Evaluation of ethnopharmacologically selected Vitex negundo L. for In vitro antimalarial activity and secondary metabolite profiling.

ETHANOPHARMACOLOGICAL RELEVANCE: Limited drugs, rise in drug resistance against frontline anti-malarial drugs, non-availability of efficacious vaccines and high cost of drug development hinders malaria intervention programs. Search for safe, effective and affordable plant based anti-malarial agents, thus becomes crucial and vital in the current scenario. The Vitex negundo L. is medicinal plant possessing a variety of pharmaceutically important compounds. The plant is used traditionally worldwide for the treatment of malaria including India and Malaysia by the indigenous tribes. In vitro studies have reported the anti-malarial use of the plant in traditional medicinal systems. AIM OF THE STUDY: The aim of the current study is to evaluate the traditionally used medicinal plants for in vitro anti-malarial activity against human malaria parasite Plasmodium falciparum and profiling secondary metabolite using spectroscopic and chromatographic methods. Chemical profiling of active secondary metabolites in the extracts was undertaken using LC-MS. MATERIALS AND METHODS: Based on the ethno-botanical data V. negundo L. was selected for in vitro anti-malarial activity against P. falciparum chloroquine-sensitive (3D7) and multidrug resistant (K1) strains using SYBR Green-I based fluorescence assay. Cytotoxicity of extracts was evaluated in VERO cell line using the MTT assay. Haemolysis assay was performed using human red blood cells. Secondary metabolites profiling was undertaken using chromatographic and spectroscopic analysis. Liquid chromatography analysis was performed using a C18, 150 X 2.1, 2.6 µm column with gradient mobile phase Solvent A: 95% (H2O: ACN), Solvent B: Acetonitrile, Solvent C: Methanol, Solvent D: 5 mM NH4 in 95:5 (H2O: ACN) at a constant flow rate of 0.250 ml/min. The LC-MS spectra were acquired in both positive and negative ion modes with electrospray ionization (ESI) source. RESULTS: The anti-malarial active extract of V. negundo L. leaf exhibited potent anti-malarial activity with IC50 values of 7.21 µg/ml and 7.43 µg/ml against 3D7 and K1 strains, respectively with no evidence of significant cytotoxicity against mammalian cell line (VERO) and no toxicity as observed in haemolysis assay. The HPLC-LC-MS analysis of the extract led to identification of 73 compounds. We report for the first time the presence of Sabinene hydrate acetate, 5-Hydroxyoxindole, 2(3,4-dimethoxyphenyl)-6, 7-dimethoxychromen-4-one, Cyclotetracosa-1, 13-diene and 5, 7-Dimethoxyflavanone in the anti-malarial active extract of V. negundo L. leaf. Agnuside, Behenic acid and Globulol are some of the novel compounds with no reports of anti-malarial activity so far and require further evaluation in pure form for the development of potent anti-malarial compounds. CONCLUSIONS: The result report and scientifically validate the traditional use of V. negundo L. for the treatment of malaria providing new avenues for anti-malarial drug development. Several novel and unknown compounds were identified that need to be further characterized for anti-malarial potential.

A phenolic glycoside from Flacourtia indica induces heme mediated oxidative stress in Plasmodium falciparum and attenuates malaria pathogenesis in mice.

BACKGROUND: Flacourtia indica is especially popular among the various communities of many African countries where it is being used traditionally for the treatment of malaria. In our previous report, we have identified some phenolic glycosides from the aerial parts of F. indica as promising antiplasmodial agents under in vitro conditions. PURPOSE: Antimalarial bioprospection of F. indica derived phenolic glycoside in Swiss mice (in vivo) with special emphasis on its mode of action. METHODS: Chloroquine sensitive strain of Plasmodium falciparum was routinely cultured and used for the in vitro studies. The in vivo antimalarial potential of phenolic glycoside was evaluated against P. berghei in Swiss mice through an array of parameters viz., hematological, biochemical, chemo-suppression and mean survival time. RESULTS: 2-(6-benzoyl-ß-d-glucopyranosyloxy)-7-(1α, 2α, 6α-trihydroxy-3-oxocyclohex-4-enoyl)-5-hydroxybenzyl alcohol (CPG), a phenolic glycoside isolated from the aerial parts of F. indica was found to exhibit promising antiplasmodial activity by arresting the P. falciparum growth at the trophozoite stage. Spectroscopic investigations reveal that CPG possesses a strong binding affinity with free heme moieties. In addition, these interactions lead to the inhibition of heme polymerization in malaria parasite, augmenting oxidative stress, and delaying the rapid growth of parasite. Under in-vivo condition, CPG exhibited significant antimalarial activity against P. berghei at 50 and 75mg/kg body weight through chemo-suppression of parasitemia and ameliorating the parasite induced inflammatory and oxidative (hepatic) imbalance in the experimental mice. CONCLUSION: CPG was found to be a potential antimalarial constituent of F. indica with an explored mechanism of action, which also offers the editing choices for developing CPG based antimalarial chemotypes.

