Parasitology International 63 (2014) 349–358

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Glabridin induces oxidative stress mediated apoptosis like cell death of malaria parasite Plasmodium falciparum Harveer Singh Cheema a,1, Om Prakash b,1, Anirban Pal a,1, Feroz Khan b,1, Dnyneshwar U. Bawankule a,1, Mahendra P. Darokar a,⁎,1 a b

Molecular Bioprospection Department, CSIR- Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow-226 015, India Metabolic and Structural Biology Department, CSIR- Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow-226 015, India

a r t i c l e

i n f o

Article history: Received 30 September 2013 Received in revised form 12 November 2013 Accepted 10 December 2013 Available online 18 December 2013 Keywords: Glabridin Reactive oxygen species P. falciparum Apoptosis Oxidative stress Pf LDH

a b s t r a c t Plants are known as the source of novel agents for developing new antimalarial drugs. Glabridin is a polyphenolic flavonoid, a main constituent in the roots of Glycyrrhiza glabra possesses various biological activities. However, its anti-plasmodial activity is unexplored. In the present work, it is for the first time demonstrated that glabridin inhibits Plasmodium falciparum growth in vitro with an IC50 23.9 ± 0.43 μM. Glabridin showed poor cytotoxicity in vitro with an IC50 246.6 ± 0.88 μM against Vero cell line and good selectivity index (9.6). In erythrocytic cycle, trophozoite stage was found to be most sensitive to glabridin. In silico study showed that glabridin inhibits Pf LDH enzyme activity by acting on NADH binding site. Glabridin induced oxidative stress by the generation of reactive oxygen and nitrogen species. Glabridin could induce apoptosis in parasite as evidenced by the depolarization of mitochondrial membrane potential (Δψm), activation of caspase like proteases and DNA fragmentation. These results indicate that glabridin exhibits antiplasmodial activity and is suitable for developing antimalarial agent from a cheap and sustainable source. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Malaria kills about a million people each year, including young children under the age of five and pregnant women [1]. Day to day increasing resistance in the malaria parasite Plasmodium falciparum against artemisinin based drugs is challenging to malaria control programs [2] and demands a non-stop attempt to develop new antimalarial leads. Plant based drugs or natural products and their derivatives represent about half of all drugs in clinical use today [3]. The discovery of novel antiplasmodial lead molecules of pharmaceutical interest from natural products is increasing after the successfulness of quinine and Artemisinin. Glabridin [(R)-4-(3,4-dihydro-8,8-dimethyl)-2H,8H-benzo[1,2b:3,4-b]dipyran-3yl)-1,3-benzenediol] is a polyphenolic flavonoid, the main ingredient of the root extract of Glycyrrhiza glabra (Licorice) plant. Licorice is a well-known medicinal herb used in Ayurveda as a

Abbreviations: ROS, Reactive oxygen species; RNS, Reactive nitrogen species; NO, Nitric oxide; pLDH, Parasite lactate dehydrogenase; Pf LDH, Plasmodium falciparum lactate dehydrogenase; Δψm, Mitochondrial membrane potential; NBT, Nitroblue tetrazolium; PES, Phenazine ethosulphate; MTT, Methylthiazolyldiphenyl-tetrazolium bromide; APAD, 3-Acetyl pyridine adenine dinucleotide; SNP, Sodium-nitroprusside; FBS, Fetal bovine serum; CM-H2DCFDA, Chloromethyl 2′,7′-dichlorodihydrofluorescein diacetate; DCF, Dichlorofluorescein; CCCP, Carbonyl cyanide 3-chlorophenylhydrazone; DEVD, L-aspartyl-Lglutamyl-L-valyl-L-aspartic acid amide. ⁎ Corresponding author. Tel.: +91 522 2718532; fax: +91 522 2342666. E-mail addresses: [email protected], [email protected] (M.P. Darokar). 1 www.cimap.res.in. 1383-5769/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.parint.2013.12.005

