Bioavailability of Eurycomanone in Its Pure Form and in a Standardised Eurycoma longifolia Water Extract.

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.

Safety Evaluation of Oral Toxicity of Carica papaya Linn. Leaves: A Subchronic Toxicity Study in Sprague Dawley Rats.

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.

Effect of selected local medicinal plants on the asexual blood stage of chloroquine resistant Plasmodium falciparum.

BACKGROUND: 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. METHODS: Each part of the plants were processed, defatted by hexane and sequentially extracted with dichloromethane, methanol and water. The antiplasmodial activities of 54 plant extracts from 14 species were determined by Plasmodium falciparum Histidine Rich Protein II ELISA technique. In order to determine the selectivity index (SI), all plant extracts demonstrating a good antiplasmodial activity were tested for their cytotoxicity activity against normal Madin-Darby Bovine Kidney (MDBK) cell lines by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Twenty three extracts derived from Curcuma zedoaria (rhizome), Curcuma aeruginosa (rhizome), Alpinia galanga (rhizome), Morinda elliptica (leaf), Curcuma mangga (rhizome), Elephantopus scaber (leaf), Vitex negundo (leaf), Brucea javanica (leaf, root and seed), Annona muricata (leaf), Cinnamomun iners (leaf) and Vernonia amygdalina (leaf) showed promising antiplasmodial activities against the blood stage chloroquine resistant P. falciparum (EC50 < 10 µg/ml) with negligible toxicity effect to MDBK cells in vitro (SI ≥10). CONCLUSION: The extracts belonging to eleven plant species were able to perturb the growth of chloroquine resistant P. falciparum effectively. The findings justified the bioassay guided fractionation on these plants for the search of potent antimalarial compounds or formulation of standardized extracts which may enhance the antimalarial effect in vitro and in vivo.

In vitro effect of important herbal active constituents on human cytochrome P450 1A2 (CYP1A2) activity.

This study was designed to investigate eight herbal active constituents (andrographolide, asiaticoside, asiatic acid, madecassic acid, eupatorin, sinensetin, caffeic acid, and rosmarinic acid) on their potential inhibitory effects on human cytochrome P450 1A2 (CYP1A2) activity. A fluorescence-based enzyme assay was performed by co-incubating human cDNA-expressed CYP1A2 with its selective probe substrate, 3-cyano-7-ethoxycoumarin (CEC), in the absence or presence of various concentrations of herbal active constituents. The metabolite (cyano-hydroxycoumarin) formed was subsequently measured in order to obtain IC50 values. The results indicated that only eupatorin and sinensetin moderately inhibited CYP1A2 with IC50 values of 50.8 and 40.2 µM, while the other active compounds did not significantly affect CYP1A2 activity with IC50 values more than 100 µM. Ki values further determined for eupatorin and sinensetin were 46.4 and 35.2 µM, respectively. Our data indicated that most of the investigated herbal constituents have negligible CYP1A2 inhibitory effect. In vivo studies however may be warranted to ascertain the inhibitory effect of eupatorin and sinensetin on CYP1A2 activity in clinical situations.

Analysis of flavone C-glycosides in the leaves of Clinacanthus nutans (Burm. f.) Lindau by HPTLC and HPLC-UV/DAD.

Clinacanthus nutans (family Acanthaceae) has been used for the treatment of inflammation and herpes viral infection. Currently, there has not been any report on the qualitative and quantitative determination of the chemical markers in the leaves of C. nutans. The C-glycosidic flavones such as shaftoside, isoorientin, orientin, isovitexin, and vitexin have been found to be major flavonoids in the leaves of this plant. Therefore, we had developed a two-step method using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC) for the rapid identification and quantification of the flavones C-glycosides in C. nutans leaves. The TLC separation of the chemical markers was achieved on silica gel 60 plate using ethyl acetate : formic acid : acetic acid : water (100 : 11 : 11 : 27 v/v/v/v) as the mobile phase. HPLC method was optimized and validated for the quantification of shaftoside, orientin, isovitexin, and vitexin and was shown to be linear in concentration range tested (0.4-200 µg/mL, r(2) ≥ 0.996), precise (RSD ≤ 4.54%), and accurate (95-105%). The concentration of shaftoside, orientin, vitexin, and isovitexin in C. nutans leave samples was 2.55-17.43, 0.00-0.86, 0.00-2.01, and 0.00-0.91 mmol/g, respectively.

