|Year : 2019 | Volume
| Issue : 4 | Page : 330-338
Leishmanicidal, cytotoxic and apoptotic effects of Gossypium hirsutum bulb extract and its separated fractions on Leishmania major
Fatemeh Sharifi1, Iraj Sharifi2, Alireza Keyhani2, Amir Asadi-Khanuki3, Fariba Sharififar3, Mostafa Pournamdari4
1 Pharmaceutics Research Center, Institute of Neuropharmacology; Research Center of Tropical and Infectious Diseases, Kerman University of Medical Sciences, Kerman, Iran
2 Leishmaniasis Research Center, Kerman, Iran
3 Herbal and Traditional Medicines Research Center, Kerman, Iran
4 Department of Medicinal Chemistry, Faculty of Pharmacy, Kerman, Iran
|Date of Submission||10-Jul-2018|
|Date of Acceptance||29-Sep-2018|
|Date of Web Publication||30-Nov-2020|
Dr. Fariba Sharififar
Herbal and Traditional Medicines Research Center, Kerman University of Medical Sciences, Kerman, Postal code–76175-493
Source of Support: None, Conflict of Interest: None
Background & objectives: Leishmaniasis is a major global health problem with no safe and effective therapeutic drugs. This study evaluated the cytotoxic and apoptotic effects of crude extract and fractions of Gossypium hirsutum bulb on Leishmania major stages using advanced experimental models.
Methods: Bulbs of G. hirsutum were collected from the Kerman province of Iran. The bulb was extracted using Soxhlet apparatus and different fractions were obtained by column chromatography (CC). Different concentrations of the extract and the fractions were evaluated against L. major and compared with Glucantime®. The cytotoxicity and apoptotic values were analysed by flow cytometry. The fractions obtained in CC were monitored by thin layer chromatography, and fractions with similar chromatographic patterns were mixed.
Results: The extract and two fractions, F4 and F5 inhibited the proliferation of L. major promastigotes and amastigotes in a dose-dependent manner at 72 h post-treatment. No significant cytotoxic effects were observed for extract and fractions, as the selectivity index was over 1000, far beyond >10. The mean apoptotic values for L. major were superior to those of Glucantime®.
Interpretation & conclusion: Both the crude extract and fractions (F4 and F5) had significant antileishmanial effects on L. major stages, and were were superior relative to Glucantime®. No cytotoxic effects were associated with the extract or fractions and they showed excellent apoptotic index, a possible mechanism behind inducing parasite death. Further investigations are essential to study the effect of G. hirsutum bulb fractions in animal model and clinical settings for planning strategies for the prevention and control of leishmaniasis.
Keywords: Apoptosis; chromatography; cutaneous leishmaniasis; cytotoxicity; Gossypium hirsutum; leishmanicidal activity
|How to cite this article:|
Sharifi F, Sharifi I, Keyhani A, Asadi-Khanuki A, Sharififar F, Pournamdari M. Leishmanicidal, cytotoxic and apoptotic effects of Gossypium hirsutum bulb extract and its separated fractions on Leishmania major. J Vector Borne Dis 2019;56:330-8
|How to cite this URL:|
Sharifi F, Sharifi I, Keyhani A, Asadi-Khanuki A, Sharififar F, Pournamdari M. Leishmanicidal, cytotoxic and apoptotic effects of Gossypium hirsutum bulb extract and its separated fractions on Leishmania major. J Vector Borne Dis [serial online] 2019 [cited 2021 Jan 21];56:330-8. Available from: https://www.jvbd.org/text.asp?2019/56/4/330/302036
Among the important infectious diseases, leishmaniasis is considered the most neglected. According to recent statistics, over 1 billion people are at risk of contracting leishmaniasis in 101 countries. Cutaneous leishmaniasis (CL) is a skin disease caused by parasites of Leishmania species. Manifestations of CL range from self-healing ulcers, long-lasting skin lesions to disfiguring disease leading to permanent scars with significant social stigmatiza- tion. At present, CL is a major health problem, affecting approximately 0.7-1.2 million patients and exposing 414 million people at risk of infection in 82 countries across the world. The two most common epidemiological and clinical forms of leishmaniasis, which constitute nearly 70-75% of the cases, are anthroponotic CL (ACL) and zoonotic CL (ZCL), caused by Leishmania major and L. tropica, respectively, in the Old World,. Iran, with approximately 58% of the provinces affected by both ZCL and ACL, is among the seven most infected countries in the world.