Antimalarial activity and safety assessment of Flueggea virosa leaves and its major constituent with special emphasis on their mode of action.

A clinical emergency stands due to the appearance of drug resistant Plasmodium strains necessitate novel and effective antimalarial chemotypes, where plants seem as the prime option, especially after the discovery of quinine and artemisinin. The present study was aimed towards bioprospecting leaves of Flueggea virosa for its antimalarial efficacy and active principles. Crude hydro-ethanolic extract along with solvent derived fractions were tested in vitro against Plasmodium falciparum CQ sensitive (3D7) and resistant (K1) strains, where all the fractions exhibited potential activity (IC50 values <10µg/mL) against both the strains. Interestingly, under in vivo conditions against P. berghei in Swiss mice, preferential chemo-suppression was recorded for crude hydro-ethanolic extract (77.38%) and ethyl acetate fraction (86.09%) at the dose of 500mg/kg body weight. Additionally, ethyl acetate fraction was found to be capable of normalizing the host altered pharmacological parameters and enhanced oxidative stress augmented during the infection. The bioactivity guided fractionation lead to the isolation of bergenin as a major and active constituent (IC50, 8.07±2.05µM) of ethyl acetate fraction with the inhibition of heme polymerization pathway of malaria parasite being one of the possible chemotherapeutic target. Furthermore, bergenin exhibited a moderate antimalarial activity against P. berghei and also ameliorated parasite induced systemic inflammation in host (mice). Safe toxicity profile elucidated through in vitro cytotoxicity and in silico ADME/T predications evidently suggest that bergenin possess drug like properties. Hence, the present study validates the traditional usage of F. indica as an antimalarial remedy and also insists for further chemical modifications of bergenin to obtain more effective antimalarial chemotypes.

Management of mycoplasma contamination in in vitro culture of Plasmodium falciparum without antibiotic treatment - a preliminary report.

The presence of foreign contamination, especially of mycoplasmas, is a major hindrance in long term in vitro cultivation of Plasmodium falciparum and may be a source of false-positive results. Efforts have been made to control mycoplasma contamination by trypsinization of P. falciparum culture. Samples of accidentally contaminated cultures were used for this study. The presence of Mycoplasma orale in contaminated culture was ascertained by a species-specific PCR-based mycoplasma detection kit (Takara; Cat. No.6601). Trypsinization was carried out using trypsin-EDTA and the growth profile of P. falciparum was monitored for more than three weeks post-trypsinization. The studies were carried out with four different P. falciparum strains, various serum supplements and human erythrocytes belonging to different blood groups. It was interesting to observe that, irrespective of the different strains of P. falciparum and the variety of serum supplements and erythrocytes, mycoplasma contamination can successfully be removed from P. falciparum culture by trypsinization. No antibiotic except gentamicin, which is routinely used, was added to the medium. Results of this study indicate that the frequent appearance of mycoplasma in continuous long-term cultures of P. falciparum can be managed by trypsinization.

Design, synthesis of 4-aminoquinoline-derived thiazolidines and their antimalarial activity and heme polymerization inhibition studies.

The present study describes the synthesis of a series of new 4-aminoquinoline-derived thiazolidines and evaluation of their antimalarial activity against a NF-54 strain of Plasmodium falciparum in vitro and N-67 strain of Plasmodium yoelii in vivo. Among the series, two compounds, 2-(4-chloro-phenyl)-thiazolidine-4-carboxylic acid [2-(7-chloro-quinolin-4-ylamino)-ethyl]-amide hydrochloride (14) and 2-(2,6-dichloro-phenyl)-thiazolidine-4-carboxylic acid [2-(7-chloro-quinolin-4-ylamino)-ethyl]-amide hydrochloride (22) exhibited significant suppression of parasitaemia in the in vivo assay. All the analogues were found to form strong complex with haematin and inhibited the ß-haematin formation in vitro. These results suggest that these compounds act on heme polymerization target.

Antimalarial activity in Xylocarpus granatum (Koen).

The antimalarial activity of Xylocarpus granatum fruits and their active constituents gedunin and xyloccensin-I were investigated using an in vitro model in this study. The chloroform fraction of X. granatum fruits was found to show promising antimalarial activity using an in vitro model of Plasmodium falciparum. On purification of the active fraction, four pure compounds were isolated and characterised, namely gedunin, photogedunin, xyloccensin-I and palmitic acid. Out of these only gedunin and xyloccensin-I were found to show activity equivalent to the parent active fraction in vitro model.

A small library of trisubstituted pyrimidines as antimalarial and antitubercular agents.

A small library of 20 trisubstituted pyrimidines were synthesized and evaluated for their in vitro antimalarial and antitubercular activities. Out of the total screened compounds, 16 compounds have shown in vitro antimalarial activity against Plasmodium falciparum in the range of 0.25-2microg/mL and 8 compounds have shown antitubercular activity against Mycobacterium tuberculosis H(37)Ra, at a concentration of 12.5microg/mL.