Comparative metabolomics analysis reveals alkaloid repertoires in young and mature Mitragyna speciosa (Korth.) Havil. Leaves.

The fresh leaves of Mitragyna speciosa (Korth.) Havil. have been traditionally consumed for centuries in Southeast Asia for its healing properties. Although the alkaloids of M. speciosa have been studied since the 1920s, comparative and systematic studies of metabolite composition based on different leaf maturity levels are still lacking. This study assessed the secondary metabolite composition in two different leaf stages (young and mature) of M. speciosa, using an untargeted liquid chromatography-electrospray ionisation-time-of-flight-mass spectrometry (LC-ESI-TOF-MS) metabolite profiling. The results revealed 86 putatively annotated metabolite features (RT:m/z value) comprising 63 alkaloids, 10 flavonoids, 6 terpenoids, 3 phenylpropanoids, and 1 of each carboxylic acid, glucoside, phenol, and phenolic aldehyde. The alkaloid features were further categorised into 14 subclasses, i.e., the most abundant class of secondary metabolites identified. As per previous reports, indole alkaloids are the most abundant alkaloid subclass in M. speciosa. The result of multivariate analysis (MVA) using principal component analysis (PCA) showed a clear separation of 92.8% between the young and mature leaf samples, indicating a high variance in metabolite levels between them. Akuammidine, alstonine, tryptamine, and yohimbine were tentatively identified among the many new alkaloids reported in this study, depicting the diverse biological activities of M. speciosa. Besides delving into the knowledge of metabolite distribution in different leaf stages, these findings have extended the current alkaloid repository of M. speciosa for a better understanding of its pharmaceutical potential.

The effects of chronic mitragynine (Kratom) exposure on the EEG in rats.

Mitragynine is the main alkaloid isolated from the leaves of Mitragyna speciosa Korth (Kratom). Kratom has been widely used to relieve pain and opioid withdrawal symptoms in humans but may also cause memory deficits. Here we investigated the changes in brain electroencephalogram (EEG) activity after acute and chronic exposure to mitragynine in freely moving rats. Vehicle, morphine (5 mg/kg) or mitragynine (1, 5 and 10 mg/kg) were administered for 28 days, and EEG activity was repeatedly recorded from the frontal cortex, neocortex and hippocampus. Repeated exposure to mitragynine increased delta, but decreased alpha powers in both cortical regions. It further decreased delta power in the hippocampus. These findings suggest that acute and chronic mitragynine can have profound effects on EEG activity, which may underlie effects on behavioral activity and cognition, particularly learning and memory function.

Kratom and Pain Tolerance: A Randomized, Placebo-Controlled, Double-Blind Study.

Background: Kratom has a long history of traditional medicine use in Southeast Asia. Consumption of kratom products has also been reported in the US and other regions of the world. Pain relief is among many self-reported kratom effects but have not been evaluated in controlled human subject research. Methods: Kratom effects on pain tolerance were assessed in a randomized, placebo-controlled, double-blind study. During a 1-day inpatient stay, participants received a randomized sequence of kratom and placebo decoctions matched for taste and appearance. Pain tolerance was measured objectively in a cold pressor task (CPT) as time (seconds) between the pain onset and the hand withdrawal from the ice bath. Health status, vital signs, objective, and subjective indicators of withdrawal symptoms, self-reported data on lifetime kratom use patterns, and assessments of blinding procedures were also evaluated. Results: Twenty-six males with the mean (SD) age 24.3 (3.4) years were enrolled. They reported the mean (SD) 6.1 (3.2) years of daily kratom consumption. Pain tolerance increased significantly 1 hour after kratom ingestion from the mean (SD) 11.2 (6.7) seconds immediately before to 24.9 (39.4) seconds 1 hour after kratom consumption (F(2,53.7)=4.33, p=0.02). Pain tolerance was unchanged after consuming placebo drinks: 15.0 (19.0) seconds immediately before and 12.0 (8.1) seconds 1 hour after consumption of placebo (F(2,52.8)=0.93, p=0.40). No discomfort or signs of withdrawal were reported or observed during 10-20 hours of kratom discontinuation. Conclusions: Kratom decoction demonstrated a substantial and statistically significant increase in pain tolerance. Further rigorous research on kratom pain-relieving properties and a safety profile is needed.

