Iran J Parasitol: Vol

Iran J Parasitol: Vol. 14, No. 2, Apr-Jun 2019, pp.258-268

Iran J Parasitol

Tehran University of Medical Open access Journal at Iranian Society of Parasitology Sciences Public a tion http://ijpa.tums.ac.ir http://isp.tums.ac.ir http://tums.ac.ir

Original Article

Metabolomics Based Study of the Antileishmanial Activity of Xanthium strumarium Leaf Extract on Promastigotes Phases of Leishmania major by Proton NMR Spectroscopy

Mohammad AHMADI 1,2, Ziba AKBARI 1, Mahbobeh ALIKHANI 1,2, Reza HAJHOSSIANI 2, Zahra ZAMANI 1, *Mohammad ARJMAND 1

1. Metabolomics Laboratory, Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran 2. Department of Biological Sciences, Payam Noor University, Tehran, Iran

Received 16 Mar 2018 Abstract Accepted 20 Jul 2018 Background: Xanthium strumarium L. is extensively used as a traditional herb to treat many diseases and is also known as a source of phytochemicals. It has been used traditionally to treat trypanosomiasis, malaria fever, eczema, cancer, ulcer, fe-

Keywords: ver, herpes headache, and skin lesion such as leishmaniasis. In this preliminary Xanthium Strumarium; study, nuclear magnetic resonance (NMR)-metabolomics approaches was used to Leishmania major; evaluate the inhibitory effects and metabolic alterations caused by leaf extract of X. NMR spectroscopy; strumarium on the stationary phases of promastigotes in Leishmania major. Metabolomics Methods: The promastigotes were cultured in Biochemistry Laboratory at Pasteur Institute of Iran in 2017, stationary phases were obtained from 5 to 6 day-old cultures and treated with different concentrations of the plant’s extract. Antileishma- 1 nial activity was assayed by MTT method and cell metabolites were extracted. H *Correspondence NMR spectroscopy was applied, and outliers were separated using multivariate sta- Email: tistical analysis. [email protected] Results: The most affected metabolic pathways in the experimental groups were limited to amino sugar and nucleotide sugar metabolism, cyanoamino acid metabolism, starch and sucrose metabolism, butanoate metabolism, and galactose metabolism. Conclusion: The ethanolic leaf extract of X. strumarium is a potent growth inhibitor of Leishmania major and can affect vital metabolic pathways of Leishmania promastigotes. The assay provided new perspectives on the development of novel treatment strategies for leishmanial activity derived from natural products.

