Exploring Potential Antimalarial Candidate from Medicinal Plants of Kheaw Hom Remedy.

The Kheaw Hom remedy is a traditional Thai medicine widely used to treat fevers. Some plant ingredients in this remedy have been investigated for their antimicrobial, antiviral, anti-inflammatory, and antioxidant activities. However, there have been no reports on the antimalarial activities of the medicinal plants in this remedy. Therefore, this study focuses on identifying potential antimalarial drug candidates from the medicinal plant ingredients of the Kheaw Hom remedy. Eighteen plants from the Kheaw Hom remedy were extracted using distilled water and ethanol. All extracts were investigated for their in vitro antimalarial activity and cytotoxicity. An extract that exhibited good in vitro antimalarial activity and low toxicity was selected for further investigation by using Peter's 4-day suppressive test and an acute oral toxicity evaluation in mice. Based on the in vitro antimalarial activity and cytotoxicity studies, the ethanolic extract of Globba malaccensis rhizomes showed promising antimalarial activity against the Plasmodium falciparum K1 strain (IC50 = 1.50 µg/mL) with less toxicity to Vero cells (CC50 of >80 µg/mL). This extract exhibited a significant dose-dependent reduction in parasitemia in P. berghei-infected mice. The maximum suppressive effect of this extract (60.53%) was observed at the highest dose administered (600 mg/kg). In a single-dose acute toxicity test, the animals treated at 2000 mg/kg died within 48 h after extract administration. In conclusion, our study indicates that the ethanolic extract of G. malaccensis rhizomes exhibited in vitro and in vivo antimalarial activities, which could serve as a promising starting point for antimalarial drug.

Evaluation of the antimalarial activity and toxicity of Mahanil-Tang-Thong formulation and its plant ingredients.

BACKGROUND: Novel potent antimalarial agents are urgently needed to overcome the problem of drug-resistant malaria. Herbal treatments are of interest because plants are the source of many pharmaceutical compounds. The Mahanil-Tang-Thong formulation is a Thai herbal formulation in the national list of essential medicines and is used for the treatment of fever. Therefore, this study aimed to evaluate the antimalarial activity of medicinal plants in the Mahanil-Tang-Thong formulation. METHODS: Nine medicinal plant ingredients of the Mahanil-Tang-Thong formulation were used in this study. Aqueous and ethanolic extracts of all the plants were analyzed for their phytochemical constituents. All the extracts were used to investigate the in vitro antimalarial activity against Plasmodium falciparum K1 (chloroquine-resistant strain) by using the lactate dehydrogenase (pLDH) method and cytotoxicity in Vero cells by using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Additionally, an extract with potent in vitro antimalarial activity and no toxicity was selected to determine the in vivo antimalarial activity with Peters' 4-day suppressive test against the Plasmodium berghei ANKA strain. Acute toxicity was evaluated in mice for 14 days after the administration of a single oral dose of 2000 mg/kg. RESULTS: This study revealed that ethanolic extracts of Sapindus rarak DC., Tectona grandis L.f., Myristica fragrans Houtt. and Dracaena loureiri Gagnep. exhibited potent antimalarial activity, with half-maximal inhibitory concentration (IC50) values of 2.46, 3.21, 8.87 and 10.47 µg/ml, respectively, while the ethanolic of the formulation exhibited moderate activity with an IC50 value of 37.63 µg/ml and its aqueous extract had no activity (IC50 = 100.49 µg/ml). According to the in vitro study, the ethanolic wood extract of M. fragrans was selected for further investigation in an in vivo mouse model. M. fragrans extract at doses of 200, 400, and 600 mg/kg body weight produced a dose-dependent reduction in parasitemia by 8.59, 31.00, and 52.58%, respectively. No toxic effects were observed at a single oral dose of 2000 mg/kg body weight. CONCLUSION: This study demonstrates that M. fragrans is a potential candidate for the development of antimalarial agents.

In Vitro and In Vivo Antimalarial Activities and Toxicological Assessment of Pogostemon Cablin (Blanco) Benth.

