J Arthropod-Borne Dis, June 2018, 12(2): 135–140 A Ramazani et al.: Antiplasmodial Property of … 135 http://jad.tums.ac.ir Published Online: June 12, 2018 Original Article Antiplasmodial Property of Glycyrrhiza glabra Traditionally Used for Malaria in Iran: Promising Activity with High Selectivity Index for Malaria *Ali Ramazani 1, Mahdi Tavakolizadeh 1, 2, Samira Ramazani 1, Hamidreza Kheiri-Manjili 1, Mehdi Eskandari 3 1Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran 2Department of Pharmacognosy, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran 3Department of Physiology and Pharmacology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran (Received 08 Oct 2016; accepted 8 May 2018) Abstract Background: Development of resistance against the frontline anti-malarial drugs has created an alarming situation, which requires intensive drug discovery to develop new, more effective, affordable and accessible anti-malarial agents. The aim of this study was to assess antiplasmodial activity of the different fractions of root extract of Glycyr- rhiza glabra. Methods: Roots of G. glabra were collected from Tarom district of Zanjan Province in 2016 and then dried root ma- terial was chopped and consecutively extracted by the percolation method using solvents of different polarity. Result- ing extracts were assessed for in vitro and in vivo anti-malarial and cell cytotoxicity activities. Results: Among the three different solvent fractions studied, water-methanol and ethyl acetate fractions showed promising in vitro antiplasmodial activity against CQ-sensitive Plasmodium falciparum 3D7 strain (IC50= 9.95 and 13µg/ml, respectively). Further, the selectivity indices (HeLa cells versus P. falciparum) for the promising water- methanol fraction showed selectivity for P. falciparum and potential safer therapy for human. Interestingly, water- methanol and ethyl acetate fractions showed a significant suppression of parasite growth (72.2% and 65%, respec- tively) in comparison with control group in mice infected with P. berghei (P< 0.05). Conclusion: The promising antiplasmodial activity of the aqueous fraction of G. glabra obtained in our study war- rant bioassay-guided fractionation of this fraction to identify active principles responsible for antiplasmodial activity. Keywords: Glycyrrhiza glabra, Malaria, Traditional medicine, Plasmodium berghei, Iran Introduction There were approximately 219 million cases of malaria all around the world and 660000 people died from this disease (1). Increasing resistance in the malaria parasite Plasmodium falciparum against artemisinin- based drugs is challenging to malaria control programs (2) and demands a wild attempt to develop novel anti-malarial drugs (3-5). Drugs containing novel structure from nat- ural source represent the main source for the discovery and development of new drugs for malaria (6). The discovery of new anti-ma- larial drugs from natural sources is increasing after the successfulness of quinine and arte- misinin. Glycyrrhiza glabra (Liquorice) is part of both western and eastern herbal traditions. It was approved to treat cough, bronchitis, and gastritis by commission E. This plant is used to treat peptic ulcers, asthma, pharyngi- tis, infections, hepatic disorders and fever (7) and it is one of the main constituents of the anti-malarial ancient Iranian remedy (8). Previously, based on our research strate- gies for malaria drug discovery from plant sources (9, 10), we reported the in vivo and in vitro antiplasmodial activity of total root *Corresponding author: Dr Ali Ramazani, E-mail: ramazania@zums.ac.ir http://jad.tums.ac.ir/ mailto:ramazania@zums.ac.ir J Arthropod-Borne Dis, June 2018, 12(2): 135–140 A Ramazani et al.: Antiplasmodial Property of … 136 http://jad.tums.ac.ir Published Online: June 12, 2018 extracts of G. glabra (9). Some active com- pounds with antimicrobial, antiviral and an- tiprotozoal properties were isolated and char- acterized from this plant (11-13). This study was designed to investigate the three solvents fractions of G. glabra with dif- ferent polarities with hexane, ethyl acetate and methanol-water (50:50). In this framework, selected plant was collected and further eval- uated for their in vitro and in vivo antiplas- modial activity and toxicity effect on HeLa cells. Materials and Methods Chemicals Reagents and materials in this study were obtained from Merck (Darmstadt, Germany) and Sigma-Aldrich (Steinheim, Germany). MTT, FBS and RPMI 1640 medium from Atocel and PAA (Austria). Plant material Roots of G. glabra were collected from Tarom district, Zanjan, Iran, at an altitude of 750m. The specimen was authenticated by Dr M Tavakolizadeh and a voucher specimen is deposited at the Herbarium of School of Pharmacy, Zanjan University of Medical Sci- ences (voucher No. 1073). Extraction Two thousand grams of dried root mate- rial was chopped and consecutively extracted by the percolation method (three times for each solvent and 72h for each time) using