© N. Bouhadi et al., Published by EDP Sciences, 2024

1 Introduction

Lentisk, Pistacia lentiscus L is an evergreen shrub or tree of the Anacardiaceae family, mainly known as Darou, dherou or Drou in North Africa (Amara et al., 2019), Listincu or Chessa in Sardinia; or Mastiha tree in Greece. Its distribution covers the whole Mediterranean area, from the Iberic Peninsula to the Middle East (Benalia et al., 2020). The use of Pistacia lentiscus berries, galls, resin, and leaves in folk medicine to treat a wide range of diseases dates back to Greek antiquity (Siano et al., 2020). Lentisk fruits, galls, resin, and leaves have been identified in almost all parts of the P. lentisk a long tradition in folk medicine dating from the times of the ancient Greeks (Dhifi et al., 2013).

The essential oil of Pistacia lentiscus is considered a promising source of natural cytotoxic compounds with anticancer properties. Phytoconstituents with pharmacological properties have been identified in almost all parts of the Pistacia lentiscus plant (Bozorgi et al., 2013). For example, the essential oil of the aerial parts (leaves, twigs and berries) of Pistacia lentiscus exhibited antibacterial and anti-inflammatory effects. Lentisk berries can be considered oleaginous drupes, as they contain high average quantities of an edible oil exceeding 40% (w/w) on a dry weight basis (Siano et al., 2020). Numerous studies have investigated the phytochemical composition of the resin, the leaves and the galls of Lentisk, as well as its essential oils (Castola et al., 2000), but the composition of the fruit oil has been little studied.

The oil extracted from ripe fruits is commonly used in Algerian traditional medicine as an anti-ulcer, healing and antiseptic agent (Nahida and Siddiqui, 2012; Ait Mohand et al., 2020). Lentisk berry oil can improve diabetes, gastric ulcers, asthma and possesses antidiarrheal, anti-helminthic, anti-inflammatory properties, as well as preventing wound infections.

The aim of this study is to evaluate the physicochemical properties and biological activities (antibacterial and antifungal) of Pistacia lentiscus oil harvested from two stations in Algeria (Boumerdes and Tizi ouzou), and to test the formulation of an antifungal emulsion based on extracted lentisk oil.

2 Materials and methods

2.1 Oil extraction

Pistacia lentiscus L. fruit were harvested from two regions in Algeria (Boumerdes 36° 46′ 00″ Nord, 3° 28′ 00″ East, and TiziOuzou 36° 43′ 30″ North, 4° 03′ 30″ East) in the second half of December 2022.

In order to obtain the best oil in terms of quantity and quality, a cold extraction press has been used to avoid any traces of the solvent. The traditionally extracted virgin fatty oil was stored in a well-filled, tightly-sealed glass bottle. It was kept cool and protect from light until use.

2.2 Methods for assessing physico-chemical parameters

The aim of this study was to determine certain physico-chemical properties of two samples of lentisk oil from two different regions. This is a qualitative study with the purpose of identifying the differences between these samples, influenced by a number of factors (geographical). The absence of standards for this oil has prompted us to compare our results with those of other vegetable oils.

2.3 Chemical analysis

This is a qualitative study to determine the main physico-chemical parameters of the extracted oils. Free acidity (ISO 660), peroxide values (ISO 6320), and UV absorption indices (K₂₃₂, K₂₇₀) were determined according to commercial standard methods for olive oil (ISO 3656).

Acidity is the percentage of free fatty acids expressed as a percentage of the molecular weight of oleic acid, palmitic acid or auric acid: 282, 256, 200 g, respectively. The acid number is the number of milligrams of potassium (KOH) needed to neutralize the acidity of 1 g of fat.

$$\%\text{Acidity}=28.2\times \text{V}\times \text{N/P}$$

Where V the volume of KOH solution used (ml); N: normality of KOH solution (0.1 N); 282.2, molecular weight of oleic acid (mol/L); P weight of oil aliquot (g).

To obtain the percentage of oleic acid, simply divide the acid number by 2.

$$\%\text{Oleic Acid}=I_{\text{a}}\text{/}2$$

The peroxide index measures a fundamental phenomenon that occurs in all fats, namely oxidation. The chemical alteration of unsaturated fats by oxygen in the air begins with the formation of peroxide by odometry. This is either the number of micrograms of peroxide per kg of fat, or the milliequivalents of active oxygen per kg of fat.