Antimonial drugs, such as meglumine antimoniate (Glucantime®) and sodium stibogluconate (Pentostam®) are first-line chemotherapeutics for visceral leishmaniasis (VL), mucocutaneous (MCL) and CL and have been used for the past seven decades. However, these suffer from drawbacks such parenteral route of administration with long sessions, insufficient efficacy, and emergence of drug resistance. Moreover, the choice/use of alternative second-line chemical compounds, such as amphotericin B, pentamidine, paromomycin, azole derivatives, and allopu- rinol alone or in combination with the first-line drugs is also limited. In the absence of an effective vaccine, control of leishmaniasis is very difficult because of the presence of numerous biological vectors and reservoir hosts implicated in the life-cycle of the parasite and epidemiology of the disease. Currently, there is no single measure to control the disease comprehensively. Therefore, it is crucial to develop new natural medicines against the disease. Ongoing research investigations show that plant extracts and plant derivative complexes have less side effects, high availability, low cost, different mechanisms of action, topical route of administration, and shorter treatment courses,. New reports indicate that approximately 80% of the world’s population uses some kind of traditional remedies for their healthcare needs. Tremendous efforts have been made to isolate and analyse different biological metabolites, active constituents and lead molecules from plants and their derivatives,.
There has been an increasing social demand to use natural products, such as plant extracts, for healthcare due to the adverse effects of synthetic drugs. Several studies have reported the effects of herbal medicines on leishmaniasis,,,,,. To further explore the novel sources of antileishmanial agents, we studied the leishmaniacidal, cytotoxic and apoptotic effects of bulbs of Gossypium hirsutum, a species of cotton plant,. It belongs to the Malvaceae family and is an annual sub-shrub that is cultured primarily for its herbal seed fibre. The seeds and roots of this genus have been used in nasal polyps, uterine fibroids (as abortifacient) and other types of cancers. Gossypol, the toxic dihydroxyphenol present in G. hirsutum seeds exhibit anticancer activity in new LL, WA, and PS- 150 tumor system. The capsules of the plant called ‘bulb’or ‘Khuze’ have been used for treating CL in Iranian folk medicine.
In the present study, a different variety of G. hirsutum plant was used to assess the effect of bulb extract and its different fractions on the promastigote and amastigote stages of L. major by colorimetric and macrophage models, respectively. Cytotoxicity (CC50) of the extract and fractions were assessed by MTT powder [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The apoptotic value of the biological components was determined by flow cytometry.
| Material & Methods|| |
Plant material and chemicals
Fresh bulbs of G. hirsutum var. Varamin were collected between September 2015 and November 2016 from Orion County, Kerman province, southeast of Iran. The identity of the plant was kindly approved by Dr. Mirtadzadini, Department of Botany, Bahonar University, Kerman, Iran. A voucher specimen (KF131) of the collected plant was deposited at the Herbarium of Faculty of Pharmacy, Kerman University of Medical Sciences. Roswell Park Memorial Institute medium (RPMI-1640), fetal calf serum (FCS), penicillin/streptomycin, and MTT powder were purchased from Sigma-Aldrich (France). Meglumine antimoniate (Glucantime®, 99% purity), used as positive control, was provided by the Ministry of Health, Iran (manufactured by Sanofi-Aventis, France). Other materials used in this study were of high grade and prepared from original sources.
Preparation of extracts and control
Initially, fresh bulbs were dried in shade and used as the source for plant extract. Based on plant constituents and the method of preparation in folk medicine, the extract of G. hirsutum bulbs were extracted for 4 h using the Soxhlet apparatus, with chloroform. The obtained extract was filtered and concentrated by a rotary evaporator under vacuum. Finally, the extract was allowed to dry in oven at 40 °C for 48-72 h to obtain completely dried extract, and stored at -20 °C until testing. The extract and Glucantime® were diluted in the RPMI 1640 medium to obtain final concentrations of 1-1000 μg/ml according to the method described elsewhere.
Preliminary phytochemical screening
Preliminary phytochemical analysis ofthe extract was performed by qualitative and quantitative methods as previously described,.
