Leishmanicidal effects of resveratrol and its derivatives: a systematic review and meta-Analysis


 Background

Leishmaniasis is one of the most important health problems worldwide. The evidence has suggested that resveratrol and its derivatives have anti-leishmanial effects; however, the results are inconsistent and inconclusive. The aim of this study was to assess the effect of resveratrol and its derivatives on the Leishmania viability through a systematic review and meta-analysis of available relevant studies.
Methods

The electronic databases PubMed, ScienceDirect, Embase, Web of Science and Scopus were queried between October 2000 and April 2020 using a comprehensive search strategy. The eligible articles selected and data extraction conducted by two reviewers. Mean differences of IC50 (concentration leading to reduction of 50% of Leishmania) for each outcome was calculated using random-effects models. Sensitivity analyses and prespecified subgroup were conducted to evaluate potential heterogeneity and the stability of the pooled results. Publication bias was evaluated using the Egger’s and Begg’s tests. We also followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines for this review.
Results

Ten studies were included in the meta-analysis. We observed that RSV and its derivatives had significant reducing effects on Leishmania viability in promastigote [24.02 µg/ml; (95% CI 17.1, 30.8); P < 0.05; I2 = 99.8%; P heterogeneity = 0.00] and amastigote [18.3 µg/ml; (95% CI 13.5, 23.2); P < 0.05; I2 = 99.6%; P heterogeneity = 0.00] stages of Leishmania. A significant publication bias was observed in the meta-analysis. Sensitivity analyses showed a similar effect size while reducing the heterogeneity. Subgroup analysis indicated that the pooled effects of leishmanicidal of resveratrol and its derivatives were affected by type of stilbenes and Leishmania species.
Conclusions

Our findings clearly suggest that the strategies for the treatment of leishmaniasis should be focused on natural products such as RSV and its derivatives. Further study is needed to identify the mechanisms mediating this protective effects of RSV and its derivatives in leishmaniasis.


Results
Ten studies were included in the meta-analysis. We observed that RSV and its derivatives had signi cant reducing effects on Leishmania viability in promastigote [24.02 µg/ml; (95% CI 17.1, 30.8); P < 0.05; I 2 = 99.8%; P heterogeneity = 0.00] and amastigote [18.3 µg/ml; (95% CI 13.5, 23.2); P < 0.05; I 2 = 99.6%; P heterogeneity = 0.00] stages of Leishmania. A signi cant publication bias was observed in the metaanalysis. Sensitivity analyses showed a similar effect size while reducing the heterogeneity. Subgroup analysis indicated that the pooled effects of leishmanicidal of resveratrol and its derivatives were affected by type of stilbenes and Leishmania species.

Conclusions
Our ndings clearly suggest that the strategies for the treatment of leishmaniasis should be focused on natural products such as RSV and its derivatives. Further study is needed to identify the mechanisms mediating this protective effects of RSV and its derivatives in leishmaniasis.

