Blutaparon portulacoides ethanolic extract reduced IL-1ß and inflammatory parameters induced by the Mycobacterium complex and carrageenan in mice.

Information on the health benefits of ethanolic extracts obtained from Blutaparon portulacoides stem (EEBP) hasn´t been consistently described in the literature until the present moment. This study investigated the antimycobacterial, anti-inflammatory and toxicological effects of EEBP in models of inflammation/infection, as well as its chemical composition. Chemical analysis of EEBP by electrospray ionization-mass spectrometry/HPLC-MS/MS identified 3,5,3'-Trihydroxy-4'-methoxy-6,7-methylenedioxy-flavone, gomphrenol, ferulic, vanillic, and caffeic acids. The minimum inhibitory concentration of EEBP and isoniazid in the presence of Mycobacterium tuberculosis was 123.4 and 0.030 µg/ml, respectively. EEBP oral administration (p.o.) (300-1000 mg/kg) or dexamethasone subcutaneous injection (s.c.) (1 mg/kg) significantly inhibited leukocytes and proteins resulting from carrageenan-induced pleurisy in Swiss mice. In the BCG-induced pleurisy model, the oral treatments performed once a day for 7 days, with EEBP (30 and 100 mg/kg) and isoniazid (25 mg/kg), inhibited the increase in plasmatic IL-1ß levels and in pleural exudate from C57BL-6 mice, and reduced M. tuberculosis growth in organs (colony forming units assays). EEBP (30-300 mg/kg, p.o.) and dexamethasone (1 mg/s.c.) significantly prevented carrageenan-induced oedema and mechanical hyperalgesia in Swiss mice. The treatments (once a day for 22 days) with EEBP (30 mg/kg, p.o.) and dexamethasone (1 mg/s.c.) substantially inhibited oedema and mechanical- and cold-hyperalgesia at 11, 16 and 22 days after the administration of Freund's Complete Adjuvant in C57bL6 mice. No evidence of physio-pathologic was observed in Wistar rats acutely treated with EEBP (2000 mg/kg, p.o.). This study confirms the anti-inflammatory and antibiotic properties of EEBP, opening possibilities for the development of safe new drugs with dual anti-inflammatory/antimycobacterial activities which could be favorable from a pharmacoeconomic perspective.

Three promising antimycobacterial medicinal plants reviewed as potential sources of drug hit candidates against multidrug-resistant tuberculosis.

Regimens of current drugs for tuberculosis are lengthy and are associated with many adverse effects. Currently, the emergence of different resistant strains has been observed. This urges a need for the discovery and development of novel drugs. The main sources of drug lead candidates are based on natural products. Zanthoxylum leprieurii, Lantana camara, and Cryptolepis Sanguinolenta are among the plants that have antimycobacterial activity. Recent technological methods, such as metabolomics, can rapidly detect and identify active compounds from medicinal plants. In this review, we aim to provide an overview and discussion of the antimycobacterial activity, phytochemical analysis and toxicity profile of these plants and their products as well as the potential of metabolomic fingerprinting of medicinal plants with a given activity on microbes, in the search for the potential drug hit molecules. The information for this review was extracted from databases such as Excerpta Medica Database, Google Scholar, Springer, and PubMed Central. Primary studies, using a combination of the keywords antimycobacterial medicinal plant, multidrug-resistant tuberculosis, phytochemistry, toxicity, Zanthoxylum leprieurii, Lantana camara, Cryptolepis sanguinolenta, and plant metabolomics/metabolic fingerprinting of plant extracts, have been considered. The above-mentioned plant species showed antimycobacterial activity against drug-resistant strains of M. tuberculosis. They may provide potential candidates for novel drugs against multidrug-resistant tuberculosis. However, extensive work is still needed. To our knowledge, there is no or limited literature that reports the metabolic fingerprints of these plants. The analysis of the metabolite fingerprints of medicinal plants with similar antimicrobial activity could be important to determine whether the activity results from common metabolites within different plant species. This review shows that these plants are potential candidates to provide drug hits against multidrug-resistant tuberculosis strains. Future studies of compound optimization, in vivo safety and efficacy, as well as of the specific mechanisms of action are however required.