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Introduction

eishmaniasis, the infection caused by their carboxylic skeletons, into xanthanolides several species of intracellular protozoa and guaianolides. STLs are believed to be the L of the genus Leishmania, is identified as active metabolites of X. strumarium and have a neglected health problem by WHO, especial- caused considerable interest because of the ly in tropical and subtropical regions. These broad range of their biological activities. Ar- infections exist in two clinical manifestations, temisinin for the treatment of malaria, xan- visceral and cutaneous leishmaniasis (1). Cuta- thatin and xanthinosin for antitumor activity, neous leishmaniasis (CL) is the most prevalent parthenolide for treating migraine and clinical form worldwide with 0.7 to 1.3 million thapsigargin for curing prostate cancer all be- new cases each year and annually causes long to terpenoids classes (8). 20,000 infection cases in Iran alone (1, 2). Leishmania parasites undergo fluctuating en- Since 1923, the chemotherapy based on pen- vironmental conditions in their life cycle; tavalent antimonials (SbV) has been the first- promastigotes proliferate in the mildly alkaline line treatment for leishmaniasis, which is far and glucose-rich environment of the midgut from satisfactory. Alternative drugs such as of sandfly and amastigotes grow in the acidic amphotericin B (amphoB), pentamidine, and glucose- poor environment of the phago- miltefosine, and drug combinations have also lysosome of macrophages. There were signifi- been recommended (3). The first indications cant changes in parasite metabolism during of drug resistance were reported in the early these developmental stages (9). The altered 80s first in India and then in Nepal, in patients metabolic fluxes undoubtedly reflect adapta- who had relapsed (4). The toxicity and the tion to differing challenges that Leishmania high cost of the available drugs due to drug naturally confronts in its two hosts. Glycolysis resistance, teratogenicity and the need for the rather than gluconeogenesis is more important hospitalization of patients have resulted in the in promastigotes than in amastigotes. Differ- loss of drug effectiveness (5). entiation of procyclic promastigotes to infec- Therefore, due to the mentioned reasons, tive metacyclic promastigotes involves chang- there is an urgent need to develop safer and es in membrane fluidity in lipophosphoglycan more effective antileishmanial drugs with high (LPG) and glycerolipids structures (10). efficacy. Folk medicine based on natural Antimonials have a complex multifactorial plants is used by approximately 80% of the mode of action such as an inhibitory effect on world population. It contains about 25% of trypanothione reductase of the parasite, and modern medicine, widely applied in the treat- sodium stibogluconate [Sb(V)] inhibits fatty ment of anti-parasitic diseases (6). acid oxidation, glycolysis, and energy metabo- Xanthium strumarium L. (Cocklebur) is an an- lism (11). nual weed, including 25 species belonging to Metabonomics is one of the omics technol- the Asteraceae family, which grows in Iran ogies, which is regarded as one of the func- between August and September with local tional responses of biological systems to path- common name, Tough or Zardineh X. stru- ophysiological stimuli or genetic modifications marium is traditionally used to treat trypano- and is relevant to drug resistance studies (12). somiasis, malaria fever, eczema, cancer, ulcer, It is defined as a promising approach to study fever, herpes, headache and skin sores such as metabolic alterations within living samples leishmaniasis (7). The species contain a class after drug treatment (13). In fact, this ap- of plant terpenoids called Sesquiterpene lac- proach directly links the metabolic profile of tones (STLs) which are classified based on an organism to its corresponding genomic

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profile, which is widely used to find new tions was applied to calculate the number of pharmaceutical compounds (12). total polyphenols. Measurements were done in Due to the lack of effective chemotherapy, tetraplicate (14). the emergence of drug resistance and the growing interest in plant compounds for the Leishmania parasites treatment of parasitic diseases, this preliminary Amastigote forms of L. major (strain study aimed to explore the inhibitory effects MRHO/IR/75/ER) were originally isolated of X. strumarium leaf extract and compare the from infected Balb/c mice followed by trans- metabolome fingerprint alterations on station- formation into promastigote forms in RPMI ary phases of promastigotes in L. major treated 1640 medium supplemented with 10-20% with this substance. heat-inactivated fetal bovine serum(FBS), 100 µg/ml streptomycin, and 100 U/ml penicillin Materials and Methods G at 23-26 ºC (Sigma Aldrich, St. Louis, MO, USA). To perform leishmanicidal assays, Sta- Plant material tionary-phase promastigotes were obtained from 5 to 6 day-old cultures and centrifuged at Leaves of X. strumarium were collected from o August- September 2014 from around Ker- 3000 rpm for 10 min at 4 C (15). manshah City, Kermanshah Province, Iran. Voucher specimens were authenticated by Assay of leishmanicidal activity Central Herbarium of Tehran University The leishmanicidal assay was carried out (TUH) under voucher specimen number following a protocol (16). The promastigote forms, in stationary phase, were seeded at 48241. 5 27°C for 24 h at 2×10 promastigotes/well in Preparation of crude plant extracts a 96-well microplate. The stock solutions of The dried and powdered leaves of X. stru- each of the test samples (150 µg/mL) were marium (40 g) were exhaustively macerated added and serial dilution was carried out with with an alcohol-water solution (80%) at room RPMI medium. After 48 hours, promastigotes temperature for 72 hours. This mixture was viability was calculated by tetrazolium-dye filtrated and concentrated under reduced pres- (MTT) colorimetric method and absorbance sure to obtain a light-green extract. The result- was measured at 545 nm in the presence of ing extract was mixed with active charcoal, reference length wave 630 and the percentage centrifuged to isolate plant pigments and of the viability of the promastigotes was stored at -20 0C until required for bioassay. measured according to IC50. The results were expressed according to the amount of the ex- Determination of total polyphenol content tract concentration (15-0.000015 µg/mL) re- Total phenolic content was determined us- quired to reduce the absorbance to half (IC50) ing the Folin-Ciocalteau reaction (2014). The that of the negative control wells. calibration curve of gallic acid standard solu-

