Goniothalamus scortechinii, Andrographis paniculata and Aralidium pinnatifidum were selected for the study based on their ethnomedicinal values. They were screened for anti-malarial activity towards Plasmodium falciparum in vitro using the lactate dehydrogenase (LDH) assay. The crude extract of G. scortechinii exhibited the most potent schizonticidal activity compared to the other extracts. It is effective against both the chloroquine resistant isolate, Gombak A and the sensitive strain, D10 of Plasmodium falciparum. Furthermore a better IC(50) value was obtained against the resistant strain, (9 microg/ml) compared to the sensitive strain, 40 microg/ml. When the crude extract was fractionated into 3 fractions, the chloroform fraction yielded the best activity, exhibiting equipotency against both strains of parasite used; IC(50) of 23.53 microg/ml against Gombak A and 21.06 microg/ml against D10.
Matched MeSH terms: Biological Assay/methods; Biological Assay/statistics & numerical data
Tocotrienols and tocopherols are isoforms of vitamin E. Vitamin E may exhibit antioxidant, prooxidant and non-antioxidant activities depending upon circumstances. In this study, the effect of tocotrienols and α-tocopherol on the activities of HMG CoA reductase and cholesterol 7 α-hydroxylase was investigated. Pure tocotrienols were isolated from palm fatty acid distillate and pure α-tocopherol was obtained commercially. Guinea pigs were treated with different dosages of tocotrienols and α-tocopherol. After the treatment period, animals were sacrificed and liver microsomes were prepared. HMG CoA reductase and cholesterol 7α-hydroxylase were assayed using tracer techniques. Our results showed that the effects of tocotrienols and α-tocopherol on the activities of both the enzymes were dose-dependent. At low dosages, both tocotrienols and α-tocopherol exhibited an inhibitory effect on both the enzymes. Moreover, tocotrienols were a much stronger inhibitors than α-tocopherol. At high dosages, on the other hand, tocotrienols and α-tocopherol showed opposite effects on the enzymes. While tocotrienols continued to exhibit an inhibitory effect, α-tocopherol actually exhibited a stimulatory effect on both the enzymes. A possible explanation for this observation is suggested.
Studies on toxicities and tolerances of cadmium (Cd), copper (Cu), lead (Pb) and zinc (Zn) in the brown alga Isochrysis galbana and in the green-lipped mussel Perna viridis were conducted by short-term bioassays using endpoints growth production and mortality, respectively. The 5-day EC(50) and 24-h LC(50) of these heavy metals were determined in the brown alga and mussel, respectively. The EC(50) values calculated for the alga were 0.74 mg/l for Cd, 0.91 mg/l for Cu, 1.40 mg/l for Pb and 0.60 mg/l for Zn. The LC(50) values for the mussels were 1.53 mg/l for Cd, 0.25 mg/l for Cu, 4.12 mg/l for Pb and 3.20 mg/l for Zn. These LC(50) values were within the concentration ranges as reported by other authors who used P. viridis as the test organism. Based on these EC(50) and LC(50) values, the alga was most sensitive to Zn, followed by Cd, Cu and Pb while the mussel was most sensitive to Cu, followed by Cd, Zn and Pb. Differences in the trophic levels, metal handling strategies, biology and ecology of the primary producer (brown alga) and the primary consumer (mussel) are believed to be the plausible causes for the different toxicities and tolerances of the metals studied.
Our recent studies on the stem bark of Calophyllum mucigerum (Guttiferae) have yielded a new coumarin mucigerin, a prenylated xanthone cudraxanthone C and the common steroidal triterpenes friedelin and stigmasterol. Structural elucidations of these compounds were achieved using 1H NMR, 13C NMR, DEPT, COSY, HETCOR and HMBC experiments while MS gave the molecular masses. Cytotoxic assays using CEM-SS cell line (T-lymphoblastic leukemia) on the crude extracts of the stem bark indicated some activity. The crude extracts were also found to be moderately toxic against the larvae of Aedes aegypti. This article reports the isolation and identification of mucigerin as well as bioassay data.