Artemisia annua and Artemisia afra extracts exhibit strong bactericidal activity against Mycobacterium tuberculosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Emergence of drug-resistant and multidrug-resistant Mycobacterium tuberculosis (Mtb) strains is a major barrier to tuberculosis (TB) eradication, as it leads to longer treatment regimens and in many cases treatment failure. Thus, there is an urgent need to explore new TB drugs and combinations, in order to shorten TB treatment and improve outcomes. Here, we evaluated the potential of two Asian and African traditional medicinal plants, Artemisia annua, a natural source of artemisinin (AN), and Artemisia afra, as sources of novel antitubercular agents. AIM OF THE STUDY: Our goal was to measure the activity of A. annua and A. afra extracts against Mtb as potential natural and inexpensive therapies for TB treatment, or as sources of compounds that could be further developed into effective treatments. MATERIALS AND METHODS: The minimum inhibitory concentrations (MICs) of A. annua and A. afra dichloromethane extracts were determined, and concentrations above the MICs were used to evaluate their ability to kill Mtb and Mycobacterium abscessus in vitro. RESULTS: Previous studies showed that A. annua and A. afra inhibit Mtb growth. Here, we show for the first time that Artemisia extracts have a strong bactericidal activity against Mtb. The killing effect of A. annua was much stronger than equivalent concentrations of pure AN, suggesting that A. annua extracts kill Mtb through a combination of AN and additional compounds. A. afra, which produces very little AN, displayed bactericidal activity against Mtb that was substantial but weaker than that of A. annua. In addition, we measured the activity of Artemisia extracts against Mycobacterium abscessus. Interestingly, we observed that while A. annua is not bactericidal, it inhibits growth of M. abscessus, highlighting the potential of this plant in combinatory therapies to treat M. abscessus infections. CONCLUSION: Our results indicate that Artemisia extracts have an enormous potential for treatment of TB and M. abscessus infections, and that these plants contain bactericidal compounds in addition to AN. Combination of extracts with existing antibiotics may not only improve treatment outcomes but also reduce the emergence of resistance to other drugs.

Antitubercular compounds isolated and characterized in Tithonia diversifolia and Couroupita guianensis.

Background: Tuberculosis (TB) has become a public health challenge in the current scenario with a single causative agent, Mycobacterium tuberculosis (MTB) causing the highest morbidity and mortality affecting almost 1.7 million of the population. Furthermore, there has been no novel drug discovery for the past five decades, and the emergence of latent, multiple drug-resistant, and extensively drug-resistant species has given rise to an alarming necessity for a novel compound/s for treating this highly untamable microbe. In developing countries, plant-based drugs have shown promising results in combating TB or its symptoms; naturally occurring secondary metabolites can act as lead-drug molecules or can be co-administered with conventional drugs. Therefore, the present study was focused to identify and characterize potential antimycobacterial compounds found in the screened ethnobotanical plants, Tithonia diversifolia (TD) and Couroupita guianensis (CG). These plants are used for treating respiratory disorders and allergies in the traditional medicinal systems. Methods: These plant leaf extracts were detected and purified using chromatographic techniques for potent antitubercular phytochemicals, and the purified eluents were tested on Mycobacterium smegmatis (MSM) as a surrogate for MTB; further, the fractions inhibiting growth of MSM were characterized through gas chromatography-mass spectrometry. A toxicity test of the purified samples was also assessed by an in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and hemolytic assays. Results: The analyzed plant extracts showed the presence of a C-15 sesquiterpene, zingiberene in TD, and a phthalate ester, bis (2-ethylhexyl) phthalate, in CG leaf extracts. The toxicity assessment proved the purified fractions to be moderately toxic at higher concentrations (≥100 µg/mL). Conclusion: Therefore, the identified compounds can be promising antitubercular agents; however, further in vivo investigations will add substantial value to the compounds being pharmacologically useful.

Fatty acids-carotenoid complex: An effective anti-TB agent from the chlorella growth factor-extracted spent biomass of Chlorella vulgaris.

