Anti-leishmanial activities of Olea europaea subsp. laperrinei extracts

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Introduction
Leishmaniasis is an infectious disease transmitted by vectors. A flagellate-bearing protozoan of the Trypanozomatidae family, Leishmania, causes it. The sand fly is the vector of the disease, and only the female is haematophagous (1). It transmits the infection to humans during a blood meal by injecting the parasite in its extracellular, flagellated promastigote form ( Figure 1A). In the epidermis, the promastigotes are phagocytosed by macrophages, which surround them with a vacuole containing dehydrogenases. As these enzymes are not specific, the parasite escapes this cellular defense (2) and assumes the amastigote form in the macrophage ( Figure 1B). After the parasite multiplies and the macrophage bursts, the amastigotes locally infect new phagocytic cells and migrate to other tissues.
The clinical expression and course of the disease depend on both the tropism of the Leishmania species involved and the human immune response (3). According to Alvar et al. (4) and Lecoeur et al.(5), infection causes severe immunopathologies in humans and animals that can be fatal. In immunocompromised individuals, leishmaniasisis considered an opportunistic disease and is associated with human immunodeficiency virus (HIV) infection (6).
In the majority of cases, the infection is asymptomatic; it leads to cutaneous lesions characteristic of cutaneous leishmaniasis (CL), which are confined to the skin. When lesions involve the ENT sphere (nose, mouth) and dermis, this is called muco cutaneous leishmaniasis (MCL) (7).
Visceral leishmaniasis (VL) affects the internal organs of the host. The entire reticulo-histiocytic system, spleen, and internal lymph nodes are affected. VL is fatal if left untreated (8). According to Solano-Gallego et al. and Messahel et al. (2,9) the parasite responsible for Mediterranean foci is Leishmania infantum. In Algeria, Phlebotomus pernicious ( Figure 1C) is recognized as the vector of this infection (10).
Currently, there is no vaccine for humans and only a few treatment options are available (13). These have many disadvantages in terms of their relative efficacy and toxicity, leading to cardiac, hepatic, nephrological, and pancreatitis complications (14). In addition, they are associated with high costs, and the long duration of treatment leads to  (15,6). The active substances contained in the olive tree are of growing scientific interest for the development of new therapeutic molecules and represent an alternative to toxic chemicals. Olive leaves are known to be rich in biophenols, which have both nutritional and therapeutic properties (16). These compounds possess antimicrobial (17,18) and antiprotozoal properties (19). The bioinsecticidal activity of olive extracts proved to be effective against the mealybug (20) and against the olive psyllid (21).
Olea europaea subsp. laperrinei(Batt. etTrab.) ( Figure  2A) is a subspecies of the olive tree in the family Oleaceae. It is endemic and naturally adapted to the dry areas of Hoggar, Tefedest, Tassili N'ajjer, and Mouyedir (Algerian Central Sahara).

Plant material
Harvesting of the petioles used in our experiment is done on adult mother plants at Ouled Hanghassi station (23°14'50.1''N, 5°29'13.7''E) in Tamanrasset region in May 2021. The olive tree is identified by its characteristics using (22). The harvested leaves are separated from the stems, sorted, and then dried in the open air, in the shade and protected from moisture. They are then ground separately (particle diameter between 0.5 and 1 mm) and stored in the dark at room temperature. The powder obtained is used for the aqueous extractions. The various analyzes began during the same year.

Biological material
The strain of Leishmania infantum responsible for VL, which was tested in our trials, belongs to the cryobank of the Parasite Ecoepidemiology Service of the Institute Pasteur (Algeria). It is the strain LIPA 1227, listed under the code MON1/ DZ /01 /LIPA1227/01.

Extraction procedure
Dissolve 20 g of plant powder in 200 ml of distilled water. After maceration for 24 hours under stirring (100 rpm) in the dark and at room temperature, the extracts are filtered on Wattman paper No. 1 and the filtrate is freezedried. The dry residues obtained are stored at 4°C. By weighing the residues, the yield of each extract can be determined.

Determination of total phenol content
Total phenols are determined according to the Folin-Ciocalteu method described by Singleton and Rossi (23). The content of total polyphenols is determined by extrapolation to the standard curve obtained from a series of dilutions of gallic acid (100 μg/ml). To each test tube, add 0.25 ml of the sample to be determined, 1.25 ml of Folin-Ciocalteu (1/10), and 1 ml of sodium carbonate (75 g/l). Then the absorbance is measured at 765 nm. The content of total phenols is given in milligrams of gallic acid equivalent per gram of dry residue (mg GAE /g DR).