This study was carried out to characterize the detection and quantitation of several paralytic shellfish poisoning (PSP) toxin congeners using a receptor binding assay (RBA). This involved competitive binding of the toxin congeners against tritium-labeled STX for receptor sites on rat brain sodium channels. Competitive binding curves were described by a four-parameter logistic equation. Half-saturation values (EC(50)) ranged from 4.38 nM for STX to 142 nM for GTX5. Receptor binding affinity was in the order STX>GTX1/4>neoSTX>GTX2/3>dcSTX>GTX5, and this was similar to the order of mouse toxicity of these congeners. Predicted toxin concentrations from observed STXeq values and EC(50) ratios relative to STX were within 20% or better of the actual concentrations used in the assay. In contrast predicted toxin concentrations using mouse toxicity ratios relative to STX did not provide a good match to actual concentrations, except for GTX1/4. This study has shown that the rat brain sodium channel RBA will provide a reliable integration of total toxicity of various PSP toxin congeners present in a sample.
The adulticidal activity of methanol extracts from three Malaysian plants namely Acorus calamus Linn., Litsea elliptica Blume and Piper aduncum Linn. against adult of Aedes aegypti (L.) were studied. Standard WHO bioassay tests were used to evaluate the effectiveness of these plant extracts. The hexane fraction from methanol extract of Acorus calamus rhizome was the most effective, exhibiting LC50 and LC90 values of 0.04 mgcm(-2) and 0.09 mgcm(-2) respectively. For L. elliptica, the methanol fraction also displayed good adulticidal property with the LC50 and LC90 values of 0.11 mgcm(-2) and 6.08 mgcm(-2) respectively. It is found that hexane fraction of the P. aduncum crude extract was the least effective among the three plants showing LC50 and LC90 values of 0.20 mgcm(-2) and 5.32 mgcm(-2), respectively. However, although A. calamus showed lowest LC values, the LT50 results indicated that the methanol fraction of L. elliptica was most potent extract among the extracts tested.
This paper outlines the past five decades of scientific interests and advances in medicinal plant research in Malaysia. Initially the prime interest of research programmes has been on phytochemical studies leading to the discovery of biologically active compounds as chemical templates to produce new drug candidates. As the Malaysian herbal medicine market experiences an extraordinary growth, the research approaches taken have recently included activities to develop herbal medicines into quality, efficacious and safe products for human consumption. Advances in chromatographic and spectroscopic techniques have had a tremendous impact on the isolation and structure elucidation of the constituents of medicinal plants. The development of a series of bioassay methodologies and utilization of bioassay-guided isolation techniques have contributed significantly to the progress of medicinal plant research in Malaysia. Research work on some medicinal plants carried out by the local scientists will be illustrated as examples.
Larvae obtained from Taman Samudera (Gombak, Selangor), Kampung Banjar (Gombak, Selangor), Taman Lembah Maju (Cheras, Kuala Lumpur) and Kampung Baru (City centre, Kuala Lumpur) were bioassayed with diagnostic dosage (0.012 mg/L) and operational dosage (1 mg/L) of temephos. All strains of Aedes aegypti and Aedes albopictus showed percentage mortality in the range of 16.00 to 59.05 and 6.4 to 59.50 respectively, after 24 hours. LT50 values for the 6 strains of Ae. aegypti and Ae. albopictus were between 41.25 to 54.42 minutes and 52.67 to 141.76 minutes respectively, and the resistance ratio for both Aedes species were in the range of 0.68 to 1.82 when tested with operational dosage, 1 mg/L temephos. These results indicate that Aedes mosquitoes have developed some degree of resistance. However, complete mortality for all strains were achieved after 24 hours when tested against 1 mg/L temephos.