Column chromatography: Approximately 80 g of activated silica gel (60-120 mesh) was dispersed into pure petroleum ether 80 to make the slurry for the stationary phase of column chromatography (CC). The slurry was immediately poured into a glass column (300 x 40 mm, containing a sintered-glass frit and a stopcock at the end) placed on a stable (without vibration) laboratory desk. The residue ofthe slurry was washed with the same solvent and transferred to the column. The stopcock ofthe column was opened, and the excess amount ofthe solvent was allowed to be drained until the stationary phase was settled. Clean sand (or grinded glass, ~ 1 mm in thickness) was added to the top of the stationary phase to provide a flat base at the top ofthe sorbent. The level ofthe solvent during drainage of slurry solvent, application ofthe sample, and elution steps were observed carefully (for stopping the fall below the level of adsorbent) to prevent the development of cracks in the adsorbent (which cannot be used for chromatography). Then, 1 g of grinded crude extract was loaded onto the top of the column until it was absorbed into the top of the stationary phase. Subsequently, another 1 mm thick sand layer was added to the top of the sample. The sand avoids the disturbance of the adsorbent, as fresh mobile phase is added to the column in the initial stages of development. Next, the column was eluted step-wise with 500 ml of petroleum ether: dichloromethane (100 : 0, 90 : 10, 70 : 30, 50 : 50, 20 : 80 and 0 : 100 % v/v); dichloromethane : t- butylmethylether (90 : 10, 80 : 20, 50 : 50, 80 : 20, 0 : 100 %v/v); t-butylmethylether : acetone (50 : 50, 0 : 100 % v/v); acetone : acetonitrile (50 : 50, 0 : 100 % v/v) and 750 ml ofacetonitrile : methanol (90 : 10, 70 : 30, 30 : 70, 0 : 100 % v/v).
Thin layer chromatography: Thin layer chromatog- raphy (TLC) was performed to monitor the eluted fractions resulting from column chromatography by UV light visualization and staining with detecting reagents. Approximately 10 μl of each sample was applied on the TLC plate and developed in two different solvent systems, chloroform : ethylacetate (9 : 1) and chloroform : ethylacetate (8 : 2). The developed plates were inspected under UV light at 254 and 365 nm and visualised after spraying with different reagents such as 2-aminoethyl diphenyl borate.
Leishmania major strain MRHO/IR/75/ER was obtained from Leishmaniasis Research Center, Kerman University of Medical Sciences. Promastigotes were sub- cultured in RPMI-1640, supplemented with 100 IU/ml penicillin and 100 μg/ml streptomycin at 25 ± 1 °C and 10% v/v heat-inactivated FCS for 30 min. It was kept at -20 °C until use. The multiplication rate of Leishmania promastigotes was observed every 24 h, and the number of parasites was determined by counting the parasites in a drop of medium (10 μl) using a Neubauer slide.
Murine macrophage cell line (J774-A1) was obtained from the Pasteur Institute of Iran, Tehran, Iran. The cells were cultured and maintained in RPMI-1640 DMEM, supplemented with Pen/Str, 10% FCS at 37 °C and 5% CO2.
The effect of crude extract and 12 separated fractions of G. hirsutum on promastigotes was evaluated by colorimetric cell viability MTT assay using the method described by Mahmoudvand et al. Briefly, 100 μl of promastigotes, containing 10 cells/ml, harvested from the logarithmic phase, was added to a 96-well microtitre plate. Next, 100 μl of various concentrations (1-1000 μg/ml) of each extract was added to each well and incubated at 25°C ± 1°C for 72 h. After incubation, 10 μl of MTT solution (5 mg/ml) was added to each well and plates were incubated under the same condition. Promastigotes were cultured in RPMI-1640 medium with no drug considered as untreated control, and medium with no promastigotes and drugs as blank. All the experiments were performed thrice. Finally, the absorbance was measured by an ELISA reader (BioTekELX800) at 490 nm. The 50% inhibitory concentrations (IC50 values) were calculated using the probit test and analysed by a SPSS software (Chicago, IL, USA).
Anti-intra-macrophage amastigote assay
Drug susceptibility of amastigotes in the macrophage cell line (J774-A1 cells, ECACC 91051511) was determined using the modification method of Chang. Before adding macrophages to the plates, 1 cm cover slips were placed in the wells of six-chamber slides (Lab-Tek, Nalge Nunc International, NY, USA). Afterwards, 200 μl of cells (10 cells/ml) was placed in each well. Following 2 h of incubation at 37 °C and 5% CO2, promastigotes at stationary phase was added to macrophages and again incubated under same conditions for 24 h. Free parasites were removed by washing with RPMI-1640 medium and infected macrophages were treated with 100 μl of various concentrations of G. hirsutum (1-1000 μg/ml) at 37 °C and 5% CO2 for 72 h. Finally, dried slides were fixed with methanol, stained by Giemsa and observed under a light microscope (Nikon, Japan). Macrophages containing amastigotes with no extract and murine macrophage medium with no parasite and extract were considered as untreated and negative controls, respectively. Glucantime® was used in similar manner as the positive standard drug (first-line drug). Anti-intra-macrophage amastigote activity of the extract was evaluated by the mean infection rate (MIR) of macrophages obtained by counting the number of intra-macrophage amastigotes in each macrophage by examining 100 macrophages. All the experiments were repeated in triplicate.