Determination of the total flavonoid content
The method used is the same as that described by Chang et al. (24). To 0.5 ml of the solution of each extract, add 1.5 ml of methanol, 0.1 ml of aluminium chloride (10 %), 0.1 ml of potassium acetate (1 M), and 2.8 ml of distilled water.
The mixture is incubated for 30 minutes at room temperature. The absorbance is measured at 415 nm. The standard curve is established with quercetin (100μg/ml) and the linear regression equation allows the calculation of the total flavonoid content expressed as milligram quercetin equivalent per gram plant powder (mg EQ /g DR).

Determination of total tannin content
The determination of tannins is carried out according to the protocol developed by Hagerman and Butler (25). 1 ml of BSA solution (1 mg/ml) is mixed with 500 μl of the extract solution. After incubation at 4°C for 24 hours, the precipitate is collected by centrifugation at 750 g for 15 minutes at 4°C and then dissolved in 2 ml of sodium dodecyl sulphate (SDS)/triethanolamine (TEA) (1%/5%) to which 500 μl of FeCl3 reagent has been added. After incubation for 15 minutes, the absorbance is measured at 510 nm.
The total tannin content, expressed as milligrams of tannic acid equivalent per gram of dry residue (mg EAT /g DR), is determined from the tannic acid calibration curve using the linear regression equation.

Chemical composition
The chemical composition of the aqueous extracts of leaves (EAF) and stems (EAT) is obtained by heating in the liquid phase under the following operating conditions: Quantitative analysis is carried out using an AGILENT 1100 series liquid chromatograph equipped with a diode array detector (DAD) with several wavelengths selected according to the maximum absorptions of the molecules sought, a quaternary pump, an on-line degasser, and an automatic injector. The chromatograph is equipped with a Hypersil BDS -C18, 5um, 250X4.6 column at 30°C. The mobile phase consists of water acidified with 0.2 % acetic acid at a pH of 3.1 and acetonitrile in a linear elution gradient over 30 minutes at 1.5 ml/min, starting with 95 % H 2 O and ending with 100 % acetonitrile. The injected volume corresponds to 5 μl of the extract dissolved in methanol at 0.16 g/ml. The percentage of each chemical compound in the extract is determined from the peak areas obtained. The phenolic compounds are identified by comparing the retention times of the different standards analyzed with the  A factorial correspondence analysis (FCA) was performed with a 5% risk. It aimed to deal with several variables at the same time in order to establish correlations between them.

Ethical Approval
The proposed study has been reviewed by the Ethics and Deontology Committee of MouloudMammeri University, Tizi-Ouzou, Algeria. The corresponding ethical approval code is UMMTO/25/01/2022/Eth-Deon-A-102.

Quantitative analysis
To characterize the aqueous extracts of leaves (EAF) and stems (EAT) of the Laperrine olive tree, a quantitative study is carried out using colorimetric methods (folin-ciocalteu, aluminium trichloride and formation of the proteintannin complex).
The total content of phenolic compounds is determined using the respective standard curves. The results obtained reveal an obvious variability in the calculated contents. Indeed, the statistical analysis shows a significant difference between the mean values in the case of total phenols (P = 2.7110 -6 ), where we obtained a value of 776.76±30.64 mg EAG/g DR for EAF and 94.45±1.64 mg EAG/g DR for EAT, and in the case of total flavonoids (P = 3.5610 -6 ), with a calculated mean value of 114.35±14.12mg EQ /g DR for EAF and 24.92±0.95 mg EQ /gDR for EAT. For total tannins, the calculated means correspond to 214.89±20.17 mg EAT /g DR for EAF and 90±1.47 mg EAT /g DR (P = 0.0004).

Qualitative analysis
The chemical composition of the two extracts was determined by high-performance liquid chromatography same procedure and by their UV spectra (20).

