In vitro study of the antihemolytic and antioxidant potential of two essential oils from Salvia officinalis L. and Curcuma longa L. against glucantime® toxicity

Amina Tabet Zatla, Amina Hammoudi, Mamoun Fellah, Dunya Mohammed Zeki, Joelle Pérard-Viret, Gamal A. El-Hiti

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To cite this version:

Amina Tabet Zatla, Amina Hammoudi, Mamoun Fellah, Dunya Mohammed Zeki, Joelle Pérard-Viret, et al. In vitro study of the antihemolytic and antioxidant potential of two essential oils from Salvia officinalis L. and Curcuma longa L. against glucantime® toxicity. Journal of engineering research = Mağallať al-abḥāt al-handasiyyať, 2024, 24, 10.1016/j.jer.2024.12.007. hal-04976616

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# *In vitro* study of the antihemolytic and antioxidant potential of two essential oils from *Salvia officinalis* L. and *Curcuma longa* L. against glucantime® toxicity

Amina Tabet Zatla<sup>a</sup>, Amina Hammoudi<sup>a</sup>, Mamoun Fellah<sup>b,*</sup>, Dunya Zeki Mohammed<sup>c</sup>, Joëlle Pérard<sup>d</sup>, Gamal A. El-Hiti<sup>e</sup>

## ARTICLE INFO

**Keywords:**

Glucantime

Hemolysis

Essential oil

*Salvia officinalis* L.

*Curcuma longa* L.

Red blood cells

Antioxidants

## ABSTRACT

Our study is a strategy aimed at improving the therapeutic index of the molecule by reducing its toxicity while maintaining its activity. To this end, we will investigate the use of *Salvia officinalis* L. and *Curcuma longa* L. essential oils, which are rich in molecules with potential therapeutic activities. These oils will be tested and evaluated *in vitro* on the red blood cells by measuring three biological parameters, including intracellular LDH concentration, potassium concentration, and extra cellular hemoglobin level, to assess their antihemolytic activity. Additionally, we will examine markers of oxidative stress, including malondialdehyde, glutathione, catalase, and protein carbonylation, to determine their antioxidant activity. *Curcuma longa* L. and *Salvia officinalis* L. essential oils have been demonstrated to decrease the destruction of red blood cells significantly. Thereby protecting erythrocytes from hemolysis induced by glucantime. Specifically, the study observed a progressive decrease in intracellular lactate dehydrogenase (LDH) concentrations, with levels dropping from 77 U/L to 44 U/L in suspensions with *Salvia officinalis* L. and from 89 U/L to 47 U/L for *Curcuma longa* L. over time. However, these decreases were not as pronounced as the reduction seen with glucantime alone, which lowered LDH levels from 67 U/L to 21 U/L. Moreover, glucantime treatment led to a significant decrease in intracellular potassium concentration, falling from 2.03 mmol/L to 1.38 mmol/L. In contrast, the presence of the essential oils diminished this effect; potassium levels decreased to 2.1 mmol/L with *Curcuma longa* L. and to 1.69 mmol/L with *Salvia officinalis* L. Furthermore, hemoglobin was detected at a low initial concentration of 0.0021 g/L in suspensions containing glucantime, which escalated to 0.201 g/L by the end of the observation period. Significantly, the hemoglobin levels were lower when the essential oils were present. Additionally, the results indicated that *Curcuma longa* L. essential oil possesses a relatively greater antihemolytic capacity compared to that of *Salvia officinalis* L. Both essential oils demonstrated antioxidant properties by mitigating oxidative stress effects caused by glucantime, as evidenced by reductions in the levels of catalase, glutathione (GSH), malondialdehyde, and carbonylated proteins. Our results are suggestive that *Salvia officinalis* L. and *Curcuma longa* L. essential oils can protect erythrocytes against glucantime-induced hemolytic phenomena. This effect is due to these essential oils' antioxidant activities.

## Introduction

Leishmaniasis comprises a range of disorders caused by protozoan parasites belonging to the genus *Leishmania*. These conditions appear in

three main forms: visceral, mucocutaneous, and cutaneous leishmaniasis [1]. Visceral leishmaniasis is the most severe and potentially fatal if not properly treated [2]. In contrast, cutaneous and mucocutaneous leishmaniasis are more common and often result in significant social

<footnote><sup>a</sup> Laboratoire de Chimie Organique, Substances Naturelles et Analyses (COSNA), Université Abou Bekr Belkaid, BP 119, 13000, Algeria</footnote>

<footnote><sup>b</sup> Mechanical Engineering Department, Abbes Laghrour-University, Khenchela, PO 1252, 40004, Algeria</footnote>

<footnote><sup>c</sup> College of Engineering Gilgamesh University, Baghdad 10022, Iraq</footnote>

<footnote><sup>d</sup> University of Paris Descartes, Faculty of Pharmacy, CNRS (UMR 8038), Paris, France</footnote>

<footnote><sup>e</sup> Department of Optometry, College of Applied Medical Sciences, King Saud University, Riyadh 11433, Saudi Arabia</footnote>

<footnote>* Corresponding author.
E-mail address: mamoune.fellah@univ-khenchela.dz (M. Fellah).</footnote>
stigma due to severe skin and mucosal lesions. Leishmaniasis remains a major health problem in more than 80 countries, particularly in tropical and subtropical areas, contributing to high levels of illness and death [3].

In Algeria, the incidence of cutaneous leishmaniasis (CL) is the second highest globally after Afghanistan. This disease has been steadily increasing in the western region of the country for several years, as reported by the Regional Health Observatory of Oran (ORS). A recent report shows that the prevalence of this disease increased from 0.6 cases per 100,000 people in 2014–2.8 cases per 100,000 people in 2019. Infection primarily affects individuals under the age of 14, with a higher prevalence observed in males [4].

Pentavalent antimony derivatives remain the most effective therapeutic approach for leishmaniasis. Glucantime® (meglumine antimoniate) (Fig. 1) is currently the recommended first-line treatment in line with the World Health Organization (WHO) [5,6]. However, these medications are considered outdated, require intravenous or intramuscular delivery, and are associated with adverse effects due to their high toxicity, such as fatigue, headache, jaundice, liver and kidney problems, especially hemolytic anemia [7,8].

Medicinal and aromatic plants are increasingly recognized as a significant reservoir of new molecules with therapeutic potential [9]. Approximately 40 % of all drugs utilized are obtained, whether directly or indirectly, from natural sources, the majority of which are derived from plants [10]. These plants and their essential oils are promising candidates for complementary and alternative treatments due to their antimicrobial properties, potential anesthetic effects, immunostimulatory characteristics, and antioxidant potential. Among these, *Curcuma longa* L. and *Salvia officinalis* L. hold notable significance [11].

Medicinal and aromatic plants, including *Curcuma longa* L. (turmeric) and *Salvia officinalis* L. (sage), have been recognized for their diverse therapeutic benefits, particularly in the fields of inflammation, antimicrobial activity, and antioxidant properties. *Curcuma longa* is well-known for its active compound, curcumin, which has anti-inflammatory, neuroprotective, and antioxidant effects [12–20]. Similarly, *Salvia officinalis* has a long history of use in traditional medicine for its antimicrobial, antitumor [21], and antioxidant effects, making it a promising candidate for various therapeutic applications [22–28]. It is traditionally used for treating bronchitis, colds, gastrointestinal disorders, tuberculosis, and inflammation [23–27].