medicine and its extracts are also used as flavoring and sweetening agents in various food products [4]. The licorice root possesses several bioactive components, including glycyrrhizin, glycyrrhetinic acid, glabridin and isoliguiritigenin etc. [5]. Glabridin is a principal constituent with multiple biological activities, such as anti-proliferative, antimicrobial, antiviral, antifungal, antihepatotoxic, anti-oxidant or radical scavenging activities, anti-inflammatory, estrogen-like activity, antiulcer, as well as neuroprotective and antiosteoporosis activities etc. [5–9]. Glabridin is reported as a novel anticancer agent inhibiting migration, invasion, angiogenesis and the Rho signaling pathway [10]. Oxidative stress is the result of imbalances in cellular redox regulation and the inability of the antioxidant defense system to regulate stress causing agents like reactive oxygen species (ROS) or other free radicals [11]. ROS and reactive nitrogen species (RNS) at the physiological level regulate intracellular signaling and act as redox messenger, while at high levels cause oxidative burst that leads to the disruption of cellular homeostasis in various cells. The accumulation of ROS induces oxidative damage of membrane lipids, nucleic acid and proteins that ultimately leads to cell death [12]. Induction of oxidative stress is a major molecular mechanism of various drugs like chloroquine, quinine, mefloquine, primaquine, artemisinin [13] and ciprofloxacin [14] etc. Previous studies showed that several molecules induce oxidative stress leading to the induction of programmed cell death (PCD) e.g. arsenic trioxide [15], 2-methoxyestradial [16], Monensin [17], Elesclomol [18], and Bilirubin [19]. Many studies reported the occurrence of programmed cell death in unicellular parasites including P. falciparum [19–22]. A putative metacaspase gene (PfMCA1) has also

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been identified with an important role in programmed cell death [23,24]. The well known drugs chloroquine and etoposide induced DNA fragmentation in a drug sensitive strain of P. falciparum [25]. In this study, we for the first time report the antiplasmodial activity of glabridin and its possible mechanism of action. We found that glabridin inhibits Pf LDH activity and shows parasitostatic effect on parasite blood stages. We showed experimental evidences suggesting that glabridin induces oxidative stress in parasite that lead to depolarization of Δψm, activation of caspase like proteases, DNA fragmentation and causes apoptosis like parasite death.

2.2. P. falciparum culture The chloroquine sensitive P. falciparum strain NF-54 was cultivated in human O+ red blood cells using RPMI-1640 medium supplemented with 25 mM HEPES, 0.2% NaHCO3, 370 μM hypoxanthine, 40 μg/mL gentamycin, 0.25 μg/mL Fungizone, and 0.5% Albumax II at 37 °C using the method described previously [26]. Culture was maintained in a standard gas mixture consisting of 5% CO2. Culture medium was changed after every 24 h and routinely monitored through Geimsa staining of thin blood smears. The culture was synchronized by 5% D-sorbitol treatment to obtain ring-stage parasites [27].

2. Material and methods

2.3. Antiplasmodial activity

2.1. Reagents

Glabridin was dissolved in DMSO and further diluted with culture medium to achieve the required concentrations (final concentration of DMSO b1%). Parasite growth was determined spectrophotometrically in control and drug-treated cultures using a parasite lactate dehydrogenase assay (pLDH) as described previously with minor modifications [28]. Briefly, a synchronous ring stage culture with 1.5% parasitemia and 2% hematocrit was incubated in flat bottom 96-well tissue culture plate with different concentrations (100 to 1 μM) of glabridin at 37 °C for 72 h. Artemisinin and Chloroquine diphosphate salt was used as positive control. After incubation, plates were subjected to three 20 minute freeze–thaw cycles to release cell content. Parasite culture was carefully mixed and aliquots of 20 μL were taken and added to another flat bottom 96-well plate containing 100 μL of Malstat reagent (0.125% Triton X-100, 130 mM L-lactic acid, 30 mM Tris buffer and 0.62 μM APAD) and 25 μL of NBT–PES (1.9 μM NBT and 0.24 μM PES) solution per well. The plate was incubated in dark

Glabridin (Cat. No-G 9548), Hypoxanthine, Triton X-100, L-lactic acid, 3-acetyl pyridine adenine dinucleotide, Nitroblue tetrazolium, Phenazine ethosulphate, Methylthiazolyldiphenyl-tetrazolium bromide, antibiotic–antimycotic solution (100 ×), Phosphate Buffered Saline, D-sorbitol, DMSO, Sodium-nitroprusside, Chloroquine diphosphate, Artemisinin, and Doxorubicin hydrochloride, were purchased form Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 medium, Albumax II, Fetal bovine serum and Fungizone were purchased from Gibco BRL (Grand Island, NY, USA). Griess reagent kit, (G-7921) Chloromethyl 2′, 7′-dichlorodihydrofluorescein diacetate, and Enzchek Caspase-3 assay kit (Cat. No-E13184) were purchased from Molecular probe/ Invitrogen (Carlsbad, CA, USA). MitoScreen Flow Cytometry kit (Cat. No-551302) and APO-BrdU apoptosis detection kit (Cat. No-556405) were obtained from BD Biosciences (Franklin Lakes, New Jersey, USA).