Effect of choline kinase inhibitor hexadecyltrimethylammonium bromide on Plasmodium falciparum gene expression.

Investigations on the fundamental of malaria parasite biology, such as invasion, growth cycle, metabolism and cell signalling have uncovered a number of potential antimalarial drug targets, including choline kinase, a key enzyme involved in the synthesis of phosphatidylcholine, an important component in parasite membrane compartment. The effect on gene expression of Plasmodium falciparum K1 strain following 72 hours exposure to 2 microM (IC50 concentration) of the choline kinase inhibitor, hexadecyltrimethylammonium bromide (HDTAB) was evaluated by DNA microarray analysis. Genes important in P. falciparum intraerythrocytic life cycle, such as invasion, cytoadherance and growth were among those affected by at least 2-fold changes in their expression levels compared with non HDTAB-treated control.

Effect of eurycomanone on cytochrome P450 isoforms CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 in vitro.

Eurycomanone, an active constituent isolated from Eurycoma longifolia Jack, was examined for modulatory effects on cytochrome P450 (CYP) isoforms CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 using in vitro assays. The IC50 value was determined to assess the potencies of modulation for each CYP isoform. Our results indicated that eurycomanone did not potently inhibit any of the CYP isoforms investigated, with IC50 values greater than 250 µg/ml. Hence there appears to be little likelihood of drug-herb interaction between eurycomanone or herbal products with high content of this compound and CYP drug substrates via CYP inhibition.

Plasmodium vivax population structure and transmission dynamics in Sabah Malaysia.

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.

High prevalence of mutation in the Plasmodium falciparum dhfr and dhps genes in field isolates from Sabah, Northern Borneo.

BACKGROUND: 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. METHODS: A total number of 619 individuals were screened from 23 study sites of which 31 were positive for P. falciparum. Analysis of restriction fragment length polymorphisms (RFLP) was used to identify polymorphism in the pfdhfr and pfdhps genes at positions 16, 51, 59, 108, 164 and 437, 540, 581, respectively. RESULTS: All samples had at least one mutation in each of the genes associated with drug resistance. The prevalence of pfdhfr 59arg, 164leu and 108asn were 100%, 80.65% and 58.06%, respectively. Pfdhps mutants 437gly and 581gly accounted for 100% and 74.19% respectively. In pfdhfr, the most common mutant genotypes were combination 59arg + 164leu (22.58%) and 59arg + 108asn + 164leu (51.61%). In pfdhps the most common genotype was 437gly + 581gly (74.19%). One individual (3.22%) harboured parasites with four pfdhfr (16 val + 59arg + 108asn + 164leu) and two pfdhps (437gly + 581gly) mutations. The highest quintuple pfdhfr/pfdhps (41.94%) was three pfdhfr (59arg + 108asn + 164gly) and two pfdhps (437gly + 581gly). CONCLUSION: The data suggest a high prevalence of genetic variations conferring resistance to SP which can predict treatment failure before becoming clinically evident. In areas like this, the use of SP may no longer be indicated.

Study on the developmental toxicity of a standardized extract of Orthosiphon stamineus in rats

Infusions of Orthosiphon stamineus Benth., Lamiaceae, leaves are widely used in Southeastern Asia to treat different illnesses. Nonetheless, no data is available on the safety of O. stamineus for pregnant women and their babies. This study was undertaken to evaluate the developmental toxicity of O. stamineus standardized aqueous extract in female Sprague Dawley rats (n=21) at 0, 250, 500, 1000 and 2000 mg/kg/day, by gavage on gestation days 6-20. Clinical signs of maternal toxicity, body weight gain, and food and water consumption were recorded. Caesarean sections were performed on gestation day 21; resorptions and living and dead fetuses were counted. Fetuses were weighed and examined for external abnormalities. Half of the fetuses from each litter were cleared and stained with Alizarin red S for skeleton evaluation. O. stamineus standardized aqueous extract did not alter pregnancy body weight gain and food and water consumption and caused no other sign of maternal toxicity. Embryolethality and prenatal growth retardation were not observed either. O. stamineus standardized aqueous extract increased a few skeleton variations and a skull bone malformation (hyoid bone absent) in a non-dose dependent manner. Anogenital distance was increased in male and female fetuses exposed to the highest O. stamineus standardized aqueous extract dose, an indication that the extract could possibly contain androgenic compounds.