The extraction process plays a crucial role in determining the bioactive potential of medicinal and aromatic plants. Different methods of extraction, such as steam distillation, solvent extraction, and cold pressing, can influence the yield, composition, and potency of essential oils. The efficiency of these methods depends on factors such as temperature, time, and solvent polarity, which in turn affect the chemical profile and biological activity of the extract. For instance, essential oils obtained through steam distillation tend to retain a higher concentration of volatile compounds, which are often responsible for the plant's antimicrobial, anti-inflammatory, and antioxidant activities. The quality of the extract can thus directly impact its therapeutic potential, making it essential to optimize extraction techniques to preserve the desired properties of the plant material. This aspect is particularly important when exploring the potential of essential oils from *Curcuma longa* L. and

*Salvia officinalis* L. to mitigate the toxic effects of meglumine antimoniate (Glucantime®). In this study, the extraction methods were carefully chosen to ensure that the essential oils used retained their bioactive components, thereby enhancing their protective effects against Glucantime®-induced hemolysis and oxidative stress.

In this study, we explore the potential of these essential oils to mitigate the toxic effects of meglumine antimoniate (Glucantime®), a first-line treatment for leishmaniasis that can cause hemolysis and oxidative stress. While the therapeutic properties of *Curcuma longa* and *Salvia officinalis* essential oils are well documented, their ability to protect red blood cell membranes from the toxicity of Glucantime® has never been explored. This study aims to fill this gap by demonstrating the protective effects of these oils against Glucantime®-induced hemolysis and oxidative stress. Through this innovative approach, we seek to provide new insights into the therapeutic potential of these oils as adjunctive treatments for leishmaniasis, thereby improving the safety and efficacy of existing therapies.

Our findings demonstrate that *Curcuma longa* L. and *Salvia officinalis* L. essential oils significantly reduce the destruction of red blood cell membranes, thereby protecting erythrocytes from glucantime-induced hemolysis. Specifically, intracellular LDH concentrations decreased from 77 U/L to 44 U/L with *Salvia officinalis* L. and from 89 U/L to 47 U/L with *Curcuma longa* L. though not as pronounced as the reduction observed with glucantime alone (67 U/L to 21 U/L). Similarly, glucantime treatment caused a significant drop in intracellular potassium levels (from 2.03 mmol/L to 1.38 mmol/L), whereas the essential oils mitigated this effect, with levels decreasing to 2.1 mmol/L (*Curcuma longa* L.) and 1.69 mmol/L (*Salvia officinalis* L.). Additionally, hemoglobin levels, which escalated from 0.0021 g/L to 0.201 g/L under glucantime treatment, were lower when the oils were present.

Both oils also displayed antioxidant properties by reducing oxidative stress markers such as malondialdehyde, protein carbonylation, catalase, and reduced glutathione (GSH). Collectively, these results highlight the potential of *Salvia officinalis* L. and *Curcuma longa* L. essential oils as protective agents against glucantime-induced hemolytic phenomena, leveraging their antihemolytic and antioxidant activities.

Although the therapeutic properties of *Curcuma longa* L. and *Salvia officinalis* L. essential oils are well-documented, particularly their antimicrobial, anti-inflammatory, and antioxidant activities, no prior study has evaluated their ability to mitigate the toxic effects of antileishmanial treatments, specifically meglumine antimoniate (Glucantime®). This research represents a pioneering effort to explore the potential of these essential oils to protect red blood cells from Glucantime®-induced hemolysis. By analyzing key parameters related to hemolysis (intracellular LDH, potassium, extracellular hemoglobin) and oxidative stress (malondialdehyde, glutathione, catalase, protein carbonylation), it introduces an innovative approach to improving the therapeutic index of meglumine antimoniate while reducing its side effects. This work thus opens new avenues for the integration of natural extracts into therapeutic protocols to enhance the efficacy and safety of treatments against leishmaniasis.

## Material and methods

This investigation evaluates the protective effects of two Tlemcen essential oils on red blood cells against glucantime-induced hemolysis and oxidative stress.

The study was conducted at the Central Laboratory of Biochemistry Services at the Hospital Center Dr. Tidjani DAMERDJI in Tlemcen from February to May 2022.

## Vegetal material and extraction

For the extraction of essential oils, 500 g of fresh plant material from each species, *Curcuma longa* (roots) and *Salvia officinalis* (aerial parts) were used. The plant specimens were identified by Dr. Choukry KAZI

<figure><img src="image_4.png" /><figcaption>Fig. 1. Chemical structure of meglumine antimoniate (glucantime®).</figcaption></figure>
TANI, a pharmacogenetic botanist at the Faculty of Pharmacy, Tlemcen University, Algeria, and are stored in the university's herbarium under the numbers *Curcuma longa* (CL-0522) and *Salvia officinalis* (SO-0522). The plants were ground prior to extraction to increase the efficiency of the process. Extraction was carried out by hydrodistillation for 6 hours using a Clevenger-type apparatus, following the guidelines of the European Pharmacopoeia (Fig. 2). The liquid-to-solid ratio used during extraction was 4:1 (2 L of water for 500 g of plant material). This apparatus allows for the recirculation of the aqueous phase by coharbation, ensuring optimal distillation. The procedure was performed in triplicate to ensure the reproducibility of the results.

### Gas Chromatography (GC) analysis

Gas Chromatography analyses were conducted with a Perkin Elmer Clarus 600 GC system (USA, MA, Walhton). This system is fitted with FID (Dual Flame Ionization and Detectors) and a single injector. It allows for simultaneous analysis on dual fused silica capillary columns (0.22 mm × 60 m, with a 0.25 µm film Size), each featuring a various

stationary phase: polydimethylsiloxane (Rtx-1) and polyethylene glycol (Rtx-Wax). The oven temperature was adjusted from 60 °C to 230 °C at a rate of 2 °C per minute and maintained for 35 minutes at 230 °C. The carrier gas was helium. The flow rate was 1 mL per minute. The detector and injector were maintained at a fixed temperature of 280 °C. The injection system used was a split injection system with a 1:50 distribution ratio and an injection volume of 0.2 µL. Perkin-Elmer software was used to determine the retention indices (RI) of the compounds [29,30].

### Analysis by gas chromatography coupled with mass spectrometry (GC-MS)

A Perkin-Elmer Turbo Mass quadrupole mass spectrometer coupled to a Perkin-Elmer Auto system XL gas chromatograph was used to analyze the samples. Two identical fused silica capillary columns were used, with the same chromatographic conditions as described above, except for a 1:80 separation ratio. The conditions for recording electron impact (EI) mass spectra are as follows: ionization energy is 70 electron-Volt, the temperature of the ion source is 150 °C, and the mass range is 35–350 Da, with a scanning interval of one second [31].

<figure><img src="image_5.png" /><figcaption>*Curcuma longa* L. Extraction of EOs by hydrodistillation *Salvia officinalis* L.</figcaption></figure>

GC and GC-MS identified essential oils.