Fig. 1. Microscopic observation after glabridin treatment at different blood stages of P. falciparum. Ring, trophozoite and schizont stage cultures at 5% parasitemia were treated with 100 μM glabridin and removed after 12 h corresponding to each stage. For continuous treatment the ring stage synchronized culture was treated at 24 μM and 48 μM concentrations of glabridin. Morphological changes were observed after 12 h interval up to 72 h. R + G = Ring stag treated with glabridin; T + G = Trophozoite stage treated with glabridin; S + G = Schizont stage treated with glabridin.

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Fig. 2. Molecular docking of glabridin with Pf LDH A. Blindly docked glabridin (yellow) and NADH (pink) in the active site pocket of Pf LDH (PDB: 1LDG); B. Targeted docking of 3,5dihydroxy-2-naphthoic acid with Pf LDH PDB: 1U5A; C. Targeted docking of glabridin with Pf LDH PDB: 1U5A at binding pocket of 3,5-dihydroxy-2-naphthoic acid.

for 30 min and absorbance was recorded at 650 nm using a microplate reader (FLUOStar Omega, BMG Labtech). The antiplasmodial activity of the glabridin was expressed as IC50s (mean ± SEM), calculated from dose–response curve data by nonlinear regression analysis.

wells was recorded using a microplate reader (FLUOStar Omega, BMG Labtech) at 570 nm. The cytotoxicity of glabridin was expressed as IC50s (mean ± SEM), calculated from dose–response curve data by nonlinear regression analysis. Doxorubicin hydrochloride was used as positive control.

2.4. Cytotoxicity

2.5. Determination of selectivity ratio

Vero cells (VERO C1008; ATCC CRL-1586) were cultured in 75-cm2 flasks in RPMI-1640 medium supplemented with 10% FBS, 0.2% NaHCO3 and 1 × antibiotic–antimycotic solution at 37 °C in an atmosphere of 95% humidity and 5% CO 2. Cytotoxicity of glabridin was assessed using MTT assay as described previously [29] with minor modification. Briefly, cells were seeded in a flat bottom 96-well tissue culture plate at a density of 2 × 104 cells/well in the RPMI complete medium. Different concentrations (100 to 1 μM) of glabridin were added after 24 h of seeding and incubated further for 48 h. After incubation 10 μL of MTT (5 mg/mL in PBS) solution was added to each well, gently mixed and incubated for another 4 h at 37 °C. After the incubation culture medium was removed, 100 μL of DMSO was added to each well and mixed gently. The absorbance of control and treated

Selectivity index (SI) was used as a parameter of clinical significance. Generally, selectivity index N2.0 is considered to be safe for natural products. SI of glabridin was calculated from the following expression as described previously [30]: SI ¼ IC50 of glabridin against Vero cells=IC50 of glabridin against P : falciparum:

2.6. Blood stage-specific effect of glabridin The effect of glabridin on blood stages of parasite was studied using the method described earlier with some modifications [31]. To determine the effect on intra-erythrocytic cycle, ring synchronized

Table 1 Docking affinity of glabridin with Pf LDH in comparison with NADH, and 3, 5-dihydroxy-2-naphthoic acid. Ligand

Binding affinity (kcal/mol)

PDB ID

Docking type

Pocket residue interacting with ligand (with H-bond)

No. of H-bonds

NADH Glabridin Naphthoic acid (control) Glabridin

−9.9 −8.2 −5.8 −7.2

1LDG 1LDG 1U5A 1U5A

Blind Blind Targeted Targeted

TYR-85, ASP-53, THR-97, ASN-140, GLY-32, HIS-195, ILE-31, MET-30 TYR-85 ASN-140 ASN-140, ARG-171

08 01 01 02

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Fig. 3. Measurement of ROS (H2O2) level. A. Level of intracellular ROS; B. percent increase in ROS level. The experiments were performed thrice (n = 3) and data expressed as mean values ± SEM. *P b 0.05, ***P b 0.001 vs. control: Tukey's test. C. Flow cytometry histograms showing increase in DCF positive cells after treatment at various concentrations of glabridin a. Untreated, b. 12 μM, c. 18 μM, d. 24 μM, e. 30 μM. Data representative of duplicate sample of two separate experiments.

