Phytochemical Composition, Biological Activity and Application of Cymbopogon citratus In Vitro Microshoot Cultures in Cosmetic Formulations

Abstract

This study investigated the phytochemical composition and biological activity of Cymbopogon citratus microshoot cultures and evaluated their suitability for incorporation into a cosmetic formulation. Microshoot cultures were established on Murashige and Skoog media supplemented with plant growth regulators and served as a reproducible source of biomass. Methanolic and ethanolic extracts were analyzed for total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. Chemical composition was characterized using LC-MS/MS analysis, which revealed the presence of phenolic acids and flavonoids, with p-coumaric, caffeic, and ferulic acids being among the most abundant detected constituents. In biological assays, the extracts inhibited murine tyrosinase in a concentration-dependent manner and exhibited antimicrobial activity against selected oral and skin-associated microorganisms, including Streptococcus mutans, Streptococcus pyogenes, and Staphylococcus epidermidis, as well as showing fungistatic and fungicidal effects against Candida albicans. Cytotoxicity analysis performed on HaCaT keratinocytes confirmed biocompatibility within the tested concentration range. To assess formulation suitability, the microshoot extract was incorporated into an oil-in-water (O/W) cream, which maintained stable pH, viscosity, and physical properties while preserving the antioxidant activity of the extract. Overall, these findings indicate that C. citratus microshoot cultures represent a reproducible source of bioactive metabolites with potential application in cosmetic formulations.

1. Introduction

Among natural resources, plants are particularly valued as renewable sources of structurally diverse bioactive compounds, many of which have long been applied in traditional medicine, nutrition, and cosmetics. Their secondary metabolites enable adaptation to abiotic and biotic stressors such as ultraviolet radiation, drought, and pathogens, while in humans they exert antioxidant, antimicrobial, anti-inflammatory, and photoprotective effects [1]. With the growing consumer demand for sustainable natural products, the cosmetics industry is increasingly focused on plant-derived bioactives with multifunctional properties, including free-radical scavenging, anti-aging, and skin-lightening activities [2,3]. In this context, polyphenolic compounds, especially phenolic acids and flavonoids, are of particular interest because of their combined antioxidant and skin-protective functions [4,5].

Cymbopogon citratus (D.C.) Stapf, commonly known as lemongrass, is a perennial aromatic grass of the Poaceae family, widely cultivated in tropical and subtropical regions, primarily for essential oil production [6]. The species has a long history of ethnomedicinal use, including applications as a stomachic, antispasmodic, carminative, anxiolytic, antihypertensive, and antipyretic agent, as well as in the treatment of malaria, rheumatism, pneumonia, and menstrual irregularities [4,7,8]. Lemongrass essential oil has also been used topically as an antiseptic, antifungal, and anti-inflammatory remedy [9]. Modern pharmacological studies support these traditional uses, reporting antioxidant, antimicrobial, anti-inflammatory, antihyperlipidemic, antidiabetic, and anticancer activities of C. citratus extracts [2,9,10,11,12,13,14,15,16].

The chemical composition of C. citratus varies depending on geographical origin, plant part, developmental stage, and extraction method [17]. Its essential oil is dominated by citral (geranial and neral), accompanied by monoterpenes such as citronellal, myrcene, geraniol, nerol, geranyl acetate, terpinolene, and methylheptenone [18]. In addition to volatile constituents, lemongrass contains phenolic acids, including caffeic, ferulic, rosmarinic, syringic, vanillic, and p-coumaric acids, as well as flavonoids such as luteolin, apigenin, quercetin, kaempferol, and isoorientin 2-O-rhamnoside [2,4]. These compounds, typically present in polar fractions of the plant, contribute to the broad biological activity of C. citratus and its relevance in food, nutraceutical, and cosmetic applications [17].

Phenolic compounds are of particular interest in cosmetic research due to their antioxidant properties and their ability to modulate enzymes involved in skin aging and pigmentation. Tyrosinase, a key oxidoreductase in melanogenesis, catalyzes the hydroxylation of monophenols and the oxidation of o-diphenols to o-quinones, leading to melanin synthesis. Inhibition of this enzyme is a common strategy in the management of hyperpigmentation and in the development of skin-lightening formulations [19,20].

The quantitative levels of total phenolic content (TPC) and total flavonoid content (TFC) in C. citratus vary substantially depending on geographical origin, harvest conditions, post-harvest processing, and extraction protocols. In addition to quantitative variability, qualitative differences in phenolic and flavonoid profiles have also been reported, with specific compounds being differentially accumulated under distinct environmental and processing conditions [21,22,23,24,25]. This inherent variability underscores the need for standardized and reproducible plant raw materials. In vitro culture systems offer such an alternative, providing sterile, controllable, and season-independent biomass production and reducing fluctuations in both quantitative yields and qualitative composition of secondary metabolites [1,26]. The availability and quality of medicinal plant materials are further affected by habitat loss, environmental pressures, and harvesting practices, as well as by genetic and agronomic variability [27,28].

In vitro propagation of C. citratus has been achieved using nodal segments, leaf bases, and shoot tips, with efficient shoot induction reported on Murashige and Skoog (MS) medium supplemented with appropriate plant growth regulators [29]. However, secondary metabolite accumulation in in vitro cultures is often lower than in field-grown plants due to the absence of abiotic and biotic stress factors that activate defense-related pathways in planta. Exposure to factors such as ultraviolet radiation, drought, pathogens, or heavy metals has been shown to enhance the biosynthesis of phenolic compounds and flavonoids under field conditions [1], while elicitation strategies are frequently required to stimulate metabolite production in vitro [26]. Despite these differences, in vitro systems offer superior reproducibility and safety, which are critical for cosmetic applications requiring consistent raw material quality [30,31].

From a formulation perspective, it is essential to verify whether plant extracts retain their biological functionality after incorporation into complex systems such as cosmetic emulsions. Previous studies have demonstrated that oil-in-water (O/W) creams containing botanical active ingredients can maintain antioxidant and antimicrobial activity after formulation, with skin-compatible pH, acceptable sensorial properties, and short-term physicochemical stability [32,33,34,35,36,37]. Moreover, although relatively few studies have examined methanolic extracts of C. citratus, available data consistently demonstrate antibacterial activity against clinically relevant Gram-positive and Gram-negative microorganisms, including Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa [38,39]. These findings highlight the importance of evaluating not only crude extracts but also finished cosmetic formulations when assessing efficacy [35].

Despite extensive research on C. citratus, information on extracts obtained specifically from in vitro microshoot cultures remains limited. The suitability of such extracts for topical formulations, including their behavior in finished oil-in-water (O/W) emulsions and their safety in human keratinocytes, is also poorly documented. Therefore, the present study aimed to provide an integrated assessment of the phytochemical composition of C. citratus microshoot cultures using LC–MS/MS analysis, together with their antioxidant and antimicrobial activity, inhibitory effects on murine tyrosinase, cytotoxicity toward HaCaT keratinocytes, and the retention of antioxidant activity after incorporation into a model oil-in-water (O/W) cosmetic emulsion.