Larvae of Aedes aegypti and Aedes albopictus obtained from 6 consecutive ovitrap surveillance (OS) in Taman Samudera and Kg. Banjar were evaluated for their susceptibility to temephos. Larval bioassays were carried out in accordance with WHO standard methods, with diagnostic dosage (0.012 mg/L) and operational dosage (1 mg/L) of temephos respectively. Aedes aegypti and Ae. albopictus obtained from six OS in Taman Samudera showed resistance to diagnostic dosage of temephos with percentage mortality between 5.3 to 72.0 and 9.3 to 56.0, respectively, while Ae. aegypti and Ae. albopictus obtained from Kg. Banjar showed resistance to temephos with percentage mortality between 16.0 to 72.0 and 0 to 50.6, respectively. Only two strains of Ae. aegypti from Kg. Banjar were susceptible to temephos with 93.3% (OS 2) and 100% (OS 3) mortality. The 50% mortality at lethal time (LT50) for all strains of Ae. aegypti and Ae. albopictus tested against operational dosage of temephos showed range between 36.07 to 75.69 minutes and 58.65 to 112.50 minutes, respectively, and complete mortality was achieved after 24 hours. Our results indicated that there is weekly variations of the resistance status for Ae. aegypti and Ae. albopictus. Aedes susceptibility to temephos is changing from time to time in these two study sites. It is essential to continue monitoring the resistance of this vector to insecticides in order to ensure the efficiency of program aimed at vector control and protection of human health.
To determine resistance level and characterize malathion and permethrin resistance in Culex quinquefasciatus, two methods were used namely: WHO procedures of larval bioassay to determine the susceptibility of lethal concentration (LC) and adult bioassay to determine the lethal time (LT) which are resistant to malathion and permethrin. These mosquito strains were bred in the Insectarium, Division of Medical Entomology, IMR. Thousands of late fourth instar larvae which survived the selection pressure to yield 50% mortality of malathion and permethrin were reared and colonies were established from adults that emerged. Larvae from these colonies were then subjected to the subsequent 10 generations in the test undertaken for malathion resistant strain (F61 - F70) and permethrin resistant strain (F54 - F63). Selection pressure at 50% - 70% mortality level was applied to the larvae of each successive generation. The rate of resistance development and resistance ratio (RR) were calculated by LC5 0 for larval bioassay and LT50 value for adult bioassay. The lab bred Cx. quinquefasciatus was used as a susceptible strain for comparison purpose. The adult bioassay test was carried out by using diagnostic dosages of malathion 5.0%, permethrin 0.75% and with propoxur 0.1%. All bioassay results were subjected to probit analysis. The results showed that LC5 0 for both malathion (F61 - F70) and permethrin (F54 - F63) resistant Cx. quinquefasciatus increased steadily to the subsequent 10 generations indicating a marked development of resistance. The adult female malathion resistant strain have developed high resistance level to malathion diagnostic dosage with resistance ratio 9.3 to 9.6 folds of resistance. Permethrin resistance ratio remained as 1.0 folds of resistance at every generation. It was obvious that malathion resistance developing at a higher rate in adult females compared to permethrin. Female adults exposed to 2 hours of exposure period for propoxur 0.1% showed presence of cross-resistance among the both strains of mosquitoes towards propoxur and it was indicated by 70%-100% mortality at 24 hours post-recovery period.
Bioassay-guided fractionation was performed on a crude dichloromethane extract of Kaempferia galanga L. using chromatography techniques. Screening of the extract for biological activity started with the brine shrimp lethality bioassay, followed by the study of its antihypertensive activity on anaesthetized rats, which involved monitoring of the extract's effect on mean arterial blood pressure. The components of the fractions obtained from the separation procedures were analyzed using gas chromatography (GC). The yield of the CH(2)Cl(2) extract was 0.29% of the crude plant extract. Analysis of the data for brine shrimp lethality test using the Finney computer program showed that this extract exhibited potent bioactivity with an ED(50) value of 7.92+/-0.13 microgml(-1). Intravenous administration of the extract induced a dose-related reduction of basal mean arterial pressure (MAP) (130+/-5 mmHg) in the anaesthetized rat, with maximal effects seen after 5-10 min of injection. The gas chromatogram showed that the common compound in the active fractions obtained from the bioassay-guided fractionation of the CH(2)Cl(2) extract was ethyl cinnamate. This vasorelaxant active compound, ethyl cinnamate, was isolated as a colorless oil. Ethyl p-methoxycinnamic acid was also isolated as white needles but did not exhibit any relaxant effect on the precontracted thoracic rat aorta.