P. falciparum culture at 2.5% parasitemia was incubated in the presence of IC 50 (24 μM) and 2 × IC50 (48 μM) of glabridin for 72 h. Parasitemia was measured by counting 1500 red blood cells. For the blood stage specific study, synchronized ring stage culture at the same parasitemia was divided in three separate wells of 24-well tissue culture plate. Each well was treated with 100 μM of glabridin after 12 h (ring stage), 24 h (trophozoite stage) and 36 h (schizont stage) and incubated for 12 h followed by removal of the glabridin through washing with complete medium by centrifugation (500 g for 2 min). Finally, culture was re-suspended in a drug free complete medium. Thin blood smears were prepared and stained with Geimsa at 12 h interval up to 72 h. Parasite morphology was evaluated by light microscopy (Nikon, Japan). 2.7. Docking study of glabridin with Pf LDH enzyme A three-dimensional structure of Pf LDH complexes with NADH as PDB: 1LDG [32] and 3, 5-dihydroxy-2-naphthoic acid as PDB: 1U5A [33], were obtained from a protein data bank. Glabridin (CID 124052) was the ligand under study for anti-malarial activity. To perform an unbiased study, nested docking was performed. Firstly glabridin was processed for blind docking with PDB: 1LDG and molecular interaction information was collected. Secondly, glabridin was further processed for targeted docking at the binding pocket of ‘3, 5-dihydroxy-2naphthoic acid’ a known inhibitor of Pf LDH as PDB: 1U5A and molecular

interaction information was analyzed. Docking was performed with AutoDock Vina, considering the Lamarckian genetic algorithm for searching the best fitting location of ligand into the protein/binding pocket. Molecular interaction was quantified as binding affinity in kcal/mol [34]. 2.8. Measurement of reactive oxygen species Intracellular ROS was measured using CM-H 2 DCFDA, a nonfluorescent dye that fluoresces on reaction with ROS in cells, as described earlier [19]. In brief, P. falciparum (12% parasitemia) culture was incubated with different concentrations of glabridin for a period of 36 h. After incubation, culture was washed twice with culture media and further incubated for 30 min in culture medium containing CM-H2DCFDA (10 μM). Culture washed twice with PBS and parasites was isolated from vehicle control and treated groups as described previously [35] and lysed by mild sonication using a bath type sonicator. ROS level was determined in lysate by measuring the formation of fluorescent dichlorofluorescein (DCF) using a microplate reader (FLUOStar Omega, BMG Labtech) at wavelengths 485–10 nm and 520 nm for excitation and emission, respectively. Each experiment was performed separately in triplicate. Intracellular ROS production was also measured by flow-cytometry using the CM-H2DCFDA dye as described previously with minor modifications [36]. Briefly, P. falciparum culture (12% parasitemia) was treated with

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2.10. Measurement of mitochondrial membrane potential Mitochondrial membrane potential was investigated using a BD MitoScreen Flow Cytometry mitochondrial membrane potential detection kit as per manufacturer's instructions with minor modifications. In brief, P. falciparum cultures (12% parasitemia, mature trophozoite stage) were treated with 18 μM, 24 μM and 30 μM concentrations of glabridin for 3 h at 37 °C in the CO2 incubator. CCCP (2.5 μM) was used as positive control. After washing by centrifugation at 500 g for 2 min, the 100 μL of packed cells was suspended in 1 × assay buffer and stained with JC-1 dye according to the manufacturer's instruction and analyzed in the flow-cytometer (LSRII BD Biosciences) equipped with the 488 nm argon laser beam as light source. Percentage of JC-1 green positive cells and mean green fluorescence intensity were calculated using FACSDiva analysis software. Simultaneously, a spectrophotometric measurement of Δψm investigating using JC-1 dye was recorded, as described previously [37]. Briefly, the P. falciparum culture treated as described above and after treatment was washed with PBS. Then parasite further incubated at 37 °C in the CO2 incubator for 30 min with JC-1 dye (5 μg/mL). Fluorescence intensities were recorded from the parasite lysate using the microplate reader (FLUOStar Omega, BMG Labtech) at wavelengths 520 nm and 590 nm for excitation and emission, respectively. All the experiments were performed in triplicate. 2.11. Measurement of caspase like activity

Fig. 4. Measurement of nitric oxide level A. NO level in terms of O.D., B. percent increase of NO level. The experiments were performed thrice (n = 3) and data expressed are mean values ± SEM. *P b 0.05, ***P b 0.001 vs. control: Tukey's test.

different concentrations of glabridin for 36 h at 37 °C in the CO2 incubator. After incubation, samples were stained as per instructions of the kit manual and analyzed using a LSRII flow-cytometer (BD Biosciences) equipped with a 488 nm argon laser as light source. Percentage of DCF positive cells and mean fluorescence intensity was calculated using FACSDiva analysis software (BD Biosciences).