2. Materials and Methods

2.1. Plant Material In Vitro Culture Establishment and Reagents

Seeds of C. citratus (W. Legutko, Jutrosin, Poland) were surface sterilized (1 min in 96% ethanol followed by 7 min in 0.1% HgCl₂), rinsed three times with sterile distilled water and germinated on Murashige and Skoog (MS) medium. The in vitro culture conditions were established based on previously reported protocols for Cymbopogon species, with modifications as described below [30,31]. The medium was adjusted to pH 5.7 prior to autoclaving (121 °C, 20 min). The culture medium used for microshoot propagation contained 1.0 mg/L 6-benzylaminopurine (BA), 0.5 mg/L 1-naphthaleneacetic acid (NAA) and 0.25 mg/L gibberellic acid (GA₃). Cultures were maintained at 25 ± 2 °C under white LED illumination (2.75 W/m²; 16/8 h photoperiod) and subcultured every 14 days. Biomass used for subsequent analyses was collected from three independent in vitro microshoot culture passages harvested at two-week intervals. All cultures were propagated under identical and strictly controlled conditions. While no quantitative yield comparison between passages was performed, the use of multiple independent passages supports the reproducibility of biomass production under the applied in vitro culture system.

Fresh in vitro-derived microshoots were immediately frozen at −80 °C and lyophilized using a FreeZone freeze dryer (LABCONCO, Kansas City, MO, USA) at −80 °C and 0.04 mbar for 48 h. The dried material was ground to a fine powder using an electric laboratory mill (IKA, Warsaw, Poland) and stored at −20 °C until extraction.

The following analytical-grade reagents were used in the study: ethanol 96% p.a. (Avantor, Gliwice, Poland); methanol, acetic acid 100% and Sabouraud agar (all from Merck, Darmstadt, Germany); fetal bovine serum (FBS; Pan Biotech, Aidenbach, Germany); Mueller–Hinton broth and Mueller–Hinton agar (BTL, Łódź, Poland); resazurin (Acros Organics, Geel, Belgium); ethanol (Honeywell, Charlotte, NC, USA); and 0.9% NaCl saline (Chempur, Piekary Śląskie, Poland). All other chemicals and biochemicals, including Folin–Ciocalteu reagent; gallic acid, catechin and other phenolic standards (rosmarinic, rutin, quercetin, syringic, ferulic, caffeic and p-coumaric acids); sodium carbonate, sodium nitrite, sodium hydroxide, aluminum chloride; DPPH (2,2-diphenyl-1-picrylhydrazyl); phosphate buffer; FeCl₃; TPTZ (2,4,6-tripyridyl-s-triazine); hydrochloric acid; acetic acid; Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid); Murashige and Skoog basal medium; 3,4-dihydroxy-L-phenylalanine (L-DOPA); kojic acid; elastase from porcine pancreas; TRIS-HCl buffer; N-succinyl-Ala-Ala-Ala-p-nitroanilide (SANA); epigallocatechin; Neutral Red dye (0.33% in DPBS); Dulbecco’s Modified Eagle Medium (DMEM); trypsin–EDTA; Dulbecco’s phosphate-buffered saline (DPBS); and Tween 80 were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Extraction Procedures

2.2.1. Ultrasound-Assisted Extraction for Antioxidant Assays, TPC, TFC, Tyrosinase Inhibition, Cytotoxicity (HaCaT) and Microbiological Activity

Dried microshoot biomass (1.0 g) was extracted with 100 mL of solvent (methanol or 70% ethanol, v/v) using ultrasound-assisted extraction for 20 min at room temperature in an ultrasonic bath (Sonic 3, Polsonic, Poland). The obtained extracts were filtered through filter paper (Whatman No. 1) and evaporated to dryness under reduced pressure. The resulting dry residues were weighed and re-dissolved in the appropriate solvent to obtain stock solutions of defined concentrations, which were used immediately for further analyses.

The selection of methanol and aqueous ethanol as extraction solvents was based on their widely reported effectiveness in the recovery of phenolic compounds and flavonoids from plant matrices. Phenolic acids and flavonoids are polar secondary metabolites, and numerous comparative studies have demonstrated that methanol and ethanol provide high extraction efficiency and significantly influence total phenolic content, total flavonoid content, and antioxidant activity of plant extracts [40,41,42]. Similar solvent-based extraction approaches have been applied in studies on C. citratus, confirming that methanolic and ethanolic extracts exhibit high antioxidant and biological activity [39].

Methanolic extracts were used for tyrosinase inhibition assays, cytotoxicity evaluation in HaCaT keratinocytes, microbiological tests, and for incorporation into the cosmetic oil-in-water (O/W) cream after complete solvent removal and reconstitution in propylene glycol (10% w/w). The glycolic extract was incorporated into the O/W cream formulation at a concentration of 2% (w/w), which falls within the range of extract concentrations previously reported for lemongrass-based cosmetic creams demonstrating acceptable physicochemical properties and retained biological activity [43]. Both methanolic and ethanolic extracts were employed for the determination of antioxidant activity (DPPH, FRAP), total phenolic content (TPC), and total flavonoid content (TFC).

2.2.2. Extraction for Chromatographic Profiling (LC-MS/MS)

The extraction procedure for chromatographic analysis of polyphenolic compounds was adapted from Bajkacz et al. [44]. Freeze-dried microshoot biomass (2.5 g) was extracted with 20 mL of methanol for 5 h at 900 rpm using a mechanical shaker. The extract was filtered, evaporated to dryness and reconstituted in 1 mL of methanol and 14 mL of acidified water (pH 3.5, adjusted with HCl). Solid-phase extraction (SPE) was carried out using Supelclean LC-18 cartridges (500 mg, 6 mL; Sigma-Aldrich, St. Louis, MO, USA) preconditioned with methanol and acidified water. The sample was loaded at a flow rate of 1 mL/min, dried under vacuum and eluted with 6 mL of methanol. The purified extract was subsequently used for LC–MS/MS qualitative profiling of phenolic compounds.

2.3. DPPH Radical Scavenging Activity of Extracts

The antioxidant activity of the extracts was determined using the DPPH radical assay, following the procedure described by Blois [45] with slight modifications. A 50 μL aliquot of extract or ethanol (blank) was added to 500 μL of 0.1 mM DPPH solution in ethanol. The mixture was vortexed and left to stand for 20 min at room temperature in the dark. The absorbance was then measured at 517 nm using a UV-1900i spectrophotometer (Shimadzu, Kyoto, Japan). The DPPH radical scavenging activity was calculated using the following formula:

DPPH scavenging activity (%) = (I₀ − I₁) × 100/I₀, where

I₀—signal intensity of the DPPH radical from the control

I₁—signal intensity of the DPPH radical from the sample

All measurements were performed in triplicate. The antioxidant activity was assessed for both the extracts and the reference compound (ascorbic acid) at an identical concentration of 100 µg/mL. The results are expressed as the mean percentage of DPPH inhibition.

2.4. Ferric Reducing Antioxidant Power (FRAP)

The reducing capacity of the extracts was assessed using the FRAP assay, according to the method of Benzie and Strain [46], with slight modifications. The working FRAP solution was freshly prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ dissolved in 40 mM HCl, and 20 mM FeCl₃·6 H₂O in a 10:1:1 ratio (v/v/v). A total of 1450 μL of FRAP reagent was combined with 50 μL of plant extract, and the mixture was incubated for 4 min at 37 °C. The absorbance was then measured at 593 nm using a UV-1900i spectrophotometer (Shimadzu, Kyoto, Japan). The antioxidant potential was expressed as µmol of Trolox equivalents per gram of dry weight (µmol TE/g DW), based on a Trolox standard calibration curve. All analyses were conducted in triplicate.