ETHNOPHARMACOLOGICAL RELEVANCE: The multidrug-resistant (MDR) pathogen, Mycobacterium tuberculosis, still remains as one of the major threat to mankind, despite the availability of a live attenuated vaccine and effective antibiotics. Marine microalgae, at all times, act as a key resource for valuable therapeutic compounds with limited side effects. AIM OF THE STUDY: The present explorative attempt is to isolate the biomolecules of pharmacological importance from the marine microalgae, Chlorella vulgaris, and to evaluate its effect on the ever dreadful disease, Tuberculosis. The study is also aimed to develop an economically feasible methodology for by-products extraction from microalgae. MATERIALS AND METHODS: Fatty acids-carotenoid complexes (FACC), namely, FACC-1 (red oil) and FACC-2 (brown oil) were isolated, in addition to lipid and lutein from the Chlorella Growth Factor (CGF, a protein fraction enriched with vitamins, minerals and carbohydrates)-extracted spent biomass through column chromatography. RESULTS: FACC-1 is a complex of fatty acids such as oleic and linoleic acids, and carotenoids such as canthaxanthin and neoxanthin. FACC-2 is a complex of oleic, linoleic and linolenic acids and carotenoids (cryptoxanthin and echinenone). Initial screening for evaluation of minimum bactericidal concentration (MBC) of FACC-1 and -2 was performed against Mycobacterium tuberculosis strains such as H37Rv, SHRE sensitive clinical isolate and SHRE resistant clinical isolate. MBC was noted at 10 µg/mL by FACC-1 and at 5 µg/mL by FACC-2, determined using colony forming and Lucipherase Reporter Mycobacteriophages (LRP) assay. Testing in the PAN sensitive isolates indicated that the MBC was noted at 5 µg/mL by FACC-1 and at 2.5 µg/mL by FACC-2. Complete inhibition (100%) was observed at 100 µg/mL by FACC-1 and at 50 µg/mL by FACC-2. Testing of FACC-1 and FACC-2 individually as well as in combination on two different types of MDR strains confirmed the efficacy of the algal oils, wherein in MDR-strain 1, FACC-1 revealed 50% inhibition at 10 µg/mL, while FACC-2 exhibited the same at 5 µg/mL. Conversely, in the case of MDR strain-2, MBC of FACC-1 was at 500 µg/mL and MBCof FACC-2 to be at 250 µg/mL. No significant synergistic effect was observed on combining both the oils. CONCLUSION: The study signifies the development of a potent therapeutic agent comprising of a complex of anti-TB agent (fatty acids) and antioxidants (carotenoids) from the CGF-extracted spent biomass of C. vulgaris.

A high content screening assay for identifying inhibitors against active and dormant state intracellular Mycobacterium tuberculosis.

The antitubercular drug development pipeline could start with an in vitro investigation of several compounds to examine their effect on active and dormant state Mycobacterium tuberculosis (Mtb). However, in vitro screening of dormant state bacilli cannot provide enough information on the simultaneous effect of a compound on the host. Therefore, we developed a live cell fluorescence based screening protocol by utilizing the high content system for determining the effect of inhibitors against active and dormant state intracellular mycobacteria. THP-1 macrophages infected with an actively growing and hypoxia derived dormant Mtb culture were standardized to develop the screening protocol. The signal to noise ratio and the Z' factor of this assay were found to be 7.5-29 and 0.6-0.8, respectively, which confirm the robustness of the protocol. The protocol was then validated with standard inhibitors. This newly developed drug screening assay offers an easy, safe, image based high content screening tool to search for novel antitubercular inhibitors against both active and dormant state intracellular mycobacteria. Therefore, this assay could fill in the gap between in vitro and in vivo latent tuberculosis drug screening programs.

Strategies in anti-Mycobacterium tuberculosis drug discovery based on phenotypic screening.