2.9. Measurement of nitric oxide P. falciparum (12% parasitemia) culture was incubated with different concentrations of glabridin for a period of 36 h and the nitrite formed was detected by the spontaneous oxidation of NO using the Griess reagent kit, according to the manufacturer's protocol with minor modifications. In brief, 150 μL of culture supernatant containing nitrite, 130 μL of deionized water and 25 μL of Griess reagent were mixed in a 96 well microplate and incubated for 30 min at room temperature. In parallel, a reference sample was prepared by mixing 20 μL of Griess reagent and 280 μL of deionized water. Sodium-nitroprusside, a NO producer was used for preparing positive control. The absorbance of the samples was measured using a microplate reader (FLUOStar Omega, BMG Labtech) at 548 nm. Percent increase in nitrite production was calculated in comparison to vehicle control. All the experiments were performed in triplicate.

Caspase-like activity was measured in the whole parasite, using an Enzchek Caspase-3 Assay Kit, according to the manufacturer's instructions with some modifications. In brief, P. falciparum culture (12% parasitemia) was incubated with different concentrations of glabridin for 36 h at 37 °C in the CO 2 incubator. After incubation, parasite culture was washed in PBS and re-suspended in 50 μL of the 1 × cell lysis buffer and incubated on ice for 30 min. Culture was pellet by centrifugation at 3000 g for 5 min. 50 μL of the supernatant was transferred from each sample to individual black micro-plate wells and 50 μL of the 2× substrate working solution (Z-DEVD-R-110, DTT) was added to each sample and vehicle control. Camptothecin (1.5 μM) was used as positive control. The microplate was covered and incubated at room temperature for 30 min. Fluorescence intensities were recorded from the lysate using the microplate reader (FLUOStar Omega, BMG Labtech) at wavelengths 485–10 nm and 520 nm for excitation and emission respectively. All the experiments were performed in triplicate. 2.12. DNA fragmentation (TUNEL) assay DNA fragmentation was measured using an APO-BrdU apoptosis detection kit (BD Biosciences) and according to the manufacturer's protocol with minor modification. In brief, P. falciparum culture (15% parasitemia) was incubated in the different concentrations of glabridin for 36 h at 37 °C in the CO 2 incubator. After incubation, parasite cultures were washed with PBS and re-suspended in 4% (w/v) para-formaldehyde in PBS (pH-7.4) to obtain 2 × 106 cells/mL for fixing the cells. Samples were stained as per kit manual and analyzed using the flow-cytometer (BD Biosciences) equipped with the 488 nm argon laser beam as light source. Percentage of FITC-BrdU positive cells and mean green fluorescence intensity were calculated using FACSDiva analysis software. Camptothecin (1.5 μM) was used as positive control. All the experiments were performed in triplicate. 2.13. Statistical analysis One-way analysis of variance (ANOVA) was used to analyze the mean values obtained for the treatment and control. Tukey's test was used to compare the treatment and control and statistical significance was set at P ≤ 0.05.

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Fig. 5. Mitochondrial membrane potential (Δψm) A. Showing change in fluorescence intensity at 590 nm; B. Ratio of fluorescence intensity at 590/530 nm after the treatment with glabridin at various concentrations. The experiments were performed thrice (n = 3) and data expressed as mean values ± SEM. ***P b 0.001 vs. control: Tukey's test. C. Flow cytometry histograms showing the shifting of population towards FITC (JC-1) green channel and increased mean fluorescence intensity after the treatment of glabridin at various concentrations a. Control, b. 18 μM, c. 24 μM, d. 30 μM and e. CCCP at 2.5 μM. Data representative of duplicate sample of two separate experiments.