The aim of this study was to investigate the antimalarial activities and toxicity of Pogostemon cablin extracts. In vitro activities against the chloroquine-resistant Plasmodium falciparum K1 strain were assessed by using the Plasmodium lactate dehydrogenase enzyme (pLDH) assay, while in vivo activity against the Plasmodium berghei ANKA strain in mice was investigated using a 4-day suppressive test. The in vitro and in vivo toxicity were determined in Vero cells and mice, respectively. The ethanolic extract possessed antimalarial activity with an IC50 of 24.49 ± 0.01 µg/ml, whereas the aqueous extract showed an IC50 of 549.30 ± 0.07 µg/ml. Cytotoxic analyses of the ethanolic and aqueous extracts revealed a nontoxic effect on Vero cells at a concentration of 80 µg/ml. Based on a preliminary study of in vitro antimalarial activity, the ethanolic extract was chosen as a potential agent for further in vivo antimalarial activity analysis in mice. The ethanolic extract, which showed no toxic effect on mice at a dose of 2000 mg/kg body weight, significantly suppressed parasitemia in mice by 38.41%, 45.12% and 89.00% at doses of 200, 400 and 600 mg/kg body weight, respectively. In conclusion, this study shows that the ethanolic P. cablin extract possesses in vitro and in vivo antimalarial activity without toxic effects.

In-vitro Studies of Anti-EGFR Tyrosine Kinase Activity of Thai nutraceutical Plants.

Functional foods have emerged as a new approach to improve human health in term of nutraceutical to prevent people from illness rather than cure patients through medical treatment. In Asian society, particularly in Thailand, the utilizations of functional ingredients have been integrated in every parts of ordinary life. In this study, the tyrosine kinase activity of epidermal growth factor receptor (EGFR) inhibiting properties of 23 Thai's herbs-ethanol extracts have been examined. The crude extracts of only four species that inhibit the activity of EGFR-tyrosine kinase, Azadirachta indica (neem, Sa-dao), Brucea javanica (L.) Merr. (Rajadad), Hibiscus sabdariffa L. (Roselle, Krachiap daeng), and Saccharum chinensis Roxb. (Red sugar cane). Moreover, only ethanol extractions from A. indica and B. javanica were also showed antitumor effect to non-small cell lung cancer, A549 cells.

Acquisition of naturally acquired antibody response to Plasmodium falciparum erythrocyte membrane protein 1-DBLα and differential regulation of IgG subclasses in severe and uncomplicated malaria

Objectives:To explore whether individuals infected with Plasmodium falciparum (P.falciparum) develop antibodies directed against PfEMP1-DBLα,and to assess their IgG subclass distribution in severe and uncomplicated malaria.Methods:The anti-PfDBLα IgG and their IgG subclass distributions in plasma of severe (SM) and uncomplicated malaria (UCM) were assessed by enzyme-linked immunoabsorbent assay.The antibody profiles to P.falciparum blood stage antigens were evaluated.CD36 binding ability was determined by static receptor-binding assays.Rosette formation was performed by staining with acridine orange.Results:Significantly higher number of UCM (86.48％) than SM (57.78％) plasma contained total acquisition of specific IgG to P.falciparum antigens (P =0.000).Similar manners were seen in response to P.falciparum DBLα with significant difference (UCM,59.46％ vs SM,40.00％;P =0.014).Anti-PfDBLα-IgG1 and-IgG3 were the predominant subclasses.Similar percentage of UCM (31.82％) and SM (33.33％) plasma contained only IgG1,while 13.64％ of UCM and 27.78％ of SM plasma contained only IgG3.AntiPfTDBLα-IgG1 coexpressed with more than one subclass was noted (UCM,27.27％;SM,16.67％).Obviously,IgG1 coexpressed with IgG3 (9.09％) was observed in only UCM plasma.IgG1 was coexpressed with IgG2 in UCM (9.09％) and SM (11.11％) plasma,while IgG1 was coexpressed with IgG4 only in UCM plasma (4.55％).IgG subclasses to P.falciparum antigens were distributed in a similar manner.Only the levels of IgG1,but not IgG3 were significantly higher in UCM than in SM.Conclusions:These data suggest that individuals infected with P.falciparum can develop the anti-PfEMP1 antibodies with the major contribution of specific IgG subclasses.The balance and the levels of anti-PfDBLα IgG subclasses play a crucial role in antibody mediated protection against severe malaria.