<figure><img src="image_6.png" /></figure>

Assessing the antihemolytic activity of essential oils by evaluating three biological parameters

<figure><img src="image_7.png" /></figure>

Intracellular LDH concentration

Potassium concentration

Extracellular hemoglobin level

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

Evaluation of antioxidant activities of Essential Oils.

<figure><img src="image_11.png" /></figure>

Measure oxidative stress with markers such as glutathione, malondialdehyde, catalase, and protein carbonylation.

<caption>Fig. 2. The experimental protocol.</caption>
## Components identification and quantification

Characterization of the components was achieved by two primary methods: (a) comparison of their gas chromatographic retention indices (RIs) on apolar columns as well as polar columns. Linear interpolation was used to calculate these indices based on the retention times of a series of n-alkanes and subsequently compared with data from reference compounds or literature sources. (b) with commercial libraries of mass spectra [32,33] and by comparing the spectra obtained with spectra from an internal laboratory database. Peak normalization was used to quantify the components in the hydrosol extract. This involved calculating percentage abundances by integrating the FID (flame ionization detector) response factors and comparing them with those of Tridecane (Internal Standard).

## Preparation of the red cell suspension

Blood specimens were collected from healthy male and female volunteers who were non-smokers and non-alcoholics. Informed consent was obtained prior to venipuncture, and samples were collected using heparinized Vacutainer tubes. The collected blood samples were gently mixed by inverting the containers and subsequently subjected to centrifugation at 4000 rpm for 6 minutes at 5 °C. This separation process allowed for the removal of plasma and buffycoat. To obtain a purified erythrocyte suspension, the patient's blood sample was subjected to three washes using 2 volumes of chilled wash solution (2 mM MgCl₂, 150 mM NaCl). Following each wash, the erythrocytes were concentrated through centrifugation at 72 × g at +5 °C for 20 minutes, and then the supernatant was carefully removed. The red blood cells were then rinsed three more times with two volumes of 10 mM of Phosphate buffered saline (1X PBS: 8.2 mM Na₂HPO₄, 1.8 mM Na₂HPO₄, 150 mM NaCl, pH 7.5), followed by centrifugation at 72 × g during 20 minutes at +5 °C, and the supernatant was carefully removed. Erythrocytes were then re-isolated in PBS to achieve a hematocrit of 10 %, stored at 5 °C, and used within 6 hours [34].

## In vitro hemolysis assay

Four sub-suspensions were prepared from the original 10 % (v/v) red blood cells suspension. The first sub-suspension consisted of 17.8 mL from the initial cell solution. The second sub-suspension contained 17.8 mL of the original cell suspension along with 75 µL of glucantime diluted to 1/10 concentration. The third sub-suspension contained 17.8 mL from the initial cell solution, 75 µL of glucantime diluted to 1/10th concentration, and 500 µL of C. longa essential oil, and a final suspension containing 75 µL of glucantime diluted to 1/10th concentration and 500 µL of Salvia officinalis L. essential oil.

These four erythrocyte suspensions were agitated continuously for 90 minutes. Samples of 2 mL were taken from the reaction solution at time intervals T = 0, 30, 45, and 90 min. To these samples, 3 mL of chilled wash solution (2 mM MgCl₂, 150 mM NaCl) was then introduced. The samples were then subjected to centrifugation at 4000 rpm at +5 °C for 6 minutes, with supernatant and cellular deposit being collected separately. The supernatants were used to determine the extracellular hemoglobin level in each cell suspension, while the pellets were used to determine other biological parameters, such as intracellular LDH and potassium concentrations, as well as oxidative stress parameters [34].

## Measurement of intracellular potassium concentration

At each time point (T₀, T₃₀, T₄₅, T₉₀), the pellet is collected in 1 mL of purified water and then vortexed to pellet lyse the cells (Fig. 2). Measurement is performed using a Flame Atomic Absorption Spectrophotometer (FAAS). After cell lysis, the supernatant is carefully collected, and the potassium concentration is quantified by comparing the absorbance to a calibration curve prepared with known potassium standards

[35].

## Measurement of intracellular LDH concentration

At each time point (T₀, T₃₀, T₄₅, T₉₀), 50 µL of the solution previously prepared for the potassium assay is collected and mixed with 450 µL of distilled water (Fig. 2). The measurement is performed using a colorimetric assay. After the lysis, the red blood cells and the supernatant were incubated with a specific substrate, and the change in absorbance at 340 nm, corresponding to the reduction of NAD⁺ to NADH, was measured. The LDH activity is calculated based on the increase in absorbance and expressed in U/L. The measurement is performed using a SIEMENS ADVIA 1800 automated system [36].

## Determination of extracellular hemoglobin concentration

Protected from light and in the cuvettes of the spectrophotometer, 500 µL of buffer solution (0.015 % H₂O₂ + DMSO) is mixed with 50 µL of chromogenic dye. After incubation for 5–10 minutes, 10 µL of supernatant was added and incubated for another 5–10 minutes. Finally, 500 µL H₂SO₄ is introduced to halt the reaction (Fig. 2). The sample absorbance and standard ([Hb] = 0.582 g/L) are determined at 450 nm after incubating for 15 minutes at ambient temperature, using distilled water as a reference blank. The percentage of extracellular hemoglobin was determined relative to the standard's absorbance value [37].

## Determination of oxidative stress parameters in erythrocytes

The pellets of the four previously prepared cell suspensions were used to measure oxidative stress parameters (Fig. 2).

## Measurement of lipid peroxidation

Lipid peroxidation in membrane lipids was estimated by measuring malondialdehyde (MDA) levels in both plasma and erythrocytes, following the method of Nourooz-Zadeh et al. MDA detection is based on its colorimetric reaction with thiobarbituric acid (TBA). After acid treatment at an elevated temperature, aldehyde compounds react with TBA to produce a chromogenic condensation product. This product consists of two molecules of thiobarbituric acid and one molecule of malondialdehyde. The absorbance of the resulting complex is measured at a wavelength of 532 nm using a spectrophotometer. The concentration of MDA is calculated using the extinction coefficient for the MDA-TBA complex (ε = 1.56 × 10⁵ mol⁻¹ l·cm⁻¹ at 532 nm) [38].

## Enzyme catalase (CAT) activity assay

Catalases are tetrameric enzymes that protect cells from oxidative damage by eliminating reactive oxygen radicals and accelerating the spontaneous dismutation reaction of hydrogen peroxide (H₂O₂). The assay is conducted following the procedure outlined by Aebi [39]. When catalase is present, the breakdown of hydrogen peroxide results in a gradual reduction in the absorbance of the H₂O₂ solution over time. The reaction mixture contains the enzyme source (the sample), H₂O₂, and 50 mmol/L of PBSat pH 7. After incubation for 5 minutes, the titanium oxide sulfate (TiOSO₄) reagent is added. The absorbance of the reaction mixture is measured at 420 nm. The remaining H₂O₂ concentrations are determined from a standard range of H₂O₂ concentrations from 0.5 to 2 mmol/l. Enzyme activity units are calculated using Eq. (1) [40].

$$A = \log A_1 - \log A_2 \quad (1)$$

A₁ is the Initial concentration of H₂O₂, while A₂ denotes the H₂O₂ concentration after incubating for 5 minutes. The specific property is quantified by U/min/mL of erythrocyte lysate.

