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Medicine
DOI: 10.21070/acopen.10.2025.10970

Anti-Leishmanial Activity of Iraqi Plant Gundelia Tournefortii and Isolation of Beta-Sitosterol


Department of Pharmacognosy, College of Pharmacy, University of Basrah, Basra
Iraq
Department of Pharmacognosy, College of Pharmacy, University of Basrah, Basra
Iraq
Department of Pharmacognosy, College of Pharmacy, University of Baghdad, Baghdad
Iraq

(*) Corresponding Author

Gundelia Tournefortii Leishmania Tropica Leishmania Donovani Pentostam IC50

Abstract

Background: Herbal medicine remains a significant focus in contemporary pharmacological research due to its therapeutic benefits and low side effects. Specific Background: Gundelia tournefortii is recognized for its medicinal potential, yet its anti-leishmanial properties are underexplored. Leishmaniasis, particularly caused by Leishmania donovani, remains a critical health challenge with no available vaccines and limited treatment options. Knowledge Gap: Despite the ethnobotanical use of G. tournefortii, its phytoconstituents, especially beta-sitosterol, have not been thoroughly investigated for anti-leishmanial activity. Aims: This study aimed to isolate beta-sitosterol from the oil extract of Iraqi G. tournefortii and evaluate the anti-leishmanial efficacy of two different extracts against Leishmania tropica and Leishmania donovani, in comparison to the standard drug, pentostam. Results: Beta-sitosterol was successfully isolated via HPLC at a concentration of 98.08 ppm. The oil extract showed superior efficacy, with IC₅₀ values of 0.042 mg/ml for L. tropica and 0.00127 mg/ml for L. donovani. Novelty: This research represents the first report on the isolation of beta-sitosterol from Iraqi G. tournefortii and its significant anti-leishmanial activity. Implications: The findings support the potential of G. tournefortii oil extract as a natural source for developing new anti-leishmanial therapies, particularly due to its sterol and triterpene content.

Highlights:

  • First isolation of beta-sitosterol from Iraqi G. tournefortii.

  • Oil extract shows high efficacy against L. donovani.

  • Potential for developing plant-based leishmaniasis treatment.

Keywords: Gundelia Tournefortii, Leishmania Tropica, Leishmania Donovani, Pentostam,  IC50

Introduction

Gundeliatournefortii L is a medicinal plant belonging to the Asteraceae (Compositae) family. It is widely found in the semi-desert parts of Western Asia, notably in the temperate Asian countries of Lebanon, Syria, Palestine, Jordan, Iraq, Iran, Azerbaijan, Armenia, and Turkey (Anatolia) [1]. The plant proliferates in several environments but flourishes in well-drained, wet soil. It is intolerant of shade and favors sandy, loamy, acidic, neutral, and alkaline soils [2]. The Gundelia, a spiny, thistle-like plant, belongs to the Gundelia L. genus. Its flowers, leaves, seeds, and stems are commonly used as food [3]. For over 2,000 years, the G. tournefortii plant has been used as a medicinal herb. It has been conventionally utilized to treat a wide range of medical conditions, including liver diseases, angina pectoris, diabetes, vitiligo, stroke, gastric disorders, hypoglycemia, laxatives, sedatives, diarrhoea, bronchitis, skin diseases, pain, respiratory diseases, digestive disorders, high blood pressure, and cancer. All plant parts have been utilized for these traditional treatments [4], [5], [6]. Gundelia spp contains terpenoids, which have a role as an anti-leishmanial effect [7]. Gundelia oil extract also contains sterols such as beta-sitosterol, stigmasterol, and campesterol [8]. Leishmaniasis is a disease caused by many species of the protozoan parasite. They can manifest with different clinical presentations, ranging from cutaneous lesions to the deadly visceral form of the disease [9]. Female sandfly bites expose humans and animals, such as rats, hyraxes, and canids, to the parasite responsible for visceral and cutaneous leishmaniasis. In some areas, women exhibit a greater case fatality rate than males as a result of gender imbalance. Untreated visceral leishmaniasis is estimated to cause 59,000 deaths annually globally. Nonetheless, cutaneous leishmaniasis often cures autonomously, although it may result in scarring [10]. Individuals afflicted by leishmaniasis often belong to disadvantaged demographics and lack the financial means to get the costly drugs required for extended treatment. Moreover, the existing chemotherapeutic interventions for leishmaniasis are very toxic, resulting in severe adverse effects, including nephrotoxicity, hepatotoxicity, and cardiotoxicity. The emergence of drug resistance in parasites exacerbates the therapy of this disease. Consequently, there is a want for more effective leishmanicidal agents. Herbal medicines have gained popularity as safe, cost-effective, and efficacious alternatives to conventional treatments owing to their reduced side effects [10], [11], [12].