2.5. Total Polyphenol Content (TPC)

The total polyphenol content was determined using the Folin–Ciocalteu colorimetric method, based on the procedure of Agbor et al. [47]. A 20 μL aliquot of each extract was mixed with 1.58 mL of distilled water and 100 μL of Folin–Ciocalteu reagent. After 3 min, 300 μL of 20% sodium carbonate was added. The reaction mixture was incubated at room temperature for 30 min in the dark. Absorbance was measured at 766 nm using a UV-1900i spectrophotometer (Shimadzu, Kyoto, Japan). The polyphenol content was calculated using a calibration curve constructed with gallic acid and is expressed as mg gallic acid equivalents per gram of dry weight (mg GAE/g DW). All determinations were performed in triplicate.

2.6. Total Flavonoid Content (TFC)

The flavonoid content was quantified using a colorimetric method described by Kim et al. [5]. A 100 μL aliquot of the extract was mixed with 1.4 mL of distilled water, followed by the addition of 60 μL of 5% sodium nitrite solution and 60 μL of 10% aluminum chloride. After 5 min of incubation, 400 μL of 1 M sodium hydroxide was added to the mixture. The final solution was thoroughly mixed, and the absorbance was measured at 510 nm using a UV-1900 i spectrophotometer (Shimadzu, Kyoto, Japan). The results were calculated using a calibration curve prepared with catechin and are expressed as mg catechin equivalents per gram of dry weight (mg CE/g DW). Each sample was analyzed in triplicate.

2.7. LC–MS/MS Analysis

The qualitative analysis of phenolic compounds was performed using a Shimadzu UHPLC system coupled with a triple quadrupole mass spectrometer equipped with an electrospray ionization source. Chromatographic separation was carried out on a Shim-pack GIST-HP C18-AQ column (2.1 mm i.d. × 50 mm, 1.9 µm) maintained at 40 °C, with a flow rate of 0.3 mL/min and an injection volume of 3 μL. The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (acetonitrile). The gradient started at 95% A and 5% B for 3 min, followed by a linear increase of solvent B to 95% from 3 to 15 min. This composition was held until 17 min, after which the system returned to the initial conditions and was re-equilibrated until 20 min.

Mass spectrometric detection was conducted in both positive and negative ion modes. Data acquisition included monitoring of precursor ions and their fragmentation patterns. Identification of phenolic compounds was based on comparison of MS/MS spectra, retention behavior, and fragmentation patterns with published LC–ESI–MS data for C. citratus and related plant matrices [4].

2.8. Murine Tyrosinase Inhibitory Activity

The assay was developed based on literature reports describing the assessment of tyrosinase activity in cell-free extracts of B16 melanoma cells with protocol-specific modifications [20]. B16F10 cells were maintained in DMEM high-glucose medium supplemented with 10% FBS at 37 °C in a humidified atmosphere with 5% CO₂. Cells (8 × 10⁶) were lysed in 5 mL of 100 mM phosphate buffer (pH 6.8) containing 1% Triton X-100 using sonication in an ice-cold water bath for 1 h. After centrifugation (10 min, 13,000 rpm), the supernatant was collected as the murine tyrosinase solution, and the protein concentration was determined using a DC Protein Assay.

To determine tyrosinase inhibitory activity, aliquots of cell lysate containing 20 µg of protein were mixed with 20 µL of the extract solution or reference compound, 40 µL of 4 mM L-DOPA, and 100 mM phosphate buffer (pH 6.8) to a final volume of 200 µL. The reaction mixtures were incubated for 4 h at 37 °C, and absorbance was measured at 450 nm. Kojic acid was used as a positive control. The control sample, containing tyrosinase solution, L-DOPA, and phosphate buffer without extract, was set as 100% enzyme activity and used to calculate the percentage of tyrosinase inhibition. Each sample was analyzed in three independent replicates.

2.9. In Vitro Cytotoxicity

The cytotoxic potential of the extracts was assessed using the neutral red uptake (NRU) assay in HaCaT keratinocytes, following the protocol described by Repetto et al. [48]. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and maintained at 37 °C in a humidified 5% CO₂ atmosphere. For the experiment, 1 × 10⁴ cells per well were seeded into 96-well microplates. After 24 h of incubation, the culture medium was replaced with fresh medium containing the extract from C. citratus microshoot cultures in four concentrations: 200, 100, 50, and 25 µg/mL, prepared in 10% DMEM. Control wells received 10% DMEM without extract. Following 48 h of treatment, the medium was removed and cells were incubated for 2 h with 33 µg/mL neutral red prepared in DMEM supplemented with 1% FBS. After staining, cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) and lysed using acidified ethanol (50% ethanol, 1% acetic acid, 49% water, v/v/v). The absorbance of the released dye was measured at 540 nm using a FilterMax F5 microplate reader. The mean absorbance value of DMSO-treated control cells was defined as 100% viability, and the viabilities of extract-treated cells were calculated accordingly. Each experimental condition was performed in three independent experiments, each with five technical replicates (n = 15).

2.10. Evaluation of Cosmetic Formulation

2.10.1. Preparation of Facial Emulsion

A facial emulsion incorporating a glycolic extract of C. citratus obtained from the dried methanolic extract of microshoot cultures was formulated and compared with a corresponding control emulsion without the plant extract. Both emulsions were prepared according to the composition outlined in

Table 1. Composition of experimental and control facial emulsions (percent by weight).

Phase	Ingredient	Control Emulsion (%)	Emulsion with C. citratus Extract (%)
Oil Phase	Sweet almond oil	15.0	15.0
	Glyceryl monostearate SE	15.0	15.0
	Cetyl alcohol	5.0	5.0
Water phase	Distilled water	74.0	72.0
	Glycerin	1.0	1.0
	C. citratus extract	-	2.0

Glyceryl monostearate SE-self-emulsifying grade of glyceryl monostearate.

.

The emulsions were prepared using standard two-phase emulsification. The oil and water phases were heated separately to approximately 70 °C in a water bath under continuous magnetic stirring to ensure complete dissolution of all ingredients. The oil phase was homogenized using a high-speed overhead homogenizer (Ultra-Turrax T25 digital, IKA^®, Staufen, Germany) to ensure uniformity. Subsequently, the heated oil phase was gradually added to the aqueous phase under vigorous stirring (600 rpm). After full integration (approximately 15 min), the system was gradually cooled while maintaining agitation. Once the emulsion temperature dropped to 40 °C, the aqueous extract of C. citratus was incorporated into the experimental formulation. The prepared emulsions were transferred into tightly closed, sterile, chemically inert polypropylene containers (capacity: 50 mL), each filled with approximately 30 g of cream to minimize headspace. The samples were stored at room temperature (22 ± 2 °C) and protected from light until further analysis. No pH adjustment was required, as the measured values were within the skin-compatible range.

2.10.2. Rheological Examination of Emulsions and pH Measurement

The viscosity of the emulsions was determined using a rotational viscometer (ViscoQC 300, Anton Paar, Graz, Austria) equipped with an L4 spindle. Measurements were performed at 25 °C with a rotation speed of 20.0 rpm for 30 s. The viscosity values were recorded after stabilization of the sample. All measurements were carried out in triplicate.

The density of the emulsions was measured at 25 °C using a digital density meter DMA 501 (Anton Paar, Graz, Austria), based on the oscillating U-tube principle. Approximately 1 mL of each sample was introduced via syringe, and the measurements were automatically corrected for viscosity and temperature. The results were expressed in g/cm³ and represent the mean of three independent determinations. The pH of the emulsions was determined using a CP-411 pH meter (Elmetron, Zabrze, Poland).