The principles are potassium iodide oxygenation and the titration of iodine released by sodium thiosulfate.

The peroxide value is calculated according to the following formula:

$$IP=(V_1-V_0)\times N\times 10/P$$

Where IP, peroxide value (meq/kg); V₁, is the volume of Na thiosulfate in the sample; V₀, is the volume required to titrate the white; N, is the exact title of Na thiosulfate used and P, oil sample in grams.

The saponification index is the number of milligrams of KOH required to neutralize the free acidity and saponify the esters of 1 g of lipid. The value of the saponification index allows us to estimate the carbon chain lengths of the fatty acids that make up the oil (Azizi et al., 2022).

Fourier Transform Infrared (FTIR) analysis of the specimen was carried out the 400–4000 cm⁻¹ rang.

2.4 Total phenol contents

Total phenol contents of samples were determined according to the method of Singleton and Rossi (1965), based on the principle of a colorimetric reaction using the Folin–Cicalteu (FC) reagent.

Folin–Ciocalteu (0.5 ml) was added to the sample (0.5 ml) and the solution mixed for five minutes. Next, 0.5 ml to 10% Na₂CO₃ was added to the solution tubes, and the final volume was made up to 5 ml with distilled water. The absorbance for total phenolic content was measured at 750 nm using a spectrophotometer, taking gallic acid (0–2 mg/ml) as the standard for the calibration curve.

2.5 Flavonoids assay

Flavonoids were quantified using the method of Zhishen et al. (1999), a calibration curve produced by a standard flavonoid, quercetin. Flavonoid content is expressed in milligrams of quercetin equivalent per gram of extract.

In a 100 mL volumetric flask, 1 mL of oil is added to 1 mL 20% AlCl₃ and 100 mL pure ethanol, then the flask is topped up with distilled water. Incubated in the shade at room temperature for 30 min. After incubation, absorbance is read at 430 nm on a blank.

2.6 Determination of anti-radical activity

The anti-radical activity was determined by the DPPH test using 2,2- diphenyl-1-picrylhydrazyl reagents.

A solution of DPPH- (0.004%) was freshly prepared. A 1300 μL volume of this solution was mixed with 100 μL of ethanolic extract prepared by dissolving 1 ml of lentisk oil in 5 ml of ethanol to recover as much of the active ingredient as possible. The solutions were shaken well and incubated in the dark for 30 min at room temperature. Absorbance was measured at 517 nm. A control was prepared by replacing the ethanolic extract with the same volume of ethanol. The percentage of radical inhibition due to the antioxidant properties of the extracts was calculated using the following formula:

$$\%\text{inhibition}=[(Ab{s}_{\text{control}}-Ab{s}_{\text{extract}})/Ab{s}_{\text{control}}]\times 100$$

Where Abs_contro absorbance of control; Abs_extract absorbance of sample.

3 Microbial strains

A number of four microbial strains, provided by the microbiology laboratory of the SAIDAL group at El-Harrach (Algiers), were tested, including one Gram-positive bacterial strain (Staphylococcus aureus ATCC6538), two Gram-negative (Escherichia coli ATCC839 and Pseudomonas aeruginosa ATCC 27853) and one yeast (Candida albicans ATCC 10231).

3.1 Evaluation of the antibacterial activity of lentiscus oil

The method adopted for the determination of antibacterial activity is that of disc diffusion (Bammou et al., 2015; Debbabi et al., 2017) This method consists of using Wattman paper discs 6 mm in diameter, impregnated with a known volume of the oil to be tested, placed on the surface of an agar medium (Muller Hinton Agar “AMH”), previously inoculated by swabbing. On the surface by the bacterial suspension and the oil diffuses radially from the disc into the agar, thus forming a concentration gradient. After incubation for 24 h at 37 °C, the results were read by measuring the diameter of the inhibition zone in (mm) produced around the discs containing the oil following the National Committee for Clinical Laboratory Standards (NCCLS, 2006).

