Assessment of antimalarial activity of crude extract of Chan-Ta-Lee-La and Pra-Sa-Chan-Dang formulations and their plant ingredients for new drug candidates of malaria treatment: In vitro and in vivo experiments.

The emergence and spread of antimalarial drug resistance have become a significant problem worldwide. The search for natural products to develop novel antimalarial drugs is challenging. Therefore, this study aimed to assess the antimalarial and toxicological effects of Chan-Ta-Lee-La (CTLL) and Pra-Sa-Chan-Dang (PSCD) formulations and their plant ingredients. The crude extracts of CTLL and PSCD formulations and their plant ingredients were evaluated for in vitro antimalarial activity using Plasmodium lactate dehydrogenase enzyme and toxicity to Vero and HepG2 cells using the tetrazolium salt method. An extract from the CTLL and PSCD formulations exhibiting the highest selectivity index value was selected for further investigation using Peter's 4-day suppressive test, curative test, prophylactic test, and acute oral toxicity in mice. The phytochemical constituents were characterized using gas chromatography-mass spectrometry (GC-MS). Results showed that ethanolic extracts of CTLL and PSCD formulations possessed high antimalarial activity (half maximal inhibitory concentration = 4.88, and 4.19 g/mL, respectively) with low cytotoxicity. Ethanolic extracts of the CTLL and PSCD formulations demonstrated a significant dose-dependent decrease in parasitemia in mice. The ethanolic CTLL extract showed the greatest suppressive effect after 4 days of suppressive (89.80%) and curative (35.94%) testing at a dose of 600 mg/kg. Moreover, ethanolic PSCD extract showed the highest suppressive effect in the prophylactic test (65.82%) at a dose of 600 mg/kg. There was no acute toxicity in mice treated with ethanolic CTLL and PSCD extracts at 2,000 mg/kg bodyweight. GC-MS analysis revealed that the most abundant compounds in the ethanolic CTLL extract were linderol, isoborneol, eudesmol, linoleic acid, and oleic acid, whereas ethyl 4-methoxycinnamate was the most commonly found compound in the ethanolic PSCD extract, followed by 3-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-chromen-4-one, flamenol, oleic acid amide, linoleic acid, and oleic acid. In conclusions, ethanolic CTLL and PSCD extracts exhibited high antimalarial efficacy in vitro. The ethanolic CTLL extract at a dose of 600 mg/kg exhibited the highest antimalarial activity in the 4-day suppressive and curative tests, whereas the ethanolic PSCD extract at a dose of 600 mg/kg showed the highest antimalarial activity in the prophylactic test.

Assessment of the Gastroprotective Effect of the Chaga Medicinal Mushroom, Inonotus obliquus (Agaricomycetes), Against the Gastric Mucosal Ulceration Induced by Ethanol in Experimental Rats.

The chaga medicinal mushroom (Inonotus obliquus) was traditionally used to treat various ailments. To establish the pharmacological properties of I. obliquus, studies were performed to show the antiulcer activity of the ethanolic extract. The ethanolic extract of I. obliquus was prepared. The antiulcer activity of I. obliquus was determined using gastric ulcerated rats (ulceration induced by ethanol). The ethanolic extract of I. obliquus (200 mg/kg) did not cause any sign of toxicity or sensitivity to rats when the extracts were administered by oral feed. Oral administration of ethanolic extract of I. obliquus exhibited antiulcer activity in all models used. The ethanolic extract of I. obliquus showed an effective antiulcer activity, which could be due to the presence of various biologically active compounds. This confirmed the traditional uses of I. obliquus in the treatment of ailments.

In vivo antiplasmodial activity and toxicological assessment of hydroethanolic crude extract of Ajuga remota.

BACKGROUND: Malaria is one of the most life-threatening health problems worldwide and treatment has been compromised by drug resistance. Identifying lead molecules from natural products might help to find better anti-malarial drugs, since those obtained from natural sources are still effective against malarial parasites. This study aimed at investigating the in vivo antiplasmodial activity of crude extract of the leaves of Ajuga remota together with its safety in mice models. METHODS: In vivo parasite growth inhibitory effect of crude extract was assessed in mice inoculated with Plasmodium berghei (ANKA strain). The in vivo antiplasmodial activity of the test extract was performed against early infection (4-day suppressive test), curative effect against established infection and prophylactic effect against residual infection. Acute and sub-acute toxicity were carried out according to OECD guidelines. RESULTS: In vivo parasite growth inhibition effect of hydroethanolic crude extract of A. remota was evaluated at 30, 50 and 100 mg/kg dose levels. It suppressed parasitaemia by 77.34% at 100 mg/kg dose level in the 4-day test. In curative and prophylactic potential tests, it suppressed parasitaemia by 66.67 and 59.66% at 100 mg/kg dose level, respectively. In vivo toxicity tests revealed no toxicity. All parasitaemia suppressions were statistically significant at P < 0.05 as compared to the vehicle-treated group. The crude extract also prolonged survival time in a dose dependent manner. CONCLUSIONS: The investigation results suggest that the leave extract of Ajuga remota possesses antimalarial activity.

