Enhanced antioxidant and antiproliferative activities of Cymbopogon

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Enhanced antioxidant and antiproliferative activities of Cymbopogon citratus (DC.) Stapf essential oils in microemulsion Yuan Li, Chengtao Wang, Zi Tao, Zhengang Zhao, Lijun You, Rui Zheng, Xiaoming Guo, and Zhanying Zhang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.9b01606 • Publication Date (Web): 15 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019

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ACS Sustainable Chemistry & Engineering

Enhanced antioxidant and antiproliferative activities of Cymbopogon citratus (DC.) Stapf essential oils in microemulsion

Yuan Lia,b,1, Chengtao Wanga,1, Zi Taob, Zhengang Zhaob,c*, Lijun Youb,d, Rui Zhengb, Xiaoming Guob, Zhanying Zhange

a

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing

Technology and Business University, No.11, Fucheng Road, Haidian District, Beijing, 100048, China.

bSchool

of Food Science and Engineering, South China University of Technology, 381 Wushan

Road, Guangzhou 510640, China.

cGuangdong

Province Key Laboratory for Green Processing of Natural Products and Product

Safety, South China University of Technology, 381 Wushan Road, Guangzhou 510640, China.

dOverseas

Expertise Introduction Center for Discipline Innovation of Food Nutrition and

Human Health (111 Center), 381 Wushan Road, Guangzhou 510640, China.

eCentre

for Tropical Crops and Biocommodities, Queensland University of Technology, 2

George Street, Brisbane, Australia

*Corresponding author: Zhengang Zhao, [email protected]. (Z. Z.), No.381 Wushan Road, South China University of Technology, Guangzhou, China.

1These

authors contributed equally to this work and share first authorship.

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract

C. citratus essential oils (C.EO) have important applications in food and pharmaceutical industries. However, the studies on microemulsions of C.EO (M-EO) are very limited though M-EO from other plants have been extensively studied. In this study, C.EO were firstly extracted by hydrodistillation and characterized by GC-MS analysis with the identification of citronellal (35.7%), citronellol (12.5%) and geraniol (16.6%) being the major oil compounds. Furthermore, M-EO were prepared using Tween 80 as a surfactant and ethanol as a cosurfactant and the pseudo-ternary phase diagrams were depicted at different surfactant/cosurfactant (Km) and surfactant+co-surfactant/essential oils (Smix) ratios. The M-EO structure changes were investigated and elaborated through the determination of electrical conductivity and viscosity of the system prepared at a Km of 2:1 and Smix of 8:2. M-EO was relatively stable after 90 days at ambient temperature of 25 ºC. Extracellular antioxidant activities of M-EO were improved by approximately 47% assessed by oxygen radical absorbance capacity (ORAC) assay and by 184% by peroxyl radical scavenging capacity (PSC) assay compared to the direct use of C.EO. The cellular antioxidant activity (CAA) of M-EO against liver cancer HepG2 cells was 151.1 ± 15.5 μmole quercetin equivalent/mg essential oils compared to negligible activity with C.EO. EC50 of the anti-proliferative activity of M-EO against HepG2 cells was 26.75 ± 2.91 μg essential oils/g, much lower than that (174.22 ± 8.20 μg essential oils/g) of C.EO. In addition, M-EO had little cytotoxicity against human normal cells L02. The results obtained 2


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from this study indicate that microemulsions are very good systems for applications of C.EO in functional food and pharmaceutical industries.

Keyword: Cymbopogon citratus, essential oils, microemulsion, antioxidant, antiproliferation, cytotoxicity

Introduction

Cymbopogon citratus (DC.) Stapf is a perennial herb of the Cymbopogon genus with natural lemon flavor. It is native to South India, Thailand, Vietnam and other Southeast Asian countries as a common seasoning material. In China, there are eight species of C. citratus plants, mainly distributed in southern region. C. citratus (DC.) Stapf is a low cost raw material for recovering bioactive compounds, which can be applied in medicine, cosmetics, flavor spices, food additives and other modern industries 1, 2 with antibacterial, antifungal, anti-carcinogenic, antiinflammatory, antioxidant and anti-rheumatic activities.3-5 C. citratus (DC.) Stapf is also recorded in Traditional Chinese Medicine Dictionary as a source of herbal medicine.