Antibacterial activity, when observed, appears as a halo of inhibition around the disc. The measured diameter gives qualitative data on inhibition, with the rule: “The greater the inhibition diameter, the greater the antibacterial activity the tested sample presents”. The discs were prepared in two different ways according to Fontanay et al. (2015): At 10 µL of an HV/DMSO mixture (V/V 50/50) per disc; At 10 µL of pure HV per disc. The aim of these two different conditions is to evaluate the impact of the dispersant (here DMSO) on the diffusion of the HV in the agar culture medium. This test was carried out to determine the resistance or sensitivity of bacteria to the two types of lentisk oil. The results are read by measuring the diameter of the inhibition zone according to the method of Ponce et al. (2003).

From 18 h of bacteria young culture, we marked suspensions by taking 3 to 5 well-isolated, identical colonies and placing them in 5 ml sterile distilled water. We vortexed for a few seconds. We used a spectrophotometer to read the optical density at a wavelength of 620 nm, which should be between (0.8 − 1) for Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, corresponding to a concentration of 10⁷-10⁸ germs/ml (Fontanay et al., 2015). From of yeast young culture of 48 h, we marked suspensions by taking 3 to 5 well-isolated and identical colonies and placing them in 5 ml of sterile physiological water, then we vortexed for a few seconds and we used a spectrophotometer to read the optical density, which should be between (0.8- 1) for Candida albicans, corresponding to a concentration.

4 Analysis by Fourier Transform Infrared Spectroscopy (FTIR)

Analysis by infrared spectrophotometry makes it possible to distinguish the bands characteristic of the main chemical functions of a food; it is an essential tool for monitoring induced chemical modifications. Thus, a sample irradiated by an infrared beam undergoes molecular vibrations and absorbs part of the incident energy. Its infrared spectrum therefore presents absorption bands whose frequencies are characteristic of the chemical nature of the compound. The device used is an Alpha brand Fourier Transform spectrophotometer from Bruker. The analysis of the spectrum is carried out in the 4000–400 cm⁻¹ wave number zone (resolution 1 cm⁻¹).

5 Formulation experimental protocol

The emulsion (W/O) was prepared according to the protocol used for the preparation of pharmaceutical emulsion (W/O) (Phanazol) in the physicochemical laboratory of the SAIDAL unit in Dar el Beida, Algiers. The formulation prepared is a water-in-oil (W/O) type emulsion.

5.1 Preparation of the emulsion (W/O)

The oil phase mixture was obtained by using acetyl alcohol as a stabilizer, stearic acid and isopropyl myristate as an oil phase constituent and stabilizer, polyoxy 40 stearates as an emulsifier. The mixture was heated at 70 °C for 05 min.

In parallel, we prepared the aqueous phase by dispersing the sodium lauroyl sulfate in purified water and heated for 05 min it at 70 °C. Then, the oily phase is dispersed and mixed with the aqueous phase at 70 °C for 15 min until an emulsion is obtained. The emulsion is transferred in a cold bath for 15 min until it is stabilized in an emulsion texture. Finally, the active ingredient is added (Tab. 1).

5.2 Physicochemical characterization of emulsion (W/O)

Emulsion characterization (W/O) is based on pH and electrical conductivity measurements, organoleptic analyzes, stability, emulsion direction (W/O) and viscosity study.

5.3 Sensory analysis

In our study, we chose to carry out a sensory analysis to evaluate the quality criteria of the emulsion (W/O) produced, by obtaining the levels of appreciation of the emulsion (W/O) by consumers. Evaluation sheets were prepared and distributed to the 20 examiners for possible sensory testing. We chose the following quality criteria: emulsibility, color, odor, viscosity, dermal allergy, homogeneity, easy to incorporate into the skin, itching, and ease of spreading on the skin.

6 Results and discussion

6.1 Physicochemical parameters

Table 2 shows the Physico-chemical characteristics of oils extracted from Pistacia lentiscus fruits in two regions.

We can observe from data on Table 2 that the peroxide index for the analyzed oil from Tizi Ouzou is 0.012 mg/kg, while that for the oil from Boumerdes is about 0.0150 mg/kg. These values comply with Codex Alimentarius standards, which sets the value below 20 meq of peroxides/kg of oil. Our values are significantly lower than those found by Siano et al. (2020), which are 6 mg/kg. The low values found confirm that the samples are not exposed to air or oxidation during storage and processing. Fatty substances can oxidize in the presence of oxygen and certain contributing factors (high temperature, water, enzymes, trace metals: Cu, Fe, etc.). Indeed, good hygiene and manufacturing practices will have a positive impact on peroxide content immediately after extraction.

