Natural mycelial biomass (MB) and exopolysaccharides (EPS) of Malaysian tiger milk mushroom Lignosus rhinocerus are considered high-end components due to their high commercial potential value in drug discovery. This study aims to evaluate the toxicity of the mushroom extracts' generated in a bioreactor using the zebrafish embryo toxicity (ZFET) model assay as a new therapy for treating asthma. Both MB and EPS extracts, at concentrations 0.16-10 mg/mL, were tested for ZFET and early development effects on Zebrafish Embryos (ZE) during 24-120 h post-fertilisation (HPF). Findings revealed that MB was deemed safe with an LC50 of 0.77 mg/mL; the EPS were non-toxic (LC50 of 0.41 mg/mL). Neither MB nor EPS delayed hatching nor teratogenic defects in the treated ZE at a 2.5 mg/mL dose. There were no significant changes in the ZE heart rate after treatments with MB (130 beats/min) and EPS (140 beats/min), compared to that of normal ZE (120-180 beats/min). Mixing both natural compounds MB and EPS did not affect toxicity using ZFET testing; thus, intimating their safe future use as therapeutic interventions. This represents the first study to have used the ZFET assay on MB and EPS extracts of L. rhinocerus for future health applications.
The pellet morphology and diameter range (DR) of Ganoderma lucidum were observed in a repeated-batch fermentation (RBF) for the trio total production of biomass, exopolysaccharide (EPS) and endopolysaccharide (ENS). Two factors were involved in RBF; broth replacement ratio (BRR: 60%, 75% and 90%) and broth replacement time point (BRTP: log, transition and stationary phase) in days. In RBF, 34.31 g/L of biomass favoured small-compact pellets with DR of 20.67 µm< d < 24.00 µm (75% BRR, day 11 of BRTP). EPS production of 4.34 g/L was prone to ovoid-starburst pellets with DR of 34.33 µm< d <35.67 µm (75% BRR, day 13 of BRTP). Meanwhile, the highest 2.43 g/L of ENS production favoured large-hollow pellets with DR of 34.00 µm< d < 38.67 µm (90% BRR, day 13 of BRTP). In addition, RBF successfully shortened the biomass-EPS-ENS fermentation period (31, 33 and 35 days) from batch to 5 days, in seven consecutive cycles of RBF. In a FTIR detection, β-glucan (BG) from EPS and ENS extracts were associated with β-glycosidic linkages (2925 cm-1, 1635 cm-1, 1077 cm-1, 920 cm-1 and 800 cm-1 wavelengths) with similar 1H NMR spectral behaviour (4.58, 3.87 and 3.81 ppm). Meanwhile, 4 mg/L of BG gave negative cytotoxic effects on normal gingival cell line (hGF) but induced antiproliferation (IC50 = 0.23 mg/mL) against cancerous oral Asian cellosaurus cell line (ORL-48). Together, this study proved that G. lucidum mycelial pellets could withstand seven cycles of long fermentation condition and possessed anti-oral cancer beta-glucan, which suits large-scale natural drug fermentation.
Eurycoma longifolia is one of the commonly consumed herbal preparations and its major chemical compound, eurycomanone, has been described to have antimalarial, antipyretic, aphrodisiac, and cytotoxic activities. Today, the consumption of E. longifolia is popular through the incorporation of its extract in food items, most frequently in drinks such as tea and coffee. In the current study, the characterisation of the physicochemical and pharmacokinetic (PK) attributes of eurycomanone were conducted via a series of in vitro and in vivo studies in rats and mice. The solubility and chemical stability of eurycomanone under the conditions of the gastrointestinal tract environment were determined. The permeability of eurycomanone was investigated by determining its distribution coefficient in aqueous and organic environments and its permeability using the parallel artificial membrane permeability assay system and Caco-2 cultured cells. Eurycomanone's stability in plasma and its protein-binding ability were measured by using an equilibrium dialysis method. Its stability in liver microsomes across species (mice, rat, dog, monkey, and human) and rat liver hepatocytes was also investigated. Along with the PK evaluations of eurycomanone in mice and rats, the PK parameters for the Malaysian Standard (MS: 2409:201) standardised water extract of E. longifolia were also evaluated in rats. Both rodent models showed that eurycomanone in both the compound form and extract form had a half-life of 0.30 h. The differences in the bioavailability of eurycomanone in the compound form between the rats (11.8%) and mice (54.9%) suggests that the PK parameters cannot be directly extrapolated to humans. The results also suggest that eurycomanone is not readily absorbed across biological membranes. However, once absorbed, the compound is not easily metabolised (is stable), hence retaining its bioactive properties, which may be responsible for the various reported biological activities.