Development of an ELISA for detection of mitragynine and its metabolites in human urine.

An enzyme-linked immunosorbent assay for detection of mitragynine, other closely related Kratom alkaloids and metabolites was developed using polyclonal antibodies. Mitragynine was conjugated to a carrier protein, cationized-bovine serum albumin using Mannich reaction. The synthesized antigen was injected into rabbits to elicit specific polyclonal antibodies against mitragynine. An enzyme conjugate was synthesized for evaluating its performance with the antibodies produced. The assay had an IC50 of 7.3 ng/mL with a limit of detection of 15 ng/mL for mitragynine. Antibody produced have high affinity for mitragynine (100%), other closely related Kratom alkaloids such as paynantheine (54%), speciociliatine (63%), 7α-hydroxy-7H-mitragynine (83%) and cross-reacted with metabolites 9-O-demethyl mitragynine (79%), 16-carboxy mitragynine (103%), 9-O-demethyl mitragynine sulfate (263%), 9-O-demethyl mitragynine glucuronide (60%), 16-carboxy mitragynine glucuronide (60%), 9-O-demethyl-16-carboxy mitragynine sulfate (270%) and 17-O-demethyl-16,17-dihydro mitragynine glucuronide (34%). It showed cross-reactivity less than 0.01% to reserpine, codeine, morphine, caffeine, methadone, amphetamine, and cocaine. Ten-fold dilution urine was used in the assay to reduce the matrix effects. The recovery ranged from 83% to 112% with variation coefficients in intraday and interday less than 8% and 6%, respectively. The ELISA turned out to be a convenient tool to diagnose mitragynine, other closely related Kratom alkaloids and metabolites in human urine samples.

Assessing physiological dependence and withdrawal potential of mitragynine using schedule-controlled behaviour in rats.

RATIONALE: Kratom is proposed to exhibit therapeutic potential as an opium substitute, but little is known about its dependence-producing profile, particularly of its main psychoactive compound, mitragynine (MG). OBJECTIVES: This study examined the dependence-producing effects of MG using operant-scheduled behaviour in rats and investigated the potential therapeutic effect of MG by comparing effects to buprenorphine in morphine-dependent rats using the same schedule-controlled behavioural task. METHODS: The effects of acutely administered MG and morphine were determined in rats trained to respond under fixed-ratio (FR) 10 schedule of food reinforcement. Next, the rats were administered MG and morphine twice daily for 14 consecutive days to determine if physiological dependence would develop by examining cessation of drug treatment and following antagonist-precipitated withdrawal. The study then examined the effects of MG substitution to suppress naloxone-precipitated morphine withdrawal effects on scheduled responding. RESULTS: Acute doses of MG did not produce dose-related decreases on FR schedules of responding compared to morphine. Unlike morphine, MG-treated rats showed no suppression of response rates following cessation of MG treatment. However, withdrawal effects were evident for MG after precipitation by either naloxone or SR141716A (rimonabant), similar to morphine-treated rats. MG in higher doses (10 and 30 mg/kg) attenuated the naloxone-precipitated morphine withdrawal effects while smaller doses of buprenorphine (0.3 and 1.0 mg/kg) were necessary to alleviate these effects. CONCLUSION: The findings suggest that MG does not induce physiological dependence but can alleviate the physical symptoms associated with morphine withdrawal which represent the desired characteristics of novel pharmacotherapeutic interventions for managing opioid use disorder (OUD).

Intestinal permeability of mitragynine in rats using in situ absorption model.

The intestinal permeability of mitragynine was investigated in situ using a single pass intestinal perfusion (SPIP) absorption model, in small intestine of rat using mitragynine in the absence/presence of the permeability markers, P-gp and/or CYP3A4 inhibitors. Mitragynine demonstrated high intestinal permeability (Peff of 1.11 × 10-4 cm/s) that is in the range of highly permeable drugs such as propranolol (Peff of 1.27 × 10-4 cm/s) indicating that it readily crosses the intestine. The addition of azithromycin (P-glycoprotein inhibitor) and ciprofloxacin (CYP3A4 inhibitor) or combination of both has no effect on intestinal permeability of mitragynine across the rat small intestine.

Long-Term Cognitive Effects of Kratom (Mitragyna speciosa Korth.) Use.