The acidity indices obtained varies from 1.136 to 0.954 (mg KOH / g) for lentisk oil from the Tizi Ouzou and Boumerdes regions respectively. These values are in line with Codex Alimentarius standards (0.8-3.3), indicating that this oil contains low levels of free fatty acids since it is freshly extracted. This low content may be due to the fruit being well preserved from harvesting to extraction and analysis, and thus to complete ripening. Our values are significantly lower than those found by Beldi et al. (2020), which are 2,24 to 8,36.

Lentiscus oil analysed from Boumerdes had a saponification index of 199.78 mg KOH/g oil and that from Tizi Ouzou had an index of 204.195 mg KOH/g oil. The values for both regions are lower than those found by Kechidi et al. (2020) which are 296.28.

Our values are below those found by Beldi et al. (2020), with saponification indices for Algerian varieties ranging from 197.75 to 213.45 mg KOH/g. On the other hand, Charef et al. (2008) (recorded a low index compared with that of the current study, where the index for oil extracted from black Pistacia lentiscus fruits is 147.8 ± 0.2 mg KOH /g of oil, while for red fruits, this exponent is 154.6 ±. 0.1 mg KOH/g oil.

The variation in the saponification index can be attributed to climatic factors and fruit maturity.

The ultraviolet (UV) absorbance values of the coefficients (K₂₃₂, K₂₇₀) are respectively (2.241, 1.064) for Tizi Ouzou oil and (2.2, 1.019) for Boumerdes oil. These values are close to those found by Karoui et al. (2020).

The UV absorbance results (Tab. 1) show that the samples analyzed have UV absorbances that comply with the values recommended by the COI standard: K232≤ 2.5; around 230 nm, while the values recorded are higher than the standard around 270 nm (K270 ≤ 0.25), indicating the presence of secondary oxidation products.

6.2 Total phenolic content

The concentration of total phenolic compounds is determined by reference to the calibration curve obtained using gallic acid as the calibration standard.

The total phenol contents of the oils from the Tizi Ouzou and Boumerdes regions are extremely different, 843.5583 meq g of gallic acid/ml for Boumerdes oil and 499.2683 meq g of gallic acid/ml for Tizi-Ouzou oil (Tab. 3). Siano et al. (2020) obtained a value of 339. Mezni et al. (2018) gave a value higher than ours, equal to 3358.08 mg.g⁻¹.

Variations in observed values may be due to difference in the degree of maturity of the lentisk seeds before crushing (early harvesting lentisk) but may also depend on the variety grown and the geographical area (García et al., 2003). The variations in content observed may also depend on the difference in the part of the plant studied (Fig. 1).

6.3 Flavonoid dosage

The quercetin calibration curve was used (Y = 0.0259X−0.0978, R2 = 0.9958) for flavonoid determination, and results are expressed in mg quercetin equivalent per mg of extract (mg/ mg extract). The results show variability in total flavonoid contents (from 17,854 mg Eq/mg extract to 39,150 mg Eq/mg oil extract) depending on the type of oil. High levels of total flavonoids were observed in Boumerdes lentisk oil extracts, while the lowest values were recorded in Tizi Ouzou oils (Fig. 1).

6.4 Antioxidant activity

DPPH, initially violet, discolors when the single electron pairs. This discoloration is representative of the phenolic compounds that can scavenge these free radicals. This test therefore provides information on the direct anti-radical power of the various phenolics present in the extracts. In our work, we chose to test the different extracts of each plant oil. The values obtained were used to draw exponential curves (Figs. 2 and 3).

From these curves, we can determine the inhibition percentages obtained as a function of the concentration used as well as the IC₅₀ value of each extract.

Fig. 1 Gallic acid calibration curve.

Fig. 2 Percentage of DPPH free radical inhibition as a function of the different concentrations used for the two oils.

Fig. 3 Percentage of DPPH free radical inhibition as a function of the different ascorbic acid concentrations.

6.5 Determination of antioxidant capacity (IC50)

The antioxidant capacity of the different extracts was determined from the IC50. This index corresponds to the concentration required to reduce 50% of the DPPH radical. The smaller the IC50 value, the greater the activity of the extract tested. The IC50 values found for all the extracts tested are shown in Table 4.

Table 4 shows the IC50 values of extracts from the two plant oils studied, compared with those of the reference antioxidant.