Carica papaya Leaves Juice Significantly Accelerates the Rate of Increase in Platelet Count among Patients with Dengue Fever and Dengue Haemorrhagic Fever.

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.

Linkage disequilibrium between polymorphisms of ABCB1 and ABCC2 to predict the treatment outcome of Malaysians with complex partial seizures on treatment with carbamazepine mono-therapy at the Kuala Lumpur Hospital.

PURPOSE: 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. METHOD: 314 patients with CPS were recruited from the Neurology Department of the KLH based on stringent inclusion and exclusion criteria, of whom 152 were responders and the other 162 were non-responders. DNA was extracted from their blood samples and Taqman technology for allelic discrimination was performed. Results were described as genotype frequencies. The SHEsis analysis platform was used to calculate linkage disequilibrium index and infer haplotype frequencies. Haploview was used to do permutation test to obtain a corrected p-value. RESULTS: Resistance to treatment with CBZ mono-therapy was significantly associated with the 2677TT and the 3435TT genotypes while it was not significantly associated with the G1249A and -24C>T polymorphisms. The GCGC haplotype combination of the 2677G>T, 3435C>T, 1249G>A and -24C>T respectively was found to be extremely significant (p = 1.10e-20) with good drug response to CBZ mono-therapy. CONCLUSION: Linkage disequilibrium between the 2677G>T, 3435C>T, 1249G>A and -24C>T SNPs may be used as a reliable screening marker to determine the treatment outcome of CBZ mono-therapy with CPS irrespective of race or gender.

Inhibitory effects of cytochrome P450 enzymes CYP2C8, CYP2C9, CYP2C19 and CYP3A4 by Labisia pumila extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Labisa pumila (LP), popularly known with its local name, Kacip Fatimah, is a well known herb grown in Indochina and Southeast Asia and is traditionally used to regain energy after giving birth in women. The propensity of LP to cause drug-herb interaction via cytochrome P450 (CYP) enzyme system has not been investigated. AIM OF THE STUDY: To evaluate the in vitro inhibitory effects of various LP extracts (aqueous, ethanol, dichloromethane (DCM) and hexane) on cytochrome P450 2C8 (CYP2C8), CYP2C9, CYP2C19 and CYP3A4 activities. MATERIALS AND METHODS: Probe substrate-based high performance liquid chromatography (HPLC) methods were established for CYP2C9, CYP2C19 and CYP3A4 whereas a fluorescence-based enzyme assay was established for CYP2C8. The metabolite formations were examined after incubation of probe substrate with respective CYP isoform in the present or absent of LP extracts. The inhibitory effect of LP was characterized with kinetic parameters IC(50) and K(i) values. RESULTS: LP extracts showed differential effect of CYP activities with the order of inhibitory potency as follows: dichloromethane>hexane>ethanol>aqueous. This differential effect was only observed in CYP2C isoforms but not CYP3A4. Both the hexane and DCM extracts exhibited moderate to potent inhibition towards CYP2C activities in different modes including non-competitive, competive and mixed-type. The DCM effect was notably strong for CYP2C8 and CYP2C9 showing K(i) values of below 1 µg/ml. The selectivity of LP for CYP2C isoforms rather than CYP3A4 may be attributed to the presence of relatively small, lipophilic yet slightly polar compounds within the LP extracts. CONCLUSIONS: The results of our study revealed that phytoconstituents contained in LP, particularly in hexane and dichloromethane extracts, were able to selectively inhibit CYP2C isoforms. The inactivation was characterized by low K(i) values, in particular, in CYP2C8 and CYP2C9. These in vitro data indicate that LB preparations contain constituents that can potently inhibit CYP2C activities and suggest that this herb should be examined for potential pharmacokinetic drug interactions in vivo.

Repeated dose 28-days oral toxicity study of Carica papaya L. leaf extract in Sprague Dawley rats.

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.

Heterologous expression of human cytochromes P450 2D6 and CYP3A4 in Escherichia coli and their functional characterization.

