|Original Article, Biomed Biopharm Res., 2022; 19(1):168-180
doi: 10.19277/bbr.19.1.284; PDF version here [+] ; Portuguese html version [PT]
Cymbopogon citratus (DC.) Stapf essential oil: Unraveling potential benefits on human skin
Sérgio Faloni de Andrade 1, Eucinário José Pinheiro 1, Catarina Pereira-Leite 1,2, Maria do Céu Costa 1,2, Ana Cristina Figueiredo 3, Luis Monteiro Rodrigues 1*
1CBIOS - Research Center for Biosciences and Health Technologies, Universidade Lusófona de Humanidades e Tecnologias, Lisboa, Portugal; 2LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal); 3Centro de Estudos do Ambiente e do Mar (CESAM Ciências), Faculdade de Ciências da Universidade de Lisboa (FCUL), Biotecnologia Vegetal (BV), Departamento de Biologia Vegetal (DBV), Lisboa, Portugal
* corresponding author:
The essential oil of Cymbopogon citratus (DC.) Stapf is widely used for the production of fragrances, cosmetics, and detergents. However, there is no information on the effects of formulations containing C. citratus essential oil (EOCC) on human skin. This study aimed to evaluate the effects of formulation containing EOCC on human skin physiology. The study involved participants of both sexes (n=12). Two areas (3 cm x 3 cm) were drawn on both forearms. One randomly chosen area was treated for 14 days, 2 times/day with polyacrylic acid gel containing 5% EOCC and the other was used as a control for the same time period. Transepidermal water loss (TEWL), hydration, epidermal lipids, and biomechanics were measured (D0 and D14). High resolution Sonography images were also obtained. Results revealed a significant decrease in TEWL, a significant increase in hydration, firmness and elasticity, and a decrease in lipids, in the areas treated with EOCC. Sonography showed an increase in echogenicity of the epidermis after applying the formulation indicating that the essential oil penetrates only the most superficial layers of the skin. Results also suggest that formulations containing EOCC are safe for topical application and can improve and protect human skin.
Keywords: Cymbopogon citratus, essential oil, citral, lemongrass
Received: 05/05/2022; Accepted: 09/06/2022
Currently, there is a great interest in natural products that might benefit human skin physiology. Many essential oils have been indicated as potentially interesting by combining beneficial effects on the skin while providing pleasant organoleptic properties to formulations. In addition, some essential oils have been used in traditional medicine for over 5,000 years, with many purposes, including anti-inflammatory, analgesic, antifungal, antimicrobial, antioxidant (1,2) and also to prevent skin aging (3,4). Due to their lipid characteristics, certain essential oils might be interesting for the treatment of cutaneous xerosis, a common condition in the elderly characterized by excessively dry skin resulting from an imbalance of lipids in the most superficial layer of the skin (5,6).
However, there are uncertainties about the safety of these products, as irritation, sensitivity, and contact dermatitis have been reported in human skin (7-9). It is important to point out that most efficacy and safety studies about the application of essential oils on the skin have been conducted in vitro (using skin cell lines) or in animal models, using multiple methodologies which are difficult to compare or extrapolate to human. Thus, the scarcity of studies on humans has limited the potential use of essential oils as effective and safe phytotherapeutic agents. Further studies are needed to verify the efficacy and safety of these products (3,10).
Cymbopogon citratus (DC.) Stapf, commonly known as lemongrass, is an important medicinal plant cultivated in different regions of the world, including Portugal. Its essential oil, whose principal component is the monoterpene citral, is widely used to produce fragrances, cosmetics, detergents, and pharmaceutical products (11,12). However, there is no scientifically-based information on the effects of formulations containing C. citratus essential oil (EOCC) on human skin. Therefore, the purpose of this study was to evaluate the effects of a formulation containing EOCC on the skin physiology of healthy volunteers.
Material and Methods
The essential oil obtained from aerial parts of Cymbopogon citratus was provided by "Cantinho das Aromáticas” (Lisbon, Portugal). The quantitative composition of the EOCC-containing gel is presented in Table 1. EOCC, glycerin (José M. Vaz Pereira, Benavente, Portugal), disodium ethylenediaminetetraacetate (Saninter, Lisboa, Portugal), and a paraben preservative solution, made of methylparaben (6% w/w, AppliChem GmbH, Darmstadt, Germany) and propylparaben (3% w/w, José M. Vaz Pereira, Benavente, Portugal) in propylene glycol (LABCHEM, Santo Antão do Tojal, Portugal), were mixed. The polymer (Carbopol® 940, José M. Vaz Pereira, Benavente, Portugal) was dispersed in distilled water and then allowed to hydrate and swell before the addition of the previously prepared mixture. Triethanolamine (José M. Vaz Pereira, Benavente, Portugal) was added under stirring until a viscous and homogeneous gel was obtained with a pH value of 4.6 ± 0.1. A control gel, in the absence of EOCC, was similarly prepared without the addition of the essential oil.