Antioxidant tests of extracts by DPPH^. method showed that extract from Tizi ouzou oil has a greater activity than extract from Boumerdes oil. Inhibitory concentrations at 50% (IC50) were respectively 919.405 mg/ml and 948.06 mg/ml for extract from Tizi ouzou and Boumerdes oil while ascorbic acid used as standard showed an IC50 value equal to 4,777 g/ml. The results thus discussed confirm that these results point that this oil is a promising source of new bioactive compounds source.

Several recent studies have proven that pistacia lentiscus fruits have been shown in vitro radical scavenging properties by using DPPH. As reported by Belyagoubi Benhammou et al. (2018), the results of Antioxidant activity; showed that oil of fruits has a height capacity to scavenge DPPH radical (EC50 = 20.619 ± 0.312 mg/mL).

6.6 Infrared analysis

Fourier transform infrared spectroscopy was carried out to identify the functional groups of lentisk oils. Figure 4 represents the comparison of the FTIR spectra of lentisk oils from Tizi Ouzou lentisk oil (TO) and Boumerdès lentisk oil (BO), (two national regions).

The spectra obtained for the two lentisk oil samples are similar in shape, but slightly different in peak intensities; the peaks observed on the infrared spectra of lentisk vegetable oils show good agreement with the literature (Souza et al., 2007). Indeed, by referring to existing infrared spectrometry databases and considering the absorption bands of the peaks obtained, it was possible to identify the characteristic groups and bonds of lentisk oil as follows:

• The carbonyl groups (C=O) present at the ends of the fatty acid ester chains show an absorption band around 1740 cm⁻¹;

• The double bond (C=C) that characterizes the establishment of oleic acid C18:1 and linoleic acid C18:2, contained in lentisk oil, shows an absorption band around 1464 cm⁻¹;

• The (C-O-C) groups present at the fatty acid/glycerol function show a corresponding peak at 1160 cm⁻¹;

• The (-CH2-)_n groups characterizing the long aliphatic chains of fatty acids show a band with an average intensity of 722 cm⁻¹;

• The (-CH₂-) groups in the adsorption band [2922–2850 cm⁻¹].

Fig. 4 FTIR spectra of TiziOuzou lentisk oil (TO) and Boumerdes lentisk oil (BO).

7 Physicochemical characterization of emulsion

7.1 Macroscopic analysis

The examination of macroscopic characteristics constitutes the first approach to the quality of the preparation, and must be the subject of an in depth study by the manufacturer, so that any anomalies can be observed. Indeed, changes recorded at the level of visual characteristics are possible indicators of degradation of the emulsion depending on various causes and factors (variation in pH, conductivity, viscosity, appearance, color, odor, texture). In this work, macroscopic analysis, stability and pH of antifungal emulsion revealed the following characteristic elements (Tab. 5).

Boumerdes emulsion 0.5, 1.5 and 2 g of lentiscus oil (B1, B2 and B3), Tizi Ouzou emulsion 0.5, 1.5 and 2 g of lentiscus oil (T1, T2 and T3) and Phanazol reference (FR)

Emulsiony texture, Odor of lentisk “Pistacia lentiscus L” and color varies from pale yellow to paler yellow (yellowish) to dark yellow. The color of the formulated emulsions varies depending on the quantity of lentisk (Pistacia lentiscus L.) vegetable oil in the emulsion.

7.2 Hydrogen potential determination

According to the data in Table 5, the reference emulsion “Phanazol” is too acidic. In contrast, antifungal emulsions formulated with Pistacia lentiscus L. vegetable oil (VO) are characterized by an acid pH profile close to skin pH, with no skin irritation. This profile is considered satisfactory for use on the face, where the pH should be between 4.5 and 8 (S.N. 1996).

Apriliana et al. (2018), stated that an over alkaline pH can cause scaly skin, while an over acidic pH can cause irritation. The pH values obtained for emulsion formulations were always considered to meet the requirements for use on the face, with pH values ranging from 4.5 to 8. A pH that is too acidic pH can cause irritation, while one that is too alkaline pH can lead to skin peeling.

7.3 Emulsion stability

The stability study was carried out at centrifugation, to assess the degree of phases separation. In this study, the stability of the antifungal emulsion formulated with Pistacia lentiscus L. vegetable oil was analyzed according to its storage time (approximately two months). The results of this test found are mentioned in Table 5 and Figure 5.