2.10.3. Stability Assessment

Preliminary Stability Assay (Centrifugation Test)

The preliminary physical stability of the emulsions was evaluated using a centrifugation stress test, which is commonly applied as a rapid screening method to predict phase separation under accelerated conditions. For each formulation, three independent samples (n = 3) were prepared by weighing 5.0 g of cream into 15 mL conical polypropylene tubes. The samples were centrifuged in a 5430R centrifuge (Eppendorf, Hamburg, Germany) at 4000 rpm for 15 min at 22 °C. After centrifugation, the emulsions were visually inspected for signs of physical instability, including creaming, sedimentation, coalescence, or phase separation, as described in standard stability evaluation procedures for emulsions [49].

Freeze–Thaw Stability Test

To further assess the physical robustness of the emulsions under temperature stress, a freeze–thaw cycling test was performed according to commonly used cosmetic and pharmaceutical stability assessment approaches [50]. For each formulation, test portions were transferred into standard cosmetic polypropylene containers with a nominal capacity of 50 g and tightly closed. The samples were sequentially stored at −17 °C for 24 h, 22 °C for 24 h, and 40 °C for 24 h (laboratory incubator ST 25, POL-EKO Aparatura, Wodzisław Śląski, Poland). This temperature cycle was repeated three times. After each cycle, the emulsions were visually evaluated for changes in appearance, phase separation, and texture.

2.10.4. DPPH Radical Scavenging Activity of Emulsions

For the determination of antioxidant activity, 5.0 g of each emulsion was homogenized with 5.0 g of analytical grade methanol and subjected to sonication for 20 min. The mixtures were centrifuged at 6500 rpm for 20 min, and the supernatants were analyzed using the DPPH radical scavenging assay, as described in Section 2.3.

To assess the repeatability of antioxidant extraction from the emulsions, the homogenization–sonication–centrifugation procedure was performed in three independent extraction replicates (n = 3) for each formulation. The resulting methanolic extracts showed consistent DPPH radical scavenging values across replicates, indicating good reproducibility of the extraction procedure.

2.11. Evaluation of the Antimicrobial Activity of the Methanolic Extract

2.11.1. Test Microorganisms

The antimicrobial activity of the tested methanolic extract was evaluated against a panel of reference microorganisms, including the fungal strain Candida albicans ATCC 10231 and the bacterial strains Streptococcus mutans ATCC 25175, Staphylococcus epidermidis ATCC 8853, Streptococcus pyogenes ATCC 19615, Staphylococcus aureus 6538P, Staphylococcus epidermidis PCM 2118, Escherichia coli ATCC 8739, and Pseudomonas aeruginosa PAO1.

2.11.2. Minimal Inhibitory Concentration (MIC)

MIC values were determined using the broth microdilution method, adapted from Sarker et al. [51]. Overnight bacterial and fungal cultures were diluted 1:10 in Mueller–Hinton broth. The dried methanolic extract (24 mg/mL) was prepared by dissolving it in Mueller–Hinton broth supplemented with DMSO and 0.05% (v/v) Tween 80 to ensure complete solubilization of the plant material. The extract was dispensed into 96-well microplates followed by serial two-fold dilutions (0.375–12 mg/mL) in inoculated media. Plates were incubated aerobically at 35 °C for 18–20 h. Resazurin (0.1 µg/mL) was subsequently added to assess microbial viability. A blue color indicated growth inhibition, whereas a pink color indicated metabolic activity.

2.11.3. Minimum Bactericidal/Fungicidal Concentration (MBC/MFC)

To evaluate MBC/MFC, 100 µL aliquots were collected from the final three MIC wells lacking visible growth and spread onto Mueller-Hinton agar. Plates were incubated at 35 °C for 18–20 h. The lowest concentration yielding a ≥99.9% reduction in colony-forming units was recorded as MBC or MFC. Triplicates were performed for each condition.

2.12. Statistical Analysis

Statistical analyses were performed using Statistica 13. Data are presented as mean ± standard deviation (SD), unless stated otherwise. For comparisons between groups and across multiple concentrations, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was applied. A significance level of p ≤ 0.05 was considered statistically significant.

For phytochemical analyses and biological activity assays (TPC, TFC, DPPH, FRAP, tyrosinase inhibition, and cytotoxicity), n refers to independent biological replicates obtained from separate extractions performed on independently produced in vitro biomass. Emulsion density and viscosity measurements were performed as repeated measurements of the same emulsion sample (technical replicates) and were therefore not subjected to inferential statistical analysis.

3. Results and Discussion

3.1. Antioxidant Activity

Oxidative stress is a pathological condition resulting from an imbalance between the production of reactive oxygen species (ROS) and the antioxidant defense mechanisms, leading to cellular and tissue damage [52]. One of the fundamental reasons for the utilization of plant-based materials is their natural antioxidant potential, which is primarily attributed to the presence of phenolic and flavonoid compounds [53].

The antioxidant activity of C. citratus microshoot extracts was evaluated using DPPH and FRAP assays (

Table 2. DPPH assays of C. citratus extracts from microshoot cultures (extract and ascorbic acid concentrations: 100 µg/mL).

C. citratus Extract	DPPH % Inhibition ± SD
ME	66.66 ± 0.76 a
EE	43.50 ± 3.33 b
Ascorbic acid	84.48 ± 0.52 c

ME, methanolic extract; EE, ethanolic extract; DPPH, 2,2-diphenyl-1-picrylhydrazyl; SD, standard deviation. Mean values marked with different letters in columns differ statistically significantly (p ≤ 0.05), n = 3.

and

Table 3. FRAP assays of C. citratus extracts from microshoot cultures (extracts concentrations: 100 µg/mL).

C. citratus Extract	FRAP [µmol TE/g DW ± SD]
ME	13.26 ± 0.53 a
EE	7.29 ± 0.11 b

ME, methanolic extract; EE, ethanolic extract; FRAP, ferric reducing antioxidant potential; TE, Trolox equivalents; DW, dry weight; SD, standard deviation. Mean values marked with different letters in columns differ statistically significantly (p ≤ 0.05), n = 3.

). At the tested concentration (100 µg/mL), the methanolic extract demonstrated higher antioxidant activity than the ethanolic extract.

A comparison with published data allows contextualization of the activity levels measured for microshoot extracts. In a recent solvent-screening study, Aouadi et al. [54] reported DPPH IC₅₀ values between 21.65 and 24.65 µg/mL for methanolic preparations and 19.22–24.87 µg/mL for ethanolic extracts, with activity closely linked to flavonoid concentration. Falah et al. [55] demonstrated that 70% ethanolic leaf extracts reached a DPPH IC₅₀ of 79.44 mg/L, whereas extracts prepared with other ethanol concentrations produced weaker effects (89.64–38.95 mg/L), confirming the sensitivity of antioxidant capacity to extraction polarity and matrix composition. Costa et al. [11] reported DPPH scavenging in the range of 10.12–26.03% (depending on extract fraction) and FRAP values up to 922.43 μM Trolox/100 g DW, illustrating the reducing potential of polar C. citratus extracts.