Anti-leishmanial activity in vitro
Promastigotes are grown on NNN medium (Novy Nicolle and Mc Neal). After centrifugation of the cultures at 2,500 rpm for 10 minutes and washing three times with physiological water, they are grown in masse in RPMI medium (Roswell Park Memorial Institute) 1640 supplemented with 10% foetal calf serum (26). Screening is performed in 96-well cell culture microplates maintained at 25°C. The promastigote forms from the culture in the exponential growth phase are diluted to a concentration of 10 6 cells/ml. The assay is performed in 96-well microplates, with each well filled with 100 μl suspension containing the parasite and the whole incubated at 25°C. After one hour, the extracts of leaves and stems of the Laperrine olive tree are added; these are dissolved in DMSO and added to each well to obtain the final concentrations of 12.5; 25; 50, and 100 μg/ml. An incubation of 72 hours at 25°C is carried out. The viability of the promastigotes is determined by the colorimetric trypan blue method and the activity of the laperrine olive extracts is evaluated by comparing the viability rates obtained at different concentrations with untreated control and positive control.

Data processing
The results are expressed as the mean with its standard deviation at a 5% risk. The comparison of means is performed to analyze the level of statistical significance between the series of means treated in pairs in the case of the tests performed.
The influence of the different extracts on the antileishmania efficacy is analyzed by a single-factor analysis of variance. The normality of the data was previously checked with the Shapiro test. Statistical analysis was performed with a 5% risk using R 3.4 software. (HPLC). We identified 22 chemical molecules, 19 in the EAF extract and only 10 in the extract from EAT (Table 1).

EAF extract EAT extract
Our results indicate the presence of hydroxytyrosol and oleuropein in the extracts, compounds typical of Olea europaea. Quercetin, rutin, vanillic acid, salicylic acid, aesculetin, cinnamic acid, and caffeic acid are found in Olea europaea leaves (21). Moreover, our results are similar to those obtained by Lahcene et al. (20) after acid hydrolysis and Djenane et al. (27) with a methanolic extract of Laperrine olive leaves.

Anti-leishmania activity
The results obtained show that both extracts of Laperrine olive have antileishmanial activity. The increase in this activity is proportional to the concentration of the extracts. However, there is a significant difference (P=3.5610 -6 ) between EAF and EAT. Exposure of the parasite to a maximum concentration (100 µg/ml) of the extract results in a percentage viability of 36% for the EAF extract and 50.33±0.58% for the EAT extract ( Figure 3). Moreover, our results show that the extracts of Laperrine olive are more efficient in eliminating promastigotes than the positive control T + corresponding to Glucantime, for which we record an average value of 68±2% at the therapeutic dose. For the negative control T -, the percentage of viability is 100%.
Anti-Leishmania activity, expressed by parasite viability, is strongly correlated with the concentrations of the extracts tested. The trend curves applied to the graphs in Figure 4 shows a very high linear fit coefficient for the extract EAF, which is 0.95. In other words, more than 95% of the viability observed in the period considered is explained by the effect of the leaf extract. We, therefore, assume that the extract is probably more effective than the extract of EAT, in eliminating the parasite. However, we note that the effect of the stem extract is not negligible.
From the resulting regression equations, the 50% lethal dose (LD50) of the extracts EAF and EAT is determined. Thus, for the leaf extract (Y = -0.133 X + 49; R2 = 0.95), a dose of 7.52 ± 2.71 µg/ml is sufficient to cause the death of 50% of the parasites. For the stem extract (Y = -0.100 X + 59.29; R2 = 0.81), the concentration required to eliminate 50 % of the promastigotes is on average 12 times higher and corresponds to a concentration of 92.9±2.3 µg/ml. These results underline the clear effect of the EAF extract on the viability of the parasites studied. This effect is explained by the polyphenol and flavonoid content of the EAF extract, which is on average 8.2 times higher for phenols and 4.6 times higher for flavonoids than that of the extract from EAT.
To explain the effect of the extracts on the viability of L. infantum, we integrated their chemical composition to interpret the results and correlate the different variables. The data were subjected to a factorial correspondence analysis (FCA) and are shown in Figure 5.
On axis 1 of the AFC ( Figure 5), a negative correlation is observed between EAF extract and LD50. It appears that a group of 18 molecules correlated with this set would explain the particular effect of EAF extract on the viability of the parasite studied.
Since the extract of EAT has a non-zero effect on the viability of promastigotes, we can say that the molecules common to both extracts, namely gallic acid, hydroxytyrosol, resorcinol, vanillic acid, rutin, and oleuropein, are the main ones responsible for eliminating 50% of the parasites.