This study aimed to express two major drug-metabolizing human hepatic cytochromes P450 (CYPs), CYP2D6 and CYP3A4, together with NADPH-cytochrome P450 oxidoreductase (OxR) in Escherichia coli and to evaluate their catalytic activities. Full length cDNA clones of both isoforms in which the N-terminus was modified to incorporate bovine CYP17α sequence were inserted into a pCWori(+) vector. The modified CYP cDNAs were subsequently expressed individually, each together with OxR by means of separate, compatible plasmids with different antibiotic selection markers. The expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. Enzyme activities were examined using high performance liquid chromatography (HPLC) assays with probe substrates dextromethorphan and testosterone for CYP2D6 and CYP3A4, respectively. Results from immunoblotting demonstrated the presence of both CYP proteins in bacterial membranes and reduced CO difference spectra of the cell preparations exhibited the characteristic absorbance peak at 450 nm. Co-expressed OxR also demonstrated an activity level comparable to literature values. Kinetic parameters, K(m) and V(max) values determined from the HPLC assays also agreed well with literature values. As a conclusion, the procedures described in this study provide a relatively convenient and reliable means of producing catalytically active CYP isoforms suitable for drug metabolism and interaction studies.

In vitro determination of the effect of Andrographis paniculata extracts and andrographolide on human hepatic cytochrome P450 activities.

We investigated the effects of Andrographis paniculata (AP) extracts and andrographolide on the catalytic activity of three human cDNA-expressed cytochrome P450 enzymes: CYP2C9, CYP2D6 and CYP3A4. In vitro probe-based high performance liquid chromatography assays were developed to determine CYP2C9-dependent tolbutamide methylhydroxylation, CYP2D6-dependent dextromethorphan O-demethylation and CYP3A4-dependent testosterone 6ß-hydroxylation activities in the presence and absence of AP extracts and andrographolide. Our results indicate that AP ethanol and methanol extracts inhibited CYP activities more potently than aqueous and hexane extracts across the three isoforms. Potent inhibitory effects were observed on CYP3A4 and CYP2C9 activities (K (i) values below 20 µg/ml). Andrographolide was found to exclusively but weakly inhibit CYP3A4 activity. In conclusion, data presented in this study suggest that AP extracts have the potential to inhibit CYP isoforms in vitro. There was, however, variation in the potency of inhibition depending on the extracts and the isoforms investigated.

In vitro effects of active constituents and extracts of Orthosiphon stamineus on the activities of three major human cDNA-expressed cytochrome P450 enzymes.

Orthosiphon stamineus (OS) has been traditionally used to treat diabetes, kidney and urinary disorders, high blood pressure and bone or muscular pain. To assess the possibility of drug-herb interaction via interference of metabolism, effects of four OS extracts of different polarity and three active constituents (sinensetin, eupatorin and rosmarinic acid) on major human cDNA-expressed cytochrome P450 (CYP) enzymes were investigated. Three substrate-probe based high-performance liquid chromatography (HPLC) assays were established to serve as activity markers for CYP2C9, CYP2D6 and CYP3A4. Our results indicate that OS extracts and constituents exhibited differential modulatory effects on different CYPs. While none of the OS components showed significant inhibition on CYP2C9, eupatorin strongly and uncompetitively inhibited CYP2D6 activity with a K(i) value of 10.2µM. CYP3A4 appeared to be the most susceptible enzyme to OS inhibitory effects. It was moderately inhibited by OS dichloromethane and petroleum ether extract with mixed-type and noncompetitive inhibitions (K(i)=93.7 and 44.9µg/mL), respectively. Correlation study indicated that the inhibition was accounted for by the presence of eupatorin in the extracts. When IC(50) values of these extracts were expressed in volume per dose unit to reflect inhibitory effect at recommended human doses from commercially available products, moderate inhibition was also observed. In addition, CYP3A4 was strongly and noncompetitively inhibited by eupatorin alone, with a K(i) value of 9.3µM. These findings suggest that co-administration of OS products, especially those with high eupatorin content, with conventional drugs may have the potential to cause drug-herb interactions involving inhibition of major CYP enzymes.

In vitro modulatory effects of Andrographis paniculata, Centella asiatica and Orthosiphon stamineus on cytochrome P450 2C19 (CYP2C19).