Kratom or Mitragyna speciosa (Korth.) is a medicinal plant of Southeast Asia. As a result of its opioid-like effects, it remains unknown whether consumption of kratom tea is associated with impaired cognitive function. We assessed the cognitive function of 70 regular kratom users and 25 control participants using the Cambridge Neuropsychological Test Automated Battery. Participants performed six neuropsychological tasks that assessed motor, learning and memory, attention and executive function. Relative to control participants, higher consumption (>3 glasses daily or mitragynine doses between 72.5 mg and 74.9 mg) of kratom tea was selectively associated with impaired performance on the Paired Associates Learning task, reflecting deficits in visual episodic memory and new learning. Overall, the performance of kratom users compared to control participants, and the performance of high (>3 glasses per day) as well as low (≤3 glasses per day) kratom using groups, were comparable on all neuropsychological domains. Higher intake of kratom juice (>3 glasses daily) did not appear to impair motor, memory, attention or executive function of regular kratom users.

Metabolomics data of Mitragyna speciosa leaf using LC-ESI-TOF-MS.

Mitragyna speciosa is a psychoactive plant known as "ketum" in Malaysia and "kratom" in Thailand. This plant is distinctly known to produce two important alkaloids, namely mitragynine (MG) and 7-hydroxymitragynine (7-OH-MG) that can bind to opioid receptors [1]. MG was reported to exhibit antidepressant properties in animal studies [2]. These compounds were also proposed to have the potential to replace opioid analgesics with much lower risks of side effects [3]. To date, there are only over 40 metabolites identified in M. speciosa [4,5]. To obtain a more complete profile of secondary metabolites in ketum, we performed metabolomics study using mature leaves of the green M. speciosa variety. The leaf samples were extracted using methanol prior to liquid chromatography-electrospray ionization-time of flight-mass spectrometry (LC-ESI-TOF-MS) analysis. This data can be useful to for the identification of unknown metabolites that are associated with alkaloid biosynthesis pathway in M. speciosa.

Assessment of gonadotropins and testosterone hormone levels in regular Mitragyna speciosa (Korth.) users.

ETHNOPHARMACOLOGICAL RELEVANCE: Mitragyna speciosa (Korth.) also known as kratom, is a native medicinal plant of Southeast Asia with opioid-like effects. Kratom tea/juice have been traditionally used as a folk remedy and for controlling opiate withdrawal in Malaysia. Long-term opioid use is associated with depletion in testosterone levels. AIM OF THE STUDY: Since kratom is reported to deform sperm morphology and reduce sperm motility, we aimed to clinically investigate the testosterone levels following long-term kratom tea/juice use in regular kratom users. METHODS: A total of 19 regular kratom users were recruited for this cross-sectional study. A full-blood test was conducted including determination of testosterone level, follicle stimulating hormone (FSH) and luteinizing hormone (LH) profile, as well as hematological and biochemical parameters of participants. RESULTS: We found long-term kratom tea/juice consumption with a daily mitragynine dose of 76.23-94.15 mg did not impair testosterone levels, or gonadotrophins, hematological and biochemical parameters in regular kratom users. CONCLUSION: Regular kratom tea/juice consumption over prolonged periods (>2 years) was not associated with testosterone impairing effects in humans.

Evaluating the hematological and clinical-chemistry parameters of kratom (Mitragyna speciosa) users in Malaysia.

ETHNOPHARMACOLOGICAL RELEVANCE: Kratom (Mitragyna speciosa Korth.) from the Rubiaceae family is an indigenous tropical medicinal tree of Southeast Asia. Kratom leaves have been used for decades in Malaysia and Thailand in traditional context for its perceived vast medicinal value, and as a mild stimulant among manual labourers. Kratom consumption has been reported to cause side-effects in kratom users. AIM OF THE STUDY: To evaluate kratom's effects towards hematological and clinical-chemistry parameters among regular kratom users in Malaysia. METHODS: A total of 77 subjects (n=58 regular kratom users, and n=19 healthy controls) participated in this cross-sectional study. All the surveys were conducted through face-to-face interview to elicit subject's socio-demographic characteristics and kratom use history. A full-blood test was also administered. Laboratory analysis was conducted using GC-MS to determine mitragynine content in the acquired kratom samples in order to relate mitragynine consumption with possible alterations in the blood parameters of kratom users. RESULTS: Findings showed that there were no significant differences in the hematological and clinical-chemistry parameters of traditional kratom users and healthy controls, except for HDL and LDL cholesterol values; these were found to be above the normal reference range for the former. Similarly, long-term kratom consumption (>5 years), and quantity of daily kratom use (≥3 ½ glasses; mitragynine content 76.3-114.8mg) did not appear to alter the hematological and biochemical parameters of kratom users. CONCLUSION: These data suggest that even long-term and heavy kratom consumption did not significantly alter the hematological and clinical-chemistry parameters of kratom users in a traditional setting.

