003e. 2025. http//doi:10.2147/CEOR.S540982\u003c/li\u003e\n\u003cli\u003eH.K Cho, C.G Park and H.B Lim. Construction of a Synergy Combination Model for Turmeric (\u003cem\u003eCurcuma longa\u003c/em\u003e L.) and Black Pepper (\u003cem\u003ePiper nigrum\u003c/em\u003e L.) Extracts: Enhanced Anticancer Activity against A549 and NCI-H292 Human Lung Cancer Cells. \u003cem\u003eCurrent Issues in Molecular Biology\u003c/em\u003e. 2024. https://doi.org/10.3390/cimb46060332.\u003c/li\u003e\n\u003cli\u003eH Schoenfelder, Y Wiedemann and D.J Lunter. Development and characterization of topical formulation for maintenance therapy containing sorbitan monostearate with and without PEG-100-stearate. \u003cem\u003eInt J Cosmet Sci\u003c/em\u003e. 2025. https://doi:10.1111/ics.13023\u003c/li\u003e\n\u003cli\u003eG Lucia, D.D Giulia, V Giulia, C.B Maria , T Lihua, L Cristina and B.R Anna Development, characterization and in vitro assessment of a novel self-microemulsifying drug delivery system to increase cannabidiol intestinal bioaccessibility. \u003cem\u003eInternational Journal of Pharmaceutics\u003c/em\u003e. 2025. https://doi.org/10.1016/j.ijpharm.2025.126284.\u003c/li\u003e\n\u003cli\u003eH.D Lee, L Sunyoung Lim, K.S Sung and K Joohee. Advancements in Skin-Mediated Drug Delivery: Mechanisms, Techniques, and Applications. \u003cem\u003eAdvanced Healthcare Materials\u003c/em\u003e \u003cem\u003e. \u003c/em\u003e2024. https://doi.org/10.1002/adhm.202302375.\u003c/li\u003e\n\u003cli\u003eT Xiao, X Ma and H Hu. Advances in emulsion stability: A review on mechanisms, role of emulsifiers, and applications in food. \u003cem\u003eFood Chem X\u003c/em\u003e. 2025. http://doi:10.1016/j.fochx.2025.102792.\u003c/li\u003e\n\u003cli\u003eH Xiang , S Xu , J Li , S Pan and X Miao . Particle Size Effect of Curcumin Nanocrystals on Transdermal and Transfollicular Penetration by Hyaluronic Acid-Dissolving Microneedle Delivery. \u003cem\u003ePharmaceuticals (Basel)\u003c/em\u003e. 2022. http://doi:10.3390/ph15020206.\u003c/li\u003e\n\u003cli\u003eZ N\u0026eacute;meth , I Cs\u0026oacute;ka and R J Semnani. Quality by Design-Driven Zeta Potential Optimisation Study of Liposomes with Charge Imparting Membrane Additives. \u003cem\u003ePharmaceutics\u003c/em\u003e. 2022. http://doi:10.3390/pharmaceutics14091798.\u003c/li\u003e\n\u003cli\u003eN Choudhary and B Sekhon. An overview of advances in the standardization of herbal drugs. \u003cem\u003eJournal of Pharmaceutical Education and Research\u003c/em\u003e. 2011(2), 55-70. \u003c/li\u003e\n\u003cli\u003eS Bose , D.V Singh , T.K Adhya , N Acharya . \u003cem\u003eEscherichia coli,\u003c/em\u003e but Not \u003cem\u003eStaphylococcus aureus,\u003c/em\u003e Functions as a Chelating Agent That Exhibits Antifungal Activity against the Pathogenic Yeast \u003cem\u003eCandida albicans\u003c/em\u003e. \u003cem\u003eJ Fungi (Basel)\u003c/em\u003e. 2023.http://doi:10.3390/jof9030286.\u003c/li\u003e\n\u003cli\u003eB.A Witika , P.A Makoni and S.K Matafwali. Biocompatibility of Biomaterials for Nanoencapsulation: Current Approaches. \u003cem\u003eNanomaterials (Basel)\u003c/em\u003e. 2020. http://doi:10.3390/nano10091649.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Khon Kaen University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Stability, Bio-functional, Nanoemulsion, Thai herbal, Efficacy","lastPublishedDoi":"10.21203/rs.3.rs-8788292/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8788292/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe objective of this study was to develop and evaluate a stable nanoemulsion for the treatment of dermatitis using the traditional Thai recipe of Khun Nithets Sukkij, consisting of turmeric, cannabidiol (CBD), and cottonseed oil. Using the cold process cream method, two emulsifiers, Viscolam and Aquamax, were compared using four formulations (Dev#1-Dev#4). The study employed several evaluation methods including accelerated stability testing (Freeze-Thaw cycles), analysis of nanoparticles with a Zeta sizer, chemical integrity with Fourier transform infrared (FTIR) spectroscopy, antimicrobial activity with the agar well diffusion method, antioxidant capacity with the DPPH assay, and in vitro safety with the MTT assay. The experiment resulted in the successful stabilization of a nanoemulsion with an optimal particle size of 162.67 nm, which was highly colloidally stable, as indicated by a zeta potential of -72 to -96 mV. The formulations showed good antimicrobial activity, with maximum inhibition zones against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (16.30 ± 2.73 mm) and \u003cem\u003eCandida albicans\u003c/em\u003e (19.17 ± 0.55 mm), but no inhibitory activity against \u003cem\u003eEscherichia coli\u003c/em\u003e. The research found that the formulated nanoemulsion, namely, formulation Dev#2, is safe (cell viability\u0026gt;80%), stable, and cost-effective topical formulation that effectively integrates traditional Thai herbal knowledge with modern dermatological applications.\u003c/p\u003e","manuscriptTitle":"Stability, Bio-functional Nanoemulsion Derived from Thai Herbal for Enhanced Topical Efficacy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-05 06:04:42","doi":"10.21203/rs.3.rs-8788292/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"52d10518-a226-4367-8e9e-a8e697498ab9","owner":[],"postedDate":"February 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":62328039,"name":"Integrative \u0026 Complementary Medicine"}],"tags":[],"updatedAt":"2026-02-05T06:04:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-05 06:04:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8788292","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8788292","identity":"rs-8788292","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}