The rise of multi- and extensively drug-resistant Mycobacterium tuberculosis (M. tb) strains and co-infection with human immunodeficiency virus has escalated the need for new anti-M. tb drugs. Numerous challenges associated with the M. tb, in particular slow growth and pathogenicity level 3, discouraged use of this organism in past primary screening efforts. From current knowledge of the physiology and drug susceptibility of mycobacteria in general and M. tb specifically, it can be assumed that many potentially useful drug leads were missed by failing to screen directly against this pathogen. This review discusses recent high-throughput phenotypic screening strategies for anti-M. tb drug discovery. Emphasis is placed on prioritization of hits, including their extensive biological and chemical profiling, as well as the development status of promising drug candidates discovered with phenotypic screening.

Pasakbumin A controls the growth of Mycobacterium tuberculosis by enhancing the autophagy and production of antibacterial mediators in mouse macrophages.

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) and remains a major health problem worldwide. Thus, identification of new and more effective drugs to treat emerging multidrug-resistant TB (MDR-TB) and to reduce the side effects of anti-TB drugs, such as liver toxicity and other detrimental changes, is urgently needed. In this study, to develop a novel candidate drug for effective TB treatment with few side effects in the host, we selected pasakbumin A isolated from Eurycoma longifolia (E. longifolia) Jack, which protected host cells against Mtb infection-induced death. Pasakbumin A significantly inhibited intracellular Mtb growth by inducing the autophagy via the ERK1/2-mediated signaling pathway in Mtb-infected macrophages. We further investigated whether pasakbumin A could be used as a potential adjuvant for TB treatment. Treatment with pasakbumin A and anti-TB drug rifampicin (RMP) potently suppressed intracellular Mtb killing by promoting autophagy as well as TNF-α production via the ERK1/2- and NF-κB-mediated signaling pathways in Mtb-infected cells. Our results suggest that pasakbumin A could be developed as a novel anti-TB drug or host-directed therapeutic (HDT) strategy to protect against host cell death and improve host defense mechanisms against Mtb infection in macrophages.

In silico identification of potential inhibitors against shikimate dehydrogenase through virtual screening and toxicity studies for the treatment of tuberculosis.

The present study attempts to identify the novel inhibitors of shikimate dehydrogenase (SD), the enzyme that catalyzes the fourth reaction in the shikimate pathway, through virtual screening and toxicity studies. Crystal structure of SD was obtained from Protein Data Bank (PDB ID 4P4G, 1.7 Å) and subjected to energy minimization and structure optimization. A total of 13,803 compounds retrieved from two public databases and used for the virtual screening based on physicochemical properties (Lipinski rule of five) and molecular docking analyses. A total of 26 compounds with good AutoDock binding energies values ranging between - 12.03 and - 8.33 kcal/mol was selected and further filtered for absorption distribution metabolism excretion and toxicity analyses (ADMET). In this, eight compounds were selected, which satisfied all the ADME and toxicity analysis properties. Three compounds with better AutoDock binding energies values (ZINC12135132, - 12.03 kcal/mol; ZINC08951370, - 10.04 kcal/mol; and ZINC14733847, 9.82 kcal/mol) were considered for molecular dynamic (MD) simulation and molecular generalized born surface area (MM-GBSA) analyses. The results of the analyses revealed that the two ligands (ZINC12135132 and ZINC08951370) had better inhibitory activities within their complexes, after the 50-ns MD simulation, which suggested that the complexes formed stable conformation. It is noteworthy that compounds identified by docking, MD simulation, and MM-GBSA methods could be a drug for tuberculosis which required further experimental validation.

Bioassay-guided isolation of a Mycobacterium tuberculosis bioflim inhibitor from Arisaema sinii Krause.

Mycobacterium tuberculosis biofilms harbour drug-tolerant bacteria. Identification of drugs that inhibit biofilm formation could enable the dramatic shortening of tuberculosis treatments using standard antibiotics. Arisaema sinii Krause is used to treat pulmonary and lymphatic tuberculosis by Dong People of China. Current study was aimed to purify the active components against M. tuberculosis biofilms from Arisaema sinii extract by using bioassay-guided isolation. (E)-2-(methyl (phenyl) amino) ethyl 2-(2-hydroxyundecanamido)-7, 11-dimethyl-3-oxotetradec-4-enoate, compound 1, was identified as the active component. It could inhibit mycobacterial biofilm formation, disperse the preformed biofilms, and disrupt the mature biofilms at concentration of 4, 8, and 32 µg/ml, respectively. At the dose of 32 µg/ml, it could potentiate the bactericidal activity of isoniazid against M. tuberculosis in mature biofilms. The results of this study indicate that compound 1 might be a novel lead compound against mycobacterial biofilm formation.