Studies on the stem of Garcinia mangostana have led to the isolation of one new xanthone mangosharin (1) (2,6-dihydroxy-8-methoxy-5-(3-methylbut-2-enyl)-xanthone) and six other prenylated xanthones, alpha-mangostin (2), beta-mangostin (3), garcinone D (4), 1,6-dihydroxy-3,7-dimethoxy-2-(3-methylbut-2-enyl)-xanthone (5), mangostanol (6) and 5,9-dihydroxy-8- methoxy-2,2-dimethyl-7-(3-methylbut-2-enyl)-2H,6H-pyrano-[3,2-b]-xanthene-6-one (7). The structures of these compounds were determined by spectroscopic methods such as 1H NMR, 13C NMR, mass spectrometry (MS) and by comparison with previous studies. All the crude extracts when screened for their larvicidal activities indicated very good toxicity against the larvae of Aedes aegypti. This article reports the isolation and identification of the above compounds as well as bioassay data for the crude extracts. These bioassay data have not been reported before.
The bioefficacy of a commercial formulation of temephos, Creek against Aedes aegypti larvae was studied in the laboratory. Earthen jars were filled with 10 L tap water each. One g of temephos (Creek) sand granule formulation was added into each earthen jar as recommended by the manufacturer. The final test concentration of Creek was 1 mg a.i./L. One earthen jar was filled with 10 L tap water and served as a test control (untreated). Thirty late 3(rd) or early 4(th) instar of lab-bred Ae. aegypti larvae were added into each earthen jar. Mortality of the larvae was recorded after 24 hours and percent mortality was calculated. Test was repeated every week. The results showed that complete larval mortality was achieved after 24 hours. The residual effect lasted 15 weeks (105 days), indicating that Creek is effective at the dosage recommended by the manufacturer which is 1 mg a.i./L.
A study was conducted on the long term effects of nine heavy metals on the Chironomus plumosus and Culicoides furens larvae. This study tested the effect of the heavy metals on several generations of the larvae to observe the formation of increased hardiness against pollutants present within the aquatic habitat. From this study it was observed that susceptibility or sensitivity to heavy metals decreased with LC50 values becoming larger indicating a decreased toxicity level. Significant variations (p < 0.05) were observed between first generation and third generation culicoides for all metals and at all concentrations. Variations between third and fourth generation culicoides were also significantly different (p < 0.05) with the exception of chromium at 25 degrees C and nickel and lead at every temperature range group. The variation between all generations 4, 5 and 6 was found to be insignificant (p > 0.05). This would indicate that metal tolerance would have occurred in these generations and the effect of metals was less toxic to the culicoides. Generation 9 was found to have LC50 values (p > 0.05) the same as the LC50 values obtained in third generation culicoides. Thus it would appear that heavy metal resistance was developed when the organisms were exposed to prolonged exposure of the heavy metals but was lost when the organisms were bred in non-contaminated water.
In this research, we modify a previously developed assay for the quantification molybdenum blue to determine whether inhibitors to molybdate reduction in bacteria inhibits cellular reduction or inhibit the chemical formation of one of the intermediate of molybdenum blue; phosphomolybdate. We manage to prove that inhibition of molybdate reduction by phosphate and arsenate is at the level of phosphomolybdate and not cellular. We also prove that mercury is a physiological inhibitor to molybdate reduction. We suggest the use of this method to assess the effect of inhibitors and activators to molybdate reduction in bacteria.