The results obtained (Tab. 5) show that all six antifungal emulsion formulations have perfect stability without any separation of the two phases (oily and aqueous) after centrifugation. The quality of the formulated emulsion was assessed using different evaluation methods. No change in physical properties were observed in formulation. The formulated emulsion showed good consistency and spreadability, a pH near to skin pH and there is no phase separation during the formulation study period. In addition, stability parameters like visual appearance, nature and viscosity of the formulated emulsion showed no variation during the study period.

Several research have shown that when phase separation occurs, all the characteristics of emulsions are affected (Casteli et al., 2008).

Fig. 5 Stability of emulsion after centrifugation (2000 rpm for 15 min) with Pistacia lentiscus L. vegetable oil from the two regions of Algeria (Tizi Ouzou (a) and Boumerdes (b)).

7.4 Emulsion direction

7.4.1 Methylene blue staining method

The result of the methylene blue emulsion staining experiment is shown in Figure 6.

This test revealed that methylene blue did not diffuse into all the emulsions prepared (Fig. 6). This could be justified by the fact that the emulsion/dye mixture is heterogeneous, so the antifungal emulsion formulated in this study does not absorb the dye. It can therefore be said that the emulsions used are of the water/oil type.

Fig. 6 Colorimetric reaction of methylene blue on the antifungal emulsion formulated with Pistacia lentiscus L. vegetable oil.

7.5 Electrical conductivity determination

In general, the electrical conductivity is the ability of a solution or all types of materials to pass an electric current. In solution, anions and cations are responsible for carrying current. The results are presented in Table 6.

According to the results obtained for this test (Tab. 6), we note that all the emulsions formulated with different percentages of lentisk “Pistacia lentiscus L.” vegetable oil have similar electrical conductivity values. These results agree with those of stability since these formulas are all stable.

On the other hand, the electrical conductivity values depend on the composition of the antifungal emulsion formulated.

7.6 Viscosity determination

This test makes it possible to study the variation in the viscosity of antifungal emulsions formulated with Pistacia lentiscus L vegetable oil as a function of the shear speed at room temperature. The results obtained are illustrated in Figure 7.

The rheological essays showed that these emulsions are non-Newtonian or pseudo-plastic (i.e., as shear rate increases, viscosity decreases) corresponding to Farah et al. (2005) and Lavaseli et al. (2012).

From the results obtained, we note that the appearance of the viscosity of the Phanazol^® (reference) is proportional to the curve of the emulsions formulated with Pistacia lentiscus L. vegetable oil from the two regions of Algeria.

Fig. 7 Rheological curve of antifungal emulsions formulated with Pistacia lentiscus L vegetable oil from the two regions of Algeria and Phanazol reference.

7.7 Infrared analysis of formulated emulsion

The results of Infrared analysis of antifungal emulsion formulations of Pistacia lentiscus L. are shown in Figure 8.

Fig. 8 IRTF spectra of control antifungal emulsions and these prepared with Pistacia lentiscus vegetable oil from two Algerian regions : (a)IRTF spectra of Tizi Ouzou lentisk oil (b) Boumerdes lentisk oil. Boumerdes emulsion 0.5, 1.5 and 2 g of lentiscus oil (B1, B2 and B3), Tizi Ouzou emulsion 0.5, 1.5 and 2 g of lentiscus oil (T1, T2 and T3) and Phanazol reference (c).

7.8 Sensory analysis

After collecting the evaluation sheets, a statistical study was carried out to obtain the evaluation levels for each criterion examined. The results obtained are represented on the histogram (Fig. 9).

Reading the data on the histogram shows that most people appreciated the quality of the emulsion with percentages of appreciation between 80-100%. Therefore, we can say that the incorporation of lentisk oil in the formulation of an antifungal emulsion does not affect the quality of the latter.

Fig. 9 Results of the sensory study, expressed as percentage of appreciation for each criterion of the emulsion formulated with Pistacia lentiscus vegetable oil from two Algerian regions.