Cytotoxicity effects on macrophages
We determined the CC50 (cytotoxicity concentration for 50% of cells) of G. hirsutum on macrophages to evaluate the cytotoxicity on murine macrophages. Macrophage cells were plated at 10 cells/ml in a 96-well Lab-Tek (Nunc, USA) and allowed to adhere for 24 h at 37 °C and 5 % CO2. After removing the non-adherent cells by washing with RPMI 1640 medium, the cells were incubated under similar conditions as previously mentioned. Afterwards, 190 μL of complete RPMI-1640 medium was added to each well, and 10 μL of extract dilutions was added. Macrophages were incubated with extract concentrations ranging from 10 to 1000 μg/ml for 72 h. The cytotoxicity rate was evaluated using the colorimetric assay with MTT test as previously defined in the promastigote susceptibility assay. All experiments were performed thrice.
Apoptosis was studied using the annexin-V FLUOS staining kit according to the manufacturer’s instructions. The L. major promastigotes were washed in cold phosphate-buffered saline (PBS) twice and centrifuged at 1,400 g for 10 min. Subsequently, they were incubated for 15 min in the dark at room temperature (25 °C) in 100 μL of annexin-V FLUOS in the presence of 7-aminoactinomicin D (7AAD). Different concentrations of the extract, Glucantime® (1, 10, 100 and 1000 μg/mL) and untreated control were used.
All experiments were performed in triplicate. Data were analysed using SPSS statistical package version 17.0. Differences between the test and control groups were analysed by t-test and ANOVA. In addition,p <0.05 was defined as statistically significant. The IC50 values were calculated by PROBIT test in SPSS. Differences between the IC50 values in two stages of L. major growth were analysed by t-test. The selectivity index (SI) was calculated at various concentrations based on the following equation of CC50 for peritoneal macrophage: cells/IC50 = SI >10 was considered non-toxic.
The study protocol (No. 94/638) was approved by the Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman, Iran
| Results|| |
Preliminary phytochemical screening of chloroformic extract of G. hirsutum indicated the presence of tannins, saponins, alkaloids, terpenoids, flavonoids and antraquinones [Table 1].
|Table 1: Preliminary phytochemical screening of Gossypium hirsutum bulb extract|
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Column chromatography: The CC system, loading of extract, elution with different solvents, and separation of different bands and excluding the extract elution were performed. The separated fractions were collected in sample tubes. A total of 295 fractions were found during CC that were monitored by TLC, and fractions with similar chromatographic patterns were mixed. Finally, 12 fractions with different compositions were obtained. These fractions were concentrated using a rotary evaporator at 40 °C and then poured into a pre-weighed beaker and evaporated to dryness under a fan.
Initially, the activity of G. hirsutum extract on L. major promastigotes was evaluated. The parasites was incubated with varying concentrations of G. hirsutum (1-1000 μg/mL) for 72 h. G. hirsutum extract (p <0.001) significantly decreased L. Major promastigote viability in a dose-dependent manner [Figure 1]. Compared with the untreated control group, the inhibitory effect was observed at several concentrations, and the overall IC50 value was 204.9 μg/ml for crude extract and 126.4 μg/ml and 184.6 μg/ml for fractions F4 and F5, respectively. Although, not significant, as evident from the IC50 values, the inhibitory effect of G. hirsutum extract and two fractions (F4 and F5) was superior to that of the positive control (Glucantime®) [Table 2]. The inhibitory activity of G. hirsutum extract and two fractions were also assessed in comparison to positive (Glucantime®) and negative (untreated control) groups. Similarly, data confirmed their potent activity in a dose-dependent manner when compared with both the control groups [Figure 2].