Cell extraction for metabolite analysis Amphotericin B(0.5 mg/ml) and pro- Stationary promastigotes (initial concentra- mastigotes with no drugs were used as position 1×109 parasites/vial) were treated with tive and negative controls, respectively IC50 concentrations of leaf extract (0.003 mg/ml-1 leaf extract/2×105parasites). After

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Ahmadi et al.: Metabolomics Based Study of the Antileishmanial Activity of …

incubation at 25 oC for 72 hours, the cells pervised technique to visualize the class- were centrifuged at 4000 rpm for 10 min at 4 specific segregation and to obtain the signifi- oC. Then pellets were harvested for the extrac- cant bins contributing to the variation across tion process of metabolites for 1HNMR spec- the classes, was used. This, in turn, led to the troscopy. In brief, pellets (1×109 pro- identification of the metabolites correspond- mastigotes/vial) were resuspended in RPMI ing to the spectral bins using Human Metabo- 1640 without FBS, centrifuged at 15000 rpm lome Database (HMDB) and LeishCyc data- for 15 min at 4oC and washed twice with PBS. base, which is the pathway/genome database 1.0 ml of chilled perchloric acid (1.8 M) was for L. major. Metabolic pathways were also added to the cell suspension, vortexed and investigated by the help of Metaboanalyst sonicated for 5 min at 4oC followed by cen- (v3.0) online software package. trifugation at 12000 rpm at 4 oC for 10 min. The pH of the supernatant was adjusted to 6.8 Results and it was kept on ice for one hour to allow the precipitation of potassium perchlorate and Total polyphenol content then was centrifuged again as above. The su- The total phenolic content (TPC) of leaf ex- pernatant was lyophilized (17). tract of X. strumarium was equated to 0.150 µg/ml. 1HNMR Spectroscopy Before NMR acquisition, lyophilized pow- Antileishmanial assay der samples (n=10) were resuspended in D2O X. strumarium stock extract ( 150 µg/mL) containing trimethylsilyl propionate (1mM) as showed the highest anti-promastigote activity, a chemical shift reference (δ=0 p.p.m.) and while a weaker inhibitory activity against this imidazole (2 mM) as pH indicator (δ=5.50 to parasite was observed in other concentrations. 8.80 p.p.m.). Samples were centrifuged (18000 In comparison, amphotericin B presented an rpm for 30 min at 4 oC). 500 µl of the extract- inhibitory activity almost similar to the stock ed samples were transferred to a NMR tube, fraction. Concerning the antileishmanial ac- analyzed on a Bruker AV-500 NMR spec- tivity, we observed that only one concentra- trometer with field gradient operating at tion could be considered as having promising 500.13 MHz for proton observation at 298K. activity with an IC50 value of 0.15 µg/ml One-dimensional 1HNMR spectra were rec- treating culture of stationary promastigotes orded using a 10-µs pulse, 0.1 s mixing time, (Table 1). There was a dose-dependent de- 3.0 s relaxation delay, 6009.6 Hz spectra width, crease in the percentage of viability. and 3000 transients with standard 1D NOE- SY pulse sequence to suppress the residual Effect of leaf extract of X. strumarium on water peak (18). metabolome profile 1D NMR assay showed that many metabolic Data analysis pathways were influenced by leaf extract of NMR spectra were preprocessed using cus- X.strumarium. In the current study, the class- tom written ProMatab function in MATLAB specific segregation can be refined using PLS- (v.7.8.0.347) environment. Following the DA. This aided in the visualization of the data standard processing steps, each 1D spectra as scores plot (Fig. 1). Outliers were also sepa- were aligned and binned in 0.005 ppm and rated by the use of loading plot (Fig. 2). Im- selected signals arising from water at 4.7 ppm portant features were identified by PLS-DA as were removed (19). Partial Least Square - Dis- variable importance of projection (VIP) criminant Analysis (PLS-DA), which is a su- (Fig.3).