Malaysia has a national goal to eliminate malaria by 2020. Understanding the genetic diversity of malaria parasites in residual transmission foci can provide invaluable information which may inform the intervention strategies used to reach elimination targets. This study was conducted to determine the genetic diversity level of P. falciparum isolates in malaria residual foci areas of Sabah. Malaria active case detection was conducted in Kalabakan and Kota Marudu. All individuals in the study sites were screened for malaria infection by rapid diagnostic test. Blood from P. falciparum-infected individuals were collected on filter paper prior to DNA extraction. Genotyping was performed using merozoite surface protein-1 (MSP-1), merozoite surface protein-2 (MSP-2), glutamate rich protein (GLURP) and 10 neutral microsatellite loci markers. The size of alleles, multiplicity of infection (MOI), mean number of alleles (Na), expected heterozygosity (He), linkage disequilibrium (LD) and genetic differentiation (FST) were determined. In Kalabakan, the MSP-1 and MSP-2 alleles were predominantly K1 and FC27 family types, respectively. The GLURP genotype VI (751-800 bp) was predominant. The MOI for MSP-1 and MSP-2 were 1.65 and 1.20, respectively. The Na per microsatellite locus was 1.70. The He values for MSP-1, MSP-2, GLURP and neutral microsatellites were 0.17, 0.37, 0.70 and 0.33, respectively. In Kota Marudu, the MSP-1 and MSP-2 alleles were predominantly MAD20 and 3D7 family types, respectively. The GLURP genotype IV (651-700 bp) was predominant. The MOI for both MSP-1 and MSP-2 was 1.05. The Na per microsatellite locus was 3.60. The He values for MSP-1, MSP-2, GLURP and neutral microsatellites were 0.24, 0.25, 0.69 and 0.30, respectively. A significant LD was observed in Kalabakan (0.495, p<0.01) and Kota Marudu P. falciparum populations (0.601, p<0.01). High genetic differentiation between Kalabakan and Kota Marudu P. falciparum populations was observed (FST = 0.532). The genetic data from the present study highlighted the limited diversity and contrasting genetic pattern of P. falciparum populations in the malaria declining areas of Sabah.
Malaria cases persist in some remote areas in Sabah and Sarawak despite the ongoing and largely successful malaria control programme conducted by the Vector Borne Disease Control Programme, Ministry Of Health, Malaysia. Point mutations in the genes that encode the two enzymes involved in the folate biosynthesis pathway, dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes confer resistance to pyrimethamine and sulfadoxine respectively, in both Plasmodium falciparum and P. vivax. The aim of the current study was to determine the mutation on both pvdhfr at codon 13, 33, 57, 58, 61, 117, and 173 and pvdhps genes at codon 383 and 553, which are potentially associated with resistance to pyrimethamine and sulfadoxine in P. vivax samples in Sabah.
Chloroquine (CQ) and fansidar (sulphadoxine-pyrimethamine, SP) were widely used for treatment of Plasmodium falciparum for several decades in Malaysia prior to the introduction of Artemisinin-based Combination Therapy (ACT) in 2008. Our previous study in Kalabakan, located in south-east coast of Sabah showed a high prevalence of resistance to CQ and SP, suggesting the use of the treatment may no longer be effective in the area. This study aimed to provide a baseline data of antimalarial drug resistant markers on P. falciparum isolates in Kota Marudu located in the north-east coast of Sabah. Mutations on genes associated with CQ (pfcrt and pfmdr1) and SP (pfdhps and pfdhfr) were assessed by PCR amplification and restriction fragment length polymorphism. Mutations on the kelch13 marker (K13) associated with artemisinin resistance were determined by DNA sequencing technique. The assessment of pfmdr1 copy number variation associated with mefloquine resistant was done by real-time PCR technique. A low prevalence (6.9%) was indicated for both pfcrt K76T and pfmdr1 N86Y mutations. All P. falciparum isolates harboured the pfdhps A437G mutation. Prevalence of pfdhfr gene mutations, S108N and I164L, were 100% and 10.3%, respectively. Combining the different resistant markers, only two isolates were conferred to have CQ and SP treatment failure markers as they contained mutant alleles of pfcrt and pfmdr1 together with quintuple pfdhps/pfdhfr mutation (combination of pfdhps A437G+A581G and pfdhfr C59R+S108N+I164L). All P. falciparum isolates carried single copy number of pfmdr1 and wild type K13 marker. This study has demonstrated a low prevalence of CQ and SP resistance alleles in the study area. Continuous monitoring of antimalarial drug efficacy is warranted and the findings provide information for policy makers in ensuring a proper malaria control.