## Measurement of glutathione (GSH)

The test consists of reacting the thiol groups of glutathione with DTNB (5,5'-Dithiobis(2-nitrobenzoic acid)), commonly known as Ellman's reagent, to give a yellow color [41,42]. When DTNB reacts with
glutathione, it is reduced to produce two molecules of TNB (5-thiol-2-nitrobenzoic acid), which is the final product responsible for the yellow coloration. The glutathione reductase enzyme reduces the resulting product, regenerating glutathione and producing additional TNB. The rate at which TNB is formed correlates with the GSH concentration in the sample. The absorbance of TNB is determined at 412 nm. This nitrobenzoic acid at pH = 9 has an optical density of 412 nm with an extinction coefficient of 13.6 $mM^{-1} cm^{-1}$.

## Measurement of carbonylated proteins

Carbonylated proteins in erythrocyte plasma, which indicate protein oxidation, are assessed by reacting with 2,4-dinitrophenylhydrazine, following the method outlined by Levine et al.[43]. The reaction produces a colored dinitrophenylhydrazone compound. Concentrations of carbonyl groups are determined spectro photometrically at 350 and 375 nm and quantified with a specified extinction coefficient (ε) of 21.5 $mmol^{-1} l^{-1} cm^{-1}$.

Curcuma longa L. Extraction of EOs by hydrodistillation Salvia officinalis L.

## Results and discussion

### GC/MS analysis of essential oils

The analysis by GC and GC-MS of Salvia officinalis L. and Curcuma longa L. essential oils allowed the characterization of 97.3 % and 98.1 % of their compositions, respectively. Identification was supported by retention indices (RI) calculated during the GC analysis, combined with mass spectra data obtained by GC-MS. This analysis identified 19 compounds for Salvia officinalis L. and 13 compounds for Curcuma longa L. Identification of all compounds was achieved by matching their electron ionization mass spectra (EI-MS) and gas chromatography retention times against entries in our laboratory's Aromes reference library. Two compounds have been confirmed by comparison of their spectral information and retention indices with literature data (Table 1).

Essential oil extracted from Salvia officinalis L. aerial parts was predominantly made up of oxygenated sesquiterpenes (82.1 %) and sesquiterpene hydrocarbons (11.9 %). Major components were germacrene D-4-ol (33.4 %), τ-muurolol (20.3 %) and τ-cadinol (14.1 %). Rhizomes of Curcuma longa L. essential oil were predominantly dominated by oxygenated sesquiterpenes (81.8 %), represented by ar-turmerone (32.8 %), along with a significant amount of α-turmerone (25.6 %) and β-turmerone (13.6 %) (Table 1).

The essential oil from Salvia officinalis was predominantly composed of oxygenated sesquiterpenes such as germacrene D-4-ol, τ-muurolol, and τ-cadinol. While the essential oil from Curcuma longa was dominated by α-turmerone, β-turmerone, and ar-turmerone. These distinct chemical profiles highlight the intrinsic variability of essential oils, influenced by botanical and environmental factors, as well as their potential for specific therapeutic applications.

The GC/MS results revealed a distinct chemical profile within the tested essential oils when compared to other compositions reported in the literature. This variability is attributed to several factors that may influence the chemical composition of essential oils, including geographical origin, harvest time, extraction techniques, and growing conditions [44]. For example, in Curcuma longa L. essential oils from Italy, India, and Algeria, terpinolene (87.8 %), α-phellandrene (53.4 %), and α-turmerone (35.0 %) were identified as major compounds [45]. Similarly, there is significant variability in the chemical composition of Salvia officinalis L. essential oils. Major compounds found in various sage essential oils include cis-thujone (up to 65.7 %), 1,8-cineol (up to 60.0 %), camphor (up to 46.7 %), trans-thujone (up to 40.0 %), α-humulene (33.9 %), and linalool (up to 34.9 %) [46]. In Iranian Salvia officinalis L. oils, α-thujone (37.2 %) was the predominant compound, with 1,8-cineol (12.7 %) and β-thujone (9.1 %) as secondary major constituents [47]. Salvia officinalis L. essential oil from Tunisia was

<caption>**Table 1**</caption>

Chemical analysis of Curcuma longa L. and Salvia officinalis L. essential oils growing in western Algeria.