A heavy-metal assay has been developed using bromelain, a protease. The enzyme is assayed using casein as a substrate with Coomassie dye to track completion of hydrolysis of casein. In the absence of inhibitors, casein is hydrolysed to completion, and the solution is brown. In the presence of metal ions such as Hg2+ and Cu2+, the hydrolysis of casein is inhibited, and the solution remains blue. Exclusion of sulfhydryl protective agent and ethylenediaminetetraacetic in the original assay improved sensitivity to heavy metals several fold. The assay is sensitive to Hg2+ and Cu2+, exhibiting a dose-response curve with an IC50 of 0.15 mg 1(-1) for Hg2+ and a one-phase binding curve with an IC50 of 0.23 mg 1(-1) for Cu2+. The IC50 value for Hg2+ is found to be lower to several other assays such as immobilized urease and papain assay, whilst the IC50 value for Cu2+ is lower than immobilized urease, 15-min Microtox, and rainbow trout.
Molybdenum-reducing activity in the heterotrophic bacteria is a phenomenon that has been reported for more than 100 years. In the presence of molybdenum in the growth media, bacterial colonies turn to blue. The enzyme(s) responsible for the reduction of molybdenum to molybdenum blue in these bacteria has never been purified. In our quest to purify the molybdenum-reducing enzyme, we have devised a better substrate for the enzyme activity using laboratory-prepared phosphomolybdate instead of the commercial 12-phosphomolybdate we developed previously. Using laboratory-prepared phosphomolybdate, the highest activity is given by 10:4-phosphomolybdate. The apparent Michaelis constant, Km for the laboratory-prepared 10:4-phosphomolybdate is 2.56 +/- 0.25 mM (arbitrary concentration), whereas the apparent V(max) is 99.4 +/- 2.85 nmol Mo-blue min(-1) mg(-1) protein. The apparent Michaelis constant or Km for NADH as the electron donor is 1.38 +/- 0.09 mM, whereas the apparent V(max) is 102.6 +/- 1.73 nmol Mo-blue min(-1) mg(-l) protein. The apparent Km and V(max) for another electron donor, NADPH, is 1.43 +/- 0.10 mM and 57.16 +/- 1.01 nmol Mo-blue min(-1) mg(-1) protein, respectively, using the same batch of molybdenum-reducing enzyme. The apparent V(max) obtained for NADH and 10:4-phosphomolybdate is approximately 13 times better than 12-phoshomolybdate using the same batch of enzyme, and hence, the laboratory-prepared phosphomolybdate is a much better substrate than 12-phoshomolybdate. In addition, 10:4-phosphomolybdate can be routinely prepared from phosphate and molybdate, two common chemicals in the laboratory.
Vegetative proteins from Malaysian strains of Bacillus thuringiensis israelensis strains (Bt 11, Bt 12, Bt 15, Bt 16, Bt 17, Bt 21 and Bt 22) and Bacillus sphaericus H-25 strains (Bs 1 and Bs 2) were screened for haemolytic, cytotoxic and larvicidal activity. SDS-PAGE profiles of the Bacillus thuringiensis strains studied consistently showed major bands of 33-37 kDa and 47 kDa. Bt 16 also showed two bands of 66 kDa and 45 kDa similar to the previously reported binary vegetative protein, Vip1Ac (66 kDa) and Vip 2Ac (45 kDa). Both the Bacillus sphaericus strains showed a 35 kDa band that was similiar to a previously reported vegetative protein, the Mtx2 protein. Bs 2 also contains a 37 kDa band, similar to another vegetative protein, the Mtx 3 protein. With the exception of Bt 17 and Bt 21, vegetative proteins from all Bacillus thuringiensis and Bacillus sphaericus strains were highly haemolytic to human erythrocytes, causing more than 75% haemolysis at the highest concentration of 200 microg/ml. High haemolytic activity was associated with high cytotoxic activity with most of the haemolytic strains being indiscriminately cytotoxic to both CEM-SS (human T lymphoblastoid) and HeLa (human uterus cervical cancer) cell lines. Interestingly, the less haemolytic vegetative proteins from Bt 17 and Bt 21 demonstrated cytotoxic activity comparable to that of the highly haemolytic vegetative proteins. Bt 21 displayed toxicity towards both cell lines while Bt 17 was more toxic towards CEM-SS cells. Bioassay against Aedes aegypti and Culex quinquefasciatus larvae revealed that vegetative proteins from the Bacillus thuringiensis strains had activity against both species of larvae but vegetative proteins from Bacillus sphaericus were weakly larvicidal towards Cx. quinquefasciatus only.