7.9 Antifungal and antibacterial activities of the lentisk emulsion

Several preliminary tests of formulation of antifungal emulsions with three percentages (0.5-1.5–2 g) of vegetable oil from two Algerian lentisk “Pistacia lentiscus L” regions (Boumerdes, Tizi Ouzou) were carried out. The results of the analysis of these emulsions, were compared to Phanazol Reference (FR) manufactured by the company Saidal and the quality of the antifungal emulsion formulated in this study was analyzed in terms of Macroscopic analysis (Texture, color, Ordor), physical appearance (stability...) and chemical characteristic of emulsion (pH, ...).

7.9.1 Antibacterial activity of emulsion

The antimicrobial action of antifungal emulsion formulated was checked against microorganisms tested (bacteria; gram positive and gram negative, yeast) by using the modified agar well diffusion method.

Our results show that these emulsions have a moderate antimicrobial effect against all the microorganisms tested. There were differences in their activities depending on the microorganism tested (Tab. 7).

The data in Table 7 showed that the antifungal emulsion formulated with 2% of the vegetable oil was able to inhibit the growth of microorganisms tested. The results of antibacterial tests of emulsions formulated with Pistacia lentiscus vegetable oil from the two Algerian regions showed moderate inhibition of bacterial growth (11 mm).

Gram-negative bacterial strains “Pseudomonas aeruginosa” and “Escherichia coli”, showed resistance to emulsions formulated with Pistacia lentiscus vegetable oil from Tizi Ouzou. Emulsions formulated with Pistacia lentiscus vegetable oil from Boumerdes showed moderate antibacterial activity (11 mm). Whereas “Escherichia coli”, showed sensitivity to the pure formulated emulsion (100%) with inhibition zones of 11 mm, and zero for the diluted emulsion (50%). While the diluted emulsion (50%) based on Pistacia lentiscus vegetable oil from the Boumerdes region, revealed average inhibition of the growth of the Gram-positive bacterial strain of Staphylococcus aureus (11 mm).

Regarding the results of the reference emulsion "Phanazole" showed a negative antibacterial effect of the pure emulsion (100%) compared to the diluted emulsion (50%) with small diameters of inhibition zones (7 mm). The three bacterial strains tested showed resistance against the reference emulsion “Phanazole”. The emulsion formulated in this study was found to have varying degrees of antibacterial activity. What remains to be done is to determine the optimal dose of vegetable oil, or to incorporate an essential oil into the emulsion formula. Indeed, several scientific studies show the antibacterial activity of essential oils is greater than that of vegetable oils.

Ours results of the bio-chemical study, revel the richness of vegetable oil of Pistacia lentiscus fruit on polyphenolic and flavonoids contents. According to Supomo et al. (2015) in Apriliana et al. (2018), the phenolic compounds have antibacterial activities such as flavonoids, saponins and tannins. In addition, Belabbas et al. (2023) determined the antibacterial effect of the phenolic extract from Pistacia lentiscus leaves (PLLPE). These extracts rich in the total polyphenols contents and flavonoids contents showed also a significant antioxidant activity (concentration-dependent) and antibacterial. Poeloengan et al. (2010) stated the antibacterial effect of saponin as an active substance that can increase membrane permeability resulting cell hemolysis, when saponins interact with germ cells, the bacteria will be lysed. Research of Cushnie and Lamb (2005) indicated the relationship between the flavonoid structure and antibacterial activity.

7.9.2 Antifungal activity of emulsion

The zones of inhibition produced by the Pistacia lentiscus L. antifungal emulsion formulations are shown in Table 7. Our results show an antifungal effect of the emulsion formulated against the yeast tested “Candida albicans”, with inhibition zones of inhibition between 9 and 12 mm diameters. These results recorded in the emulsions developed are comparable to those found for the reference emulsion Phanazol® (11-12 mm). This emulsion shows the good antimicrobial action against the gram positive and gram-negative bacteria.

According to Hartanto (2013), Tannin can react on cell membranes, inactivate enzyme, and inactivate fungi genetic material. Furthermore, tannin can shrink the cell wall or cell membrane so that interferes the permeability of the cell, which ultimately causes the cell cannot perform life activities so that the growth is hampered or even death (Hartanto, 2013). It is known that Candida albicans is yeast that can naturally colonize the skin, (oral, intestinal and vaginal mucosa). It is an opportunistic pathogen fungus, which causes many infections (Moussaid et al., 2019). Since the vegetable oil did show activity against bacteria and fungi, it was therefore considered necessary to include these doses of vegetable oil in antifungal emulsion formula.