<table><thead><tr><th rowspan="2">N<sup>oa</sup></th><th rowspan="2">Compounds</th><th rowspan="2">IRI<sup>b</sup><sub>a</sub></th><th rowspan="2">RI<sup>c</sup><sub>a</sub></th><th rowspan="2">RI<sup>d</sup><sub>p</sub></th><th colspan="2">Essential oils</th></tr><tr><th>C. longa</th><th>S. officinalis</th></tr></thead><tbody><tr><td>1</td><td>Linalool</td><td>1078</td><td>1075</td><td>1280</td><td>-</td><td>0.4</td></tr><tr><td>2</td><td>Pulegone</td><td>1213</td><td>1216</td><td>1640</td><td>-</td><td>1.5</td></tr><tr><td>3</td><td>Thymol</td><td>1266</td><td>1259</td><td>1648</td><td>-</td><td>0.6</td></tr><tr><td>4</td><td>α-Copaene</td><td>1379</td><td>1366</td><td>1985</td><td>-</td><td>0.5</td></tr><tr><td>5</td><td>Trans-3-isopropyl-2-(3-methylcyclopent-2-enyle) tetrahydrofuran</td><td>1394</td><td>1379</td><td>2007</td><td>-</td><td>0.3</td></tr><tr><td>6</td><td>E-β-Caryophyllene</td><td>1424</td><td>1418</td><td>1583</td><td>-</td><td>0.7</td></tr><tr><td>7</td><td>Cis-muurola-4(14),5-diene</td><td>1455</td><td>1450</td><td>2112</td><td>-</td><td>0.2</td></tr><tr><td>8</td><td>Germacrene D</td><td>1480</td><td>1474</td><td>1692</td><td>-</td><td>1.6</td></tr><tr><td>9</td><td>Bicyclogermacrene</td><td>1494</td><td>1485</td><td>1725</td><td>-</td><td>1.1</td></tr><tr><td>10</td><td>α-Muurolene</td><td>1496</td><td>1492</td><td>1709</td><td>-</td><td>1.2</td></tr><tr><td>11</td><td>Cubebol</td><td>1509</td><td>1502</td><td>1823</td><td>-</td><td>8.2</td></tr><tr><td>12</td><td>γ-Cadinene</td><td>1507</td><td>1506</td><td>1750</td><td>-</td><td>5.2</td></tr><tr><td>13</td><td>Trans-Calamenene</td><td>1512</td><td>1510</td><td>1810</td><td>-</td><td>1.1</td></tr><tr><td>14</td><td>β-Sesquiphellandrene</td><td>1516</td><td>1517</td><td>2037</td><td>11.5</td><td>-</td></tr><tr><td>15</td><td>Tans-Caryophyllene</td><td>1521</td><td>1518</td><td>2156</td><td>0.5</td><td>-</td></tr><tr><td>16</td><td>(E)-α-Bisabolene</td><td>1532</td><td>1536</td><td>2015</td><td>1.3</td><td>-</td></tr><tr><td>17</td><td>Cis-Hydrate sesquisabinene</td><td>1565</td><td>1564</td><td>2078</td><td>2.5</td><td>-</td></tr><tr><td>18</td><td>Germacrene-B</td><td>1553</td><td>1559</td><td>1830</td><td>1.2</td><td>-</td></tr><tr><td>19</td><td>trans-Sabinene hydrate</td><td>1565</td><td>1562</td><td>2076</td><td>0.5</td><td>-</td></tr><tr><td>20</td><td>Germacrene D-4-ol</td><td>1573</td><td>1566</td><td>2156</td><td>-</td><td>33.4</td></tr><tr><td>21</td><td>Caryoplèneoxyde</td><td>1576</td><td>1578</td><td>1982</td><td>2.1</td><td>-</td></tr><tr><td>22</td><td>Veridiflorol</td><td>1591</td><td>1579</td><td>2144</td><td>-</td><td>4.3</td></tr><tr><td>23</td><td>Zingiberenol 1</td><td>1599</td><td>1599</td><td>2110</td><td>1.6</td><td>-</td></tr><tr><td>24</td><td>Zingiberenol 2</td><td>1613</td><td>1614</td><td>2192</td><td>3.1</td><td>-</td></tr><tr><td>25</td><td>Epi cubenol</td><td>1624</td><td>1597</td><td>2563</td><td>-</td><td>0.7</td></tr><tr><td>26</td><td>τ-Cadinol</td><td>1627</td><td>1627</td><td>2111</td><td>-</td><td>14.1</td></tr><tr><td>27</td><td>τ-Muurolol</td><td>1634</td><td>1641</td><td>2158</td><td>-</td><td>20.3</td></tr><tr><td>28</td><td>Ar-turmerone</td><td>1639</td><td>1642</td><td>2256</td><td>32.8</td><td>-</td></tr><tr><td>29</td><td>α-Turmerone</td><td>1649</td><td>1656</td><td>2178</td><td>25.6</td><td>-</td></tr><tr><td>30</td><td>β-Bisabolol</td><td>1653</td><td>1660</td><td>2141</td><td>0.5</td><td>-</td></tr><tr><td>31</td><td>β-Turmerone</td><td>1680</td><td>1685</td><td>2244</td><td>13.6</td><td>-</td></tr><tr><td>32</td><td>14-Hydroxy-δ-cadinene</td><td>1789</td><td>1780</td><td>2530</td><td>-</td><td>1.1</td></tr><tr><td colspan="2">% identification</td><td></td><td></td><td></td><td>97.3</td><td>98.1</td></tr><tr><td colspan="2">Sesquiterpene hydrocarbons %</td><td></td><td></td><td></td><td>14.5</td><td>11.9</td></tr><tr><td colspan="2">Monoterpene hydrocarbons %</td><td></td><td></td><td></td><td>-</td><td>4.1</td></tr><tr><td colspan="2">Oxygenated sesquiterpene %</td><td></td><td></td><td></td><td>81.8</td><td>82.1</td></tr></tbody></table>

a Elution order on a non-polar column (Rtx-1)

b Retention indices on a non-polar column according to the bibliography

c Retention indices on non-polar column Rtx-1

d Retention indices on polar column Rtx-wax

e RI: Retention Index; MS: Mass Spectrometry using electron impact mode.

distinguished by its high content of camphor (33.6 %), 1,8-cineol (22.2 %), and α-thujone (21.4 %) [48].

Conversely, it is noteworthy that Curcuma and Sage oils from the western Algerian region, as described in our study, exhibit interesting characteristics and are characterized by the predominance of oxygenated sesquiterpenes combined with sesquiterpene hydrocarbons.

Essential oils, such as those extracted from sage and turmeric, are recognized for their various therapeutic benefits, including anti-hemolytic, antioxidant, and anti-inflammatory properties. Specific compounds such as germacrene D-4-ol, τ-muurolol, and τ-cadinol in sage or compounds such as β-turmerone, ar-turmerone, and α-turmerone found in turmeric demonstrate that the components present in essential oils may impact their therapeutic effects. Sesquiterpenes, whether oxygenated or hydrocarbonized, play a central role in these properties. Hydroxyl sesquiterpenes, for example, have shown significant effects in reducing inflammation. Additionally, several recent investigations have elucidated the mechanisms underlying the anti-inflammatory and antioxidant effects of these essential oils.

A recent review reported that ar-turmerone may exert anti-inflammatory power by suppressing pro-inflammatory cytokine
production, positioning it as a potential treatment for inflammatory conditions [49]. Similarly, sesquiterpenes found in *Salvia officinalis* L. essential oils are known for their potential to attenuate oxidative stress and enhance antioxidant enzyme activity [50]. It is also worth noting that synergistic interactions between different compounds can amplify their biopharmacological effects. One study concluded that the combination of several sesquiterpenes in essential oils may offer more robust protection against oxidative damage [51].

While several activities of these two essential oils have been studied, no studies have examined their anti-hemolytic and antioxidant activities against the toxicity of glucantime®.

## Study of the biochemical parameters responsible for the antihemolytic activity of essential oils

### LDH levels

The recorded values of the control suspensions stabilize within a narrow range of concentrations close to 92 U/L. The presence of glucantime causes a gradual decrease in concentrations over time, leading to a significant drop to 42 U/L at T30. This decrease is also observed with the presence of *Curcuma longa* L. and *Salvia officinalis* L. essential oils, but it is less pronounced compared to the decrease caused by glucantime.

The measurement of LDH concentration is crucial for evaluating hemolytic activity, as it not only indicates the presence of potential hemolysis but also helps to assess the severity of the hemolytic condition. In this study, we monitored the intracellular LDH concentration in our blood samples at various time points (T₀, T₃₀, T₄₅, and T₉₀). Upon reviewing the results, we observed that intracellular LDH concentrations in control suspensions remained stable and within a narrow range over time. This stability suggests that the experimental conditions were consistent, and any variations due to external factors were minimized. Therefore, we could exclude the presence of potential hemolysis caused by manipulation or mechanical agitation.

Numerous studies confirm that lactate dehydrogenase (LDH) is an enzyme found in erythrocytes at significantly higher concentrations than in blood plasma [52]. As a result, the rupture of erythrocytes leads to the release of LDH into the plasma, causing a significant elevation in plasma LDH concentrations and, consequently, a reduction in intracellular LDH levels [53].

Thus, the decrease in intracellular LDH concentrations in the glucantime-containing suspensions, ranging from 67 U/L at the beginning of the study (T₀) to 21 U/L after 90 minutes (T₉₀) (Fig. 3), can be explained by the onset of hemolysis, leading to the rupture of red blood

cells and the subsequent release of LDH into the plasma. Research on glucantime toxicity on red blood cells has shown a gradual decrease in intracellular LDH concentrations over time, as well as other hematological effects. For example, the study by Mahmoudi et al. determined the effect of *Salvia officinalis* L. essential oil on peroxynitric acid-induced hemolysis in human red blood cells. The results showed that this essential oil exhibited significant antihemolytic activity, thus protecting red blood cells from oxidative damage.