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Leishmaniasis is a global health problem worldwide which refers to a broad range of disease including cutaneous lesion, muco-cutaneous and visceral forms. This disease is endemic in 98 countries with approximately 12 million infected cases and 350 million people in regions with catching risk of leishmaniasis [1]. The annually estimated incidence of cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) is 1-1.5 million and 500,000 cases, respectively [2]. CL caused by L. major, L. amazonensis, L. panamensis and L. tropica species and L. donovani, L. chagasi and L. infantum are associated with visceral leishmaniasis (VL). Leishmania parasite has digenetic life cycle which includes the extracellular promastigote in sand-y and the intracellular amastigote into the mammalian hosts.
Despite various studies such as genomic [3], proteomics [4,5], metabolomics [6] and protein network [7] analysis to identify proteins as new drug and vaccine targets for leishmaniasis treatment, no vaccine is still available for the disease. Currently, chemotherapy using pentavalent antimonials such as sodium stibogluconate (pentostam) and meglumine antimoniate (glucantime) is main and rst-line treatment method for leishmaniasis [8]. The functional mechanism of these drugs is not fully clear. However, some studies have suggested that these drugs led to reduction of ATP production through inhibition of glycolytic and oxidative activity of fatty acids [9]. Unfortunately, because of high toxicity, high cost and side effects of these drugs, and also, appearance of drug-resistant strains, the use of alternative therapies is essential [10]. From 2002, miltefosine has been used for visceral leishmaniasis in India but e cacy of this drug is also low against cutaneous leishmaniasis [11]. According to the limitation of chemotherapy and lack of effective vaccine against leishmaniasis, furthermore the search for new drugs with better effectiveness and without serious side effect is necessary [7]. The use of herbal medicines has recently gained popularity in the world, and medical capacity of these natural compounds has been repeatedly demonstrated in various in vitro and in vivo systems [12]. In several studies have been shown that resveratrol (3, 5, 4-trihydroxy-trans-stilbene) contain antibacterial [13], antiviral [14], and antiparasitic [15] properties. Resveratrol is a polyphenol micronutrients compound produced by different plant species including pines, berries, and peanuts which mainly found in the skin of grapes and red wine [16].
Resveratrol represents Cis and Trans isomeric forms in nature that biological activity of it associated with Tran's form [17]. Despite several studies about the potential activity of resveratrol against both promastigotes and amastigotes of Leishmania parasites [18,19], associated studies with antileishmanial activity of resveratrol still is in its infancy. Moreover, resveratrol or analogues could also be a novel potential drug in leishmaniasis management and need to be further investigated in future. Various surveys have been carried out to assess anti-leishmanial activity of resveratrol and its derivatives in the worldwide, however, considering the fact that these studies are individuals. One way of achieving a conclusive result is the formulation of a meta-analysis study, which mathematically combines and analyses the results of different studies to achieve a more reliable outcome [20]. However, in spite of previously researches about leishmanicidal effects of resveratrol and other stilbene derivatives, there has been no comprehensive review of published data. In the present study, we conducted the rst systematic review and meta-analysis for evaluating the anti-leishmanial activity of resveratrol and its derivatives.
The meta-analysis was conducted in accordance with the guidelines of the 2009 Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement [21].

Search protocol
We performed an exclusive search from October 2000 to April 2020 through Medline, Embase, Scopus, Science Direct, Web of Science, EMBASE, and Cochrane Library. All of eligible studies related to the leishmanicidal effects of resveratrol and its derivatives were selected. Furthermore, Gray literature and reference lists were reviewed to identify relevant studies. We searched databases using Mesh terms and keywords ("Leishmania" OR "leishmaniasis" OR "Leishmania species") OR ("L. major" OR "L. tropica" OR "L. infantum" OR "L. amazonesis" OR "L. braziliensis" OR "L. donovani" OR "L. aethiopica") AND ("Resveratrol" OR "resveratrol derivatives" OR "resveratrol analogs" OR "trans-resveratrol" OR "3, 5, 4'trihydroxystilbene"). Furthermore, Gray literature, Conference abstracts and reference lists were manually searched to identify potentially relevant studies. Searches were limited to the English language. Two reviewers (NA. D and MK) evaluated each article separately, any disagreement between extracted data were resolved by agreement and discussion with a third party (NA). Ethical approval and informed consent will not be applied for because of the relevant data we extracted which does not involve any individual privacy.

Selection Criteria Of Studies
Articles were selected carefully if they met the following criteria: report outcome (the effect of resveratrol and resveratrol analogues on promastigote and amastigote stages of Leishmania, English language, published studies in a number of internationally indexed journals and having su cient information and repetitive articles, studies without limitations in time and the form of resveratrol usage and also studies that reported the IC50 (The compound concentration causing 50% reduction in parasite viability) of resveratrol and its analogues and control's IC50. The lack of adequate details of study methodology were excluded.

Quality Assessment
A systematic assessment of bias in the included studies was performed independently by two expert authors using Newcastle-Ottawa Quality Assessment Scale (NOS) [22]. In cases of disagreement, a third author would examine such articles. Authors discussed the results comprehensively until they agreed on the accuracy and usefulness of data. The items used for the assessment of each study were composed of main three section coupled with questions within each section as follows: 1) selection, 2) comparability and 3) exposure. NOS score ≥ 5 were considered high quality.

Extracted Data Of Studies
We extracted data from the nal included articles using specially-designed data extraction Form. The form piloted and tested against 3 articles in which all authors will extract data from. The form modi ed according to this pilot test. Data extraction form initially including information about: the rst author, the year of publication, origin country, parasite species, type of stilbenes (resveratrol and its derivatives), dose of resveratrol and its derivatives (mg/ml), IC 50 value of resveratrol, IC50 values of resveratrol derivatives and exposure time, control's name, control's IC50 and quality assessment score. All extracted data summarized for promastigote and amastigote stages of Leishmania in Table 1 and Table 2, respectively.