The main bioactive compounds of C. citratus are the volatile extractives, namely, C. citratus essential oils (C.EO) which contain aldehydes (e.g., citronellal, citral) and terpenes (e.g., Dlimonene, β-elemene, eugenol, linalool, geraniol). In addition to the human health beneficial functions,6 many of these compounds also have antibacterial and antioxidant activities.3, 7

Despite the bioactivities of C.EO, their applications in functional foods and/or as 3



pharmaceutical formula are hindered due to their poor stability, high volatility and low solubility in aqueous solutions. Microemulsions improve the thermodynamic stability and the solubility of hydrophobic bioactive compounds, leading to enhanced bioavailability of bioactive compounds and have been used to improve the performance of essential oil compounds from plants.8-10 For example, Hamed et al.9 found that water-based microemulsion systems improved antioxidant and antimicrobial activities of the oils from clove bud and the major oil ingredient eugenol. Xu et al.11 reported a more significant antifungal activity of a microemulsion consisting of cassia oils/ethanol/Tween 20 (10:30:60) against Geotrichum citriaurantii than direct use of cassia oils. Shaaban et al.12 found that essential oils of black cumin in microemulsion was highly effective against some pathogens. Pascoa et al.13 reported that microemulsion consisting of “sucupira“ oils/ethoxylated Castor Oil (surfactant and cosurfactant)/water at a ratio of 10:15:75 had a very effective anti-inflammatory activity compared to the direct use of the “sucupira“ oils.

In addition, microemulsions consisting of essential oils/surfactant (and co-surfactant)/water were also used for delivery of drugs or independent bioactive compounds. A recent study reported an optimized microemulsion system consisting of rose oils/Tween 20/water at a ratio of (5:20:70) improved the solubility and antifungal activity of Ketoconazole against C. albicans and the microemulsion was non-toxic against human lymphocyte at low usage.14 Lv et al. prepared microemulsions consisting of Cremophor EL/1,2-propanediol/essential oils (47:23:30, 4


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w/w) with the use of six types of essential oils (peppermint oi, clove oil and rosemary oil in one study; rose oil, jasmine oil and lemon oil in the other study).15, 16 The microemussions improved the solubility, pH stability, antioxidant activity, photostability and skin permeation of quercetin15 and of trans-resveratrol.16 Furthermore, the trans-resveratrol loaded microemulsions prepared from rose oils and jasmine oils demonstrated lower cytotoxicity against HaCat cells and more significant protection of the cells from ultraviolet B damage than the one based on lemon oils.16

Despite the significant commercial value of the C. citratus essential oils and increasing interest in microemulsions for pharmaceutical, cosmetic and food applications, the studies on the development of microemulsion systems with improved solubility, stability and bioactivities for application of C.EO are very limited if not missing. In this study, first, Tween 80, ethanol, water and C.EO were mixed to develop M-EO along with the establishment of the pseudoternary phase diagram for better understanding the M-EO systems. Furthermore, several M-EO were studied in terms of stability and particle size distribution. In addition, antioxidant activities of one selected M-EO were determined through extracellular oxygen radical absorbance capacity (ORAC) and peroxyl radical scavenging capacity (PSC) assays as well as the cellular antioxidant activity (CAA) assay. Finally, the antiproliferative activity of the M-EO on human liver cancer HepG2 cells was compared with those of the direct use of C.EO and the cytotoxicity of the M-EO against both HepG2 was compared with human normal cells L02 were evaluated. 5



Materials and Methods Materials

Dry C. citratus was purchased from a local wholesale market in Guangzhou (China) and it was sliced into pieces with approximately 4 cm width and stored in sealed bags under shade for extraction of essential oils. The sliced samples had a moisture of 13.2 ± 0.4%.

Tween 80 and ethanol were purchased from Aladdin (Shanghai, China), 6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid (Trolox), gallic acid, quercetin, 2,2’-azobis (2amidinopropane) dihydrochloride (ABAP), dichlorofluorescin diacetate (DCFH-DA) and 1,1Diphenyl-2-picrylhydrazylradical (DPPH) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hank’s Balanced Salt Solution (HBSS) was purchased from Haoyang Biological Manufacture Co., Ltd (Tianjin, China). Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biotechnology Co. (Hangzhou, China). Dulbecco’s modified Eagle’s medium (DMEM), Williams’ Medium E (WME), trypsin and other regents for cell culture were purchased from Gibco U.S. Biotechnology Co. (Carlsbad, CA, USA). Deionized water was obtained from Millipore System (Bedford, MA. USA). And all the other chemicals and reagents used in this work were of analytical grade.