This is like the earlier reports from previous studies (Eichie et al., 2011), noted that this low sensitivity of these organisms to the emulsion may be attributed to dilution of the vegetable oil doses by the antifungal emulsion base. Or possible partitioning of the active principles between two phases of the emulsion (aqueous and organic) during formulation. This implies that a higher concentration of the extract will be required in formulating the emulsion to obtain an effective concentration that will render a beneficial therapeutic effect. The results showed that the vegetable oil of Pistacia lentiscus can be formulated in antifungal emulsion dosage form. Emulsion formulae were more effective against yeast than bacteria. The antifungal emulsion dosage form formulated in this study meets the best physical properties at 2%.

This present study is carried out for the first time to produce an antifungal emulsion using vegetable oil of Pistacia lentiscus from two Algerian regions (Boumerdes and Tizi Ouzou).

8 Conclusion

The aim of this work is to substitute the synthetic active ingredient with a natural antioxidant, and thus to contribute to the development of vegetable oil extracted from the Pistacia lentiscus.

In this study, we first undertook the physical and chemical characterization of vegetable oil extracted from the lentisk fruit. The results show that this oil has a significant antioxidant activity due to its polyphenol content (843.55 and 499.26 meq g gallic acid /ml oil from the Boumerdes and Tizi Ouzou regions, respectively) and flavonoids (39.15 and 17.85 mg Eq / mg oil extract from the Boumerdes and Tizi-Ouzou regions, respectively), recognized as secondary metabolites with antioxidant properties.

Next, we formulated an antifungal emulsion using this oil in the first time, then characterized it physically and chemically, and evaluated its biological activity.

The formulated emulsion showed good consistency and spreadability, a pH close to that of the skin (between 4,178 and 4,626), and there was no phase separation during the formulation study period. In addition, stability parameters such as visual appearance, nature and viscosity of the formulated emulsion showed no variation over the study period. The results of the physicochemical analyses obtained showed that the quality indices are in accordance with standards and are consistent with those found in previous studies. Also, emulsions formulated with lentisk vegetable fruit oil have remarkable antioxidant activity, due to their high polyphenols (843.5583 meq g of gallic acid/ml for Boumerdes oil and 499.2683 meq g of gallic acid/ml for Tizi Ouzou oil) and flavonoids content (from 17,854 mg Eq/mg extract to 39,150 mg Eq/mg oil extract). The latter are secondary metabolites with strong antioxidant power.

The formulated emulsion has shown encouraging antifungal effects but still needs to optimize the quantities of oil to be incorporated alone or in synergy with other active ingredients before it can be marketed.

References

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All Tables

All Figures

	Fig. 1 Gallic acid calibration curve.
In the text

	Fig. 2 Percentage of DPPH free radical inhibition as a function of the different concentrations used for the two oils.
In the text

	Fig. 3 Percentage of DPPH free radical inhibition as a function of the different ascorbic acid concentrations.
In the text

	Fig. 4 FTIR spectra of TiziOuzou lentisk oil (TO) and Boumerdes lentisk oil (BO).
In the text

	Fig. 5 Stability of emulsion after centrifugation (2000 rpm for 15 min) with Pistacia lentiscus L. vegetable oil from the two regions of Algeria (Tizi Ouzou (a) and Boumerdes (b)).
In the text

	Fig. 6 Colorimetric reaction of methylene blue on the antifungal emulsion formulated with Pistacia lentiscus L. vegetable oil.
In the text

	Fig. 7 Rheological curve of antifungal emulsions formulated with Pistacia lentiscus L vegetable oil from the two regions of Algeria and Phanazol reference.
In the text

	Fig. 8 IRTF spectra of control antifungal emulsions and these prepared with Pistacia lentiscus vegetable oil from two Algerian regions : (a)IRTF spectra of Tizi Ouzou lentisk oil (b) Boumerdes lentisk oil. Boumerdes emulsion 0.5, 1.5 and 2 g of lentiscus oil (B1, B2 and B3), Tizi Ouzou emulsion 0.5, 1.5 and 2 g of lentiscus oil (T1, T2 and T3) and Phanazol reference (c).
In the text

	Fig. 9 Results of the sensory study, expressed as percentage of appreciation for each criterion of the emulsion formulated with Pistacia lentiscus vegetable oil from two Algerian regions.
In the text