Evaluation of the anti-malarial activity and cytotoxicity of 2,4-diamino-pyrimidine-based kinase inhibitors.

A series of 2,4 diamino-pyrimidines have been identified from an analysis of open access high throughput anti-malarial screening data reported by GlaxoSmithKline at the 3D7 and resistant Dd2 strains. SAR expansion has been performed using structural knowledge of the most plausible parasite target. Seventeen new analogs have been synthesized and tested against the resistant K1 strain of Plasmodium falciparum (Pf). The cytotoxicity of the compounds was assessed in Vero and A549 cells and their selectivity towards human kinases including JAK2 and EGFR were undertaken. We identified compound 5n and 5m as sub-micromolar inhibitors, with equivalent anti-malarial activity to Chloroquine (CQ). Compounds 5d and 5k, µM inhibitors of Pf, displayed improved cytotoxicity with weak inhibition of the human kinases.

Computational analyses of curcuminoid analogs against kinase domain of HER2.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) has an important role in cancer aggressiveness and poor prognosis. HER2 has been used as a drug target for cancers. In particular, to effectively treat HER2-positive cancer, small molecule inhibitors were developed to target HER2 kinase. Knowing that curcumin has been used as food to inhibit cancer activity, this study evaluated the efficacy of natural curcumins and curcumin analogs as HER2 inhibitors using in vitro and in silico studies. The curcumin analogs considered in this study composed of 4 groups classified by their core structure, ß-diketone, monoketone, pyrazole, and isoxazole. RESULTS: In the present study, both computational and experimental studies were performed. The specificity of curcumin analogs selected from the docked results was examined against human breast cancer cell lines. The screened curcumin compounds were then subjected to molecular dynamics simulation study. By modifying curcumin analogs, we found that protein-ligand affinity increases. The benzene ring with a hydroxyl group could enhance affinity by forming hydrophobic interactions and the hydrogen bond with the hydrophobic pocket. Hydroxyl, carbonyl or methoxy group also formed hydrogen bonds with residues in the adenine pocket and sugar pocket of HER2-TK. These modifications could suggest the new drug design for potentially effective HER2-TK inhibitors. Two outstanding compounds, bisdemethylcurcumin (AS-KTC006) and 3,5-bis((E)-3,4-dimethoxystyryl)isoxazole (AS-KTC021 ),were well oriented in the binding pocket almost in the simulation time, 30 ns. This evidence confirmed the results of cell-based assays and the docking studies. They possessed more distinguished interactions than known HER2-TK inhibitors, considering them as a promising drug in the near future. CONCLUSIONS: The series of curcumin compounds were screened using a computational molecular docking and followed by human breast cancer cell lines assay. Both AS-KTC006 and AS-KTC021 could inhibit breast cancer cell lines though inhibiting of HER2-TK. The intermolecular interactions were confirmed by molecular dynamics simulation studies. This information would explore more understanding of curcuminoid structures and HER2-TK.

Purification, crystallization and preliminary X-ray analysis of recombinant betaine aldehyde dehydrogenase 2 (OsBADH2), a protein involved in jasmine aroma, from Thai fragrant rice (Oryza sativa L.).

Fragrant rice (Oryza sativa L.) betaine aldehyde dehydrogenase 2 (OsBADH2) is a key enzyme in the synthesis of fragrance aroma compounds. The extremely low activity of OsBADH2 in catalyzing the oxidation of acetaldehyde is believed to be crucial for the accumulation of the volatile compound 2-acetyl-1-pyrroline (2AP) in many scented plants, including fragrant rice. Recombinant fragrant rice OsBADH2 was expressed in Escherichia coli as an N-terminal hexahistidine fusion protein, purified using Ni Sepharose affinity chromatography and crystallized using the microbatch method. Initial crystals were obtained within 24 h using 0.1 M Tris pH 8.5 with 30%(w/v) PEG 4000 and 0.2 M magnesium chloride as the precipitating agent at 291 K. Crystal quality was improved when the enzyme was cocrystallized with NAD(+). Improved crystals were grown in 0.1 M HEPES pH 7.4, 24%(w/v) PEG 4000 and 0.2 M ammonium chloride and diffracted to beyond 2.95 Å resolution after being cooled in a stream of N(2) immediately prior to X-ray diffraction experiments. The crystals belonged to space group C222(1), with unit-cell parameters a = 66.03, b = 183.94, c = 172.28 Å. An initial molecular-replacement solution has been obtained and refinement is in progress.