Other studies have confirmed this observation, including those by Bougherra and Jafari et al. [54–56]. Limited studies have been conducted on the antihemolytic effect of *Curcuma longa* L. essential oil. However, numerous studies indicate that its chemical composition provides important antioxidant properties [57]. Additionally, other research shows that essential oils with strong antioxidant activity also possess potential antihemolytic effects by protecting red blood cells from oxidative damage that triggers hemolysis. Based on the results from the above studies and our findings, we confirm the antihemolytic effect of the studied essential oils (*Curcuma longa* L. and *Salvia officinalis* L.).

When comparing intracellular concentrations at each time point (T₀, T₃₀, T₄₀, and T₉₀), we observed that the concentrations in suspensions treated with *Curcuma longa* L. essential oil were slightly higher than those obtained with *Salvia officinalis* L. essential oil. These results suggest that *Curcuma longa* L. essential oil has a slightly stronger protective effect than *Salvia officinalis* L. essential oil.

On the other hand, considering that the initial LDH level (T₀) recorded from control suspensions was 95 U/L, we conclude that both *Salvia officinalis* L. and *Curcuma longa* L. essential oils exhibit remarkable protective potential, capable of reducing the intensity of hemolysis manifestations, such as hemolytic anemia, agranulocytosis, and occasionally significant bleeding [58].

At each time point, the LDH levels in suspensions containing only glucantime were higher compared to those in suspensions treated with essential oils. This indicates that *Salvia officinalis* L. and *Curcuma longa* L. essential oils demonstrate significant anti-hemolytic activity, effectively mitigating the effects of glucantime.

The mechanism of action of these essential oils is likely due to the bioactive compounds they contain, particularly sesquiterpenes and phenolic constituents recognized for their cytoprotective properties and antioxidant activities in various studies [59,60]. For example, sage essential oil has been described as having the potential to prevent oxidative damage to cells, which may explain its protective effect on erythrocyte membranes [61].

Furthermore, turmeric essential oil, which is rich in curcuminoids, has been shown to possess anti-inflammatory and cell protective properties, reinforcing its role in protecting against glucocorticoid-induced hemolysis [62]. These results suggest that combining the two essential oils may represent a new therapeutic strategy to mitigate the adverse effects of glucantime in the treatment of leishmaniasis.

### Intracellular potassium levels

The results show that at each time point (T₀, T₃₀, T₄₅, T₉₀), the potassium levels in suspensions containing *Curcuma longa* L. and *Salvia officinalis* L. essential oils were consistently higher than those recorded in suspensions with only the glucantime molecule. Monitoring intracellular potassium concentrations is critical for assessing the antihemolytic protective effects of the two essential oils studied. The recorded values at different time points revealed downward trends in potassium concentration, except for the control suspensions, where the values remained stable within a narrow range of approximately 2.52 mmol/L. At T₀, suspensions containing glucantime had a potassium concentration of 2.03 mmol/L, similar to the concentration in the presence of *Salvia officinalis* L. essential oil (2.04 mmol/L). However, a higher value of 2.4 mmol/L was recorded in suspensions containing *Curcuma longa* L. essential oil.

At T₃₀, a noticeable reduction in potassium concentration was

<figure><img src="image_12.png" /><figcaption>Fig. 3. Intracellular LDH concentration in the presence of glucantime, *Curcuma longa* L. and *Salvia officinalis* L. essential oils.</figcaption></figure>
observed in all suspensions, with the most significant decrease in the glucantime-only suspensions, which dropped from 2.03 mmol/L to 1.89 mmol/L. In contrast, suspensions with *Salvia officinalis* L. and *Curcuma longa* L. essential oils recorded higher concentrations, 2.38 mmol/L, and 1.96 mmol/L, respectively.

As time progressed from T₃₀ to T₄₅ and T₉₀, potassium concentrations continued to decrease in all suspensions. At T₄₅, glucantime suspensions had a concentration of 1.66 mmol/L, which further dropped to 1.38 mmol/L at T₉₀. Suspensions with *Curcuma longa* L. essential oil exhibited values of 2.32 mmol/L at T₄₅ and 2.15 mmol/L at T₉₀, while *S. officinalis* suspensions showed a decrease from 1.82 mmol/L at T₄₅ to 1.69 mmol/L at T₉₀ (Fig. 4).

These findings indicate that glucantime causes a significant decrease in intracellular potassium concentration. However, the harmful effect of glucantime is mitigated to a lesser extent by the presence of *Curcuma longa* L. and *Salvia officinalis* L. essential oils. This trend correlates with the results of the LDH assay, which also showed a reduction in intracellular concentrations, confirming the hemolytic effect of glucantime and the protective role of the two essential oils.

The decrease in potassium concentration caused by glucantime could be attributed to the inhibition of the sodium-potassium pump and the subsequent rupture of the erythrocyte membrane or a combination of both mechanisms [63,64]. Previous studies have suggested that agents causing membrane instability often result in potassium efflux due to compromised cell integrity. For example, it has been reported that cytotoxic agents disrupt the function of the Na⁺/K⁺ ATPase pump, leading to significant potassium loss [63]. Moreover, studies have indicated that certain essential oils, including those from *Salvia officinalis* L. and *Curcuma longa* L., could stabilize membrane integrity, thereby preserving intracellular potassium levels in the presence of toxic agents [64]. These findings highlight the protective effects of *Salvia officinalis* L. and *Curcuma longa* L. essential oils in reducing the hemolytic action of glucantime, likely due to their antioxidant and membrane-stabilizing properties.

### Extracellular hemoglobin concentration level

The values obtained from the control suspensions were zero for all time measurements. In the suspensions containing glucantime, the presence of hemoglobin was detected from the beginning of the reaction, with a level of 0.0021 g/L at T₀. This level then gradually increased over time. The hemoglobin levels recorded in the presence of *Salvia officinalis* L. and *Curcuma longa* L. essential oils are significantly lower than those obtained in the presence of glucantime alone.

Measurement of extracellular hemoglobin appears to be a critical

parameter for detecting hemolytic conditions in blood suspensions. When cells are lysed, hemoglobin is released into the external medium, thereby increasing its concentration in the suspension under study. In the context of this study, the measurement of extracellular hemoglobin allowed us to evaluate the protective effect of *Salvia officinalis* L. and *Curcuma longa* L. essential oil on the erythrocyte treated with a 1/10 dilution of 75 µL of glucantime. The values obtained from control suspensions were consistently zero for all time points (T₀, T₃₀, T₄₅, and T₉₀). On the other hand, the presence of hemoglobin was detected in suspensions containing glucantime from the first moments of the reaction, with a concentration of 0.0021 g/L at T₀. This concentration then progressively increased with time, reaching levels of 0.07 g/L at T₃₀, 0.155 g/L at T₄₅, and 0.201 g/L at T₉₀ (Fig. 5).