Polysaccharide rich extract (PRE) from Tinospora cordifolia inhibits the intracellular survival of drug resistant strains of Mycobacterium tuberculosis in macrophages by nitric oxide induction.

Plethora of clinical and scientific information obtained in recent past has strengthened the idea that targeting critical constituents of host immune system may have beneficial outcomes for the treatment of tuberculosis. Macrophages being the primary host for Mycobacterium tuberculosis, offer an attractive target for modulation. Owing to their negligible toxicity, plant derived polysaccharides with the ability to activate macrophages; are suitable candidates for immunomodulation. In the present study, effects of polysaccharide rich extract (PRE) isolated from Tinospora cordifolia, on the survival of intracellular MTB strains and activation of macrophages were investigated. PRE treatment up regulated the expression of pro-inflammatory cytokines such as IL-ß, TNF-α, IL-6, IL-12, and IFN-γ in RAW 264.7 cell line. Up regulation in the expression of NOS2 was observed along with concomitant enhanced nitric oxide production post PRE treatment. Surface expression of MHC-II and CD-86 was up regulated after PRE treatment. Above results suggested the classical activation of macrophages by PRE treatment. Furthermore, PRE treatment led to the activation of all the three classes of MAPK i.e p38, ERK and JNK MAPKs. Further, PRE up regulated the expression of cytokines, NOS-2, MHC-II and CD-86 in MTB infected macrophages. PRE treatment inhibited the intracellular survival of drug resistant MTB in macrophages which was partially attributed to PRE mediated NO induction. Thus our data demonstrate classical activation of macrophages by PRE treatment and killing of intracellular MTB by NO induction.

The Deconstructed Granuloma: A Complex High-Throughput Drug Screening Platform for the Discovery of Host-Directed Therapeutics Against Tuberculosis.

Mycobacterium tuberculosis (Mtb) continues to be a threat to Global Public Health, and its control will require an array of therapeutic strategies. It has been appreciated that high-throughput screens using cell-based assays to identify compounds targeting Mtb within macrophages represent a valuable tool for drug discovery. However, the host immune environment, in the form of lymphocytes and cytokines, is completely absent in a chemical screening platform based on infected macrophages alone. The absence of these players unnecessarily limits the breadth of novel host target pathways to be interrogated. In this study, we detail a new drug screening platform based on dissociated murine TB granulomas, named the Deconstructed Granuloma (DGr), that utilizes fluorescent Mtb reporter strains screened in the host immune environment of the infection site. The platform has been used to screen a collection of known drug candidates. Data from a representative 384-well plate containing known anti-bacterial compounds are shown, illustrating the robustness of the screening platform. The novel deconstructed granuloma platform represents an accessible, sensitive and robust high-throughput screen suitable for the inclusive interrogation of immune targets for Host-Directed Therapeutics.

Antitubercular evaluation of root extract and isolated phytochemicals from Lophira lanceolata against two resistant strains of Mycobacterium tuberculosis.