3. Results 3.1. Glabridin inhibits P. falciparum growth in vitro Glabridin inhibited P. falciparum growth in vitro in a dosedependent manner. The percent growth inhibition of the parasite was observed to be 73.1 ± 0.80%, 66.0 ± 0.72%, 58.1 ± 0.54%, 27.3 ± 0.58%, 15.2 ± 0.60%, and 7.2 ± 0.49% at 120 μM, 60 μM, 30 μM, 15 μM, 7.5 μM, and 3.7 μM, respectively. The IC50 value of glabridin was found to be 23.9 ± 0.43 μM. The IC50 for chloroquine and artemisinin was 0.021 ± 0.002 μM and 0.007 ± 0.004 μM respectively. Cytotoxicity of glabridin was evaluated at various concentrations against Vero cells exhibiting an IC50 value of 246.6 ± 0.88 μM with selectivity index 9.6 that is considered to be a good safety profile. 3.2. Glabridin arrests the parasite growth at the trophozoite stage To study the effect of glabridin on the parasite blood stages, a highly synchronized ring stage culture (2.5% parasitemia) was incubated in the presence of glabridin at 24 μM and 48 μM concentrations for 72 h. At the 24 μM concentration, the growth of the parasite was found to be arrested at trophozoite and schizont stages (Fig. 1). Similar observations were recorded at the 48 μM concentration, however, parasitemia was found to be reduced as compared to the 24 μM concentration. For further confirmation of stage specificity and reversibility of drug effect, synchronized ring, trophozoite and schizont stage cultures were treated

separately with 100 μM concentration of glabridin for 12 h and further incubated in a drug free culture medium up to 72 h. It was observed that, glabridin treated ring stage parasite developed into trophozoite after 24 h and further to schizont after 36 h and new rings appeared at 48 h, however parasite growth rate was slow as compared to control. Parasite treated at the trophozoite stage did not recover up to 60 h, however some healthy trophozoite could be seen at 72 h. At schizont stage, the parasite could develop into ring and trophozoite stages at 60 h and 72 h respectively, with a slow growth rate (Fig. 1). 3.3. Glabridin inhibits Pf LDH activity by acting on the NADH binding site To investigate the effect of glabridin on Pf LDH activity, a docking study was performed. In nested docking, firstly whole Pf LDH was used while in targeted docking, binding pocket with LYS-102, ASN140, PRO-141, ASP-143, LEU-167, ASP-168, ARG-171, LYS-173, HIS195, GLY-196, LYS-198, ALA-236, GLU-238, PRO-246, and PRO-250 residues that were visualized within a 15 Angstrom region were considered. Further within a 4 Angstrom region, it was observed that the amino acid TYR-85 was shared by glabridin (− 8.2 kcal/mol) with NADH (−9.9 kcal/mol) in blind docking (Fig. 2A). While in the case of targeted docking, glabridin shared another amino acid ASN-140 with NADH with a binding affinity of −7.2 kcal/mol (Fig. 2C) which is also the binding site of 3,5-dihydroxy-2-naphthoic acid having a binding affinity of −5.8 kcal/mol (Fig. 2B). Detailed observations are summarized in Table 1.

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3.4. Glabridin inducing formation of intracellular ROS in P. falciparum To study the effect of glabridin on ROS formation in parasite, intracellular level of H 2 O 2 was measured fluorometrically and flowcytometrically. In the fluorometric study, the level of ROS at 18 μM, 24 μM and 30 μM concentrations was significantly increased while at a lower concentration (12 μM) it was found to be decreased. The percent increases in ROS level were 4.7 ± 0.20%, 26.7 ± 0.14% and 34.7 ± 0.37% at concentrations of 18 μM, 24 μM and 30 μM respectively (Fig. 3). The flow cytometric analysis was also in agreement with the fluorometric data that at higher concentrations of glabridin the number of DCF positive cells increased. The DCF positive cells were 8.0% 11.5%, 13.3% and 12.2% at 12 μM, 18 μM, 24 μM and 30 μM respectively, as compared with control (10.9%). A shift of cell population towards the green channel was also observed with higher mean green fluorescence intensity as compared to the control (Fig. 3).

3.5. Glabridin induces generation of nitric oxide in P. falciparum In order to assess the effect of glabridin on nitric oxide (NO) generation, the NO level was measured after the incubation with different concentrations of glabridin. Interestingly, glabridin increased the production of NO in a dose dependent manner and caused 6.2 ± 0.50%, 11.2 ± 1.04%, 17.2 ± 1.6%, and 29.7 ± 1.0% increases at 12 μm, 18 μm, 24 μm, and 30 μm respectively (Fig. 4).