An antiplasmodial screening of Phyllanthus debilis and Phyllanthus urinaria was carried out. The medicinal plants were extracted and evaluated for in vitro antiplasmodial activity against D10 (chloroquine-sensitive, CQS) and Gombak A (chloroquine-resistant, CQR) strains of Plasmodium falciparum. The methanolic crudes from the soxhlet extraction were active against both strains however, P. urinaria (IC50 8.9 μg/ml with CQR strain) exhibited better anti-malarial activity compared to P. debilis (IC50 12.2 μg/ml with CQR strain). Furthermore, the methanolic crude of P. urinaria obtained by the cold extraction has good anti-malarial activity towards CQS (IC50 4.1 μg/ml). The concentration of macronutrients (calcium and magnesium) and trace metals (copper, manganese, iron and zinc) from three Phyllanthus species i.e. P. debilis Klein ex Wild., Phyllanthus niruri L., P. urinaria L. and Alpinia conchigera Griff. were determined using microwave digestion method and analyzed by Flame Atomic Absorption Spectroscopy. Standard Reference Material 1547 (peach leaves) was used to validate the method throughout this study. The recovery values were in the range of 80% to 120% which were in very good agreement with the certified values. The three Phyllanthus species and leaves of A. conchigera showed the highest concentration of calcium compared to other metals and macronutrients studied. The significant presence of all the important macronutrients and trace metals which are essential for human health and well-being substantiate their use medicinally in traditional practices.
The development of resistant to current antimalarial drugs is a major challenge in achieving malaria elimination status in many countries. Therefore there is a need for new antimalarial drugs. Medicinal plants have always been the major source for the search of new antimalarial drugs. The aim of this study was to screen selected Malaysian medicinal plants for their antiplasmodial properties.
The subchronic toxicity effect of the leaf extract of Carica papaya Linn. in Sprague Dawley (SD) rats was investigated in this study. The extract was prepared by dissolving the freeze dried extract of the leaves in distilled water and was administered orally to SD rats (consisted of 10 rats/sex/group) at 0 (control), 0.01, 0.14, and 2 g/kg body weight (BW) for 13 weeks. General observation, mortality, and food and water intake were monitored throughout the experimental period. Hematological and biochemical parameters, relative organ weights, and histopathological changes were evaluated. The study showed that leaf extract when administered for 13 weeks did not cause any mortality and abnormalities of behavior or changes in body weight as well as food and water intake. There were no significant differences observed in hematology parameters between treatment and control groups; however significant differences were seen in biochemistry values, for example, LDH, creatinine, total protein, and albumin. However, these changes were not associated with histopathological changes. In conclusion, the results suggested that daily oral administration of rats with C. papaya leaf extract for 13 weeks at a dose up to fourteen times the levels employed in traditional medicine practice did not cause any significant toxic effect.
Based on the collected ethnobotanical data from the Traditional Medicine and Materia Medica Research Center (TMRC), Iran, Myrtus communis L. (myrtle) was selected for the assessment of in vitro and in vivo antimalarial and cytotoxic activities. Methanolic extract of myrtle was prepared from the aerial parts and assessed for antiplasmodial activity, using the parasite lactate dehydrogenase (pLDH) assay against chloroquine-resistant (K1) and chloroquine-sensitive (3D7) strains of Plasmodium falciparum. The 4-day suppressive test was employed to determine the parasitemia suppression of the myrtle extract against P. berghei in vivo. The IC50 values of myrtle extract were 35.44 µg/ml against K1 and 0.87 µg/ml against 3D7. Myrtle extract showed a significant suppression of parasitaemia (84.8 ± 1.1% at 10 mg/kg/day) in mice infected with P. berghei after 4 days of treatment. Cytotoxic activity was carried out against mammalian cell lines using methyl thiazol tetrazolium (MTT) assay. No cytotoxic effect on mammalian cell lines up to 100 µg/mL was shown. The results support the traditional use of myrtle in malaria. Phytochemical investigation and understanding the mechanism of action would be in our upcoming project.