Method validation in quantitative analysis of phase I and phase II metabolites of mitragynine in human urine using liquid chromatography-tandem mass spectrometry.

A method using solid phase extraction and liquid chromatography-tandem mass spectrometry to quantitatively detect mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine in human urine samples was developed and validated. The relevant metabolites were identified using multiple reaction monitoring in positive ionization mode using nalorphine as an internal standard. The method was validated for accuracy, precision, recovery, linearity, and lower limit of quantitation. The intra- and inter-day accuracy and precision were found in the range of 83.6-117.5% with coefficient of variation less than 13%. The percentage of recovery for mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine was within the range of 80.1-118.9%. The lower limit of quantification was 1 ng/mL for mitragynine, 2 ng/mL for 16-carboxy mitragynine, and 50 ng/mL for 9-O-demethyl mitragynine. The developed method was reproducible, high precision and accuracy with good linearity and recovery for mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine in human urine.

Chronic mitragynine (kratom) enhances punishment resistance in natural reward seeking and impairs place learning in mice.

Kratom (Mitragyna speciosa) is a widely abused herbal drug preparation in Southeast Asia. It is often consumed as a substitute for heroin, but imposing itself unknown harms and addictive burdens. Mitragynine is the major psychostimulant constituent of kratom that has recently been reported to induce morphine-like behavioural and cognitive effects in rodents. The effects of chronic consumption on non-drug related behaviours are still unclear. In the present study, we investigated the effects of chronic mitragynine treatment on spontaneous activity, reward-related behaviour and cognition in mice in an IntelliCage® system, and compared them with those of morphine and Δ-9-tetrahydrocannabinol (THC). We found that chronic mitragynine treatment significantly potentiated horizontal exploratory activity. It enhanced spontaneous sucrose preference and also its persistence when the preference had aversive consequences. Furthermore, mitragynine impaired place learning and its reversal. Thereby, mitragynine effects closely resembled that of morphine and THC sensitisation. These findings suggest that chronic mitragynine exposure enhances spontaneous locomotor activity and the preference for natural rewards, but impairs learning and memory. These findings confirm pleiotropic effects of mitragynine (kratom) on human lifestyle, but may also support the recognition of the drug's harm potential.

5-Methoxytryptamine reacts with natural food flavour to produce 6-methoxy tetrahydro-ß-carbolines: in vitro investigation of their antioxidant and cytotoxicity properties.

Various 6-methoxytetrahydro-ß-carboline derivatives, namely BEN (6-methoxy-1-phenyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole), ANI (6-methoxy-1-(4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole), ACE (6-methoxy-1-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole) and VAN (2-methoxy-4-(6-methoxy-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-l)phenol), were prepared via the Maillard reaction using food flavours and 5-methoxytryptamine in aqueous medium and were investigated for their in vitro antioxidant and cytotoxicity properties. These derivatives were found to exhibit moderate antioxidant properties, based on a combination of DPPH, ABTS and FRAP assays. The results suggested that the Maillard reaction could be used to generate ß-carboline antioxidants. It was beneficial that VAN showed the highest antioxidant activity but the least cytotoxic activities on non-tumourous cell lines of NIH/3T3, CCD18-Co and B98-5 using MTT assay. ACE, ANI and BEN showed mild toxicity at effective antioxidative concentrations derived from DPPH and ABTS assays. Furthermore, they are safer compared to 5-fluorouracil, cisplatin and betulinic acid on NIH/3T3, CCD18-Co and B98-5 cells. In conclusion, the antioxidant and cytotoxicity properties of 6-methoxytetrahydro-ß-carbolines were demonstrated for the first time.

Understanding the physicochemical properties of mitragynine, a principal alkaloid of Mitragyna speciosa, for preclinical evaluation.