1. Purpose of the Study

This research is to isolate beta-sitosterol from oil extract and assess the antileishmanial effects of two extracts of the Iraqi Gundeliatournefortii against Leishmania tropica and Leishmania donovani compared with the official treatment, pentostam® [13].

Methods

A. Plant Materials

The aerial portion of G. tournefortii was collected from the Sulaymaniyah Governorate in northern Iraq. The freshly harvested plant was meticulously gathered in May 2023, washed, shade-dried for several days at ambient temperature, ground into medium-sized particles, and preserved in airtight containers.

B. Extraction Procedure

The bioactive materials were extracted from the powdered G. tournefortii using two extraction methods.

C. Extraction Method for Oil

The Soxhlet apparatus was used to extract the oil fraction from the plant. Twenty grams of the powdered plant were extracted with 200 mL of 80% ethanol for 12 hours at 80°C. The solvent was removed using a rotary evaporator. The dry extract was resuspended in distal water and transported to a separatory funnel [14]. Then, an equal amount of petroleum ether was added and mixed well. The mixture was left at room temperature to stand until two distinct layers (aqueous and organic) were formed. The organic layer (petroleum ether) was collected, and the petroleum ether technique was repeated three times. The collected petroleum ether fraction was dried and stored in a glass container at 4°C.

D. Defat Extraction Method for Phytoconstituents (Methanol Extract)

The defatting procedure was conducted with petroleum ether. The marc was subjected to extraction with 250 cc of 80% methanol for 12 hours at 65°C using Soxhlet apparatus. A rotary evaporator removed the solvent. The resulting extract was preserved in a dark glass jar at 4°C [15]'

E. Phytochemical Screening by Chemical Tests

The phytochemical tests thoroughly examined the presence of triterpene, sterol, phenol, and flavonoids according to [16].

1. Tests for Sterol and Triterpene

a. Liberman Test

A few drops of acetic anhydride were added to the alcoholic extract and mixed thoroughly. Then, 1 ml of concentrated sulphuric acid was added slowly from the side of the test tube. The appearance of a reddish-brown ring indicated the presence of sterol.

b. Salkowski Test

A few drops of concentrated sulphuric acid were added to the alcoholic extract, shaken well, and allowed to stand until the appearance of a golden yellow indicated the presence of triterpenes.

2. Tests for Phenol and Flavonoids

Lead acetate, ferric chloride, and Shinoda tests: The Lead acetate test methodology included the addition of a few drops of aqueous basic lead acetate solution to the alcoholic extract; the emergence of a yellow precipitate signified the presence of flavonoids. A few drops of neutral ferric chloride solution were introduced to a tiny volume of alcoholic extract during the ferric chloride test. The emergence of a blackish-green hue indicated the existence of flavonoids. In the Shinoda test, a segment of magnesium ribbon and strong hydrochloric acid were introduced to the alcoholic solution extract. The emergence of red to pink hues after a few minutes indicated the presence of flavonoids.

3. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

The phytochemical screening by GC-MS was performed at Basra Oil Company Laboratory using an Agilent Technologies 7890B GC instrument paired with an Agilent Technologies 5977A MSD equipped with an EI signal detector. They utilized an HP-5ms 5% phenyl, 95% methyl siloxane (30m*250um*0.25) and precisely set the oven temperature at 40°C for 5 minutes. Then, ramped it up to 300°C at a consistent rate of 8°C/min for 20 minutes. The helium carrier gas flow rate was maintained at 1 ml/min, with a 3 ml/min purge flow. The injection mode was pulsed splitless with an injection temperature of 290°C. The injection sample volume was 0.5 microliter. The mass spectrometer used an Ion Source Temperature of 230°C, a scan speed of 1562(N2), and a mass range of 44-750 m/z. The data was run through the NIST 2014, 2020 Library database as an additional tool to confirm the identity of compounds [17].

4. Isolation of Beta-Sitosterol by HPLC

Using preparative high-performance liquid chromatography (HPLC) to separate the beta-sitosterol with mode SYKAM (Germany), C18 (250 mm x 4.6 mm, five μm particle size) column, using gradient mode with mobile phase including A = acetonitrile, B= 5 % acetic acid = (0 – 5 min) A: 30 %, (6 – 15 min) A = 50 %. Gradient elution was carried out for 20 minutes at a flow rate of 1 mL/min. The injection volume was 1mL, the detection was recorded with a UV detector at λ 220 nm, and the isolated constituent was recrystallized using hot methanol for purification[18].