In Vitro Assessment of Extracts of the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Higher Basidiomycetes) Against Different Plant Pathogenic Fungi.

Five isolates of the lingzhi or reishi medicinal mushroom Ganoderma lucidum (GL-1, GL-2, GL-3, GL-4, GL-5) were collected from different locations within and surrounding Lahore, Pakistan, to study the antifungal potential of their bioactive compounds. After studying morphology, different concentrations of the extracts were prepared in methanol and water using a Soxhlet extractor. Different cultures of fungal pathogens were acquired from the First Fungal Culture Bank of Pakistan, University of the Punjab, Lahore. The antimicrobial potential of 5 G. lucidum samples against 5 fungal pathogens (Fusarium oxysporum, Aspergillus niger, A. flavus, Penicillium sp., and Alternaria alternata) was observed. The lowest biomass reduction (7%) was observed in 1% and 2% concentrations of a methanolic extract and 6% in the case of a water extract. Major inhibition was observed using higher concentrations of the methanolic extract (3% and 4%). These extracts significantly suppressed fungal biomass up to 38% and 56% in A. niger, 47% in A. flavus, 58% in ,i>Penicillium sp., 46% in A. alternaria, and 45% in F. oxysporum compared with the control. It was concluded from these studies that methanolic extracts of G. lucidum showed better activity against all plant fungal pathogens when compared with the water extracts.

Global assessment of Antrodia cinnamomea-induced microRNA alterations in hepatocarcinoma cells.

Recent studies have demonstrated a potent anticancer potential of medicinal fungus Antrodia cinnamomea, especially against hepatocarcinoma. These studies, however, were performed with prolonged treatments, and the early anticancer events remain missing. To probe the early anticancer mechanisms of A. cinnamomea, we treated SK-Hep-1 liver cancer cell with A. cinnamomea fruiting body extract for 2 and 4 hours, sequenced RNA samples with next-generation sequencing approach, and profiled the genome-wide miRNA and mRNA transcriptomes. Results unmistakably associated the early anticancer effect of A. cinnamomea fruiting body extract with a global downregulation of miRNAs which occurred solely in the A. cinnamomea fruiting body extract-treated SK-Hep-1 cells. Moreover, the inhibitory effect of A. cinnamomea fruiting body extract upon cancer miRNAs imposed no discrimination against any particular miRNA species, with oncomirs miR-21, miR-191 and major oncogenic clusters miR-17-92 and miR-106b-25 among the most severely downregulated. Western blotting further indicated a decrease in Drosha and Dicer proteins which play a key role in miRNA biogenesis, together with an increase of XRN2 known to participate in miRNA degradation pathway. Transcriptome profiling followed by GO and pathway analyses indicated that A. cinnamomea induced apoptosis, which was tightly associated with a downregulation of PI3K/AKT and MAPK pathways. Phosphorylation assay further suggested that JNK and c-Jun were closely involved in the apoptotic process. Taken together, our data indicated that the anticancer effect of A. cinnamomea can take place within a few hours by targeting multiple proteins and the miRNA system. A. cinnamomea indiscriminately induced a global downregulation of miRNAs by simultaneously inhibiting the key enzymes involved in miRNA maturation and activating XRN2 protein involved in miRNA degradation. Collapsing of the miRNA system together with downregulation of cell growth and survival pathways and activation of JNK signaling unleash the extrinsic and intrinsic apoptosis pathways, leading to the cancer cell death.

Sweet potatoes as a basic component in developing a medium for the cultivation of lactobacilli.

A sweet potato medium (SPM) was formed with extract from baked sweet potatoes supplemented with 0, 4, or 8 g/L of each nitrogen source (beef extract, yeast extract, and proteose peptone #3) to form SPM1, SPM2, and SPM3 respectively. Lactobacilli MRS was used as control medium. Ten Lactobacillus strains containing an average of 2.34 ± 0.29 log CFU/mL were inoculated individually into batches of MRS, SPM1, SPM2, and SPM3. The growth patterns for the tested Lactobacillus strains growing in SPM2 and SPM3 were found to be similar to that in MRS. The average final population after 24 h of incubation in MRS, SPM2, and SPM3 reached 10.41 ± 0.35, 10.59 ± 0.27, and 10.72 ± 0.19 log CFU/mL respectively. SPM2 and SPM3 maintained higher pH values throughout the incubation period than MRS. These findings indicate that SPM2 can be a suitable medium for the growth of Lactobacillus and can provide an alternative at low-cost.

Alveolar macrophages play a key role in cockroach-induced allergic inflammation via TNF-α pathway.