The essential oil was characterized by Gas Chromatography (GC) and Gas Chromatography coupled to Mass Spectrometry (GC/MS). GC analysis was performed using a Perkin Elmer Clarus 400 Gas Chromatograph equipped with two Flame Ionization Detectors (FID), a split–splitless injector (split ratio of 1:40), and data processing system. Two columns of differents polarity were used (i) DB-1 fused-silica capillary column coated with 100% polydimethylsiloxane [(30 m x 0.25 mm, 0.25 μm thickness); J & W Scientific Inc., Rancho Cordova, CA, USA] and (ii) DB-7HT fused-silica capillary column coated with phenylmethylsilicone [(50% phenyl)- methylpolysiloxane, 30 m x 0.25 mm, 0.15 μm thickness) J&W Scientific Inc.]. The oven temperature was programmed to increase from 45°C to 175°C at 3°C/min, and subsequently at 15°C/min until 300°C, at which time the temperature was held (isothermal) for 10 min. The total run time was 61 min. Hydrogen was used as carrier gas, adjusted to a linear velocity of 30 cm/s. The temperatures of the injector and detector were kept at 280°C and 290°C, respectively. The injection volume of essential oil was approximately 0.01µL.The percentage composition of essential oil was determined by the normalization method, without using correction factors, according to ISO 7609. Values presented correspond to the mean value of two injections.
GC-MS analysis was performed on a Clarus 600T Mass Spectrometer equipped with a DB-1 fused silica column [(100% polydimethylsiloxane, 30 m x 0.25 mm, 0.25 μm thickness) J & W Scientific Inc.] connected to a Perkin-Elmer Turbomass (program version 22.214.171.1247, Perkin Elmer, Shelton, CT, USA). The oven temperature was programmed from 45°C to 175 °C, in increments of 3 °C/min, and subsequently at 15 °C/min until 300 °C, at which time the temperature was kept isothermal for 10 min. The injector temperature was 280°C, the transfer line temperature was 280 °C, and the ionization chamber temperature was 220°C. Helium was used as carrier gas, adjusted to a linear velocity of 30 cm/s, with a flow sharing ratio of 1:40. The ionization energy was 70 eV, ionization current 60 μA, collecting over a mass range of 40-300 a.m.u. with a 1 s scan time. The identity of the compounds was determined by comparing their retention rates, in relation to those of the n-alkanes and mass spectra, with standards synthesized in the laboratory, commercial standards, and by comparison with a library of mass spectra also developed in the laboratory.
The study included 12 healthy participants (8 women and 4 men; mean age 36.2 ± 16.3 years old) without any skin or other systemic diseases. Volunteers received a detailed explanation of the study and provided written informed consent. All procedures observed the principles of good clinical practice from the Helsinki Declaration and respective amendments (13) and were previously approved by the institution’s Ethics Committee (approval number 04/13).
A primary cutaneous tolerance assay using the ‘open-test’ methodology was performed (14) for 24h before the experiments.
For the efficacy test, two areas (3 cm x 3 cm) were marked in both forearms. Applications were randomized. The trial was designed for 14 days with standardized applications (0.1 mL) using a syringe two times a day (morning and evening) at home. The formulation containing EOCC was applied in one area and the control gel (without EOCC) in the other. The study was single-blinded.
Skin functions were assessed before application of the products (D0) and at the end of the trial (D15), assessing transepidermal water loss (TEWL, Tewameter® CK electronics, Germany) a measure of the epidermal "barrier" (15), the superficial and deep hydration of the epidermis (Moisturemeter SC and Moisturemeter® Dtec, Finland) (16), the biomechanical behaviour of the skin (Cutiscan® CK electronics, Germany) expressed in terms of firmness (V1) and elasticity (V3) (17) and the assessment of skin lipids (Sebumeter SM 815, CK electronics, Germany) (18). High resolution ultrasound images (HRS, DermaScan C Cortex Technology, Denmark) were obtained at D0 and D15) (19). The color images were converted into a grey scale image for further analysis processed by software ImageJ® (NIH, Bethesda, Maryland, USA) (19). All data were obtained in a controlled environment (humidity ~ 50%, temperature 21 ± 2 °C) and participants were acclimatized for at least 30 min before starting the tests. During the study period, subjects were instructed to not apply detergents, emollients, moisturizers, or any cosmetics in the testing areas.
Data were reported as mean ± standard error of the mean (SEM) and compared by Mann–Whitney test using GraphPadPrism 5® software (GraphPad Software, San Diego, CA, USA). A p value <0.05 was considered significant.
The chromatographic analysis of the EOCC allowed the identification of 24 compounds, accounting for 96.1% of the total oil. The main components identified in the essential oil were geranial (trans-citral) (42.3%) and neral (cis-citral) (33.2%). These data are summarized in Table 2.
The open skin tolerance test indicated that the formulation containing EOCC was well tolerated. No signs of irritation, such as erythema, dryness, edema, or itching, were observed. At any time during the 14-day treatment period.
Regarding skin parameters, a significant decrease in TEWL was observed in the site treated with the formulation containing EOCC, as well as a significant increase in superficial and deep epidermal hydration and a decrease in skin lipids (Table 3). Comparing skin biomechanical parameters revealed an increase in V1 (firmness) and V3 (elasticity) relative to the control gel (Table 4).