5. Antileishmanial Activity

a. Preparation of Inoculum

Leishmania donovani and Leishmania tropica were obtained from the Center of Biological Technology Research at the College of Sciences at Al-Nahrain University. These were isolated from the bone marrow of an infected child and several confirmed cases, respectively, in Baghdad. The evaluation of the antileishmanial activity of the plant extract started by first propagating the organism. This was done by incubating them in Roswell Park Memorial Institute media (RPMI) enriched with 12% fetal calf serum at 25°C for 5 days to reach an average of 10^5 parasites/ml in a hemocytometer[19], [20].

b. Preparation of Plant Extract Concentrations

Several dilutions of the oil sample and methanolic sample (defatted) were made in dimethyl sulfoxide (DMSO) at the following concentrations: 1000, 900, 800, 700,600,500,400,300, and 200μg/ml to identify the antileishmanial activity. Furthermore, pentavalent antimonial (antileishmanial medicine) was used as a positive control [21].

c. Evaluation of the Samples’ Activity Against Leishmania Tropica and Leishmania Donovani

The antileishmanial activity was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [21]. 200µL of fresh promastigotes culture (10^5 cell/ml) was transformed to each well of a flat-bottom plate containing 96 wells, except four blank wells containing culture medium only. Subsequently, 200ul of each plant extract concentration (prepared previously) was added to all plate wells except the blanks. Whereas four wells contained culture only as a negative control, four wells contained sodium stibogluconate as a positive control, and the last four wells were blank. The plate was incubated at 25°C ± 1°C for 24 hours. Afterward, 10µL of MTT dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well. The plate was incubated for an additional 4 hours at 25°C ± 1°C to assess metabolic activity. DMSO was added to each well as a solubilizing agent to dissolve the MTT purple dye inside the living matter, enabling the scanning process. An enzyme-linked immunosorbent assay (ELISA) reader at 490 nm was used to detect the optical density of each well. Finally, findings were compared with both positive and negative control. All tests were repeated in a tetraplicate. Calculating the IC50 The IC50 of each analytical sample was calculated using the following procedure: At each of the two locations, inhibition ratios (y) were plotted against sample concentrations (x). The associated regression line (y=ax+b) was generated. The IC50 value can be calculated using interpolation by connecting the two points surrounding the 50% inhibition point with a straight line. After selecting two points surrounding a 50% inhibition ratio, a regression line (y=ax+b) was created. The x (sample concentration) was determined by substituting 50 for y in the regression equation y=ax+b [22] [23]

d. Statistical Analysis

The % inhibition rate for each concentration gradient was computed, and a mean comparison was conducted to validate the significance of the results. A one-way ANOVA test was conducted using IBM software to compute the p-value and Least Significant Differences (LSD) to evaluate if the inhibition rate substantially varies from sodium stibogluconate.

Results and Discussion

A. Results

1. Phytochemical Screening by Chemical Tests

The phytochemical contents were analyzed to assess the compound groups present in each extraction process, potentially linked to the observed bioactivity. All produced extracts, including oil and methanol extracts, exhibited the presence of numerous bioactive components, as detailed in Table 1. The Liberman test confirmed the presence of sterols by producing a reddish-brown ring upon the addition of a few drops of acetic anhydride and 1 ml of concentrated sulfuric acid. The Salkowski test indicated the presence of triterpenes by yielding a golden-yellow color after the addition of a few drops of concentrated sulfuric acid. The presence of phenols and flavonoids was established through Lead acetate, Ferric chloride, and Shinoda's tests.

Test Name Oil Sample Methanol Sample (Defatted)
Liberman test + _
Salkowski test + _
Lead acetate test _ +
Ferric chloride test _ +
Shinoda test _ +
Table 1.The Result of the Phytochemical Tests

2. Gas Chromatography-Mass Spectrometry (Gc-Ms) Analysis

The GC-MS chromatograms of the oil and methanolic extracts (in Figure 1) display the GC-MS profiles of the detected chemicals. Gas Chromatography-Mass Spectrometry (GC-MS Analysis) is based on the elution sequence on the chromatographic column. By comparing the mass spectra and retention time Rt of the plant samples with the available references in the library, a qualitative analysis was carried out to identify the molecule [24]. The particular chemicals, the present area, and the (Rt) are listed in Tables 2 and 3 and Figures 1. The results of the qualitative chemical analysis using the GC-MS device were as follows: the oil extract appears to contain lupeol, beta-amyrin, and beta-Cameron, which are not found in the methanolic sample. Oil also seems to contain higher percentages of lupeol acetate and stigmasterol than the methanol extract.