ETHNO PHARMACOLOGICAL RELEVANCE: Andrographis paniculata (AP), Centella asiatica (CA) and Orthosiphon stamineus (OS) are three popular herbs traditionally used worldwide. AP is known for the treatment of infections and diabetes and CA is good for wound healing and healthy skin while OS is usually consumed as tea to treat kidney and urinary disorders. Interaction of these herbs with human cytochrome P450 2C19 (CYP2C19), a major hepatic CYP isoform involved in metabolism of many clinical drugs has not been investigated to date. AIM OF THE STUDY: In this study, the modulatory effects of various extracts and major active constituents of AP, CA and OS on CYP2C19 activities were evaluated. MATERIALS AND METHODS: S-mephenytoin, the CYP2C19 substrate probe, was incubated in the presence or absence of AP, CA and OS components. The changes in the rate of metabolite (hydroxymephenytoin) formation were subsequently determined by a high-performance liquid chromatography (HPLC)-based enzyme assay to characterize the modulatory effects. RESULTS: Among the herbal extracts studied, AP ethanol extract and CA dichloromethane extract exhibited mixed type inhibition towards CYP2C19 with K(i) values of 67.1 and 16.4 µg/ml respectively; CA ethanol extract and OS petroleum ether extract competitively inhibited CYP2C19 activity (K(i)=39.6 and 41.5 µg/ml respectively). Eupatorin (a major active constituent of OS) was found to significantly inhibit CYP2C19 by mixed type inhibition (K(i)=7.1 µg/ml or 20.6 µM). CONCLUSIONS: It was observed that AP, CA and OS inhibited CYP2C19 activity with varying potency. While weak inhibitory effect was observed with AP, moderate to strong inhibition was observed with CA dichloromethane extract and eupatorin, the major OS constituent. Therefore care should be taken when these CA and OS components are co-administered with CYP2C19 substrates (such as omeprazole, proguanil, barbiturates, citalopram, and diazepam).

High prevalence of pfcrt K76t mutants among Plasmodium falciparum isolates from Sabah, Malaysia.

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.

In vitro modulatory effects on three major human cytochrome P450 enzymes by multiple active constituents and extracts of Centella asiatica.

ETHNOPHARMACOLOGICAL RELEVANCE: Centella asiatica (CA) has been widely cultivated as a vegetable or spice in China, Southeast Asia, India, Sri Lanka, Africa, and Oceanic countries and traditionally used for wound healing and maintaining normal blood pressure. AIM OF THE STUDY: The present study was carried out to examine the potential modulatory effects of three commercially available active components (asiaticoside, asiatic acid and madecassic acid) and four extracts (aqueous, ethanol, dichloromethane and hexane) of CA on three major cDNA-expressed human cytochrome P450 (CYP) isoforms. MATERIALS AND METHODS: High-performance liquid chromatography (HPLC)-based enzyme assays, namely tolbutamide 4-methyhydroxylase, dextromethorphan O-demethylase and testosterone 6beta-hydroxylase assays were developed to probe activities of CYP2C9, CYP2D6 and CYP3A4, respectively. Probe substrates were incubated with or without each active component and extract for each isoform, followed by examination of the kinetics parameters, IC(50) and K(i), to characterize modulatory effects. RESULTS: CYP2C9 was more susceptible to inhibitory effects by CA extracts compared to CYP2D6 and CYP3A4. Moderate degree of inhibition was observed in ethanol (K(i)=39.1 microg/ml) and dichloromethane (K(i)=26.6 microg/ml) extracts implying potential risk of interaction when CYP2C9 substrates are consumed with CA products. The two extracts however showed negligible inhibition towards CYP2D6 and CYP3A4 (IC(50)'s of 123.3 microg/ml and above). Similarly CA aqueous and hexane extracts did not significantly inhibit all three isoforms investigated (IC(50)'s of 117.9 microg/ml and above). Among the active constituents investigated, asiatic acid and madecassic acid appeared to selectively inhibit CYP2C9 and CYP2D6 more than CYP3A4. Of particular interest is the potent inhibitory effect of asiatic acid on CYP2C9 (K(i)=9.1 microg/ml). This signifies potential risk of interaction when substrates for this isoform are taken together with CA products with high asiatic acid content. Inhibitions of asiatic acid with the other isoforms and that of madecassic acid with all isoforms were only moderate (K(i)'s ranged from 17.2 to 84.4 microg/ml). On the other hand, the IC(50) values for asiaticoside were high (1070.2 microg/ml or above) for all three isoforms, indicating negligible or low potential of this compound to modulate CYP enzymatic activity. CONCLUSION: Centella asiatica extracts and active constituents inhibited CYP2C9, CYP2D6 and CYP3A4 activities with varying potency with CYP2C9 being the most susceptible isoform to inhibition. Significant inhibition was observed for asiatic acid and CA ethanol and dichloromethane extracts, implying involvement of semipolar constituents from CA in the effect. This study suggested that CA could cause drug-herb interactions through CYP2C9 inhibition.