Similarly, Soares et al. [56] showed that methanolic leaf extracts achieved 86.10% DPPH inhibition, substantially exceeding the activity of ethanolic extracts; the accompanying FRAP value was 1.45 mg AAE/g DW, again emphasizing solvent effects on phenolic recovery. Additional studies highlight the contribution of specific phenolic constituents to antioxidant behavior. Cheel et al. [9] demonstrated that purified flavonoids such as orientin and isoorientin exhibit potent DPPH-scavenging activity (IC₅₀ = 9–10 μM), while caffeic and chlorogenic acids showed IC₅₀ values of 54–68 μM, indicating that these compounds substantially influence extract activity. Adeyemo et al. [57] further reported DPPH inhibition of 60–70% in methanolic extracts, with increased activity in phenolic-enriched fractions, supporting the strong quantitative link between total phenolics and radical-scavenging performance. Evaluation of volatile and non-polar fractions by Aly et al. [58] revealed measurable but lower antioxidant efficiency (e.g., 10.18 ± 0.43 mg TE/g DPPH activity for essential oil and 6.86 ± 0.81 mg TE/g for n-hexane extract), demonstrating that antioxidant constituents are present across fractions of varying polarity, though with markedly different potencies.

The antioxidant activity of C. citratus microshoot extracts corresponds to the lower-to-moderate range reported for polar extracts of field-grown leaves. The clear solvent-dependent pattern observed in this study, with methanol outperforming ethanol, is consistent with trends widely described in the literature. Although microshoot-derived extracts show lower activity than optimized or phenolic-enriched preparations, their radical-scavenging and reducing capacities follow the characteristic antioxidant profile documented for C. citratus. These findings indicate that microshoot cultures are able to synthesize antioxidant metabolites typical of the species and that their activity reflects the phenolic composition inherent to in vitro-grown biomass.

3.2. TPC, TFC

As shown in

Table 4. TPC and TFC of C. citratus extracts prepared with methanol or ethanol from microshoot cultures.

C. citratus Extract	TPC [mg GAE/g DW ± SD]	TFC [mg CE/g DW ± SD]
ME	15.74 ± 0.18 ᵃ	5.98 ± 0.11 ᵃ
EE	13.26 ± 0.12 ᵇ	4.66 ± 0.09 ᵇ

ME, methanolic extract; EE, ethanolic extract; GAE, gallic acid equivalent; CE, catechin equivalent; SD, standard deviation; DW, dry weight. Mean values marked with different letters in columns differ statistically significantly (p ≤ 0.05), n = 3.

, both TPC and TFC were higher in the methanolic extract (ME) than in the ethanolic extract (EE) of C. citratus microshoot cultures. The ME contained 15.74 ± 0.18 mg GAE/g DW and 5.98 ± 0.11 mg CE/g DW, whereas the EE reached 13.26 ± 0.12 mg GAE/g DW and 4.66 ± 0.09 mg CE/g DW. These statistically significant differences (p ≤ 0.05) indicate superior extractability of phenolic acids and glycosylated flavonoids in methanol compared with 70% ethanol, which is in accordance with the higher polarity and hydrogen-bonding capacity of methanol.

Comparison with literature data for field-grown C. citratus shows that the TPC and TFC values obtained for microshoot extracts fall within the lower spectrum of reported totals, which is consistent with data indicating that phenolic levels in field material are typically higher. Guleira et al. [59] reported 26.1–32.1 mg GAE/g DW and 14.6–16.47 mg QE/g DW in aqueous extracts of fresh and dried leaves, indicating that phenolic totals in field material are often higher. Park et al. [36] showed that EtOAc and 80% MeOH extracts displayed markedly elevated totals (TPC 90.25–132.31 mg CAE/g extract; TFC > 104 mg NE/g extract), reflecting enrichment of polyphenols in mid- and high-polarity organic fractions. Although extraction conditions and units differ, the general pattern is consistent: organic solvents extract more phenolics than water, and field-grown leaves usually contain higher totals than in vitro tissues.

Additional reports strengthen this interpretation. Costa et al. [60] quantified phenolic acids and flavonoids in different polar extracts and confirmed that caffeic, p-coumaric, ferulic and quinic acid derivatives together with luteolin C-glycosides constitute the major extractable phenolic pool. Soares et al. [56] demonstrated that methanol and ethanol extract more phenolics than water, while Adeyemo et al. [57] showed that drying increases total phenolic accumulation. These findings demonstrate that TPC and TFC vary strongly with solvent, plant material and processing method, which explains the wide ranges reported for field-grown plants.

Taken together, the phenolic and flavonoid levels detected in microshoot cultures are lower than those typically reported for field-grown leaves; however, the overall extraction patterns and qualitative phenolic composition remain comparable. The reduced levels are consistent with the controlled, stress-free conditions of in vitro culture, which lack the environmental stimuli known to promote secondary metabolite accumulation in plants [1].

From an application-oriented perspective, this further illustrates a practical trade-off between absolute phenolic yield and reproducibility. While field-grown C. citratus generally exhibits higher and more variable TPC and TFC values, in vitro microshoot cultures provide a controlled, season-independent biomass source with stable and reproducible phytochemical profiles. Such reproducibility is particularly advantageous for cosmetic applications, where batch-to-batch consistency, safety, and standardization of raw materials are critical.

3.3. Phenolic Profile of C. citratus Microshoot Extracts

The qualitative phenolic profile of C. citratus microshoot extracts determined by LC–MS/MS is presented in

Table 5. Phenolic compounds in C. citratus microshoot extract determined by LC–MS/MS.

Phenolic Compound	Content [mg/kg DW]
Epicatechin	0.020
Catechin	0.051
Myricetin	0.161
Rutin	0.035
Apigenin	0.044
Luteolin	0.392
Vanillic acid	2.325
p-Coumaric acid	18.628
Isorhamnetin	0.019
trans-Ferulic acid	9.395
Caffeic acid	8.863
Kaempferol	0.158
Protocatechuic acid	0.612
Syringic acid	0.498
4-Hydroxybenzoic acid	1.070
Rosmarinic acid	0.101

. The phenolic profile of C. citratus microshoot extracts corresponded to the major phenolic groups typically described for field-grown lemongrass, including hydroxycinnamic acids (caffeic, p-coumaric, ferulic), hydroxybenzoic acids (syringic, vanillic, protocatechuic) and flavonoids such as rutin, kaempferol, luteolin and apigenin [4,8,24,60,61,62]. The detection of these same groups in microshoots demonstrates that the core phenylpropanoid pathway remains active under in vitro conditions.

Qualitative LC–MS/MS profiling revealed that hydroxycinnamic acids constituted the predominant phenolic class in the microshoot extracts. In particular, p-coumaric, ferulic, and caffeic acids showed the highest relative abundance, whereas flavonoids such as luteolin, kaempferol, rutin, and apigenin were detected with comparatively lower abundance. This qualitative distribution pattern is consistent with previous MS-based profiling studies of C. citratus tissues and extracts. In comparison with aqueous preparations described in the literature, microshoot extracts exhibited a lower relative abundance of phenolic acids. For example, decoctions prepared from field-grown C. citratus leaves have been reported to contain syringic and caffeic acids at relatively high abundance, whereas microshoot cultures showed reduced accumulation of these compounds. Nevertheless, LC–MS/MS analysis confirmed the presence of hydroxycinnamic acids (p-coumaric, caffeic, and ferulic acids), hydroxybenzoic acids (protocatechuic, vanillic, and 4-hydroxybenzoic acids), and flavonoids including luteolin, apigenin, kaempferol, and rutin. These compounds are characteristic constituents of C. citratus and have been consistently reported in MS-based phenolic profiling studies [4,8,62]. Although the overall phenolic abundance in microshoot extracts was lower than that reported for methanolic extracts of field-grown leaves, the relative proportions of individual phenolic groups remained comparable. This observation is expected, as organic-solvent extractions of mature plant tissues typically yield higher amounts of phenolic metabolites than mild maceration procedures applied to in vitro-derived biomass [60,61].