CONTEXT: The roots of Lophira lanceolata Van Tiegh. Ex Keay (Ochnaceae) have numerous medicinal values in the Central African region. Even though the MeOH extract of the roots has shown antimycobacterial activities, the constituents responsible for this inhibitory activity remain unknown. OBJECTIVE: Phytochemical investigation of the MeOH root extract of L. lanceolata and determination of the antimycobacterial activities of that extract and constituents against the growth of Mycobacterium tuberculosis. MATERIALS AND METHODS: Column chromatography was used to provide bioactive phytoconstituents. Those compounds were elucidated using MS and NMR spectroscopic data. Antimycobacterial screening of the extract (4.882-5000 µg/mL in DMSO during 24 h at 37 °C) and isolated compounds (0.244-250 µg/mL in DMSO during 24 h at 37 °C) was performed by microplate alamar blue assay (MABA) against two mycobacterial strains. RESULTS: The investigation of L. lanceolata MeOH roots extract provided of mixture of unseparated biflavonoids with a newly described one, dihydrolophirone A (1a) associated to lophirone A (1b). The bioactive compounds that effectively inhibited the growth of M. tuberculosis AC45 were found to be compounds 1 and 2. They exhibited MIC values of 31.25 and 15.75 µg/mL, respectively, and their MIC was found to be 62.5 µg/mL against resistant strain AC83. DISCUSSION AND CONCLUSIONS: It is clearly evident from the results obtained that the mycobacterial activity of L. lanceolata could be related mainly to its steroid and flavonoid contents. Therefore, this study suggests the potential of the above-mentioned classes of compounds as promising candidate agents for developing new anti-tuberculosis drugs.

Antitubercular and anti-inflammatory properties screening of natural products from Plectranthus species.

AIM: Confirm the use of Plectanthus spp. plants in traditional medicine, particularly as anti-inflammatory and anti-infective agents. MATERIALS & METHODS: Compounds previously isolated from Plectranthus spp. were studied for their anti-inflammatory activity using the SNAP assay and RAW 264.7 cells, by the quantification of nitric oxide. An halimane diterpene and its derivatives were tested in infected macrophages with M. tuberculosis H37Rv, using CFU counts assay, at their minimum inhibitory concentration values. Results: The isolated compounds tested at noncytotoxic concentrations, did not reveal nitric oxide scavenging in the S-nitroso-N-acetylpenicillamine and the cellular assays. On the other hand, promising results were obtained regarding one semisynthetic halimane derivative (11R*,13E)-halima-5,13-diene-11,15-diol), previously prepared (2.1 × 105 CFU/mL), with an effect similar to the antitubercular drugs ethambutol (2.0 × 105 CFU/mL) and isoniazid (1.2 × 105 CFU/mL). CONCLUSION: The present report demonstrates the relevance of Plectranthus spp. in medicinal chemistry drug development for TB and other infective respiratory complaints. Also, this work suggests that further studies involving other inflammatory mediators are needed to validate the anti-inflammatory use of these medicinal plants.

Quercetin 3-O-glucoside recovered from the wild Egyptian Sahara plant, Euphorbia paralias L., inhibits glutamine synthetase and has antimycobacterial activity.

Tuberculosis remains a major health problem accentuated by the rise of resistance to all available drugs. Therefore, this study was launched to discover a novel antituberculosis agent from wild Egyptian Sahara plants. Twelve such plants were screened, in vitro, for their activity against various Mycobacterium species. The most active plant, Euphorbia paralias, was further fractionated with different organic solvents, and the activity of the obtained fractions was determined by the agar diffusion and broth microdilution methods. The methanol fraction was the most active against Mycobacterium spp., and was non-toxic in doses up to 10 g/kg of animal weight. Its main component was separated by column chromatography, and then identified by ultraviolet spectroscopy and nuclear magnetic resonance analysis as quercetin-3-O-ß-D-glucoside. Docking analysis suggested that quercetin-3-O-ß-D-glucoside inhibits the glutamine synthetase enzyme, a promising target for the development of antituberculosis drugs. This prediction was confirmed by an in vitro glutamine synthetase biosynthetic assay. To the best of our knowledge, and based on bioinformatics mining of the BioPhytMol database, this is the first report on the antimycobacterial activity of Euphorbia paralias plant. It is also the first report on the inhibition of mycobacterial glutamine synthetase by the flavonoid quercetin.

Analogues of Disulfides from Allium stipitatum Demonstrate Potent Anti-tubercular Activities through Drug Efflux Pump and Biofilm Inhibition.