|Figure 1: Comparison of mean optical density of different separated fractions of G. hirsutum (F), crude extract (CE), Glucantime® (G) as positive control and untreated control (UN) against L. major promastigotes at (a) 10μg/ml; (b) 100 μg/ml; and (c) 1000μg/ml.|
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|Table 2: IC50 values of G. hirsutum extract, effective fractions and Glucantime® (positive control) against the growth of promastigotes and intra-macrophage amastigotes of Leishmania major|
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|Figure 2: Cell viability percentage of different fractions of Gossypium hirsutum (F), crude extract (CE), Glucantime® (G) as positive control and untreated control (UN) against promastigotes of Leishmania major at concentrations: (a) 10μg/ml; (b) 100 μg/ml; and (c) 1000μg/ml.|
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Anti-intra-macrophage amastigote activity
Similar to the promastigote stage, G. hirsutum extract and fractions demonstrated a significant leishmanicidal activity against intracellular amastigotes in a dose-dependent manner (p <0.001) compared with the untreated control and Glucantime® [Figure 3]. Such potent activities were confirmed by the IC50 values of G. hirsutum extract (18.9 μg/mL), F4 (16.3 μg/mL) and F5 (21.3 μg/mL) compared with 85.5 μg/ml for Glucantime® (Table 2).
|Figure 3: Cell viability percentage of different fractions of Gossypium hirsutum (F), crude extract (CE) and Glucantime® (G) as positive control and untreated control (UN) against amastigotes of Leishmania major at concentrations: (a) 10μg/ml; (b) 100 μg/ml; and (c) 1000μg/ml.|
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Cytotoxicity effect on macrophages
Cytotoxicity of the G. hirsutum extract was evaluated on murine macrophages (J774 cells). In vitro susceptibility assay displayed no significant cytotoxic effect (981. 1 μg/ml and 624.9 μg/ml for G. hirsutum and Glucantime®, respectively) [Figure 4]. The SI was calculated for G. hirsutum extract, two effective fractions and Glucantime®, which was >10 (actual value >1000 μg/mL) for mammalian cells for crude extract and factions, whereas it was 7.3 for Glucantime® (Table 2).
|Figure 4: Cell viability of different concentrations of Gossypium hirsutum extract against murine macrophages (J774 cells) in comparison with Glucantime® as positive control and untreated control.|
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Flow cytometry was performed after labeling with annexin-V FLUOS and the levels of viable and apoptotic cells were determined for each concentration of the extract, with Glucantime® as positive control. Fluorescein- conjugated annexin-V is used to detect the externalised phospholipid classes as it has a high binding affinity to this phospholipid component. Moreover, annexin-V FLUOS allows characterisation of apoptotic and surviving cells. The levels of apoptotic promastigotes using 1 to 1000 μg/ ml of G. hirsutum extract were 48.11, 51.9, 50.08 and 56.86%, whereas these values for Glucantime® were 37.77, 45.35, 50.14 and 57.56% in the presence of annexin- V and 7AAD, indicating that the extract and Glucantime® had similar apoptotic and necrotic activities in comparison to the untreated control. Fractions (F4 and F5) were only tested at 100 μg/ml and had apoptotic activity similar to that of crude extract [Figure 5]a and [Figure 5]b.
|Figure 5: (a): Flow cytometry analysis of promastigotes treated with the Gossypium hirsutum extract and then labeled with annexin-V and 7AAD – (i) Untreated control without extract or any drug. (ii); (iii); (iv); and (v) Different concentrations of G. hirsutum extract (1000, 100, 10 and 1 μg/ml, respectively); (vi) and (vii) F4 and F5 fractions at concentration 100 μg/ml; (b) Flow cytometry analysis of promastigotes following treatment with Glucantime® and labeled with annexin-V and 7AAD — (i) Untreated control without any drug; (ii); (iii); (iv); and (v) Different concentrations of Glucantime® (1000, 100, 10 and 1 μg/ml, respectively); and (c) Apoptotic and necrotic values for promastigotes in the presence of different concentrations (1–1000 μg/ml) of G. hirsutum extract (CE) and concentration of 100 μg/ml of two effective fractions (F4 and F5) in comparison with positive control (Glucantime®) (G) and untreated control (UN) and annexin-V and 7AAD.|
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| Discussion|| |
This is the first study on the leishmanicidal activity of G. hirsutum extract that demonstrated marked inhibitory effects, and was more potent than that of the first-choice drug, Glucantime®. We had previously performed a preliminary screening work using different varieties of G. hirsutum bulb (Khordad var.), collected in November 2007 from Sabzevar County in Khorasan Razavi Province, northeastern Iran. Previously, the activity of the crude extract was only performed on promastigotes ofL. major. The chloroform extract displayed fairly good index of activity. Moreover, this product demonstrated a remarkable anti-cancer activity against several cell lines. Plant extracts are well known for their biological activities such as antimicrobial, antifungal, antioxidant and anticancer activities. In the present study, we studied the activity of chloroform extract and its fractions on L. major promastigotes and the clinical stage (Leishman bodies) by experimental models. In addition, cytotoxicity, apoptosis and necrotic values were described.