Human liver cancer cells (HepG2) and human normal hepatocytes (L02) between 8 and 25 passages were used in this study. The both cancer cell lines was purchased from ATCC 6


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company. HepG2 was grown in WME, containing an addition of 5% (v/v) fetal bovine serum (FBS), 5 μg/mL insulin, 2 μg/mL glucagons and 0.05 μg/mL hydrocortisone. L02 cell line was cultured in DMEM, which contain 10% (v/v) FBS. Both cell lines were cultured in a humidified atmosphere with 5% CO2 at 37 °C.17

Extraction of C.EO

Sliced C. citratus (10 kg raw mass, 8.8 kg dry mass) was mixed in 100 kg water and was kept at room temperature for 24 h. Then the mixture was transferred into a large-scale distillation device (KaiHong Flavor & Fragrance Co. Ltd, Guangzhou, China), and boiling for 3 h at 100 °C to collect the condensate. Then, the condensate (approximately 1.5 L) was transferred to a 5 L conical flask, followed by extraction of the volatile compounds using petroleum ether (1.5 L × 2). The petroleum ether fractions were transferred to a 5 L conical flask, dehydrated over anhydrous sodium sulfate for 30 min and filtered through a mid-speed filter paper. The dewatered petroleum ether fractions were distilled at 45 °C under vacuum to remove petroleum ether in a rotary evaporator (Hei-VAP, Heidolph, Germany). The concentrated and petroleum ether-free yellow solution, namely C.EO, was collected, weighed and kept at 4 °C for further analysis.

Gas chromatography-Mass spectrometry (GC-MS) analysis

An Agilent 7890A-5975C GC-MS system (Agilent Technologies Co. Palo Alto, CA, USA), 7



equipped with an Agilent DB-5ms column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies Co. Palo Alto, CA, USA), was used to identify the main volatile compounds of the C.EO. For the analysis, the sample injection volume was 0.2 μL with the injector temperature at 250 °C. High purity helium was used as a carrier gas with a flow rate at 1 mL/min in a split mode of 30: 1. The oven temperature was held at 50 °C for 3 min at the beginning, then programmed to 120 °C at the rate of 3 °C/min, to 180 °C at the rate of 4 °C/min, and to 292 °C at the rate of 15 °C/min, followed by 15 min at 292 °C.

Mass spectrometry conditions were as follow: ion sources with electron impact ionization (70 eV and 230 °C) were utilized; the temperature of interface and quadrupole were 280 °C and 150 °C, respectively; quality scanning had a range from 33 to 450 amu (atomic mass unit). The compounds of C.EO were identified by comparison of the mass spectra with the GC-MS database (Nist 08) and the relative percentages were calculated based on the peak areas.

Preparation of essential oil microemulsions (M-EO)

M-EO were prepared using the method described previously.18, 19 Briefly, pseudo-ternary phase diagram of Tween 80/ethanol/water/M-EO was firstly constructed with the water titration method at ambient temperature, followed by determination of the microemulsion formations boundaries with achieving a clear and transparent appearance of the solution.20 Three surfactant/co-surfactant ratios, namely Km ratios (1:2, 1:1 and 2:1) were selected for further 8


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study based on pre-experiment with a series of formulas. Firstly, surfactant and co-surfactant mixture (S+Cos) was prepared by adding Tween 80 and ethanol at different Km ratios. Each S+Cos mixture was mixed with C.EO at S+Cos/C.EO mass ratios (Smix) of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1, respectively. Then the S+Cos/C.EO mixture was stirred constantly with a magnetic stirrer with the addition of different amounts of distilled water till the formation of a clear solution for each mixture. The water contents were 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%, respectively. The solutions were then balanced for 24 h at room temperature and kept in shade.

Physiochemical characterization of M-EO

Electrical conductivity (EC) of M-EO was measured by electrical conductivity meter (DDS11A, INESA Scientific Instrument Co., Shanghai, China). Viscosity of M-EO was determined by a viscometer (DV-III+, Brookfield Engineering Labs, MA, USA) with a shear stress ranging from 0 to 400 mPa·s.