A notable feature of the microshoot extracts was the presence of luteolin and apigenin predominantly as aglycones, whereas field-grown plants typically accumulate these flavonoids mainly as glycosides. Reduced glycosylation is a well-recognized characteristic of in vitro cultures, which is commonly attributed to limited tissue differentiation and attenuated environmental signaling.

Despite the lower overall phenolic abundance, the microshoots reproduced all major phenolic groups characteristic of C. citratus. Reduced metabolite accumulation is consistent with the physiology of in vitro cultures, which lack environmental elicitors such as UV radiation, mechanical damage, or pathogen exposure that normally stimulate phenylpropanoid biosynthesis in mature plants. Nevertheless, preservation of the complete qualitative phenolic fingerprint indicates that microshoot cultures constitute a chemically representative and biologically relevant model for studies on phenolic biosynthesis, elicitation, and metabolic modulation in C. citratus.

3.4. Tyrosinase Inhibitory Activity

Tyrosinase is a key oxidoreductase that catalyzes two distinct reactions in melanogenesis: the hydroxylation of monophenols and the oxidation of o-diphenols to o-quinones, ultimately leading to melanin synthesis. The inhibition of this enzyme plays a crucial role in dermatological and cosmetic applications, particularly in treating hyperpigmentation disorders and in developing skin-lightening formulations. Natural inhibitors of tyrosinase are increasingly explored due to their lower toxicity compared to synthetic compounds such as hydroquinone and kojic acid [63].

In this study, methanolic extract from microshoot cultures was evaluated using murine tyrosinase derived from B16F10 melanoma cell lysate at three concentrations (0.25, 0.5, and 1.0 mg/mL),

Table 6. Tyrosinase inhibition ability of methanolic extracts from C. citratus.

Enzyme Activity [% ±SD]
Concentration [mg/mL]	0.25	0.5	1.0
C. citratus microshoot extract	69.64% ± 3.81	42.94% ± 3.68	34.87% ± 2.71
Kojic acid	35.98% ± 3.75	28.58% ± 4.67	24.57% ± 5.39

. A clear concentration-dependent decrease in tyrosinase activity was observed, with the enzyme retaining only 34.87 ± 1.71% of its activity at 1.0 mg/mL. At this concentration, the extract produced approximately 65% inhibition, indicating a biologically relevant anti-melanogenic effect under the applied conditions. Comparable tyrosinase inhibitory effects of C. citratus have also been reported in the literature, although most studies employed mushroom tyrosinase or alternative enzyme models. Kim et al. [36] demonstrated that the butanol extract of lemongrass inhibited monophenolase activity by 36.92% and the hexane extract inhibited diphenolase by 25.16% at 100 μg/mL, while arbutin (positive control) achieved 33.03% and 23.50% inhibition at the same concentration. Masuda et al. [63] identified geranic acid as a potent tyrosinase inhibitor in the ethyl acetate fraction of C. citratus, with IC₅₀ values of 0.14 mM (trans-isomer) and 2.3 mM (cis-isomer). These findings indicate that monoterpenes such as geranic acid and citral, abundant in lemongrass essential oil, contribute significantly to its enzyme-inhibitory potential.

It should be emphasized, however, that the present study addresses a different chemical fraction of C. citratus. While tyrosinase inhibition reported for lemongrass essential oil has been primarily attributed to volatile monoterpenes, the activity observed in this work arises from a polar extract obtained from in vitro microshoot cultures. Therefore, the observed inhibition should not be interpreted as directly comparable to essential oil–mediated effects, but rather as complementary evidence that non-volatile metabolites of C. citratus are also capable of modulating tyrosinase activity.

Beyond essential-oil constituents, the inhibitory activity observed for the methanolic extract may also be influenced by the presence of phenolic compounds, which are known to interact with tyrosinase through multiple mechanisms, including redox modulation and metal-binding effects, which are relevant for both plant-derived and mammalian tyrosinase systems. Evidence from other plant systems indicates that phenolic constituents can contribute to tyrosinase inhibition even when present in complex extract matrices. For example, a polyphenol-rich cocoa pod extract containing phenolic acids and flavonoids showed measurable inhibitory activity in vitro [64]. Consistent with the role of phenolic structures in modulating melanogenic enzymes, Njikam et al. [19] demonstrated that phenolic compounds isolated from Psorospermum aurantiacum exhibited marked tyrosinase-inhibitory activity in vitro, confirming that non-volatile polyphenols can effectively suppress enzymatic oxidation of L-DOPA.

The inhibition levels recorded in this study are within the range reported for various botanical extracts tested under comparable conditions, indicating that C. citratus microshoot cultures are capable of producing metabolites with relevant enzyme-modulating properties. Despite this, direct comparison across studies remains challenging due to substantial methodological differences, particularly in extraction procedures, sample preparation, substrate selection, and assay parameters. Harmonization of analytical protocols would greatly improve the reliability of cross-study evaluation and support the development of reproducible C. citratus-based cosmetic formulations.

3.5. In Vitro Cytotoxicity

Evaluating the cytotoxic potential of plant-derived extracts is essential for determining their suitability for dermatological or cosmetic applications. In the present study, the methanolic extract obtained from C. citratus microshoot cultures did not induce cytotoxicity in HaCaT keratinocytes within the tested concentration range of 25–200 µg/mL (

Table 7. Viability of HaCaT keratinocytes after exposure to C. citratus microshoot extract.

Extract Concentration [µg/mL]	Cell Viability [% ± SD]
200	88.81 ± 2.45
100	92.65 ± 3.20
50	93.36 ± 2.90
25	112.06 ± 9.80

SD, standard deviation.

), demonstrating good cellular tolerance under the applied experimental conditions. Absorbance values obtained for extract-treated cells were normalized to the mean absorbance of the DMSO control and expressed as percentages. Absorbance values were also recorded for untreated cells cultured in complete medium without the addition of solvent or extract. These wells served to confirm baseline cell viability and proper culture conditions.

Published studies on C. citratus extracts support a similar safety profile for polar leaf preparations. Fitria et al. [65] investigated the cytotoxicity and cytoprotective activity of a 70% ethanol extract of lemongrass leaves using fibroblasts exposed to H₂O₂-induced oxidative stress. At 10 ppm, the extract maintained cell viability above 80% and reduced intracellular ROS levels, with effects comparable to those observed for ascorbic acid. Higher concentrations (20–50 ppm) did not markedly increase cell viability, suggesting a protective effect at low doses and a plateau of activity at higher ones. Supandi et al. [66] similarly reported that lemongrass leaf extract preserved the viability of BHK-21 fibroblasts, supporting its biocompatibility in mammalian cell models. Jiang et al. [67] demonstrated that lemongrass essential oil reduced oxidative damage and DNA lesions in human embryonic lung fibroblasts, with the strongest protective effect observed at the lowest tested concentrations.

In contrast to polar leaf extracts, the essential oil of C. citratus displays a different toxicity profile. Weshahi et al. [68] assessed the toxicity of an Omani lemongrass oil using the Artemia salina lethality assay and reported mortality rates increasing from 10% to 100% across the concentration range of 125–1000 µg/mL, with an LC₅₀ value of 194.63 µg/mL. The authors attributed this cytotoxicity mainly to the high content of citral and β-citral. These findings indicate that while appropriately diluted essential oil may exhibit antioxidant and protective effects, concentrated preparations can be toxic and must be used with caution in formulations intended for topical application.