The activity of the serine protease in the German cockroach allergen is important to the development of allergic disease. The protease-activated receptor (PAR)-2, which is expressed in numerous cell types in lung tissue, is known to mediate the cellular events caused by inhaled serine protease. Alveolar macrophages express PAR-2 and produce considerable amounts of tumor necrosis factor (TNF)-α. We determined whether the serine protease in German cockroach extract (GCE) enhances TNF-α production by alveolar macrophages through the PAR-2 pathway and whether the TNF-α production affects GCE-induced pulmonary inflammation. Effects of GCE on alveolar macrophages and TNF-α production were evaluated using in vitro MH-S and RAW264.6 cells and in vivo GCE-induced asthma models of BALB/c mice. GCE contained a large amount of serine protease. In the MH-S and RAW264.7 cells, GCE activated PAR-2 and thereby produced TNF-α. In the GCE-induced asthma model, intranasal administration of GCE increased airway hyperresponsiveness (AHR), inflammatory cell infiltration, productions of serum immunoglobulin E, interleukin (IL)-5, IL-13 and TNF-α production in alveolar macrophages. Blockade of serine proteases prevented the development of GCE induced allergic pathologies. TNF-α blockade also prevented the development of such asthma-like lesions. Depletion of alveolar macrophages reduced AHR and intracellular TNF-α level in pulmonary cell populations in the GCE-induced asthma model. These results suggest that serine protease from GCE affects asthma through an alveolar macrophage and TNF-α dependent manner, reflecting the close relation of innate and adaptive immune response in allergic asthma model.

In vitro antibacterial and time-kill assessment of crude methanolic stem bark extract of Acacia mearnsii de wild against bacteria in shigellosis.

Shigellosis is an important cause of worldwide morbidity and mortality among young children and old people for which treatment with antimicrobial agents is limited. Hence, the need for curative potentials obtainable from medicinal plants becomes inevitable. This study was carried out to assess the antibacterial potentials of crude methanolic extract of the stem bark of Acacia mearnsii against some selected bacteria of clinical importance in shigellosis. The bacteria were inhibited by the extract to produce concentration dependent inhibition zones. The extract exhibited a varied degree of antibacterial activity against all the tested isolates. The MIC values for Gram negative (0.0391-0.3125) mg/mL and those of Gram positive bacteria (0.0781-0.625) mg/mL indicated that the Gram negative bacteria were more inhibited by the extract than the Gram positive bacteria. Average log reduction in viable cell count in time-kill assay ranged between -2.456 Log10 to 2.230 Log10 cfu/mL after 4 h of interaction, and between -2.921 Log10 and 1.447 Log10 cfu/mL after 8 h interaction in 1× MIC and 2× MIC of the extract. The study provided scientific justification for the use of the crude methanolic extract from the stem bark of A. mearnsii in shigellosis. The degree of the antibacterial activity indicated that the crude extract is a potential source of bioactive compounds that could be useful for the development of new antimicrobial agents capable of decreasing the burden of drug resistance and cost of management of diseases of clinical and public health importance in South Africa.

Assessment of the immunomodulatory activity of cheese extracts by a complete and easy to handle in vitro screening methodology.

A simple and rapid screening methodology based on the in vitro culture of murine hybridoma and human T-lymphocytes was developed to assess the potential immunomodulatory activity of water-soluble extracts (WSE) from cheese. The two immune cell lines were cultured in microplates with or without cheese WSE. The proliferation and the metabolic activity of cells were monitored at their different growth phases by the BrdU (5-bromo-2'-deoxyuridine) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide] assays, respectively. WSE from Abondance cheese enhanced DNA synthesis and the metabolic activity of the hybridoma cells (1.2-and 1.3-to 1.6-fold of the control, respectively) and also of the T lymphocytes (1.5-fold of the control) for almost all of the dilutions tested. To evaluate the validity of the results obtained following this screening methodology, hybridoma and T-lymphocyte cells were cultivated in 50 ml flasks: the maximal cell density was increased by about 10-16% in presence of cheese WSE for both cell lines and the antibody production by the hybridoma cells was increased by 50%.

Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections.

BACKGROUND: Double-stranded RNA (dsRNA) is a potent initiator of gene silencing in a diverse group of organisms that includes plants, Caenorhabditis elegans, Drosophila and mammals. We have previously shown and patented that mechanical inoculation of in vitro-transcribed dsRNA derived from viral sequences specifically prevents virus infection in plants. The approach required the in vitro synthesis of large amounts of RNA involving high cost and considerable labour. RESULTS: We have developed an in vivo expression system to produce large amounts of virus-derived dsRNAs in bacteria, with a view to providing a practical control of virus diseases in plants. Partially purified bacterial dsRNAs promoted specific interference with the infection in plants by two viruses belonging to the tobamovirus and potyvirus groups. Furthermore, we have demonstrated that easy to obtain, crude extracts of bacterially expressed dsRNAs are equally effective protecting plants against virus infections when sprayed onto plant surfaces by a simple procedure. Virus infectivity was significantly abolished when plants were sprayed with French Press lysates several days before virus inoculation. CONCLUSION: Our approach provides an alternative to genetic transformation of plant species with dsRNA-expressing constructs capable to interfere with plant viruses. The main advantage of this mode of dsRNA production is its simplicity and its extremely low cost compared with the requirements for regenerating transgenic plants. This approach provides a reliable and potential tool, not only for plant protection against virus diseases, but also for the study of gene silencing mechanisms in plant virus infections.