Statistical analysis
We evaluated the mean and 95% con dence intervals of the half-maximal inhibitory concentration (IC 50 ) values by pooling the results from all the selected articles. Considering the existing heterogeneity among studies, we performed this meta-analysis using the random-effects model. In this meta-analysis we used from both Q-Cochran test (p < 0.1 indicate heterogeneity) and I 2 method (I 2 < 50% no heterogeneity and I 2 > 50% indicate heterogeneity) for detect heterogeneity. Publication bias was evaluated using Funnel plot and Begg and Egger tests. The trim-and-ll analysis was used to adjust any signi cant publication bias detected. To establish the possible sources of heterogeneity between the studies on meta-analysis outcome, we performed subgroup analyses based on Leishmania species. We also conducted sensitivity analyses to evaluate the in uence of individual articles on the pooled results. Meta-analysis was conducted using Comprehensive Meta-Analysis (CMA) V2 software (Biostat, NJ) [30].

Results
The results of literature search The search identi ed 97 records. These included 8 duplicate articles, 5 reviews and 1 editorial, which were removed, leaving 83 unique articles to be screened by title and abstract. Out of the 83 articles screened, 53 were excluded as they did not meet the eligibility criteria: 22 articles were performed on other parasites, 31 articles were not measured the leishmanicidal impact of resveratrol and its derivatives. The full text of 30 articles were then evaluated out of which 21 articles were excluded for the following reasons: i) incomplete reported data, and ii) brief report and review article. Finally, 9 studies met the inclusion criteria for meta-analysis (Fig. 1).

Characteristics Of Eligible Studies
We identi ed 9 studies that reported the IC 50 of leishmanicidal effects of RSV and other stilbenes along with its derivatives as an outcome. Tables 1 and 2  The quality assessment of studies included in our meta-analysis is summarized in Table 1 and Table 2.
All the included studies were con rmed by a low to high risk in each three parts of NOS checklist. Taken together, the quality of included studies was low to high.

Quantitative Data Synthesis
Leishmanicidal effects of resveratrol and its derivatives

Sensitivity Analysis
We performed sensitivity analysis, when any study was excluded from this meta-analysis. In the sensitivity analysis, we have found that the outcome (mean of IC 50 ) was not signi cantly changed by removing one study by means of "leave-one-out method" from the analysis compared to overall effect size for the leishmanicidal effects of resveratrol and other stilbene derivatives in promastigote [ (Fig. 4). Therefore, this pooled analysis outcome could be regarded with a higher degree of certainty.

Subgroup Analysis
Subgroup analysis was performed according to Leishmania species (L. major, L. amazonesis, L. braziliensis, L. donovani and L. infantum) and type of stilbenes (resveratrol and its derivatives and stilbenes derivative). In the subgroup analysis, signi cantly, increased levels of the leishmanicidal effects of resveratrol and its derivatives were observed in L.  (Table 3). Taken together, subgroup of resveratrol and its derivatives in both stages of Leishmania signi cantly reduced Leishmania viability.