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Table 1: In vitro viability of L. major freidlin promastogotes against Xanthium strumarium extract activity

Concentration(µg/ml) Promastigote viability (%) (Test- blank/negative control*100) ` Xanthium Strumarium Amphotricin B (Positive control) Negative Control 85 80 150(Stock) 9 3 15 23 20 1.5 36 28 0.15 56 49 0.015 62 58 0.0015 76 76 0.00015 83 80 0.000015 82 79

Fig. 1: PLS-DA scores plot of stationary Fig. 2: PLS-DA loading plot of stationary phases of L.major 1HNMR. One-color dots phases of L.major.This plot shows the relative represent the sample available in a group. Red contribution of bins/spectral variables to the triangle = L.major –untreated and green cross clustering of experimental and control groups. = L. major-treated. The explained variance are Each point in the figure represents a bin. The shown in brackets loading [2] axis represents the correlation of the bin towards the predictive variation shown in Figure 1. The loading [1] axis represents the magnitude of the spectral bins

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Ahmadi et al.: Metabolomics Based Study of the Antileishmanial Activity of …

Pathway analysis Human Metabolome Database (HMDB) and LeishCyc were used to identify metabolites corresponding to the spectral bins. Also, metabolic pathways relating to these separated outliers were determined by generic databases, such as KEGG pathway database and Metaboanalyst online pathway analysis tools for metabolomics (Table 2 and Fig. 4). Five pathways with p value less than 0.5 were used in our

Fig. 3: A VIP variable score is the measure- discussion and other pathways were kept ment of the variable importance in PLS-DA aside. model. The red color indicates the increase, and the green color stands for the decrease in variable concentrations

Fig. 4: Topology map of altered biochemical pathways in experimental and control group according to the degree of centrality. The geometric position of each node in the topology map is presented by - Log(p)

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Table 2: Metabolome pathway analysis results

Metabolic pathway Metabolites Total Hits Raw p Amino sugar and nucleotide sugar metabolism D-Fructose, 21 6 0.349 Glucosamine6- phosphate, uridine diphosphate galactose, Fructose 6- phosphate, N-Acetyl-D- Glucosamine6- phosphate Cyanoamino acid metabolism L-Aspartic acid, 6 2 0.419 L-Asparagine Starch and Sucrose metabolism Glucose1- phosphate, 6 2 0.419 D-Fructose Butanoate metabolism 3-Hydroxybutyric acid, 11 3 0.483 Butanol 1- Butanol Galactose metabolism Glucose1-phosphate, 7 2 0.506 Uridine diphosphate galactose Phenylalanine, tyrosine and tryptophan biosyn- L-phenylalanine 4 1 0.650 thesis, Pantothenate and CoA biosynthesis, Pantothenic acid 10 2 0.711 L-valine Synthesis and degradation of ketone bodies 3-Hydroxy butyric acid 5 1 0.731 Glycerolipid D-glyceraldehyde 11 2 0.762 Cysteine and methionine metabolism Cysteic acid 22 4 0.786 S-Adenosyl homocyste- ine L-Serine L-Cystathionine Alanine, aspartate and glutamate metabolism Aspartic acid 17 3 0.791 L-Asparagine glucosamine 6- phosphate Pentose and glucuronate interconversions glucose -1- phosphate 6 1 0.794 Valine, leucine and isoleucine biosynthesis L-Valine 6 1 0.794 Aminoacyl-tRNA biosynthesis L- Asparagine 46 8 0.873 L- Histidine L-Phenylalanine L- Arginine L- Aspartic acid L-serine L-valine L-Lysine