Retention Time (RT) Area % Chemical Constituent
21.86 13.2455 n-Hexadecanoic acid
24.779 13.060 9,12-Octadecadienoic acid (Z,Z)-
35.885 8.7642 lupeol
37.542 19.9395 Lup-20(29)-en-3-ol, acetate, (3.beta.)-
35.261 4.8733 Beta-amyrin
34.765 0.9174 Beta-amyrone
33.916 7.4264 stigmasterol
34.643 2.9788 Beta-sitosterol
23.917 0.277 Heptadecanoic acid
Table 2.Chemical Constituents of the Oil Sample
RT Area % Chemical Constituent
22.948 15.609 n-Hexadecanoic acid
24.572 9.310 9,12-Octadecadienoic acid (Z,Z)-
13.08 16.106 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-
29.095 2.109 1,3,12-Nonadecatriene
14.526 10.266 1-Deoxy-d-arabitol
33.985 7.381 stigmasterol
34.672 4.143 Beta-sitosterol
37.339 4.043 Lup-20(29)-en-3-ol, acetate, (3.beta.)-
9.726 2.688 2-Furancarboxaldehyde, 5-methyl-
Table 3.Chemical Constituents of Methanol Sample (Defatted)

Figure 1.Gc-Ms Spectrum of the (a) Oil Sample and (B) Methanol Sample.

3. Isolation of Beta-Sitosterol

The HPLC results indicate that the isolation and purification of beta-sitosterol from the oil sample of Iraqi G. tournefortii will be executed using preparative HPLC was chosen for its efficacy, versatility, and cost-effectiveness [25]. The peaks corresponding to compounds were identified based on their retention times through comparison with beta-sitosterol standards. The sample obtained from PHPLC was dried using anhydrous sodium sulfate, weighed, and reserved for subsequent analysis after the primary peaks were collected by the fraction collector based on temporal monitoring, as indicated in Table 4 and Figures 2 and 3.

Standards Name RT Value of the Standard RT Value in an Oil Sample RT Value of Isolated
Beta-sitosterol 3.80 3.83 3.83
Table 4.Retention Time (Rt) Values for Oil Sample of G.tournefortii Compared to Standard and Isolated Beta-Sitosterol.

Figure 2.HPLC Chromatogram for Oil Sample

Figure 3.HPLC Chromatogram for (a) Beta-Sitosterol Standard and (B) Isolated Beta-Sitosterol.

4. Quantity Estimation of Beta-Sitosterol

The calibration curve for quantitative analysis was constructed by plotting the area under the curve (AUC) against four concentration levels of the beta-sitosterol standard. The concentration of the suggested beta-sitosterol in the oil sample was determined using a linear equation, as seen in Figure 4. HPLC measured the concentration of beta-sitosterol in the oil sample at 98.08 ppm.

Figure 4.Calibration Curves for Beta-Sitosterol.

5. Antileishmanial Activity in Vitro Assay

Using the Optical Density (OD) data obtained from ELISA, the following formula was used to determine the percentage of organisms killed by each concentration of each tested fraction: Percent Inhibition Rate equals (OD Control-OD Test/OD Control) x 100. For every concentration of each fraction, the percentage inhibition rate was computed and compared to the results of the positive and negative controls. An ANOVA analysis was also performed to determine the concentration at which there is no significant mean difference between this and the positive control (i.e., the null hypothesis is to be satisfied) at p > 0.05. Oil samples on Leishmaniatropica and Leishmaniadonovani have more activity than methanol samples, especially A1, A2, A8, A9, C1, C2, C3, C4, C5, C8, and C9, in which p-values are more than 0.05, as in Table 5.

Sample number Concentration A B C D
P value 1 1 0.795 0.001 0.758 0.002
2 0.9 0.762 0.001 0.394 0.000
3 0.8 0.001 0.001 0.738 0.000
4 0.7 0.001 0.001 0.918 0.000
5 0.6 0.001 0.001 0.088 0.000
6 0.5 0.045 0.001 0.003 0.000
7 0.4 0.001 0.001 0.029 0.000
8 0.3 0.137 0.013 0.678 0.000
9 0.2 0.072 0.001 0.400 0.000
Table 5.P-Value of Each Concentration Gradient of Samples A, B, C, and D on Leishmania Tropicaand Leishmania Donovania: Oil Extract on Leishmania Tropica, B: Methanol Extract on Leishmania Tropica, C: Oil Extract on Leishmania Donovani, D: Methanol Extract on Leishmania Donovani.