As indicated by these results, the absence of hemoglobin in control suspensions allows us to exclude the presence of mechanical hemolysis that could occur during handling or mechanical agitation of the working suspensions. Therefore, the presence of extracellular hemoglobin in the supernatants obtained from suspensions containing glucantime could be due to cell lysis, likely caused by the interaction of glucantime with the erythrocyte cell. Furthermore, we observed that the duration of contact between glucantime and erythrocytes influenced the intensity of hemolysis. The increase in concentrations from T₀ to T₉₀ suggests that a longer exposure time between glucantime and the erythrocyte leads to greater interaction between glucantime and the erythrocytes, resulting in greater destruction of the cell or an intensified hemolytic effect [65].

This correlation between the interaction of glucantime with red blood cells and the intensity of hemolysis was also highlighted in another study, where the results showed that glucantime could induce dose-dependent hemolysis of red blood cells by disrupting the membrane structure and inducing pore formation in the membrane [65]. The mechanism of hemolysis induced by glucantime might involve the generation of reactive oxygen species (ROS), leading to oxidative stress and subsequent damage to the cell membrane [66].

In contrast, the recorded hemoglobin amounts were considerably reduced in the presence of *Salvia officinalis* L. and *Curcuma longa* L. essential oils, indicating that these oils can minimize the damage resulting from glucantime molecules, particularly the destruction of the red blood cell. They are, therefore, able to protect erythrocytes against the hemolytic phenomena induced by this molecule [67]. The antioxidant effects of these essential oils could be fundamental to their protective effects. Both *Salvia officinalis* L. and *Curcuma longa* L. have revealed considerable antioxidant properties, which can mitigate oxidative damage to cell membranes [68–70].

Many studies have investigated the antihemolytic effect of *Salvia officinalis* L. essential oil. For example, one investigation explored the

<figure><img src="image_13.png" /><figcaption>**Fig. 4.** Intracellular potassium concentration in the presence of glucantime, *Salvia officinalis* L. and *Curcuma longa* L. essential oils.</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>**Fig. 5.** Extracellular hemoglobin level in the presence of glucantime, essential oils of *Salvia officinalis* L. and *Curcuma longa* L.</figcaption></figure>
impact of this essential oil on hydrogen peroxide, which causes hemo-
lysis of human erythrocytes [57]. The findings indicated that *Salvia
officinalis* L. essential oil had a significant antihemolytic activity by
reducing the intensity of hemolysis. Similarly, another study showed
that the antioxidant compounds in *Salvia officinalis* L. can stabilize the
erythrocyte membrane and prevent hemolysis induced by various
oxidative agents [71].

Other studies also support the anti-hemolytic effect of the phyto-
chemical constituents in *Curcuma longa* L. [72]. The principal active
compound in *Curcuma longa* L., curcumin, has been demonstrated to
safeguard erythrocytes from oxidative damage and hemolysis by
enhancing the activity of enzymes involved in oxidative defense [73].
Our findings corroborate the results reported in these studies. Never-
theless, the concentrations of extracellular hemoglobin obtained in the
presence of the essential oil of *Curcuma longa* L. were relatively lower
than those obtained with *Salvia officinalis* L. essential oil. This suggests
that the anti-hemolytic protective effect is different between these two
essential oils. This result leads us to speculate that *Curcuma longa* L.
essential oil has a relatively stronger antihemolytic capacity compared
to *Salvia officinalis* L. essential oil.

This variation in activity may be attributed to the distinct chemical
composition of each essential oil, potentially containing a higher con-
centration of specific bioactive compounds or synergy between different
components, as well as to the characteristic chemotypes of each essential
oil evaluated. For instance, *Curcuma longa* L. essential oil contains high
levels of turmerone and zingiberene, demonstrating potent antioxidant
properties [74]. In contrast, the primary compounds in the essential oil
of *Salvia officinalis* L., including 1,8-cineole and camphor, may exert
different mechanisms of action regarding membrane stabilization [68].
Thus, we conclude that the presence of *Curcuma longa* L. and *Salvia
officinalis* L. essential oils demonstrated a significant protective capacity
by reducing the intensity of hemolysis.

Study of biochemical parameters: malondialdehyde of MDA, reduced glutathione (GSH), carbonylated proteins and catalase responsible for oxidative stress

The results indicate a decrease in MDA levels in the presence of *Curcuma longa* L. or *Salvia officinalis* L. essential oil compared to the concentration of Glucantime® alone. This suggests that the essential oils protect against oxidative stress caused by H₂O₂ (Fig. 6).

The findings of our study demonstrate that *Curcuma longa* L. and *Salvia officinalis* L. essential oils have significant protective effects against oxidative damage. This is demonstrated by a notable reduction in Malondialdehyde (MDA) amounts when these oils are administered

compared to the use of Glucantime® alone. MDA is a well-known
biomarker for lipid peroxidation, which results from oxidative damage
to cellular membranes. The observed decrease in MDA concentration
indicates that both essential oils can effectively combat oxidative dam-
age due to hydrogen peroxide (H₂O₂), potentially preserving the integ-
rity of erythrocyte membranes.

The antioxidant benefits of *Curcuma longa* L. are mainly attributed to its active component, curcumin. This compound has been shown to neutralize free radicals and enhance the capacity of endogenous antioxidant defense systems [75]. Curcumin's capacity to regulate molecular pathways related to oxidative stress responses further emphasizes its therapeutic potential [76].

On the other hand, *Salvia officinalis* L. is rich in bioactive constitu-
ents, including compounds such as rosmarinic acid and carnosic acid,
both of which exhibit notable antioxidant properties [77]. While these
acids are not the focus of our study, they are commonly associated with
the antioxidant effects of *Salvia officinalis* L. essential oil. Therefore,
while the current study does not specifically examine these compounds,
it supports the general understanding of *Salvia officinalis* L. essential oil's
potential for inhibiting lipid peroxidation and protecting against
oxidative damage, which aligns with our findings.

The enhanced protective effects of *Salvia officinalis* L. essential oil
may result from its increased amounts of camphor and 1,8-cineole,
which are recognized for their neuroprotective and antioxidant quali-
ties [78]. In addition, a recent study found that *Curcuma longa* L.
essential oil significantly reduced oxidative stress markers in a
dose-dependent manner, further supporting our findings [79].

Furthermore, the potential synergistic effects of these essential oils
suggest a multifaceted approach to combating oxidative stress. The
combined use of both oils may enhance their antioxidant capacities,
providing a more comprehensive defense against oxidative damage
compared to the use of either oil alone. This correlates with recent
findings indicating that co-administration of multiple antioxidants can
yield improved protective effects against oxidative stress [80].

In summary, both *Curcuma longa* L. and *Salvia officinalis* L. essential oils exhibit significant protective effects against oxidative damage caused by H₂O₂, as evidenced by the decrease in MDA levels. This underscores their potential as natural antioxidant agents for therapeutic applications, particularly in conditions associated with oxidative stress.