Despite significant progress in the control of malaria in Malaysia, the complex transmission dynamics of P. vivax continue to challenge national efforts to achieve elimination. To assess the impact of ongoing interventions on P. vivax transmission dynamics in Sabah, we genotyped 9 short tandem repeat markers in a total of 97 isolates (8 recurrences) from across Sabah, with a focus on two districts, Kota Marudu (KM, n = 24) and Kota Kinabalu (KK, n = 21), over a 2 year period. STRUCTURE analysis on the Sabah-wide dataset demonstrated multiple sub-populations. Significant differentiation (F ST = 0.243) was observed between KM and KK, located just 130 Km apart. Consistent with low endemic transmission, infection complexity was modest in both KM (mean MOI = 1.38) and KK (mean MOI = 1.19). However, population diversity remained moderate (H E = 0.583 in KM and H E = 0.667 in KK). Temporal trends revealed clonal expansions reflecting epidemic transmission dynamics. The haplotypes of these isolates declined in frequency over time, but persisted at low frequency throughout the study duration. A diverse array of low frequency isolates were detected in both KM and KK, some likely reflecting remnants of previous expansions. In accordance with clonal expansions, high levels of Linkage Disequilibrium (I A (S) >0.5 [P<0.0001] in KK and KM) declined sharply when identical haplotypes were represented once (I A (S) = 0.07 [P = 0.0076] in KM, and I A (S) = -0.003 [P = 0.606] in KK). All 8 recurrences, likely to be relapses, were homologous to the prior infection. These recurrences may promote the persistence of parasite lineages, sustaining local diversity. In summary, Sabah's shrinking P. vivax population appears to have rendered this low endemic setting vulnerable to epidemic expansions. Migration may play an important role in the introduction of new parasite strains leading to epidemic expansions, with important implications for malaria elimination.
Carbamazepine (CBZ) is used as the first line of treatment of complex partial seizures (CPS) in Malaysia. While this drug is known to be effective for the treatment of CPS, more than 30% of patients remain drug resistant to CBZ mono-therapy. We examined a possible relationship between patients' response to CBZ mono-therapy and the G2677T SNP of the ABCB1 gene. Three hundred and fourteen patients with CPS were recruited from the Neurology Department of the Kuala Lumpur Hospital, of whom 152 were responders and the other 162 were non-responders to CBZ mono-therapy. DNA was extracted from blood samples and real-time PCR was performed to detect the G2677T SNP of the ABCB1 gene. Results were described as genotype frequencies and compared by logistic regression analysis. Among the 152 responders, 74% had the GG genotype. However, among the 162 non-responders, 26.5% had the GT genotype and 39% had the TT genotype. There was a significant difference in genotype frequency (TT vs GG; odds ratio 4.70; 95% confidence interval, 2.70-8.20) between responders and non-responders. The presence of the T allele of the G2677T SNP appears to be a useful screening marker to determine if a patient is going to be resistant to CBZ as a single drug therapy in the treatment of CPS.
Carbamazepine (CBZ) is used as the first line of treatment of Complex Partial Seizures (CPS) in the Epilepsy Clinic, Neurology Department of Kuala Lumpur Hospital (KLH). More than 30% of the patients remain drug resistant to CBZ mono-therapy. CBZ is transported by the P-glycoprotein (P-gp). The P-gp encoded by the ABCB1 and ABCC2 genes are expressed in drug resistant patients with epilepsy. A few studies have shown significant association between CBZ resistant epilepsy and Linkage Disequilibrium (LD) with adjacent polymorphisms of these genes. Our study is aimed at determining the correlation between patients' response to CBZ mono-therapy to Single Nucleotide Polymorphisms G2677T and C3435T of the ABCB1 gene as well as G1249A and -24C>T of the ABCC2 gene.
Sulphadoxine-pyrimethamine (SP) has been in use for the treatment of uncomplicated falciparum malaria in Malaysia since the 1970s and is still widely employed in spite of widespread clinical resistance. Resistance to SP is known to be mediated by mutations in the pfdhfr and pfdhps genes. The aim of the present study was to investigate the distribution of pfdhfr and pfdhps gene polymorphism in Plasmodium falciparum field isolates from Kalabakan, Sabah, in northern Borneo.