3.6. Glabridin causes depolarization of mitochondrial membrane potential (Δψm) in P. falciparum The Δψm was measured using the mitochondrial membrane potential sensitive dye JC-1. In the functionally active mitochondria, JC-1 accumulated and formed an aggregate that emits an orange red fluorescence at 590 nm, but in a depolarized state, the dye remains in the cytoplasm which emits green fluorescence at 520 nm. The ratio of 590/530 and shifting of fluorescence intensity from red to green is the measure of loss of Δψm [42]. The ratio of fluorescence intensity at 590/530 was observed to be 0.72 ± 0.04, 0.67 ± 0.003, 0.67 ± 0.02, and 0.57 ± 0.01, at 12 μM, 18 μM, 24 μM, and 30 μM concentrations respectively which was significantly (P N 0.001) lower than the control. This ratio was 1.06 ± 0.01 in the case of vehicle control and 0.34 ± 0.001 in the case of the CCCP treated cells (Fig. 5). Flow cytometric data was also in accordance and showed mitochondrial uptake of JC-1 dye as indicated by an increase in mean green fluorescence intensity. There was a shift in the distribution of cells towards the right side of the FITC green channel (Fig. 5) suggesting decreased Δψm at 18 μM, 24 μM, and 30 μM concentrations of glabridin.

3.7. Glabridin induces caspase like activity in P. falciparum The activation of caspase and other related proteases is a critical step of the programmed cell death induction [38]. To investigate whether glabridin is able to activate caspase in malaria parasite, fluorometric assay was carried out for measuring DEVD-specific protease activities (Caspase-3) using rhodamine 110 bis-(N-CBZ-L-aspartyl-Lglutamyl-Lvalyl-L-aspartic acid amide—Z-DEVD-R110) as substrate. Upon enzymatic cleavage, the nonfluorescent bisamide substrate is converted to fluorescent monoamide and then to the more fluorescent R110. A significant increase of 5.4 ± 1.9%, 22 ± 1.3% and 28.9 ± 0.50% in the activity of Caspase-3 (CED3/CPP32 group of proteases) was observed after 36 h treatment of glabridin at 18 μM, 24 μM, and 30 μM concentrations respectively as compared to control. Camptothecin was found to increase Caspase-3 activity (32.4 ± 0.34%) at the 1.5 μM concentration (Fig. 6).

Fig. 6. Determination of caspase like activity. A. Caspase like activity in terms of fluorescence units; B. percent increase in caspase activity after the treatment of glabridin at various concentrations. The experiments were performed thrice (n = 3) and data expressed as mean values ± SEM. *P b 0.05, ***P b 0.001 vs. control: Tukey's test.

3.8. Glabridin induces DNA fragmentation in P. falciparum DNA fragmentation is a hall mark and the last event of programmed cell death [39]. To investigate DNA fragmentation in glabridin treated parasite a flow cytometry based TUNEL assay was performed. As evident from the histogram (Fig. 7), a shift in the distribution of cells towards the right side of FITC green channel was observed suggesting increase in green fluorescence intensity that is proportional to fragmented DNA. It was observed that after 36 h of treatment at 18 μM, 24 μM, and 30 μM concentrations, TUNEL-positive cells were increased up to 7.3%, 8.9%, and 16% respectively. A positive control Camptothecin was found to increase TUNEL-positive cells up to 10.2% at the 1.5 μM concentration. 4. Discussion Glabridin being a natural product and with known safety in human is a suitable candidate for drug development [40]. The present study demonstrates for the first time that glabridin inhibits P. falciparum growth in vitro and exhibits parasitostatic effects on intra-erythrocytic cycle. Glabridin inhibited P. falciparum growth in a dose-dependent manner with a good selectivity index indicating a fine safety profile and its suitability for further validation as an antimalarial drug

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Fig. 7. Determination of DNA fragmentation through TUNEL assay. Flow cytometry histograms showing shifting of population towards FITC-BrdU green channel and increased mean fluorescence intensity after treatment of glabridin at various concentrations. A. Control, b. 18 μM, c. 24 μM, d. 30 μM and e. Camptothecin 2.5 μM. Data representative of duplicate sample of two separate experiments.

candidate. Parasite growth was significantly arrested at the trophozoite stage in the presence of glabridin. In a drug reversible experiment, parasite culture treated at ring and schizont stages,

no significant inhibition was observed and most of the parasite was recovered within 12 h of drug removal. Interestingly, culture treated at trophozoite showed the slowest recovery after drug