Discussion
A study by Katanilic et al. (28), in which 70 medicinal plants were classified according to their richness in total phenols quantified from an aqueous extract, showed that the extracts of Laperrine olive are very rich in polyphenols. Comparison of our results with those of Nashwa et al. (29) from a methanolic extract of Oleaeuropaea leaves, which had a value of 90.48±0.16 mg EAG/g residue, shows that the polyphenol content in Laperrine olive leaves is very high. Phytochemical analysis carried out on the two ex-   tracts revealed the presence of two important fractions in phenolic compounds, namely flavonoids and tannins, whose contents are particularly high. This abundance seems to be a response of the species to the conditions of its environment and clearly shows the close relationship between the biosynthesis of secondary metabolites and the extreme environmental conditions to which the Laperrine olive tree is exposed. It should be noted, however, that compared to the extract of EAT, the content of phenols and total flavonoids in the EAF extract is significantly higher.
The total flavonoid content of Laperrine olive leaves is higher than that calculated by Abaza et al. (30) from Olea europaea leaves of the Chetoui variety, where they found an average of 6.23 ± 0.62 mg EC /g dry weight for an aqueous extract and 15.83 ± 1.26 mg EC /g dry weight for an ethanolic extract. For Botsoglou et al. (31), the content calculated from an ethanolic extract corresponds to 12.46 ± 0.58 mg EC /g dry weight of Olea europaea L. leaves.
The total tannin content is higher in the leaves of Laperrine olive than in the leaves of Olea europaea L., corresponding to a value of 79.70 mg EC /100g dry weight for the Chemlal variety and a value of 73.05 mg EC /100g dry weight for the Neb jmel variety, Neb jmel, 2013. However, the tannin content of Olea laperrinei is close to 22.79 g EAT /kg DM, a value determined by Mebirouk-Boudechiche et al. (32) for the leaves of Olea europaea.
The results obtained in the antileishmania test are consistent with the work of Sifaoui et al. (33) and Koutsoni et al. (34), who highlighted an inhibitory effect of polyphenols and flavonoids against the promastigote form of the parasite. According to Kheirandish et al. (7), phenolic structures damage parasite membranes. The application of Laperrine olive leaf extract on promastigotes had better results than Vitris vinifera, Arbutus unedo, and Myrtus nivellei leaf extract ( Table 2) However, the greater efficacy of EAF extract compared to EAT extract could be explained by the exclusive presence of caffeic acid, rosmarinic acid, and quercetin. These molecules, according to Carter et al. (41), inhibit arginase, which is important for the multiplication and infectivity of the parasite. On the other hand, hydroxyquinone, catechin, aesculin, orientin, O anisic acid, salicylic acid, naringenin 7 glucoside, manic acid, and cinnamic acid, which could play a synergistic role in enhancing the effect studied.

Conclusion
As far as we know, no study has yet been conducted on the antiparasitic effects of Laperrin olive. Therefore, the results presented in this report show for the first time the efficacy of Laperrine olive tree extracts as a potential leishmanicidal.
The leishmania-fighting activity demonstrated in vitro is directly related to the phenolic content of the extracts studied and depends on the various interactions (synergy or antagonism) that determine it. Thus, the leishmanicidal potential is in favour of the EAF extract, whose phenolic content is higher than that of the EAT extract. The results obtained in our study clearly show that the olive leaf extract from Laperrine has a strong leishmanicidal activity against the promastigote form of Leishmaniainfantum. This is due to the combined presence of oleuropein, hydroxytyrosol, caffeic acid, rutin, quercetin, gallic acid, and rosmarinic acid, whose anti-leishmanial activity has been demonstrated by many authors.
The richness and diversity of phytochemical molecules contained in the Laperrine olive tree make it a good candidate to serve as a potential source of new biomolecules. It could be used as an alternative to harmful chemicals and as a source of biomolecules with leishmanicidal activity. However, purification of the bioactive molecules and separate or combined assessments of their activity is needed to better understand their mechanism of action on the parasite.

Funding
No funding.

Institutional Review Board Statement
The proposed study has been reviewed by the Ethics and Deontology Committee of Mouloud Mammeri University, Tizi-Ouzou, Algeria. The corresponding ethical approval code is UMMTO/25/01/2022/Eth-Deon-A-102.

Informed Consent Statement
Not applicable.

Data Availability Statement
Data is available with the investigators and may be provided upon reasonable request.