Disulfides from Allium stipitatum, commonly known as Persian shallot, were previously reported to possess antibacterial properties. Analogues of these compounds, produced by S-methylthiolation of appropriate thiols using S-methyl methanethiosulfonate, exhibited antimicrobial activity, with one compound inhibiting the growth of Mycobacterium tuberculosis at 17 µM (4 mg L-1) and other compounds inhibiting Escherichia coli and multi-drug-resistant (MDR) Staphylococcus aureus at concentrations ranging between 32-138 µM (8-32 mg L-1). These compounds also displayed moderate inhibitory effects on Klebsiella and Proteus species. Whole-cell phenotypic bioassays such as the spot-culture growth inhibition assay (SPOTi), drug efflux inhibition, biofilm inhibition and cytotoxicity assays were used to evaluate these compounds. Of particular note was their ability to inhibit mycobacterial drug efflux and biofilm formation, while maintaining a high selectivity towards M. tuberculosis H37Rv. These results suggest that methyl disulfides are novel scaffolds which could lead to the development of new drugs against tuberculosis (TB).

Survey on medicinal plants traditionally used in Senegal for the treatment of tuberculosis (TB) and assessment of their antimycobacterial activity.

ETHNOPHARMACOLOGICAL RELEVANCE: In West Africa, populations are used to taking traditional medicine as a first aid against common health problems. In this aspect, many plants are claimed to be effective in the treatment of Tuberculosis (TB), which according to the World Health Organization (WHO) remains one of the world's deadliest communicable diseases. AIM OF THE STUDY: The main aim of this study was to identify plants used to treat TB-symptoms by the population of Senegal and to evaluate their possible concomitant use with clinically approved TB-drugs. This approach allowed the selection of plants effectively used in traditional medicine. In order to verify if the usage of some of these plants can be rationalized, the activity of their traditional preparations was assessed with both an intracellular and extracellular antimycobacterial host-pathogen assays. MATERIALS AND METHODS: An ethnopharmacological survey conducted on 117 TB-patients and 30 healers in Senegal from March to May 2014. The questionnaires were focused on the use of medicinal plants to treat common TB -symptoms (cough longer than 2 weeks, fever, night sweats, weight loss and bloody sputum). Local plant names, utilized organs (herbal drugs) and traditional formulations of the plants were recorded. Extracts were prepared by mimicking the traditional decoction in boiling water and screened for their antimycobacterial activity using Mycobacterium marinum, as a validated TB surrogate, and an Acanthamoeba castellanii - M. marinum whole-cell based host-pathogen assay, to detect anti-infective activities. RESULTS: By the end of the survey, nearly 30 plants were cited and the 12 most cited herbal drugs were collected and their usage documented by extensive literature search. Extracts of the chosen herbs were screened with the described assays; with a main focus on traditional formulas (mainly herbal decoctions). Two of the water extracts from Combretum aculeatum and Guiera senegalensis showed significant antimycobacterial activities when compared to the positive control drug (rifampin). These extracts showed no observable toxicity against amoeba host cells (Acanthamoeba castellanii). CONCLUSIONS: This study demonstrates that most of the patients do not concomitantly use plants and TB drugs (~90% of informants) but, instead, most are treated with medicinal plants before they are admitted to a hospital (41%). Interestingly, among the aqueous extracts assayed, two extracts (Combretum aculeatum (Combretaceae) and Guiera senegalensis (Combretaceae)) collected within this survey demonstrate antimycobacterial activities on the validated whole-cell based host-pathogen assay. Both extracts showed significant activities against intracellular and extracellular - M. marinum growth presenting IC50 lower than 0.5mg/ml compared to the reference drug Rifampin (IC50 of 0.4 and 7µg/ml). No toxicity was observed for amoebae cells at concentration until 0.8mg/ml.