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removal suggesting that glabridin inhibits the parasite growth at the trophozoite stage. In silico studies showed that glabridin works as a Pf LDH inhibitor by acting on the NADH binding site and the binding affinity of glabridin was found to be significantly higher as compared to 3, 5-dihydroxy-2naphthoic acid, a known inhibitor of Pf LDH, however it was less than NADH. Lactate dehydrogenase is the crucial enzyme of the anaerobic glycolysis pathway and it plays an important role in the carbohydrate metabolism and energy generation in the anaerobic conditions during the intra-erythrocyte cycle [41]. The inhibition of lactate dehydrogenase causes bioenergetic and oxidative stress leading to cell death in cancer cells and is found to increase ROS generation after treatment with LDH inhibitors [36,42]. During the trophozoite stage more energy is required for metabolic activities and at the same time Pf LDH inhibition by glabridin causes oxidative stress and the parasite undergoes starved conditions. In this study, a consistent increase in ROS generation upon the treatment of glabridin at various concentrations was observed. However at 12 μM, ROS generation was less than the control which may be attributed to the radical scavenging property of glabridin at lower concentrations [5–8]. These observations are in accordance with earlier reports of natural products inducing ROS generation such as Quercetin, dietary flavonoids [43] and Withaferin A, a natural alkaloid [44]. Interestingly, a dose dependent increase in nitric oxide level was also observed in parasite treated with different concentrations of glabridin. Nitric oxide (NO) is the major signaling molecule in the cell and it was reported previously that it is one of the mediators of cytotoxicity in macrophages due to the interaction between nitric oxide and oxygen free radicals [45]. These observations clearly indicate that glabridin causes homeostatic imbalance in parasite and induces oxidative stress through the inhibition of Pf LDH, an essential enzyme for energy generation and overproduction of ROS and RNS. Mitochondria are the main site of energy generation and also the main target of injury after stresses leading to mitochondrial dysfunction and failure causing cell death [37,46,47]. Overgeneration of ROS may cause mitochondrial dysfunction which is a marker of apoptotic cell death. The loss of Δψm with the increased concentration of glabridin in both fluorometric and flow cytometric assays indicated that glabridin depolarizes the mitochondrial membrane. Nitric oxide also exerts its effect directly though the inhibition of mitochondrial respiration and inhibition of the citric acid cycle or indirectly by reacting with superoxide [46]. ROS mediates the depolarization of mitochondrial membrane promoting membrane permeabilization, which triggers the activation of caspase proteases that lead to the downstream events of apoptosis [38,46,47]. In this study, a significant increase of Caspase-3 (CED3/CPP32 group of proteases) activity was observed in glabridin treated parasite as compared to control which indicated that caspase proteases also play an important role in parasite death. The decreased caspase activity at 12 μM may be due to the decreasing ROS level at this concentration as evident in Fig. 3. The caspase-like proteases such as metacaspase (Gene ID PF14_0363) are reported in P. falciparum and play an important role in the induction of apoptosis [21,23,24]. However, the P. falciparum caspase protease was not well characterized till now and further studies are needed to confirm its role in parasite cell death. Oxidative stress mediated cell death was also reported in another unicellular parasite Leishmania donovani which lacks the classical caspase, but was reported to possess caspase 3 like proteases [19]. DNA fragmentation is a hallmark of apoptotic cell death. Previous evidences suggest that ROS and other free radicals cause oxidative DNA damage in various cells [48,49]. Caspase like proteases also cause DNA damage. However, the caspase mediated DNA fragmentation pathway is not well understood in apicoplexan parasites. In our study a significant increase in TUNEL positive cells was observed after glabridin treatment at various concentrations. Our results are

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in agreement with earlier reports that showed that drugs like chloroquine and etoposide cause DNA fragmentation in malaria parasite [25]. Based on the above facts and the previous reports [15–19], it is concluded that glabridin causes oxidative stress and induces apoptosis in P. falciparum. The present study demonstrates that glabridin, a natural flavonoid, inhibits P. falciparum growth and shows parasitostatic effect on parasite development. Glabridin inhibits Pf LDH activity, an essential enzyme for energy generation and causes oxidative stress. Further, it induces apoptosis like cell death that is evident by a change in mitochondrial membrane potential, activation of caspase proteases and DNA fragmentation. Glabridin being a natural product and with known safety in human is a suitable candidate for lead optimization that may result in the development of a novel therapeutic agent for combating malaria.

Acknowledgments The authors are thankful to the Director, CSIR-CIMAP for the R&D facilities. The authors are thankful to Dr. S K Puri and Dr. K Srivastava, CSIR—Central Drug Research Institute, Lucknow, India for providing the P. falciparum (NF-54). The help of Mr. Brijesh Sisodia in establishing the initial culture of parasite is gratefully acknowledged. Two of us (HSC and OP) are grateful to ICMR and CSIR for providing a Senior Research Fellowship. This work was a part of in-house project OLP-17 of CSIR-CIMAP, Lucknow, India with partial funding from the CSIR Network Program BSC0106 at CSIR-CIMAP, Lucknow.

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