Taken together, the available evidence shows that polar extracts of C. citratus-including the methanolic extract produced in this study-demonstrate low cytotoxicity and may even exert cytoprotective effects in oxidative stress models, whereas the essential oil requires careful dose control due to its higher intrinsic cytotoxicity at elevated concentrations.

3.6. Evaluation of Cosmetic Formulation

3.6.1. Rheological Examination of Emulsions and pH Measurement

The emulsions exhibited acidic pH values of 5.4 for the formulation containing C. citratus extract and 5.6 for the control. Representative macroscopic images of the emulsions after preparation are shown in Figure S1 (Supplementary Materials). Both values are consistent with the recommended 4–6 range for facial formulations, which supports skin-barrier compatibility and microbiome stability [69]. The results of density and viscosity measurements are summarized in

Table 8. Comparison of density and viscosity parameters for control emulsion and formulation enriched with C. citratus extract.

Emulsion	Density [g/cm3]	Viscosity [mPa·s]
Emulsion with C. citratus	0.7931	27,600
Control emulsion	0.7413	26,850

. Three repeated readings were obtained from the same emulsion sample, and a representative value is presented. The oscillating U-tube method reports the bulk density of the tested sample, which in the case of semi-solid emulsions may be influenced by internal structure and entrapped air rather than reflecting the density of the aqueous phase alone. O/W cosmetic emulsions are generally reported to exhibit densities close to that of water, depending on oil-phase content; however, in semi-solid cream formulations, the measured density may be influenced by formulation structure and air incorporation [49]. Regarding rheology, both emulsions exhibited viscosities on the order of 10³–10⁵ mPa·s, which is the same order of magnitude reported for stable topical emulsions in experimental studies. The small difference observed between the extract-containing and control emulsions did not suggest a pronounced change in macroscopic physicochemical behavior under the tested conditions [70]. Density and viscosity measurements were included to provide descriptive physicochemical characterization of the emulsions and were not intended for inferential comparison.

3.6.2. Stability Assessment

The stability evaluation demonstrated that both the emulsion containing the C. citratus extract and the corresponding control formulation remained stable under the applied test conditions, with no evidence of phase separation. During the temperature cycling test, the control emulsion without extracts also retained homogeneity.

3.6.3. DPPH Radical Scavenging Activity of Emulsions

The incorporation of C. citratus extract into the emulsion markedly increased its radical-scavenging capacity. The extract-containing cream showed 18.58 ± 0.17% inhibition in the DPPH assay, whereas the base emulsion reached only 2.47 ± 0.08%, confirming that the lemongrass extract contributed substantially to the antioxidant activity of the final formulation (

Table 9. DPPH radical-scavenging activity of emulsions containing C. citratus microshoot extract and control formulation.

Emulsion	% Inhibition ± SD
Emulsion with C. citratus	18.58 ± 0.17 a
Control emulsion	2.47 ± 0.08 b

SD, standard deviation. Mean values marked with different letters in columns differ statistically significantly (p ≤ 0.05), n = 3.

).

Similar behavior has been reported for other cosmetic emulsions containing plant-derived antioxidants. In the study by Sykuła et al. [32], O/W creams with commercial green tea extracts were compared with an emulsion without extract using ABTS and DPPH assays. After ethanol extraction of the creams, formulations enriched with green tea showed 59.65 ± 18.67% to 77.43 ± 5.64% antioxidant activity in the ABTS assay and 19.53 ± 3.39% to 30.82 ± 1.36% in the DPPH assay, whereas the emulsion without green tea extract reached only 12.69 ± 1.45% (ABTS) and 13.72 ± 1.45% (DPPH).

A pronounced extract-dependent effect was also observed in an O/W cream containing Euphorbia hirta herb extract. In that study, the base cream without extract showed negative DPPH scavenging values (e.g., 67.09 ± 3.21% at 100 µg/mL), while the cream with 10% E. hirta extract reached 21.65 ± 0.88% inhibition at 25 µg/mL, 48.28 ± 0.13% at 50 µg/mL and 87.89 ± 0.53% at 100 µg/mL [71].

In a rice-bran cream model, Suhery et al. [72] formulated an O/W base cream and two creams with 3.1% rice bran extract from different white-rice varieties. The base cream showed only 31.225% DPPH inhibition in the first week and 30.101% after eight weeks, while the cream with Kalpatali rice bran exhibited 87.231% and 86.032%, respectively, and the cream with Rice-64 bran showed 64.085% and 54.603% inhibition. A comparison cream containing vitamin E reached 49.016% inhibition.

Concentration-dependent enhancement of antioxidant activity was also demonstrated for O/W sunscreen creams with Calendula officinalis oil. In the work of Mishra et al. [73], diluted creams containing 1–5% calendula oil showed DPPH radical-scavenging activities of 29%, 43%, 61%, 75% and 86%, respectively, whereas the blank sample reached only 5%, and a marketed sunscreen cream exhibited 69% inhibition.

Taken together, these studies show that emulsions enriched with botanical extracts commonly achieve DPPH inhibition in the 20–90% range, with much lower values for the corresponding base formulations or blank samples. Against this background, the increase from 2.47 ± 0.08% to 18.58 ± 0.17% observed for the C. citratus cream represents a clear but moderate enhancement of antioxidant capacity. The lower magnitude compared with some highly active formulations (e.g., rice bran or Euphorbia hirta creams above 60–80% inhibition) may reflect differences in extract concentration, composition, and the specific assay conditions used for the creams.

3.7. Antimicrobial Activity

As shown in

Table 10. MIC and MBC/MFC values of extract in mg/mL.

Test Microorganism	MIC	MBC/MFC
Candida albicans ATCC10231	1.5	12
Streptococcus mutans ATTC 25175	0.375	12
Staphylococcus epidermidis ATTC 8853	0.375	12
Streptococcus pyogenes ATTC 19615	0.375	6
Staphylococcus aureus 6538P	>12	>12
Staphylococcus epidermidis PCM 2118	0.375	6
Escherichia coli ATCC 8739	>12	>12
Pseudomonas aeruginosa PAO1	>12	>12

MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; MFC, minimum fungicidal concentration.

, the methanolic extract obtained from C. citratus microshoot cultures exhibited a selective antimicrobial profile, with clear inhibition of Streptococcus mutans, Streptococcus pyogenes and Staphylococcus epidermidis (MIC 0.375 mg/mL; MBC 6–12 mg/mL), moderate antifungal activity against Candida albicans (MIC 1.5 mg/mL; MFC 12 mg/mL), and a lack of activity against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa up to 12 mg/mL. This pattern, characterized by high susceptibility of Gram-positive cocci and markedly reduced sensitivity of Gram-negative rods, is consistent with numerous reports on C. citratus extracts obtained from field-grown plants.

A direct comparison is possible with the methanolic leaf extract studied by Zulfa et al. [38], who reported MIC values in the low mg/mL range (approximately 0.08–0.63 mg/mL) against Bacillus cereus, Staphylococcus aureus, Klebsiella pneumoniae, E. coli O157:H7 and C. albicans, with corresponding MBC/MFC values ranging from 1.25 to 2.50 mg/mL. In their study, C. albicans was inhibited at 0.16 mg/mL and killed at 1.25 mg/mL, whereas S. aureus showed a MIC of 0.31 mg/mL. In comparison, the microshoot-derived extract investigated here required a higher concentration to inhibit C. albicans (1.5 mg/mL) and did not inhibit S. aureus within the tested range, but showed very low MIC values for Streptococcus spp. and S. epidermidis (0.375 mg/mL). Thus, although the most susceptible species differ between the two studies, both extracts display low-mg/mL activity against Gram-positive bacteria and yeasts, confirming that C. citratus derived methanolic preparations can provide potent antimicrobial effects.