The extract inhibited the growth of L. major promastigote and amastigote stages in a dose- dependent manner at 72 h post-treatment. The IC50 values, representing antileishmanial effect, were 96.3 μg/ml and 18.9 μg/ml for promastigotes and amastigotes, respectively. Importantly, these biological activities were not attributed to cytotoxic effects, as no CC50 in access of the untreated control group was obtained. The SI was 51.9, which represented an extremely safe value to be used in higher models,. Therefore, there would be no serious adverse effects associated with the regular use of the extract in a preclinical murine model or clinical setting.
The overall leishmanicidal potency of bulb extract was higher than that of Glucantime® for both promastigotes (96.3 μg/ml vs. 204.9 μg/mL, respectively) and amastigotes (18.9 μg/ml vs. 85.5 μg/mL, respectively). In the present experimental study, the activity of bulb extract in intra- cellular amastigotes was significantly more profound when compared with the extracellular form of the organism (promastigote). This finding is consistent with numerous previous results reported by the other investigators who demonstrated that promastigotes of Leishmania species are significantly less susceptible to pentavalent antileishmanial agents than the respective amastigotes,,. The exact mechanism for such difference is not clearly defined. A possible explanation could be biochemical and physiological nature of two stages with respect to sensitivity to drugs. In general, it appears that the insensitivity of extracellular form of the organism to drugs compared with the intracellular stage at the same concentration was multifactorial. Moreover, such susceptibility could be in part related to the accumulation of product as sequestered by the harboring phagocytic cells.
In the present study, L. major promastigotes were treated with varying concentrations of the extract and positive control. We investigated the role of phosphati- dylethanolamine (PE) by flow cytometry using annexin V and 7-AAD, respectively. The results demonstrated that the mean apoptotic values for the bulb extract were higher than those ofthe positive control (Glucantime®). A negligible effect was recorded for untreated control (background effect). As obviously exhibited, the inhibitory effect of the bulb extract and F4 and F5 fractions was partially mediated through apoptosis, as evident from the externalisation of PE, in part a possible mechanism of parasite death. Although, the mechanism of action remains elusive, it seems that apoptosis induced by the bulb extract of G. hirsutum as anti-proliferative effect could play a crucial role in promoting parasite death.
A previous study has reported anti-cancer and antioxidant activities of the G. hirsutum bulb extract. Moreover, the extract contains high levels of antioxidants along with potent anti-cancer activity. The findings in this study likely showed that the extract possessed polyphenol constituents which might, in part, enhanced the antioxidant effect. Antioxidants consist of diverse groups of natural products, such as vegetables, fruits and plant metabolites that have a broad range of biological effects against cancer, microbial infections and degenerative disorders. The role of G. hirsutum bulb components as a potent antioxidant combined with other properties in inhibiting L. major stages provided a rationale for an immunomodulatory role to shift the immune response towards T helper 1 (Th-1) cells. This mechanism in turn contributes to parasite death, which could be another possible mode of action.
At present, the effect of synthetic therapeutic drugs against leishmaniasis is limited by serious toxicity, high cost, emerging resistance and insufficient therapeutic modality. There is a great interest in developing new medicinal plant products that are effective and safe with higher potency, simple route of administration, with different mechanisms of action and abundant and access in countries where leishmaniasis is endemic.
| Conclusion|| |
In conclusion, the present study demonstrated significant antileishmanial effects by G. hirsutum extract against promastigote and amastigote stages of L. major (superior to the drug of choice, Glucantime®). Furthermore, the findings revealed that the extract displayed no cytotoxicity with fairly good index of apoptotic values. Therefore, we highly suggest further experimental works to isolate, analyse and identify the constituents of G. hirsutum extract to evaluate the biological activity in animal models and clinical settings.
Conflict of interest: None
| Acknowledgements|| |
The study received financial support from the Vice Chancellor for Research, Kerman University of Medical Sciences, Kerman, Iran. The authors would also like to thank the Kerman Leishmaniasis Research Center personnel for their help in performing the study.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]