Biochemical and enzymatic study of rice BADH wild-type and mutants: an insight into fragrance in rice.

Betaine aldehyde dehydrogenase 2 (BADH2) is believed to be involved in the accumulation of 2-acetyl-1-pyrroline (2AP), one of the major aromatic compounds in fragrant rice. The enzyme can oxidize ω-aminoaldehydes to the corresponding ω-amino acids. This study was carried out to investigate the function of wild-type BADHs and four BADH2 mutants: BADH2_Y420, containing a Y420 insertion similar to BADH2.8 in Myanmar fragrance rice, BADH2_C294A, BADH2_E260A and BADH2_N162A, consisting of a single catalytic-residue mutation. Our results showed that the BADH2_Y420 mutant exhibited less catalytic efficiency towards γ-aminobutyraldehyde but greater efficiency towards betaine aldehyde than wild-type. We hypothesized that this point mutation may account for the accumulation of γ-aminobutyraldehyde/Δ(1)-pyrroline prior to conversion to 2AP, generating fragrance in Myanmar rice. In addition, the three catalytic-residue mutants confirmed that residues C294, E260 and N162 were involved in the catalytic activity of BADH2 similar to those of other BADHs.

Sequence variation of PfEMP1-DBLalpha in association with rosette formation in Plasmodium falciparum isolates causing severe and uncomplicated malaria.

BACKGROUND: Rosetting and cytoadherence of Plasmodium falciparum-infected red blood cells have been associated with severity of malaria. ICAM-1 and CD36 are the main host cell receptors, while PfEMP1-DBLalpha is a major parasite ligand, which can contribute to rosette formation. This study is aimed at demonstrating whether the highly polymorphic PfEMP1-DBLalpha sequences occurring among Thai isolates causing severe and uncomplicated malaria are associated with their ability to form rosettes and reflected the clinical outcome of the patients. METHODS: Two hundred and ninety five PfEMP1-DBLalpha sequences from Thai clinical isolates causing severe and uncomplicated malaria were evaluated by sequencing and direct comparison using the specific text string analysis functions in Microsoft Excel and Perl. The relationships between the PfEMP1-DBLalpha sequences were also analysed by network analysis. The binding abilities of parasitized red blood cells (PRBCs) to CD36, wild type ICAM-1, ICAM-1Kilifi and ICAM-1S22/A under static condition were included. RESULTS: Two hundred and eighty one non-identical amino acid sequences were identified (< 95% sequence identity). When the distributions of semi-conserved features (PoLV1-4 and sequence group) within the rosetting domain PfEMP1-DBLalpha were observed, close similarity was found between isolates from the two disease groups. The sequence group 1 representing uncomplicated malaria was significantly different from the sequence group 3 representing the majority of severe malaria (p = 0.027). By using a simple non-phylogenetic approach to visualize the sharing of polymorphic blocks (position specific polymorphic block, PSPB) and cys/PoLV among DBLalpha sequences, the sequence group 1 was split from the other five sequence groups. The isolates belonging to sequence group 5 gave the highest mean rosetting rate (21.31%). However, within sequence group 2 and group 6, the isolates causing severe malaria had significantly higher rosetting rate than those causing uncomplicated malaria (p = 0.014, p = 0.007, respectively). CONCLUSION: This is the first report of PfEMP1-DBLalpha analysis in clinical Thai isolates using semi-conserved features (cys/PoLV and PSPBs). The cys/PoLV group 5 gave the highest rosetting rate. PfEMP1-DBLalpha domains in Thai isolates are highly diverse, however, clinical isolates from severe and uncomplicated malaria shared common sequences.