Varied pharmacological responses have been reported for mitragynine in the literature, but no supportive scientific explanations have been given for this. These studies have been undertaken without a sufficient understanding of the physicochemical properties of mitragynine. In this work a UV spectrophotometer approach and HPLC-UV method were employed to ascertain the physicochemical properties of mitragynine. The pKa of mitragynine measured by conventional UV (8.11 ± 0.11) was in agreement with the microplate reader determination (8.08 ± 0.04). Mitragynine is a lipophilic alkaloid, as indicated by a logP value of 1.73. Mitragynine had poor solubility in water and basic media, and conversely in acidic environments, but it is acid labile. In an in vitro dissolution the total drug release was higher for the simulated gastric fluid but was prolonged and incomplete for the simulated intestinal fluid. The hydrophobicity, poor water solubility, high variability of drug release in simulated biological fluids and acid degradable characteristics of mitragynine probably explain the large variability of its pharmacological responses reported in the literature. The determined physicochemical properties of mitragynine will provide a basis for developing a suitable formulation to further improve its solubility, stability and oral absorption for better assessment of this compound in preclinical studies.

Behavioural and Electrophysiological Evidence of Impaired Learning and Memory in Male Sprague Dawley Rats following Subchronic Exposure to Standardised Methanolic Extract of Mitragyna speciosa Korth.

BACKGROUND: Mitragyna speciosa (MS) or ketum is primarily found in Southeast Asia, particularly in northern Malaysia and Thailand. The medicinal value of this plant has attracted significant attention from both herbal medicine practitioners and scientists worldwide. Despite having illegal consumption status, the plant merits study. We conducted a series of experiments to test our hypothesis that ketum impairs both learning and memory in rats. METHODS: Ketum leaves were extracted using methanol and standardised for the amount of its pure compound, mitragynine. Rats were divided into groups for a passive avoidance task and long-term potentiation (LTP) extracellular recording. In the extracellular recording condition, rats were grouped into control, MS100 (100 mg/kg of ketum extract), MS200 (200 mg/kg of ketum extract), and MS500 (500 mg/kg of ketum extract) groups. An additional group that received morphine was included in the passive avoidance task (10 mg/kg), and there were six animals per group. Rats received daily treatments orally for 28 days for both experiments. RESULT: Using a passive avoidance task, our data revealed that the rats' memory significantly increased with increasing doses of MS compared to the morphine-treated group. Our findings from LTP recordings showed that LTP was fully blocked by the higher doses of MS. CONCLUSION: We speculate on the possibility that additional factors were involved in the passive avoidance task because it was an in vivo animal study, while the LTP experiment solely involved brain slices.

Evaluation of antinociceptive effect of methanolic leaf and root extracts of Clitoria ternatea Linn. in rats.

AIM: Clitoria ternatea Linn. (C. ternatea) is an Ayurvedic herb traditionally used as medicine to relieve inflammatory, rheumatism, ear diseases, fever, arthritis, eye ailments, sore throat and body ache. This study aims to evaluate and elucidate the possible mechanism underlying the antinociceptive action of methanolic extracts of C. ternatea leaf and root using several antinociception models. MATERIALS AND METHODS: The different antinociception models such as hot plate, tail-flick and formalin tests were used along with naloxone (a non-selective opioid antagonist) to establish the antinociceptive activity of both leaf and root extracts. RESULTS: Both C. ternatea leaf and root extracts markedly demonstrated antinociceptive action in experimental animals. Results of formalin test showed that the antinociceptive activity of the extracts may be mediated at both central and peripheral level. Moreover, the results of hot plate and tail-flick tests further implies that C. ternatea root extract mediates antinociceptive activity centrally at supraspinal and spinal levels whereas, the C. ternatea leaf extract's antinociceptive activity is mediated centrally at supraspinal level only. It is believed that the opioid receptors are probably involved in antinociceptive activity of both C. ternatea root extract. CONCLUSIONS: Our studies support the traditional use of C. ternatea leaf and root against pain. The extracts can also be utilised as a new source of central analgesics in treatment of pain.

Anxiolytic-like effects of mitragynine in the open-field and elevated plus-maze tests in rats.