The morphology of M-EO was recorded using transmission electron microscopy (TEM) (JEM100CXII, Hitachi Co. Ltd., Japan). Briefly, a drop of M-EO was placed on a copper grid with carbon film, and then stained for 2 min by 2 wt% phosphotungstic acid solution. The stained droplet was air-dried for 10 min, following by TEM analysis under an acceleration voltage of 200 kV. The mean particle sizes and polydispersity index (PDI) of M-EO were detected by

9



dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern Instrument Ltd., UK).

The M-EO solutions were also diluted (100 and 200 times) in deionized water, stored at ambient temperature for 90 days and stored at various temperatures (4 °C, 25 °C and 45 °C) for 30 days. The diluted and stored samples were further characterized using the methods described above.

Antioxidant activity assays

Antioxidant activities of C.EO and M-EO were determined using oxygen radical absorbance capacity (ORAC) and peroxyl radical scavenging capacity (PSC) assays as described previously.21, 22 The samples were prepared one-day in advance prior to the assays. In brief, for ORAC assay, the standard Trolox was dissolved and diluted to in 500 μM PBS (KH2PO4K2HPO4 buffer, 75 mM, pH 7.4) as a stock solution and was kept at -80 °C. Before use, the stock solution was diluted to a serial of Trolox standard solutions with concentrations of 6.25, 12.5, 25, 50 and 100 μM. 20 μL of test samples diluted in PBS and Trolox standards were added to a black 96-well microplate and incubated at 37 °C for 10 min, followed by addition of 200 μL of fluorescein sodium salt (0.96 μM) to each well. The mixtures were kept at 37 °C for 20 min. Then, 20 μL of ABAP (119 mM) was added to each well and mixed well to start the reaction. The black microplate was immediately put into multimode-microplate reader (Filter Max F5, Molecular Devices, CA, USA) and the optical densities were monitored every 5 min for 150 min at dual wavelengths 485 nm (excitation) and 535 nm (emission). Finally, the ORAC

10


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results were calculated using a regression equation and were expressed as μmole Trolox equivalent per gram C.EO or M-EO (μmole TE/g), representing the activity for scavenging ROO· radicals.

For PSC assay, the standard ascorbic acid (VC) was the standard was dissolved and diluted in 75 mM PBS. 20 μL of test samples (C.EO and M-EO) diluted in PBS and ascorbic acid standards were added to a black 96-well microplate, followed by addition of 100 μL of 13.26 μM DCFH-DA (diacetate) to each well which DCFH-DA was hydrolyzed with 1 mM KOH for 5 minute to remove the diacetate (DA) moiety. The wells were further added with 50 μL of 200 mM ABAP solutions to start the reaction. The fluorescence was monitored dynamically every 5 min for 40 min at dual wavelengths of 485 nm (excitation) and 535 nm (emission) in the multimode microplate reader. Finally, the PSC results were expressed as μmole ascorbic acid equivalent per gram C.EO or M-EO (μmole VCE/g).

Determination of biological activities by cell models Cellular antioxidant activity (CAA) CAA assay was conducted using model cell lines with the method described previously.17 In brief, HepG2 cells were inoculated at a density of 6 × 104 per well in the growth medium on a 96-well black microplate. The microplate was maintained at 37 °C with 5% CO2 for 24 h. After removing the growth medium and washing cells with PBS (100 μL), test samples (M-EO and 11



C.EO) and the quercetin (standard compound) solutions (100 μL) with various concentrations were diluted in antioxidant treatment medium containing 50 μM DCFH-DA and then were added to the wells in triplicate. After1 hour of incubation at 37 °C, the HepG2 cells were then washed with PBS (100 μL). Then, 100 μL HBSS containing 600 μM ABAP was added to each well. The 96-well microplate was placed into the multimode microplate reader and the absorbance was recorded at 37 °C every 5 min for 1 h under dual wavelengths of 485 nm (excitation) and 535 nm (emission). CAA results were calculated and expressed as quercetin equivalent per milligram C.EO or M-EO (μmole/mg). Cellular proliferation inhibiting test of C.EO and M-EO on HepG2 cells The antiproliferative activities of M-EO and C.EO were measured using HepG2 cells with the modified methylene blue assay reported previously.17 Briefly, 100 μL of HepG2 cells in growth medium was inoculated at a density of 2.5 × 104/well on 96-well microplate and incubated at 37 °C for 4 h. The growth medium was discarded, followed by addition of C.EO or M-EO solutions diluted in culture medium at a series of concentrations and 100 μL of growth medium without C.EO or M-EO was added to the wells as control. The cells were incubated at 37 °C h for 72 h with 5% CO2. After incubating and removing the medium, 50 μL of methylene blue solution in HBSS (1.25% glutaraldehyde and 0.6% methylene blue) was added to each well to stain the living cells, and incubated at 37 °C for 1 h, followed by removal of the methylene blue solution to terminate the staining. The cells were washed by deionized water and elution buffer 12