Antioxidant, anti-inflammatory, antiproliferative and antimycobacterial activities of the essential oil of Psidium guineense Sw. and spathulenol.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaves from Psidium guineense Sw. are used in popular medicine for the treatment of inflammatory disease. However, there is no scientific evidence demonstrating this activity. AIM OF THE STUDY: To evaluate the antioxidant, anti-inflammatory, antiproliferative and antimycobacterial activities of the essential oil of P. guineense and spathulenol (a major constituent). The study was conducted in part to provide evidence supporting the ethnobotanical use of the leaves of this species. MATERIAL AND METHODS: The essential oil (EOPG) was extracted from the leaves of P. guineense by hydrodistillation and analysed by gas chromatography-mass spectrometry (GC-MS). The major compound, spathulenol (PG-1), was isolated in a chromatographic column and characterized by nuclear magnetic resonance (NMR). EOPG and PG-1 were evaluated in vitro for antioxidant activity by DPPH, ABTS and MDA methods; anti-inflammatory potential was assessed using two models, including pleurisy and oedema, in mice. The impact of EOPG and PG-1 on cell proliferation was determined via spectrophotometric quantification of the cellular protein content using a sulforhodamine B assay, and anti-Mycobacterium tuberculosis activity was determined using the REMA method. RESULTS: A total of 38 components were identified from the EOPG, with the sesquiterpenic alcohol spathulenol (PG-1) (80.7%) being the major constituent. EOPG and PG-1 exhibited the highest antioxidant activities in the DPPH and MDA system compared with reference standard, with IC50 values ranging from 26.13 to 85.60µg/mL. Oral administration of EOPG and PG-1 showed significant inhibition in the Cg-induced mice paw oedema and pleurisy model. The EOPG (GI50 = 0.89µg/mL) and PG-1 (GI50 = 49.30µg/mL) were particularly effective against the ovarian cancer cell line. Both showed moderate antimycobacterial activity. CONCLUSION: For the first time, this study demonstrated the antioxidant, anti-inflammatory, antiproliferative and antimycobacterial properties of the essential oil of P. guineense (leaves were collected in Dourados-MS) and spathulenol, collaborating the etnhopharmacologycal use of this plant due to its an anti-inflammatory effect.

Chlorflavonin Targets Acetohydroxyacid Synthase Catalytic Subunit IlvB1 for Synergistic Killing of Mycobacterium tuberculosis.

The flavonoid natural compound chlorflavonin was isolated from the endophytic fungus Mucor irregularis, which was obtained from the Cameroonian medicinal plant Moringa stenopetala. Chlorflavonin exhibited strong growth inhibitory activity in vitro against Mycobacterium tuberculosis (MIC90 1.56 µM) while exhibiting no cytotoxicity toward the human cell lines MRC-5 and THP-1 up to concentrations of 100 µM. Mapping of resistance-mediating mutations employing whole-genome sequencing, chemical supplementation assays, and molecular docking studies as well as enzymatic characterization revealed that chlorflavonin specifically inhibits the acetohydroxyacid synthase catalytic subunit IlvB1, causing combined auxotrophies to branched-chain amino acids and to pantothenic acid. While exhibiting a bacteriostatic effect in monotreatment, chlorflavonin displayed synergistic effects with the first-line antibiotic isoniazid and particularly with delamanid, leading to a complete sterilization in liquid culture in combination treatment. Using a fluorescent reporter strain, intracellular activity of chlorflavonin against Mycobacterium tuberculosis inside infected macrophages was demonstrated and was superior to streptomycin treatment.

Rauvolfianine, a new antimycobacterial glyceroglycolipid and other constituents from Rauvolfia caffra. Sond (Apocynaceae).

The chemical investigation of the extract of the dried leaves of Rauvolfia caffra (Sond) (synonym Rauvolfia macrophylla) (Apocynaceae) led to isolation of a new glycoside derivative, rauvolfianine (1) as well as six known compounds: oleanolic acid (2), sitosterol-3-O-ß-D-glucopyranoside (3), betulinic acid (4), vellosimine (5), sarpagine (6) and D-fructofuranosyl-ß-(2→1)-α-D-glucopyranoside (7). Compounds 1, 2, 3, 4 and 7 were evaluated for antitubercular activity. Compounds 1 and 2 were the most active (MIC = 7.8125 and 31.25 µg/mL) towards the Isoniazid resistant strain of Mycobacterium tuberculosis AC45. Their structures and relative stereochemistry were elucidated by spectroscopic methods.