The effects of mitragynine on anxiety-related behaviours in the open-field and elevated plus-maze tests were evaluated. Male Sprague-Dawley rats were orally treated with mitragynine (10, 20 and 40 mg/kg) or diazepam (10 mg/kg) 60 min before behavioural testing. Mitragynine doses used in this study were selected on the basis of approximately human equivalent doses with reference to our previous literature reports. Acute administration of mitragynine (10, 20 and 40 mg/kg) or diazepam (10 mg/kg) increased central zone and open arms exploration in the open-field and elevated plus-maze tests respectively. These anxiolytic-like effects of mitragynine were effectively antagonized by intraperitoneal administration of naloxone (2 mg/kg), flumazenil (10 mg/kg), sulpiride (0.5 mg/kg) or SCH 23390 (0.02 mg/kg) 15 min before mitragynine treatments. These findings reveal that the acute administration of mitragynine produces anxiolytic-like effects and this could be possibly attributed to the interactions among opioidergic, GABAergic and dopaminergic systems in brain regions involved in anxiety.

A simple HPLC-DAD method for the detection and quantification of psychotropic mitragynine in Mitragyna speciosa (ketum) and its products for the application in forensic investigation.

Mitragyna speciosa, a native plant of Thailand and Malaysia known as 'ketum', is a plant of considerable interest. It exhibits strong antinociceptive effect and yet, acts like a psychostimulant. Due to the affordability and its ease of availability, the abuse of this plant as a substitute for other banned narcotics has become a major concern in many societies. In countries such as Thailand, Myanmar, Australia and Malaysia, the use of ketum is illegal. However, for a person to be charged for possessing or selling ketum, a reliable analytical method is needed in order to detect and identify the plant and its products. Mitragynine is the major alkaloid of ketum. This compound manifests its antinociceptive effects by acting on the opioid receptors. Since M. speciosa contain large quantity of mitragynine and it is exclusive to the species, the present analytical method is developed and validated for the purpose of screening ketum products based on this unique compound as the analytical marker. The method uses a HPLC-DAD system with Inertsil C8 (4.6 mm × 150 mm, 5 µm) as the column and a mixture of acetonitrile and formic acid, 50:50 (v/v), as the mobile phase. This method not only detects mitragynine, it can also be used to quantify the amount of mitragynine in the sample. The limit of detection is 0.25 µg/ml, while the limit of quantification is 0.50 µg/ml. The method is quick, simple and reliable with an accuracy of 97.27-101.74% and coefficient of variations of between 0.91 and 3.96%. The method has been tested and found suitable for the identification and quantification of mitragynine in dried plants, a variety of ketum extracts, as well as ketum drink obtained from the market.

Mitragyna speciosa Korth leaves extracts induced the CYP450 catalyzed aminopyrine-N-demethylase (APND) and UDP-glucuronosyl transferase (UGT) activities in male Sprague-Dawley rat livers.

BACKGROUND: Mitragyna speciosa leaves have been abused by drug addicts as some of the alkaloids (mainly mitragynine) from the plant possess opiate and cocaine-like effects. These bring to its prohibition in Malaysia in 2004 as consumption of M. speciosa leaves has been perceived to lead to the abuse of other drugs such as cannabis and heroin. METHODS: In the current study, the in vitro and in vivo effects of M. speciosa methanolic, aqueous and total alkaloid leaves extracts on drug metabolizing enzymes, namely, cytochrome P450s (CYP450s) and UDP-glucuronosyl transferase (UGT) had been evaluated in rat liver cytosolic fraction and microsomes. Aminopyrine and p-nitrophenol (pNP) were employed as probe substrates in aminopyrine N-demethylase (APND) and UGT enzyme assays, respectively. Furthermore, mitragynine was also tested in vitro for its likelihood to inhibit APND and UGT activity. The assessment of the enzyme activity was conducted using spectrophotometric methods. RESULTS: In vitro, the IC50 value could only be obtained for the methanolic extract in APND study (595.30±30.78 µg/mL) and not in other studies due to the enzyme percentage inhibitions being <70%. In contrast to the in vitro study, the oral treatment of male Sprague-Dawley rats for 14 days with 50, 100 and 200 mg/kg of methanolic and aqueous extracts and with 5, 10 and 20 mg/kg of total alkaloid extract showed a profound increment on the APND and UGT activities. CONCLUSIONS: The current findings showed that possibilities exist for herb-drug interaction with increased clearance of drugs, which are primarily metabolized by CYP450s and UGT1A6 among M. speciosa leaves extract users.