solvents of different polarities such as hex- ane, ethyl acetate and methanol-water (50: 50). Extractions were performed at room tem- perature and solutions were concentrated and dried by rotary evaporation under reduced pressure at 40 °C. The dried samples were stored in a freezer at 4 °C for further use in antiplasmodial and cell toxicity assays. In vitro cultivation of Plasmodium falciparum Chloroquine (CQ) sensitive strain (3D7) of P. falciparum was used and cultured ac- cording to the methods (9, 10, 14, 15). Briefly, P. falciparum parasites was cultured on hu- man erythrocytes (blood group O+) in RPMI 1640 medium supplemented with 0.5g/100ml AlbuMax I, 25mM HEPES, 19mM sodium carbonate, and 30μg/ml gentamicin sulfate, at pH 7.2 in a gas mixture of 91% N2, 6% CO2 and 3% O2. The medium was changed each day. In vitro antiplasmodial assays The extracts of experimental plants were evaluated for their antiplasmodial activity against 3D7 strain of P. falciparum. For drug screening, SYBR green I-based fluorescence assay was used (16). Briefly, sorbitol syn- chronized parasites (100μl) were incubated under normal culture conditions at 2% haem- atocrit and 1% parasitaemia in the presence or absence of different plant extracts (100, 50, 25, and 12.5µg/ml). CQ was used as positive controls, while 0.4% (v/v) DMSO was used as the negative control. After 48h of incuba- tion, 100μl of SYBR Green I lysis buffer was added to each well and after mixing incubat- ed in the dark at 37 °C for 1h. Fluorescence was measured using an ELISA plate reader (Infinite M200, Tecan) with excitation and emission wavelength bands cantered at 485 and 530nm, respectively. The background read- ing for an empty well was subtracted to yield fluorescence counts for analysis. The counts were plotted against the logarithm of the drug concentration and curve fitting by nonlinear regression to yield the drug concentration that produced 50% of the observed decline from the maximum counts in the drug-free control wells (IC50). The results were validated micro- scopically by examination of Giemsa stained smears of extract treated parasite cultures. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2018, 12(2): 135–140 A Ramazani et al.: Antiplasmodial Property of … 137 http://jad.tums.ac.ir Published Online: June 12, 2018 Cytotoxic assay on HeLa cells using MTT assay The cytotoxic effects of active plant extract fractions (ethyl acetate and water-methanol) on HeLa cells were assessed by using MTT (3–2, 5 diphenyl tetrazolium bromide) assay (17-20). The HeLa cells cultured in RPMI me- dium containing 10% foetal bovine serum and incubated at 37 °C with 5% CO2 and 96% humidity. Briefly, cells (104 cells in 100μl of culture medium) were distributed in 96-well flat-bottom plates in complete medium. Drug solutions were added after 24h of seeding and incubated for 48h. After 48h, 20μl of a stock solution of MTT (4mg/ml in phosphate buff- ered saline) was added to each well, gently mixed and incubated for another 3h. Then su- pernatant was removed and 100μl of DMSO (stop agent) was added. The absorbance of each well measured at 540nm using an ELI- SA plate reader (Infinite M200, Tecan). The 50% cytotoxic concentration (TC50) of test samples was determined by analysis of dose- response curves. Therapeutic index was cal- culated as a ratio of TC50 HeLa /IC50 3D7. In vivo antimalarial assay In vivo antimalarial activity of different solvent fractions of G. glabra assessed using the 4day suppressive test against P. berghei infection in mice (21). Female Swiss albino mice, weight 18–20g were inoculated with P. berghei (ANKA strain). Each mouse re- ceived 106 infected erythrocytes by intra-per- itoneal (IP) injection on the first day of the experiment. Groups of five mice were dosed daily by IP injection (200mg/kg) for 4 con- secutive days. On day 5 of the test, a blood smear was taken from the mice. Percentage suppression of parasitaemia for the fractions was calculated as 100− [(mean parasitaemia treated/mean parasitaemia control) ×100. For comparison of average parasitaemia, one- way ANOVA and two-tailed Student’s t-test were used (SPSS 21.0 Inc., USA) with P< 0.05 being considered significant. CQ at 25mg/kg was used as a positive control. The solvent (20% DMSO in PBS solution) was used as negative control. The study was approved by the Institu- tion Animal Ethical Committee. Results In vitro anti-plasmodial assay and cytotoxic study All the three fractions of G. glabra root extracts were screened for in vitro anti-plas- modial activity against the CQ-sensitive (3D7) P. falciparum strain (Table 1). The water- methanol and ethyl acetate fractions showed promising antiplasmodial activity with IC50 values of 9.95 and 13µg/ml, respectively. The n-hexane fraction did not show admissible ac- tivity. The two above mentioned active frac- tions (water-methanol and ethyl acetate) fur- ther analyzed for their toxicity on HeLa cells. The selectivity index (SI) is defined as the ra- tio of the HeLa cells toxicity to the antiplas- modial activity and is determined by divid- ing the TC50 values for the HeLa cells by the IC50 value for P. falciparum. The water-meth- anol fractions showed low toxicity against HeLa cells with higher selectivity against ma- laria (Table 1). In vivo anti-plasmodial assay The fractions that showed promising an- tiplasmodial activity further analyzed for their in vivo anti-plasmodial property (Table 2). The water-methanol and ethyl acetate fractions showed a significant suppression of parasi- taemia (P< 0.05). In comparison to control group, the water-methanol and ethyl acetate inhibited 72.2% and 65% of the growth of the parasite, respectively. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2018, 12(2): 135–140 A Ramazani et al.: Antiplasmodial Property of … 138 http://jad.tums.ac.ir Published Online: June 12, 2018 Table 1. Antiplasmodial activity, cytotoxicity and selectivity of water-methanol and ethyl acetate extracts of Glycyrrhiza glabra n Extract fraction P. falciparum IC50 (µg/ml) HeLa cell IC50 (µg/ml) Selectivity indices 1 Water-methanol 9.95 > 100 > 10 2 Ethyl acetate 13 21 1.61 3 n-hexane 215 - - (-) not tested Table 2. In vivo activities of plant extracts against Plasmodium berghei Group Dose (mg/kg) Mean Parasitemia (SD*) % Suppression of parasitemia P-value Negative control (20% DMSO in PBS solution) 11.55 (2.58) Water-methanol 200 3.15 (1.08) 72.2 0.03 Ethyl acetate 200 4.04 (2.36) 65 0.02 CQ 25 0 100 SD: Standard deviation Discussion Medicinal plants have a wide diversity of medicinal properties including for malaria therapy as two of the most important anti- malarial agents, namely quinine and artemis- inin, with plant origin. Traditional plants could be attractive for drug discovery as they are widespread and also a large population. In the present study, different fractions of G. glabra root extract known for its traditional medicinal usage (8) and anti-malarial activi- ty (9, 22) was further evaluated for its differ- ent solvent fractions antiplasmodial activity against CQ-sensitive P. falciparum 3D7 and P. berghei ANKA strain and their toxicity against HeLa cell line (Table 1). Among the three different solvent fractions studied, wa- ter-methanol and ethyl acetate fractions showed promising in vitro antiplasmodial activity against CQ-sensitive 3D7 strain (IC50= 9.95 and 1µg/ml, respectively). Further, the selec- tivity indices (HeLa cells versus P. falcipa- rum) for the promising water-methanol frac- tion showed selectivity for P. falciparum and potential safer therapy for human. Interestingly, water-methanol and ethyl acetate fractions showed a significant suppression of parasitem- ia in comparison with control group (P< 0.05). The suppression rate of parasite growth in- duced by water-methanol and ethyl acetate fractions were 72.2% and 65%, respectively (Table 2). In our previous study (9), the hydro-al- coholic extraction of G. glabra root extract showed promising antiplasmodial activity with IC50 values of 13.56µg/ml against P. falcipa- rum 3D7 strain and also suppressed the growth of the P. berghei parasite by 65% in vivo at a dose of 400mg/kg. In this study, further frac- tionation resulted in better antiplasmodial prop- erty in vitro and in vivo. Water-methanol frac- tion exhibited in vitro antiplasmodial activity with IC50 value of 9.9µg/ml and suppressed the growth of the P. berghei parasite by 72.2 % at a dose of 200mg/kg in comparison with the control group. The active ingredients of plant accumulated in water-methanol fraction. The ethyl acetate fraction also showed prom- ising in vitro and in vivo antiplasmodial ac- http://jad.tums.ac.ir/ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4340493/table/Tab1/ J Arthropod-Borne Dis, June 2018, 12(2): 135–140 A Ramazani et al.: Antiplasmodial Property of … 139 http://jad.tums.ac.ir Published Online: June 12, 2018 tivity, but this fraction had low selectivity in- dex in comparison with water-methanol frac- tion. Licorice (G. glabra) is used for the treat- ment of several diseases in traditional medi- cine. Licorice was shown to have a number of biological properties such as antiviral, an- ti-inflammatory, antimicrobial, and anticancer activities (13). Several active components from G. glabra were isolated and some cellular and molecular mechanisms of these compounds have been elucidated (11, 12, 22). One of these compounds is Licochalcone A, separated from the roots of Chinese licorice, showed prom- ising in vitro antiplasmodial property against both CQ-susceptible (3D7) and CQ-resistant (Dd2) P. falciparum strains. This compound protected the mice from the lethal P. yoelii infection (22). 18beta-glycyrrhetinic acid, an- other anti-malarial agent isolated from Indian licorice, showed significant in silico, in vitro and in vivo antimalarial activity (12). Conclusion The water-methanol fraction of G. glabra has higher selectivity index for malaria than ethyl acetate fraction. 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