The predominance of Gram-positive susceptibility is also supported by the work of Nyamath et al. [74], who evaluated aqueous, ethanolic and methanolic extracts of fresh and dried C. citratus leaves using an agar well diffusion assay. In their study, the largest inhibition zones were consistently recorded for S. aureus, while E. coli and P. aeruginosa produced much smaller zones, particularly at lower extract concentrations. Although agar diffusion data cannot be directly compared with MIC values obtained by broth microdilution, the relative pattern is similar: Gram-positive cocci are more responsive to lemongrass extracts than Gram-negative rods. The complete lack of susceptibility of E. coli and P. aeruginosa to the microshoot extract up to 12 mg/mL is therefore in line with the reduced inhibition of these species described for leaf extracts.

Further insight is provided by the study of Ajijolakewu et al. [75], who investigated aqueous and ethanolic extracts of field-grown C. citratus leaves against clinical isolates of S. aureus, E. faecalis, P. aeruginosa, E. coli and C. albicans. Their aqueous extract inhibited S. aureus at 2 mg/mL, E. faecalis at 6.25 mg/mL, P. aeruginosa at 12.5 mg/mL, E. coli at 25 mg/mL and C. albicans at 50 mg/mL, whereas the ethanolic extract was active only against S. aureus (150 mg/mL) and P. aeruginosa (12.5 mg/mL), and no bactericidal endpoints were observed for any strain. In comparison, the microshoot-derived methanolic extract demonstrated substantially lower MIC values for Gram-positive cocci (0.375 mg/mL) and for C. albicans (1.5 mg/mL), and, importantly, achieved bactericidal or fungicidal effects at 6–12 mg/mL. These differences highlight the impact of extraction solvent and plant material on antimicrobial potency and indicate that microshoot cultures can yield extracts that match or exceed the inhibitory activity of some aqueous leaf extracts from field-grown plants.

The antifungal component of the present results also fits within the broader context of lemongrass-derived antifungals. Alsudani et al. [76] demonstrated that aqueous and alcoholic extracts of C. citratus leaves markedly inhibited the radial growth of storage fungi such as Aspergillus niger, Fusarium graminearum and Rhizopus stolonifer, reaching 75–87 m% growth reduction at concentrations of 30–40 mg/mL and showing an effect comparable to that of a commercial fungicide. These values are higher than the MIC and MFC determined here for C. albicans, but the experimental systems and target fungi are different. When considered together, both studies indicate that C. citratus extracts can exert fungistatic and fungicidal effects against diverse fungal species, including human-associated yeasts and plant pathogens, with effective concentrations typically in the low- to mid-mg/mL range.

Recent work on C. citratus extracts has further supported a strong link between phenolic composition and antimicrobial activity. Tazi et al. [77] investigated aqueous, ethanolic and methanolic extracts of cultivated Moroccan lemongrass and showed that fractions with higher total phenolic and flavonoid contents exhibited stronger antibacterial effects, particularly against E. coli, again with MIC values in the low-mg/mL range. Likewise, Boeira et al. [25] evaluated C. citratus extract as a natural antioxidant and antimicrobial additive in fresh sausage, demonstrating that the extract reduced microbial growth in a complex food matrix. Although these studies used field-grown material and different test systems, they support the concept that phenolic-rich lemongrass extracts consistently display measurable antimicrobial activity and can be incorporated into applied formulations.

Comparable findings have also been reported for closely related Cymbopogon species. Mokhtar et al. [78] examined a methanolic extract of C. schoenanthus and found MIC values of 12.5 mg/mL for S. aureus and 25 mg/mL for B. cereus, K. pneumoniae and E. coli, with MBC values ranging from 25 to 50 mg/mL. These values are approximately one order of magnitude higher than those obtained here for Streptococcus spp. and S. epidermidis (0.375 mg/mL), again emphasizing that the microshoot derived extract of C. citratus is positioned at the more active end of the spectrum reported for methanolic extracts against Gram-positive bacteria.

Mechanistic studies suggest that the antimicrobial effects of C. citratus extracts are mediated by phenolic acids, tannins and flavonoids, which can disrupt cell membranes, increase permeability and interfere with oxidative balance and essential metabolic pathways. Zulfa et al. [38] linked the antibacterial and antifungal effects of their methanolic leaf extract to the presence of phenolic and flavonoid compounds.

Taken together, the antimicrobial behavior of the microshoot-derived methanolic extract is consistent with the qualitative susceptibility patterns described for C. citratus extracts obtained from field-grown plants: higher susceptibility of Gram-positive cocci, variable but often moderate susceptibility of yeasts, and reduced responsiveness of Gram-negative rods. Quantitative differences in MIC, MBC and MFC values between microshoot-based and leaf-based extracts are expected due to differences in plant material, extraction protocol and test strains; however, the common trends across independent studies indicate that C. citratus microshoot cultures constitute a reproducible and microbiologically relevant source of bioactive metabolites. In the context of dermocosmetic applications, the strong inhibition of S. mutans, S. pyogenes and S. epidermidis, combined with measurable activity against C. albicans, supports the potential utility of such extracts as functional ingredients aimed at modulating selected skin and oral associated microbial communities.

3.8. Strengths and Limitations of the Study

This study has several strengths as well as some limitations that should be acknowledged. A major strength is the integrated approach combining phytochemical characterization of Cymbopogon citratus in vitro microshoot extracts with the evaluation of their antioxidant, antimicrobial, and enzyme-inhibitory activities, followed by verification of antioxidant retention after incorporation into a model oil-in-water cosmetic emulsion. The use of in vitro cultures ensured controlled and reproducible biomass quality, which is particularly important for cosmetic raw material development. However, the biological activity was assessed at the extract level, and no direct biological testing of the finished cosmetic formulation was performed. In addition, the stability evaluation focused on short-term physical stability, while long-term storage stability was beyond the scope of the present work. Finally, the absence of elicitation strategies in the in vitro culture system may have contributed to lower phenolic levels compared to field-grown plants. These limitations indicate directions for future research, including elicitor-assisted culture optimization, extended stability studies, and formulation-level biological evaluation.

4. Conclusions

The present work demonstrates that microshoot cultures of C. citratus constitute a reproducible source of plant biomass suitable for cosmetic applications. The extracts obtained from microshoot cultures contained detectable phenolic acids and flavonoids, as revealed by LC–MS/MS profiling, and their antioxidant properties were confirmed using DPPH and FRAP assays. The methanolic extract inhibited murine tyrosinase activity in a concentration-dependent manner and exhibited selective antimicrobial activity, particularly against Streptococcus mutans, Streptococcus pyogenes and Staphylococcus epidermidis, together with fungistatic and fungicidal effects against Candida albicans. Importantly, the extract showed no cytotoxicity toward HaCaT keratinocytes within the tested concentration range, supporting its dermal safety.

The incorporation of the extract into an oil-in-water emulsion did not adversely affect pH, viscosity, density or short-term physicochemical stability, and the antioxidant activity of the extract was retained after formulation. These results indicate that C. citratus microshoot-derived extracts combine favorable biological activity with formulation compatibility, supporting their potential use as functional ingredients in cosmetic products. Further studies may include long-term stability testing, in-depth skin cell-based mechanistic assays, and optimization strategies aimed at enhancing metabolite accumulation in microshoot cultures.