In Figure 5, we can see the inhibition rate % IR against concentrations and compare the result with the positive control used.

Figure 5.% IR of Each Concentration Gradient of Samples A, B, C, and D on Leishmania Tropica and Leishmania Donovani A: Oil Extract on Leishmania Tropica, B: Methanol Extract on Leishmania Tropica, C: Oil Extract on Leishmania Donovani, D: Methanol Extract on Leishmania Donovani.

The IC50 of each analytical sample was calculated using the following procedure: At each of the five locations, inhibition ratios (y) were plotted against sample concentrations (x), and a regression line (y=ax+b) was generated. IC50 for oil and methanol extracts on Leishmaniatrropica was 0.042mg/ml and 0.1805 mg/ml, respectively. In comparison, the IC50 for oil and methanol extracts on Leishmaniadonovani was 0.00127mg/ml and 1.606 mg/ml, as shown in Figure 6.

Figure 6.The IC50 for Samples A, B, C, and Don Leishmania Tropica and Leishmania donovani.a: Oil Extract on Leishmania Tropica, B: Methanol Extract on Leishmania Tropica, C: Oil Extract on Leishmania Donovani, D: Methanol Extract on Leishmania Donovani.

B. Discussion

Owing to the exceptional diversity of chemical structures, natural products such as plant extracts, whether in the form of pure compounds or standardized extracts, provide immense promise for the discovery of innovative and targeted pharmaceuticals [26]. This research evaluated the antileishmanial efficacy of oil extract and methanolic (defatted) extract against Leishmania tropica and Leishmania donovani in comparison to pentostam, the preferred antileishmanial treatment. The findings of our investigation indicated that the growth rate of the promastigote forms of L. tropica and L. donovani was strongly suppressed (p>0.05) by Gundelia tournefortii, particularly its oil extract. The efficacy of plant extracts is influenced by several aspects, including the plant's attributes, its geographical origin, the climatic circumstances of its growth, the precise section of the plant used, and the extraction solvent employed. This is attributable to the varied plant ingredients, which may fluctuate based on various conditions. The chemical composition of G.tournefortii oil has been extensively studied. Its composition displays variability based on the geographic region of production and the extraction methods employed. However, terpenoid compounds are consistently found in most regions. Terpenoids have demonstrated antiparasitic effects on various Leishmania species [28]. In the current investigation, our findings reveal that the principal terpenoids present in G.tournefortii oil consist of lupeol, lupeol acetate, b-amyrin, and beta-amyron, all of which fall into the triterpenes category, as indicated in Table 2 in GC mass analysis. The antileishmanial activity of the oil is attributed to the triterpenes present. The leishmanicidal activity of triterpenes can be associated with significant changes in the mitochondria, nucleus, and intracellular compartments that may be linked to programmed cell death; through DISC3-mediated fluorometric analysis, it became evident that lupeol (triterpenes) exerts its leishmanicidal action by disrupting the cytoplasmic membrane of L. donovani promastigote [29]. Lupeol demonstrates significant biological potential against leishmaniasis, particularly Leishmania donovani, without exhibiting any toxic effects on normal cells. This contrasts with the toxic effects observed in amphotericin B and conventional treatment [30]. B-amyrin is also responsible for the antileishmanial activity of Gundeliatournefortii [31]. Several research studies have suggested that sterols exhibit antiparasitic properties, including stigmasterol, campesterol, and beta-sitosterol. Of particular note, beta-sitosterol demonstrates significant anti-leishmanial effects [32]

Conclusion

This work effectively extracted beta-sitosterol from the oil sample, representing the first occurrence of this accomplishment in Iraqi G. tournefortii, and measured it using a calibration curve at a concentration of around 98.8 ppm. Research indicates that oil derived from the aerial components of Iraqi G. tournefortii demonstrates superior antileishmanial efficacy compared to the methanol extract, attributable to its diverse array of triterpenes, including lupeol, lupeol acetate, beta-amyrin, and sterols such as stigmasterol, beta-sitosterol, and campesterol. These compounds have antileishmanial characteristics comparable to pentostam therapy, impacting both Leishmania tropica and Leishmania donovani. The results clearly indicate that Gundelia oil extract has significant potential for the development of antileishmanial pharmaceuticals.

Acknowledgement

The authors are grateful to the Pharmacognosy Department, College of Pharmacy at the University of Basra, for giving important assistance and facilities for the study

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