Discussion
Leishmaniasis is one of the health problems worldwide, for reducing this parasitic disease, many strategies are considered including polyphenols such as resveratrol. Previous published studies have reported the con icting results regarding the leishmanicidal effects of RSV and other stilbenes in promastigote and amastigote stages of Leishmania. In total, nine eligible studies were included for promastigote and amastigote. In the current investigation, a combination of relevant studies via a systematic review and meta-analysis demonstrates that Leishmania viability signi cantly decreased after treatment with RSV and other stilbenes, in both promastigote and amastigote stages of Leishmania. Interestingly, we observed signi cant leishmanicidal effects of stilbenes types (RSV and other stilbenes) not only on Leishmania species but also in promastigote and amastigote stages of each parasite species.
In the present meta-analysis, the funnel plot, Begg rank correlation and Egger tests showed a potential publication bias among all studies included in this meta-analysis. Moreover, there is a signi cant heterogeneity in this meta-analysis. Therefore, in order to explore the obvious heterogeneity, subgroup analyses based on Leishmania species (L. major, L. amazonesis, L. infantum and L. braziliensis) and type of stilbenes (RSV and other stilbenes) were performed. Initially, we analyzed subgroups of Leishmania species and type of stilbenes on each stage of Leishmania separately. The results showed a signi cant increase in the leishmanicidal effects of RSV and other stilbenes in species of L. major, L. amazonesis and L. infantum of promastigote stage of Leishmania, while, the leishmanicidal effects of RSV and other stilbenes on amastigote stage of Leishmania in species of L. major and L. amazonesis were signi cant. In addition, subgroups of RSV and other stilbene derivatives in both of stages of Leishmania signi cantly reduced the Leishmania viability. Subsequently, these ndings con rm the leishmanicidal effects on promastigote and amastigote stages of Leishmania following treatment with RSV and other stilbene derivatives.
Resveratrol is a natural polyphenol with potential health bene ts. There is increasing evidence showing the bene cial effects of RSV on alleviating the pathological status of metabolic diseases [16]. Apart from that, resveratrol is effective for the treatment of leishmaniasis. As a result, our data in this meta-analysis demonstrated that Leishmania viability reduced after resveratrol treatment, which is in accordance with some of studies treated with resveratrol [23,24]. Thus, the ndings of this meta-analysis collectively implicate RSV and its derivatives as a potential agent in the diminishing of Leishmania viability.
Promastigotes are extracellular forms and amastigotes are intracellular forms of Leishmania parasites that resveratrol and its analogs effects on these two stages of Leishmania evaluated in several study. Leishmania have a dimorphic life cycle consisting of an extracellular agellated promastigote stage within the midgut of a sand y vector, and a morphological distinct intracellular amastigote stage within macrophages of a mammalian host [31]. Several studies have demonstrated that resveratrol and its derivatives have an anti-leishmanial activity [29,32]. Ferreira et al. (2014) reported that resveratrol have an anti-leishmanial activity against L. amazonesis in both promastigote and amastigote stages [24]. The study of Coimbra et al. (2016) with evaluation of leishmanicidal effects of resveratrol analogs showed that these compounds have different effects against Leishmania species [26]. To that end, Kedzierski et al. (2007) reported the effects of resveratrol and its hydroxylated analogues against L. major. Their results demonstrated the leishmanicidal effects of resveratrol on promastigotes and intracellular amastigotes stages while, its hydroxylated analogues only showed anti-leishmanial activity against promastigotes [23]. Moreover, Study on anti-promastigote activity of trans-stilbenes and terphenyl compounds have shown that one of trans-stilbene derivatives have anti-promastigote effect similar to pentostam as an important drug against leishmaniasis [17]. Tolomeo et al. revealed the antipromastigote and anti-amastigote activities of TTAS (Trans-3, 4 , 5-trimethoxy-3 -amino-stilbene) as one of the stilbene derivatives [29]. Likewise, in a study the effect of four resveratrol analogs including pterostilbene, piceatannol, polydatin and oxyresveratrol evaluated against L. amazonensis, and its results showed that among four RSV analogues the piceatannol analog could be potential compound in further studies for leishmaniasis treatment [25]. In this regard, our data support the leishmanicidal effects of RSV and other derivatives of stilbenes on promastigote and amastigote stages of Leishmania.