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(49% PBS, 50% ethanol, and 1% acetic acid) was added into the microplate. Then absorbance was measured at 570 nm in a microplate reader (SpectraMax 190, Molecular Devices, USA). The antiproliferative activities of C.EO/M-EO against HepG2 cells were calculated as follows: Cell proliferation %= (As/Ac) × 100% (As is the absorbance of cells with C.EO/M-EO wells minus blank and Ac is the absorbance of control wells minus blank). EC50 of C.EO/M-EO was used to assess the anti-proliferative activities of C.EO/M-EO. Cytotoxicity test of M-EO on HepG2 and L02 cells The cytotoxicity of M-EO was measured using HepG2 and L02 cells with a modified methylene blue assay method.17 Briefly, HepG2 cells or L02 cells were inoculated at a density of 4.0× 104 /well on a 96-well microplate and incubated at 37 °C for 24 h. Then growth medium was removed and the wells were added with three concentrations of M-EO solutions (low: 65 μg essential oils/g, medium: 80 μg essential oils/g, high: 95 μg essential oils/g) and 100 μL of growth medium without M-EO was added to the wells as the control. The cells were cultured at 37 °C with 5% CO2 for 24 h. After that，the cells were stained and the absorbance was measured using the method described above. The results were shown as the microscopic images of HepG2 cells and L02 cells.

Statistic analysis

Data were expressed as mean ± standard deviation (SD) for triplicate analysis. Two-tailed t-test 13



and Duncan’s multiple range tests were used to assess the significant difference between different experimental groups at a level of p < 0.05 using SPSS 22 software (IBM Corporation, NY, USA). Dose-effect analysis was performed using Calcusyn 2.0 software (Biosoft, Cambridge, UK).

Results and discussion C.EO extraction and GC-MS analysis

In this study, lemongrass sample of 8.8 kg (dry mass) was firstly soaked in water for 24 h and the hydro-distillation was collected to collect the C.EO-rich condensate (~1.5 L). Furthermore, the C.EO in the condensate was extracted and purified by petroleum ether, followed by distillation to collect C.EO. The C.EO was bright golden with a yield of 1.76% per kg dry C. citratus. This yield was in the range of the reported yields using hydro-distillation methods.23, 24

However, the scale of the preparation process (8.8 kg dry biomass) in this study was much

larger than those reported processes (no more than 100 g).23, 24 It is worth noting in addition to extraction methods, the yield is also affected by the feedstock properties (variety, tissue types, size, etc) and preparation methods (drying methods, solvents, etc). Compared to direct extraction of C.EO by organic solvents, such as methanol and ethanol,2, 3 the hydro-distillation process could reduce the usage of organic solvents significantly. Overall, there were 64 compounds identified in C.EO by GC-MS, accounting for 96.6% of the total volatile substances (the relative percentage based on the peak areas) (Fig. S1). Table 1 shows 16 volatile 14


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compounds with contents of more than 1%. The total content of these 16 compounds was 91.78%. Aldehydes, alcohols, and terpenes accounted for 79.73% of the total volatile compounds. (R)-3,7-Dimethyl-6-octenal (citronellal), (E)-3,7-dimethyl-2,6-octadien-1-ol (geraniol), 3,7-dimethyl-6-octen-1-ol (citronellol) and D-limonene were the predominant compounds with contents of 35.69%, 16.56%, 12.47% and 3.77%, respectively. Previously, steam distillation of essential oils of lemon grass cultivated in Bida city of Algera led to the extraction of geranial (citral), neral and β-myrcene of 42.2%, 31.5% and 7.5% in essential oils, respectively.25 Extraction of leaves of lemon grass growing in Botucatu city of Brazil by hydrodistillation led to the identification of geranial, neral and β-myrcene at contents of 51.46%, 19.83% and 16.5% in essential oils, respectively.1 In another study, extraction of essential oils from leaves of lemon grass growing in Navsari of India by microwave extractionhydrodistillation led to geranial contents ranging from 41.16% to 47.10%, neral contents from 37.71% to 40.67% and α-myrcene from 3.59% to 10.83%, respectively, under different microwave conditions.26 Different essential oils profiles and component contents are likely related to the lemon grass species, the tissues (leaves, stems or whole plants), growth conditions and extraction methods.