Since, many pathways were tested at the tiple testing. Total is the total number of same time; the statistical p values from en- compounds in the pathway; Hits, the actually richment analysis are further adjusted for mul- matched number from the user upload data;

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Raw p, the original P value calculated from the cose and fructose uridine diphosphate metab- enrichment analysis by using Metaboanalyst olites in amino sugar and nucleotides sugar (v.3) online software. metabolic pathways. Collectively, it is implied that the plant ex- Discussion tract, by affecting these metabolites, causes complex glycocalyx destruction, which in turn The toxicity profile of antimony justified the causes protective surface coat formation and search for safer drugs in treating leishmaniasis. mediates essential host-parasite interactions. Antimony drugs inhibit energy metabolism In the present study, the major alterations and macromolecular biosynthesis via inhibi- of amino acids in the cyanoamino metabolic tion of glycolysis and fatty acid beta-oxidation pathway were confined to two metabolites, (20). 1HNMR spectroscopy comparison of namely aspartic acid and asparagine. In an- metabolome profile of L. major promastigotes other investigation, to find a novel drug, there treated with leaf extract of X. strumarium were 97 unique metabolic reactions in L. do- showed that there were important variations novani, which cyanoamino acid was one of in some levels of individual metabolites and them (24). Asparagine amino acid had an in- their corresponding pathways. The most sig- hibitory effect on the autophagosome path- nificantly altered pathways between the two ways of Leishmania (25). Increasing the groups were amino sugar and nucleotide sugar amount of asparagine and decreasing the metabolism, cyanoamino acid metabolism, amount of aspartic acid by inhibiting asparagi- starch and sucrose metabolism, butanoate me- nase enzyme, could enhance the inhibitory tabolism, and galactose metabolism. effect of phagolysosome fusion and conse- Leishmania parasites contain a variety of nu- quently inhibit promastigotes infection in the cleotide sugars (21). One of the most distinc- host body (26). In addition, the biological active roles of these nucleotide sugars is the bio- tivity and the role of asparagine synthase en- synthesis of glycoconjugates, forming the cell zyme are more emphasized today, due to the surface coat (22). This dense glycocalyx is role of asparagine. Asp and Asn amino acids composed of glycosylphosphatidylinositol-like can be synthesized from oxaloacetate by mito- structures including lipophosphoglycans chondrial enzymes of aspartate aminotransfer- (LPGs), GPI-anchored glycoproteins, proteo- ase and asparagine synthase which were pre- phosphoglycans(PPGs) and, free GPI glycoli- sent in L. major (27). pids which are essential for parasite survival in There are two different A and B forms of the sandfly vector and the mammalian host asparagine synthase enzyme. Form A is (23). strongly dependent on ammonium and form The distinct role of sugar nucleotides in B uses glutamine. Form A of this enzyme is Leishmania molecular adjustment has been re- not observed in humans while it is considered ported (22). Leishmania requires uridine di- a potential drug target in trypanosomes (28). phosphate glucose (UDP-GLC) in the nucleus Form A of this enzyme is necessary for the to synthesize an unusual DNA base called survival and infectivity of L. donovani. There- base J (β- D-glucosyl-hydroxymethyluracil) fore, it is considered a drug target (28). Previ- which plays an important role in regulating the ous studies revealed the important role of onset and end of transcription (22). In the these two metabolites. Therefore, it is likely present study, treatment of Leishmania pro- that X. strumarium leaf extract inhibits the mastigotes with leaf extract of X. strumarium growth of promastigotes by cyanoamino acid was associated with changes in N- metabolic pathway alteration through increas- acetylglucosamine 6-phosphate, glucose-1 ing asparagine and reducing aspartic acid. phosphate, glucosamine-6 phosphate and glu-