The study was conducted to investigate the platelet increasing property of Carica papaya leaves juice (CPLJ) in patients with dengue fever (DF). An open labeled randomized controlled trial was carried out on 228 patients with DF and dengue haemorrhagic fever (DHF). Approximately half the patients received the juice, for 3 consecutive days while the others remained as controls and received the standard management. Their full blood count was monitored 8 hours for 48 hours. Gene expression studies were conducted on the ALOX 12 and PTAFR genes. The mean increase in platelet counts were compared in both groups using repeated measure ANCOVA. There was a significant increase in mean platelet count observed in the intervention group (P < 0.001) but not in the control group 40 hours since the first dose of CPLJ. Comparison of mean platelet count between intervention and control group showed that mean platelet count in intervention group was significantly higher than control group after 40 and 48 hours of admission (P < 0.01). The ALOX 12 (FC = 15.00) and PTAFR (FC = 13.42) genes were highly expressed among those on the juice. It was concluded that CPLJ does significantly increase the platelet count in patients with DF and DHF.
Carica papaya L. leaves have been used in ethnomedicine for the treatment of fevers and cancers. Despite its benefits, very few studies on their potential toxicity have been described. The aim of the present study was to characterize the chemical composition of the leaf extract from 'Sekaki' C. papaya cultivar by UPLC-TripleTOF-ESI-MS and to investigate the sub-acute oral toxicity in Sprague Dawley rats at doses of 0.01, 0.14 and 2 g/kg by examining the general behavior, clinical signs, hematological parameters, serum biochemistry and histopathology changes. A total of twelve compounds consisting of one piperidine alkaloid, two organic acids, six malic acid derivatives, and four flavonol glycosides were characterized or tentatively identified in the C. papaya leaf extract. In the sub-acute study, the C. papaya extract did not cause mortality nor were treatment-related changes in body weight, food intake, water level, and hematological parameters observed between treatment and control groups. Some biochemical parameters such as the total protein, HDL-cholesterol, AST, ALT and ALP were elevated in a non-dose dependent manner. Histopathological examination of all organs including liver did not reveal morphological alteration. Other parameters showed non-significant differences between treatment and control groups. The present results suggest that C. papaya leaf extract at a dose up to fourteen times the levels employed in practical use in traditional medicine in Malaysia could be considered safe as a medicinal agent.
Chloroquine (CQ) remains the first line drug for the prevention and treatment of malaria in Malaysia in spite of the fact that resistance to CQ has been observed in Malaysia since the 1960s. CQ-resistance is associated with various mutations in pfcrt, which encodes a putative transporter located in the digestive vacuolar membrane of P. falciparum. Substitution of lysine (K) to threonine (T) at amino acid 76 (K76T) in pfcrt is the primary genetic marker conferring resistance to CQ. To determine the presence of T76 mutation in pfcrt from selected areas of Kalabakan, Malaysia 619 blood samples were screened for P. falciparum, out of which 31 were positive. Blood samples were collected on 3 MM Whatman filter papers and DNA was extracted using QIAmp DNA mini kit. RFLP-PCR for the detection of the CQ-resistant T76 and sensitive K76 genotype was carried out. Twenty-five samples were shown to have the point mutation in pfcrt whereas the remaining samples were classified as CQ-sensitive (wild-type). In view of the fact that CQ is the first line anti-malarial drug in Malaysia, this finding could be an important indication that treatment with CQ may no longer be effective in the future.
Studies have shown that the barks and roots of some Apocynaceae species have anticancer and antimalarial properties. In this study, leaf extracts of five selected species of Apocynaceae used in traditional medicine (Alstonia angustiloba, Calotropis gigantea, Dyera costulata, Kopsia fruticosa and Vallaris glabra) were assessed for antiproliferative (APF) and antiplasmodial (APM) activities, and analysed for total alkaloid content (TAC), total phenolic content (TPC) and radical-scavenging activity (RSA). As V. glabra leaf extracts showed wide spectrum APF and APM activities, they were further screened for saponins, tannins, cardenolides and terpenoids.
The acute toxicity of standardized extract of Orthosiphon stamineus was studied in Sprague Dawley rats. The rats were administered a single dose of 5000 mg/kg body weight (BW) orally on Day 0 and observed for 14 days. There were no deaths recorded and the animals did not show signs of toxicity during the experimental period. The effect of the extract on general behavior, BW, food and water intake, relative organ weight per 100 g BW, hematology and clinical biochemistry were measured. All the parameters measured were unaffected as compared to the control. The acute toxicity LD(50) was estimated to be > 5000 mg/kg BW.