Reduced glutathione is an important antioxidant present in cells that
helps protect them from oxidative damage. In the context of the study
above, GSH levels may indicate the effect of *Curcuma longa* L. and *Salvia
officinalis* L. essential oils on the ability of cells to maintain sufficient
levels of reduced glutathione to protect against oxidative damage
(Fig. 7)

<figure><img src="image_15.png" /><figcaption>Fig. 6. Malondialdehyde (MDA) levels in the presence of glucantime® and the essential oils.</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>Fig. 7. Reduced glutathione (GSH) content in the presence of glucantime® and the essential oils.</figcaption></figure>
Carbonylated proteins are the result of oxidative stress due to free radicals, which can lead to cellular dysfunction. The results indicate that the administration of glucantime® alone at 0.022 mmol/L produced a marked increase in the amounts of carbonylated proteins. However, when *Salvia officinalis* L. essential oils at 0.014 mmol/L and *Curcuma longa* L. at 0.0018 mmol/L were added in combination with glucantime®, the levels of carbonylated proteins decreased considerably. This suggests that the essential oils offer protection from oxidative stress induced by glucantime (Fig. 8).

Oxidative stress can result from a decrease in the activity of certain enzymatic antioxidants, such as catalase (CAT). A catalase assay was conducted to evaluate the effect of essential oils on this antioxidant enzyme. The results show a significant increase in catalase activity with the addition of *Curcuma longa* L. essential oil, reaching 0.638 U/min/mL in the presence of glucantime®. In contrast, *Salvia officinalis* L. essential oil, when combined with 0.836 U/min/mL of glucantime®, caused a slight decrease in catalase activity compared to the control samples, which had a glucantime® concentration of 0.661 U/min/mL (Fig. 9).

Oxidative stress refers to the body's inability to defend itself against free radical attack [81]. It is typically associated with a disruption in the balance between free radical (FR) production and antioxidant protection (peroxidase, catalase, superoxide dismutase, glutathione, and antioxidant vitamins) [82]. Oxidative stress induced by glucantime® treatment may lead to elevated production of reactive oxidative molecules that damage cell membranes and proteins, resulting in cellular dysfunction. In our study, several parameters of oxidative stress were measured. The results of the catalase and GSH assays showed an increase in their activities in the presence of glucantime. Research by Silvana Lopez et al. investigated the reactivity of pentavalent antimony with human blood by assessing the redox effects. It showed that a concentration of 200 mg/mL, which is about 1000 times less than the WHO-recommended dose, was capable of inducing a redox imbalance and an increase in free radicals [83,84].

However, the presence of *Curcuma longa* L. and *Salvia officinalis* L. essential oils induced a considerable reduction in GSH and catalase [85-92]. Observed concentration values suggest the potential influence of *Curcuma longa* L. and *Salvia officinalis* L. essential oils in the ability of cells to maintain adequate levels of GSH and catalase. We observed an increase in malondialdehyde (MDA) levels and carbonylated proteins in the suspension containing glucantime®. In contrast, the levels of these markers of lipid and protein peroxidation were significantly lower with *Curcuma longa* L. oil versus with *Salvia officinalis* L. oil. This suggests that *Curcuma longa* L. oil may have more potent antioxidant activity than *Salvia officinalis* L. oil under these experimental conditions [85-92]. The

<figure><img src="image_17.png" /><figcaption>**Fig. 8.** Carbonylated protein content in the presence of glucantime® and the essential oils.</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>**Fig. 9.** Effect of glucantime® and essential oils on catalase enzymatic activity.</figcaption></figure>

results are promising for the prospective employment of these two essential oils as antioxidants and emphasize their important role in defending against oxidative damage [85-92].

## Conclusions

The present study evaluated the *in vitro* effects of *Salvia officinalis* L. and *Curcuma longa* L. essential oils on the toxicity of meglumine antimoniate (glucantime), the primary treatment for leishmaniasis in Algeria. Our findings provide strong evidence of the protective effects of these essential oils on erythrocytes exposed to glucantime-induced toxicity. The following key outcomes summarize our results:

- Both *Salvia officinalis* L. and *Curcuma longa* L. essential oils significantly reduced hemolysis in red blood cells exposed to glucantime. Intracellular lactate dehydrogenase (LDH) levels decreased progressively from the starting point ($T_0$) to the final point ($T_{90}$). For *Salvia officinalis* L., LDH levels decreased from 77 U/L at $T_0$ to 44 U/L at $T_{90}$, and for *Curcuma longa* L. from 89 U/L at $T_0$ to 47 U/L at $T_{90}$. In contrast, glucantime alone caused LDH levels to drop from 67 U/L at $T_0$ to 21 U/L at $T_{90}$. This suggests that both essential oils help to mitigate glucantime-induced toxicity, though the decrease in LDH levels was less pronounced with the oils compared to glucantime alone.

- The essential oils mitigated glucantime-induced potassium loss. Potassium levels decreased to 2.1 mmol/L in the presence of *Curcuma longa* L. and to 1.69 mmol/L with *Salvia officinalis* L., compared to a significant reduction from 2.03 mmol/L to 1.38 mmol/L in glucantime-only treatments.

- The essential oils effectively reduced extracellular hemoglobin levels, demonstrating their ability to prevent hemolysis. Hemoglobin concentrations, which increased substantially to 0.201 g/L with glucantime treatment alone, were lower in samples containing the essential oils.

- Both essential oils displayed significant antioxidant effects. They enhanced catalase activity, increased glutathione concentrations, and reduced malondialdehyde (MDA) levels, demonstrating a decrease in lipid peroxidation and oxidative stress. Additionally, protein carbonylation, a key marker of oxidative damage, was significantly reduced, indicating the ability of the essential oils to protect protein integrity.

- Among the two essential oils, *Curcuma longa* L. exhibited a relatively greater antihemolytic capacity than *Salvia officinalis* L., as evidenced by its superior ability to preserve LDH levels and potassium retention.

These results underscore the potential of *Salvia officinalis* L. and
**Curcuma longa** L. essential oils as protective agents against the hemolytic and oxidative effects of glucantime. Their significant antihemolytic and antioxidant properties make them promising candidates for use as adjuvants to glucantime therapy. This approach could enhance the clinical efficacy of leishmaniasis treatment while mitigating the side effects associated with glucantime. Further research is needed to clarify the underlying mechanisms of these protective effects and to explore their potential application in clinical settings.

## Ethics approval (for research involving humans and/or animals, include appropriate

None.

## Ethical approval

‘Not Applicable’

## CRediT authorship contribution statement

**Amina Tabet Zatla:** Writing – original draft. **Amina Hammoudi:**
Visualization, Funding acquisition. **Joëlle Pérard:** Resources, Methodology. **Dunya Zeki Mohammed:** Data curation, Writing – original draft. **Gamal A. El-Hiti:** Resources, Writing – review & editing. **FELLAH Mamoun:** Writing – review & editing, Visualization, Validation, Supervision, Project administration, Investigation.

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

The authors acknowledge the support from the Directorate-General for Scientific Research and Technological Development (DGRSDT), Algeria. The authors acknowledge the support from the Researchers Supporting Project (number RSP2024R404), King Saud University, Riyadh, Saudi Arabia.

## Source of biological material (for research involving humans and/or animals)

None.

## Statement on animal welfare (following internationally recognized guidelines)

None.

## Data and code availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request

## Supplementary information

‘Not Applicable’

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