The High-Resolution Sonography has shown that the epidermis was more echogenic after applying the EOCC formulation, suggesting that essential oil penetrates the most superficial layers of the skin (Table 5, Figure 1).
In this study we investigated effects of C. citratus essential oil (EOCC) on skin physiology to evaluate its potential interest for use in skin care formulations. EOCC has been reported to have several benefits, including topical anti-inflammatory, antifungal (20), anxiolytic (21), anti-ulcer (22), and antihypertensive (23) effects. The main component of EOCC is the monoterpene citral, which can be found in two isomeric forms (cis, known as neral; or trans, known as geranial) (11, 24) and to which most of the beneficial effects of EOCC have been attributed (25). Chemical analysis of the essential oil used in this work revealed mostly citral in its composition. Lulekal et al. (26) reported no skin irritant effect and no systemic toxicity when a formulation containing 10% EOCC was administered to mice, suggesting that this essential oil has a good safety profile. We evaluated the safety of our formulation, which contained a lower concentration of EOCC, in human skin through the "open test" methodology. No signs of irritation or toxicity were observed after application, allowing the continuation of the experimental efficacy protocol.
Our results have shown that the application of the formulation containing EOCC significantly increased the epidermis superficial and deep hydration (p < 0.05 and p < 0.01, respectively) and reduced the transepidermal water loss (TEWL) (p < 0.05). Water is essential for normal skin functionality, especially its outermost layer, the stratum corneum (SC). Water retention in the SC is dependent on the presence of two main factors (1) naturally occurring hygroscopic substances collectively referred to as natural moisturizing factors (NMFs), and (2) intercellular lipids organized to form a barrier that prevents transepidermal water loss (TEWL) (27). Sufficient hydration of the epidermis is necessary for the proper maturation of the EC and physiological peeling of the skin. Thus, if the water content in the epidermis falls below a critical level the enzymatic functions required for physiological peeling are impaired, and excessive adhesion of corneocytes occurs with their accumulation on the skin surface. This process leads to the appearance of dry, rough, and flaking skin (28). The inverse correlation between TEWL and skin hydration has been well demonstrated, as higher levels of TEWL, a marker of skin barrier function, are often correlated with lower water content in the SC (29). Thus, the results regarding TEWL and hydration suggest that the formulation containing EOCC improves the water balance in the skin, among other factors, by increasing the barrier function of the skin leading to decreased TEWL and increasing the water content of the epidermis.
The image analysis obtained by High Resolution Sonography showed that the formulation containing EOCC significantly increases the epidermis echogenicity (p<0.05), suggesting that the formulation is retained mainly in this layer of the skin, which corroborates the evidence that the lipidic character of the essential oil could combine with the lipids of the SC and increase the integrity of this structure, thus providing greater barrier integrity with a consequent decrease in TEWL. In addition, the permanence of the oil in the epidermal layer is evidence of their safety, as their effects would be restricted to the skin. Similar results have recently been described by our research group with formulations containing essential oils of Lavandula angustifolia Mill.(Lavender) and Salvia officinalis L. (Sage) (30).
The biomechanical properties of the skin were evaluated using CutiScan®. A significant decrease in V1 values (p < 0.05) and an increase in V3 values (p < 0.05) were observed at the site exposed to the formulation containing EOCC. V1 is related to the firmness and seems to indicate the ability of the skin to resist displacement; the firmer the skin, the lower the V1. V3 has been related to the ability of the skin to resist displacement versus its ability to return to its original position; the higher the V3, the better the elasticity (17,31). Skin firmness is related to the ability of collagen and elastic fibers to elongate and is inversely proportional to their thickness and rigidity (32). Elasticity is considered an indicator of age and overall skin health (33) and is also related to the content of collagen and elastin fibers (34,35). Thus, our results suggest that the application of the formulation containing EOCC favors the accumulation of these protein fibers in the skin and improves its biomechanical characteristics.
Finally, the repeated use of the formulation did not increase the lipid content of the epidermal surface. However, it is possible to speculate about the potential enhancement of these compounds on epidermal cohesion, as suggested by the previously detected effects (30,36). Regardless, there appears to be a consistent decrease in epidermal lipid content (p < 0.05) after repeated exposure to the EOCC-containing formulation. This additional aspect would reinforce the interest in using these compounds in skin health products (37).
This work is funded by national funds through FCT - Foundation for Science and Technology, I.P., under projects UIDB/04567/2020, UIDP/ 04567/2020 and UIDP/50017/2020+UIDB/50017/2020+LA/P/0094/2020.
SFA, EP, MCC, LMR planned and performed experiments; CPL prepared the formulations; ACF has undertaken Chromatographic analysis; SFA and LMR wrote and corrected the manuscript.
Conflict of Interests
Editors involved in this manuscripts’ authorship had no participation in the review or decision process. All authors have stated that there are no financial and/or personal relationships that could represent a potential conflict of interest.
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