A study was conducted to determine the suitability of using selected aquatic dipterian larvae for biomonitoring bioassays. The organisms included a member of the biting midge family that was identified as Culicoides furens and a member of the non-biting midge family, identified as Chironomus plumosus. Median lethal toxicity tests were conducted to observe the variation between metal sensitivities between the two larval forms and how variations in temperature could affect the experimental setup. Nine heavy metals were used in the study. It was observed that the 96 h LC(50) (in mg/L) for the different metals was found to be Zn-16.21 (18.55 +/- 13.87); Cr-0.96 (1.08 +/- 0.84); Ag-4.22 (6.87 +/- 1.57); Ni-0.42 (0.59 +/- 0.25); Hg-0.42 (0.59 +/- 0.25); Pb-16.21 (18.31 +/- 14.11); Cu-42.24 (45.18 +/- 39.30); Mn-4.22 (7.19 +/- 1.25); Cd-0.42 (0.59 +/- 0.25) for the Chironomus plumosus and Zn-4.22 (6.56 +/- 1.88); Cr-0.42 (0.54 +/- 0.30); Ag-0.42 (0.54 +/- 0.30); Ni-0.42 (0.54 +/- 0.30); Hg-0.04 (0.07 +/- 0.01); Pb-0.42 (0.54 +/- 0.30); Cu-42.24 (45.18 +/- 39.30); Mn-4.22 (6.56 +/- 1.88); Cd-0.42 (0.54 +/- 0.30) in the case of the Culicoides furens. With temperature as a variable the LC(50) values were observed to increase from 2.51 mg/L at 10 degrees C to 4.22 ppm at 30 degrees C and to reduce slightly to 3.72 mg/L at 35 degrees C as seen in the case of Zn. It was also observed that at 40 degrees C thermal toxicity and chemical toxicity overlapped as 100% mortality was observed in the controls. This trend was observed in all metals for both C. plumosus and C. furens. Thus indicating temperature played an important role in determining LC(50) values of toxicants.
House flies were collected from April 2007-April 2008 from two poultry farms (Balik Pulau and Juru) in the state of Penang. The resistance level of the first generation offspring was evaluated against DDT, malathion, propoxur, and permethrin using the topical application method. The resistance ratio (RR) of the Balik Pulau strain house flies for propoxur, malathion and DDT ranged from 10.28 to 99.00, 7.83 to 47.01 and 6.05 to 31.10, respectively. Resistance to propoxur and malathion in house fly was attributed to cross resistance to organophosphate insecticides used in the farm. Increased metabolic detoxification might be the mechanism involved in DDT resistance due to excessive application of cypermethrin formulation. The RR of the Juru strain for propoxur, malathion and DDT was in a decreasing pattern throughout the study period, ranging from 5.58 to 83.38, 15.19 to 27.82, and 10.04 to 22.69, respectively. Permethrin appeared to be the most potent insecticide in controlling house fly in both the Balik Pulau (RR=0.50 to 1.96) and Juru poultry farms (RR=0.64 to 2.40). The fluctuations of insecticides resistance in house fly was also found to correlate with climatic factors due to its rapid breeding. Relative humidity exhibited positive correlation indices with the changes in the resistance level for DDT (r=0.481, p<0.05), malathion (r=0.698, p<0.01), and permethrin (r=0.580, p<0.05) in Balik Pulau. Similarly, relative humidity in Juru also showed strong correlation with the RR for DDT (r=0.900, p<0.01), malathion (r=0.762, p<0.05), permethrin (r=0.760, p<0.05), and propoxur (r=0.897, p<0.01).