There are several possible mechanisms for the leishmanicidal effects of RSV and other stilbene derivatives on the promastigote and amastigote stages of Leishmania parasite. Of these, apoptosis mediated by phospholipids could be considered one of the mechanisms by which the Leishmania death process is initiated. Phospholipids are major types of lipids in membrane of eukaryotic cells. Among phospholipids, Phosphatidylserine (PS) has low abundance in most biological membranes and in normal condition it is present only in the inner plasma membrane of eukaryotic cells [29]. PS also plays a role in the infectivity of Leishmania [33]. A central point in the host-parasite interaction involves the adhesion to and invasion of host macrophages, by the metacyclic promastigotes and amastigotes. Both of the promastigotes and amastigotes use receptor-dependent phagocytosis for invasion. Furthermore, according to studies, exposure of PS on the cell surface of the parasite mimics apoptosis and encourages the macrophages in the host organism to phagocytose the parasite [34]. Investigations on amastigotes of Leishmania amazonensis showed that the signaling via exposed PS is a critical mechanism for Leishmania establishment in the mammalian host. The surface PS of amastigotes lead to inhibition of macrophage in ammatory activity. This lipid using interacting with macrophages, contributes in parasite internalization and induces an anti-in ammatory response by inhibition of macrophage NO activity, increasing IL-10 message and TGFβ-1 secretion [35]. In addition, treatment of infected mice with PS-targeting monoclonal antibody ameliorated parasite loads and lesion development and also enhanced dendritic cell (DC) activation and antigen presentation in vitro. Therefore, the recognition of PS exposed on the surface of amastigotes plays a role in down-modulating DC functions, in a matter similar to that of apoptotic cell clearance [36]. Also, it can be concluded that PS can modulate interactions between Leishmania organisms and host cells and may be key factor for the result of the clinical course of leishmaniasis.
In a study that conducted on the L. amazonensis, the promastigotes were treated or not with piceatannol and incidental death measured using the expression of annexin-V. The results showed that the piceatannol signi cantly increased the expression level of annexin-V compared to untreated control [25].
Another study on L. infantum promastigotes was shown that treatment of parasites with 10 µg/ml compound 3 (resveratrol derivate) for 48 h induced PS externalization suggesting that apoptosis activation is the cause of parasites death, while this compound has not shown apoptotic effects on macrophage cells [18]. Ferreira et al. (2014) reported a 2.9-fold increase in the percentage of annexin Vpositive L. infantum promastigotes upon resveratrol treatment which suggests leishmanicidal activity of resveratrol [24]. TTAS (Trans-3, 4 , 5-trimethoxy-3 -amino-stilbene) as a new stilbene derivate that induced PS externalization which sign apoptosis activation in L. infantum. TTAS showed an LD 50 on normal CFu-GM of 17.7 µg/ml more than 6 times higher than that showed on the L. infantum strain [37]. Results of several studies in recent years indicate stilbenes and its analogs altered cell cycle of different species of Leishmania parasite with increase of parasites in sub G0/G1 phase of cell cycle [15]. Presence of cells in sub G0-G1 phase indicates the occurrence of apoptosis. Other investigation showed that TTAS induced the arrest of Leishmania prevalently in G2-M phase of the cell cycle and an increase of the sub-G1 apoptotic peak [29].
The second mechanism mediated by stilbenes is mitochondrial-induced apoptosis. Leishmania and mammalians apoptosis share similar characteristics, such as internucleosomal DNA fragmentation, phosphatidylserine exposure on the external surface of the plasma membrane, and loss of mitochondrial transmembrane potential [38]. Since maintenance of mitochondrial transmembrane potential is essential for the survival of a single mitochondrion-parasite, studies of the effects of resveratrol on Leishmania spp., evaluated mitochondrial integrity of promastigotes testing the mitochondrial membrane potential.
Previous research ndings showed a reduction of mitochondrial membrane potential in parasites treated with resveratrol or it's derivate in comparison with untreated parasite populations [25].
Our meta-analysis has several advantages. First, this meta-analysis comprehensively summarizes the evidence data on an anti-leishmanial activity resveratrol and its derivatives. Second, the included studies were conducted in different countries that are including the high prevalence of leishmaniasis. Third, our search strategy was very detailed and spanned multiple databases.
This systematic review and meta-analysis have several limitations: First, due to the small number of included studies for stilbenes and its derivatives, we were not able to better evaluate the leishmanicidal effects of stilbenes and its derivatives. Second, we had a substantial heterogeneity between included studies which does not allow a reliable assessment, although we used random-effect models to combine the pooled mean of IC 50 among included studies. Third, the sample sources for eligible studies were countries which may differ in the prevalence of Leishmania. Finally, given the limitations the ndings should be interpreted with caution, because the reliability of this meta-analysis is limited by scarcity of large studies.  Figure 1 Flow chart of the number of studies detected and selected into the meta-analysis Forest plot evaluating mean of IC50 and 95% con dence intervals for the leishmanicidal effects of RSV and other stilbenes derivatives in A) promastigote and B) amastigote stages of leishmania. Metaanalysis was performed using a random-effects model with inverse variance weighting.

Figure 3
Random-effects Funnel plot detailing publication bias in the studies investigating the leishmanicidal effects of RSV and other stilbenes derivatives in A) promastigote and B) amastigote stages of leishmania after trimming and lling. Circles represent observed published studies; closed circles represent imputed unpublished studies.