Preparation and phase study of M-EO

Tween 80 and ethanol were used as surfactant and co-surfactant, respectively, for preparation of microemulsions in this study. Tween (polysorbate) is a type of relatively safe non-ionic 15



surfactants and is widely in food and cosmetic industries as an emulsifier.27, 28 Polysorbate, produced from sorbitol, fatty acid and ethylene oxide, is a relatively sustainable and safe solvent and additive.29 Ethanol is produced from glucose by yeast fermentation and a renewable bioproduct. In addition to direct consumption of food-grade ethanol drinks by human beings, ethanol with different grades is widely used in food, pharmaceutical, cosmetic, energy and chemical industries as a solvent. Tween 80 has a good hydrophilic lipophilic balance (HLB) value comparing to Tween 20 and Tween 60, which makes it perform stronger hydrophilicity to increase the elasticity of interface film to form a pleasant microemulsion system.30 The function of co-surfactants is to reduce the surface tension of water-oil interface by combining surfactants to form a layer of mixed adsorption film. Alcohols such as aliphatic alcohols (ethanol, 1-propanol, isopropanol, etc.) and polyols (glycerol, polyethylene glycol, etc.) are used as co-surfactant as they have the hydrophilic and hydrophobic groups, can increase the flexibility and mobility of interface membrane, lower the bending energy, and regulate the HLB value so as to facilitate the formation of emulsion.31-33

When different Smix ratios were tested, clear and transparent solutions were only observed at Smix of 9:1 and 8:2. In order to load more essential oils, a Smix of 8:2 was used to for the study of pseudo-ternary phase diagrams. Fig. 1 shows the pseudo-ternary phase diagrams of M-EO. Transparent and low viscous microemulsion region with water titration method and magnetic stirring at room temperature was presented as “M-EO”, whilst the rest part of the phase 16


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diagrams was expressed as turbid or layered traditional emulsion. The M-EO areas were calculated at different mixing weight ratios of the surfactant and co-surfactant (Km) as 2:1, 1:1 and 1:2. In this study, the M-EO region area decreased from 42.19% to 35.94% and 17.19% with Km values decreased from 2:1 to 1:1 and to 1:2, respectively (Fig.1). A higher content of surfactant may lead to smaller particles of microemulsions but would also combine more essential oils, weakening water solubilization in oil phase, leading to the interface imbalance, and then resulting in a smaller M-EO area.34 A co-surfactant exists in the interface membrane under low concentration and increasing the concentration co-surfactant could increase the microemulsion stability.35 However, excessive co-surfactant could break the interface membrane between the oil phase and water phase, leading to the demulsification of micromulsions.35 Indeed, a very high or very low Km value has an adverse impact on the formulation of microemulsions. Because the relative larger M-EO region, a Km ratio of 2:1 was used for the follow studies.

Microstructural transition of microemulsions

The changes in EC and viscosity were monitored to determine the microstructural transition changes from water in oils (W/O) microemulsions to bicontinuous (B.C) structures, and from B.C structures to oils in water (O/W) microemulsions following a method described previously.18

17



The effects of water content on EC and viscosity are shown in Fig. 2. EC increased from 0 to about 40 μs/cm with water contents from 0% to 30%, followed by a slightly sharp increase from the water content of 30%. EC increased almost linearly with water contents from 30% to 60%. The EC peaked at the water content of 70%, followed by significant decrease with water contents from 70% to 90%. In non-ionic microemulsions with low water contents, discrete reverse micelles are formed within a continuous oil phase and the system would have EC similar to that of the oil phase.36 The increase in EC with further increasing water content is possibly due to the self-ionization of co-surfactants such as ethanol and the mobility of remaining polar molecules in essential oils as well as the presence of impurities.37 The reduction in EC at high water contents may be related to the poor conductivity of pure water. The EC changes were in line with the observation in a previous publication, which used Tween 80 as a surfactant and soybean oil as a co-surfactant to improve the dilutability of essential oil microemulsions.18

Viscosity is another parameter to understand the structure changes of microemulsions. In combination with the EC data, the changes in microstrutures can be explained. The low viscosity at water contents of

Enhanced antioxidant and antiproliferative activities of Cymbopogon

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