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In addition, the present study indicates the ATP supply. Our results are supported by alteration of two metabolites, fructose, and previous finding (31). glucose 1 phosphate, in the pathway of starch UDP-Gal can be interconverted to UDP- and sucrose metabolism. Thus, sucrose is an galactofuranose (UDP-Galf) by the flavor de- intermediate metabolite in this metabolic pendent enzyme UDP-galactopyranose mu- pathway. The presence of a sucrase enzyme, tase (33). The LPGs were the most abundant namely fructofuranose, α-glucosidase, α- glycoconjugates on the surface of Leishmania amylase, and cellulose in L. major was proven promastigotes, about 6×106 copies per cell while it was absent in a few trypanosomes (34), which play an important role in protec- specie (29). tion against the hydrolytic enzymes of blood- L. major promastigotes experience glucose- fed phlebotomines (22). In L. major, The LPG rich conditions in the digestive tract of their galactose side chains are progressively capped sandfly vector, as the sugar meals consumed with b1,2-linked D-arabinose during the tran- from the plant sap were digested by amylase, sition from procyclic to metacyclic pro- kinase and sucrose enzymes (30). mastigotes. These changes lead to an increase In addition, the plant starch is hydrolyzed by in the thickness of the surface glycocalyx, α-amylase enzyme and converted to maltose, which results in additional resistance to com- and maltose is often converted to glucose by plement-mediated lysis (32).The defective ga- α-glucosidase enzyme. Therefore, given that lactose metabolism present in altered gly- the main fuel of promastigotes is ATP pro- cocalyx was associated with parasite attenua- duction through glucose glycolysis, and since tion (35). an important quantity of parasite glucose is Therefore, the importance of enzymes in- provided through the pathway of starch and volved in the biosynthesis of Galf might pro- sucrose metabolism, it can be concluded that vide new targets for the development of effec- any change in the metabolites of this pathway tive drugs to combat Leishmaniasis (21). Dele- can inhibit parasite growth by interfering with tion of the UGM gene in L.major resulted in the energy of the promastigotes, which is con- the complete depletion of β-Galactofuranose sistent with our results. (β-Galf ) in the structure of repeated units of In the current study, butanoate profile alter- phosphoglycans in GIPLs and LPGs. Fur- ations are confined to three metabolites,3- thermore, mice infection by this mutant of L. hydroxy-butyric acid and butanal and 1- major was significantly attenuated (36). Moreo- butanol. No significant study was observed on ver, galactose metabolism acts as a key factor the role and probable importance of butanol in the occurrence of African sleepiness caused and butanal in the maintenance and survival of by Trypanosome brucei (37). In the present study, promastigotes. The previous metabolomics- two key metabolites of glucose 1-phosphate based investigation, confirmed 3-hydroxybuty- and uridine diphosphate galactose in the galac- ric alteration in mice treated with new ampho- tose metabolic pathway changed in pro- tericin B drug compound (31).This drug com- mastigotes treatment of promastigotes with pound had less toxic effects on patients with leaf extract of X. stramarium caused disruption Leishmaniasis (32). Therefore, it can be postu- or change in the glycoprotein cover of the lated that butanol and butanal metabolites, parasite, by disrupting or changing the galac- especially 3-hydroxybutyric acid, play an im- tose metabolism. Therefore, increased suscep- portant role in the parasite metabolism and tibility to host complement and oxidative the leaf extract of X.stumarium inhibits the stress, caused promastigotes attenuation. growth of Leishmania promastigotes by disrupting this metabolic pathway and inhibiting

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