The effect of flavonols in Anacardium occidentale L. leaf extracts on skin pathogenic microorganisms.

Cashew (Anacardium occidentale L.) leaf is traditionally used to treat skin infections. Although many flavonols have been identified from its leaf extract, their inhibitory effects on skin pathogens are not yet determined. The aims of this study were to determine the antimicrobial (against skin pathogenic microbes) and antioxidant activities of four flavonol glycosides from the crude extract and three flavonol aglycones from the hydrolyzed extract. The hydrolyzed extract was found to show higher activities than the crude extract. Myricetin showed the highest activity against all the tested bacteria and yeast with the lowest Minimum Inhibition Concentration (MIC) of 7.81 µg/mL on Corynebacterium minutissimum ATCC23348. Myricetin also exhibited good primary antioxidant activities with the effective concentration with 50% of activity (EC50) values ranged between 2.23 µg/mL and 6.40 µg/mL. The highest secondary antioxidant activity was indicated by myricetin-3-O-rhamnoside. Thus, myricetin can be considered as a bioactive compound of the hydrolyzed extract.

Cytochrome P450 inhibition activities of non-standardized botanical products.

ETHNOPHARMACOLOGICAL RELEVANCE: R-tab, H-tab and E-cap botanical products are used for the treatment of various ailments. R-tab is traditionally prescribed for improving urination, H-tab is for relieving piles, hemorrhoids, fissures, and rectal inflammation and E-cap is for regulating menstruation. AIMS OF THE STUDY: To extract the botanical products and determine their potential interaction with the cytochrome P450 (CYP1A2, CYP2D6 and CYP3A4) enzymes. MATERIALS AND METHODS: R-tab, H-tab and E-cap botanical products were first extracted using solvents and analyzed using HPLC and LC-MS/MS. The effects of methanol extracts on the cytochrome induction and inhibition activities were determined using a series of in vitro assays, including multiplex RT-qPCR, CYP activity assays (P450-Glo™) and LC-MS/MS-based assays. For the CYP induction assay, omeprazole, rifampicin and dexamethasone were used as CYP1A2, CYP2D6 and CYP3A4 inducers, respectively. Ketoconazole and acetaminophen were used as positive and negative controls for the CYP3A4 inhibition assay, whereas furafylline and ketoconazole were used as positive and negative controls for the CYP1A2 inhibition assay. RESULTS: All three botanical products did not show any significant induction in CYP1A2, CYP2D6 and CYP3A4 mRNA expression. By contrast, R-tab inhibited the mRNA expression of CYP1A2 significantly from the lowest concentration of 0.01 µg/mL, while, H-tab inhibited the mRNA expression of CYP1A2 and CYP3A4 from 0.1 µg/mL. Based on the P450 Glo assays, E-cap extract inhibited the metabolic activity of CYP1A2 with an IC50 value of 37.24 µg/mL. On the other hand, R-tab, H-tab and E-cap showed inhibitory effects on the CYP3A4 enzymatic activity with IC50 values of 17.42, 18.20 and 20.60 µg/mL, respectively. However, using the LC-MS/MS-based methods, the concentration-dependent effects of R-tab and H-tab extracts on the metabolism of testosterone appeared to be more prominent, with IC50 values of 51.90 and 56.90 µg/mL as compared with the rest of the results, which were all above 100 µg/mL CONCLUSION: The CYP3A4 mRNA and enzymatic activity were moderately inhibited by R-tab and H-tab. Methanol extract of botanical products in solid dosage forms can be evaluated for their herb-drug interaction risks using in vitro assays and may provide the minimum data required for safety labeling.

Regular Kratom ( Mitragyna speciosa Korth.) Use and Its Association With Endoplasmic Reticulum Stress Response.

OBJECTIVES: This study determined the association between expression of the endoplasmic reticulum (ER) stress sensor mRNA in the peripheral leukocytes and the patterns of kratom use and evaluated the correlations between the levels of the ER stress sensor mRNA and the severity of kratom dependence and kratom induced depressive symptoms among people who use kratom (PWUK). METHODS: A total of 20 PWUK and 20 age matched non-kratom using healthy controls were recruited. Data collected from PWUK included patterns of kratom use, severity of kratom dependence, and severity of depressive symptoms during abstinence from kratom. The mRNA expression of binding immunoglobulin protein ( BiP ), X-box binding protein 1, activating transcription factor 4, and C/-EBP homologous protein ( CHOP ) (major indicators of ER stress response) were analyzed using quantitative reverse transcription polymerase chain reaction in leucocyte-derived total RNA sample of the participants. RESULTS: PWUK regardless of their pattern of kratom use recorded significantly higher expression of BiP mRNA compared with controls. Expression of CHOP mRNA was only significantly higher in those who first consumed kratom at the age of 18 years and above and those who have been using kratom for longer than 6 years, compared with controls. Higher expression of BiP , ATF4 , and CHOP mRNA were significantly positive correlated with greater severity of kratom dependence. Although only higher expression of BiP and CHOP mRNA were significantly positively correlated with greater severity of depressive symptoms. CONCLUSIONS: Regular kratom consumption may activate the ER stress pathway and there may be a link between altered ER stress response and kratom dependence and kratom induced depressive symptoms.

The influence of chemical composition of potent inhibitors in the hydrolyzed extracts of anti-hyperuricemic plants to their xanthine oxidase activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Anti-hyperuricemic plant parts that were selected for this study, are traditionally used to treat gout in Malaysia. Caffeic acid (a hydroxycinnamic acid), apigenin (a flavone), myricetin, quercetin and kaempferol (flavonols), were reported to act as potent xanthine oxidase inhibitors. These compounds can be found in some of the selected ethnomedicinal plants. However, there is still lack of published research works on the quantification of these inhibitors from these urate-lowering phytotherapies. AIMS OF THE STUDY: The compounds were quantified from 21 hydrolyzed extracts of the phytotherapies for gout. The activity-content contributions of the compounds to the potent extracts were determined. MATERIALS AND METHODS: The anti-hyperuricemic activities of the extracts and the compounds were determined using a xanthine oxidase inhibitory assay. Ultra-Performance Liquid Chromatography (UPLC) coupled with Photodiode Array Detector (PDA) was used to quantify the compounds in the extracts. RESULTS: The results revealed higher activity of the hydrolyzed extracts. The hydrolyzed extract of the flower bud of Syzygium aromaticum Merr. & L.M.Perry exhibited the highest activity (EC50 = 39.58 ± 0.10 µg/mL) due to the highest content of myricetin (42,297.55 ± 159.47 µg/g). The activity-content contribution of myricetin was 7.69%. Due to the highest activity of apigenin (EC50 = 3.27 ± 0.09 µg/mL), the highest contribution of this flavone (29.96%) to the hydrolyzed extract of Orthosiphon aristatus (Blume) Miq. was observed. CONCLUSION: The results revealed different contents and activities of xanthine oxidase inhibitors in the hydrolyzed extracts of anti-hyperuricemic plants can play a major role to influence the activity.

Beetroot as a Potential Functional Food for Cancer Chemoprevention, a Narrative Review.

Patients with cancer are prone to several debilitating side effects including fatigue, insomnia, depression and cognitive disturbances. Beetroot (Beta vulgaris L.) as a health promoting functional food may be potentially beneficial in cancer. As a source of polyphenols, flavonoids, dietary nitrates and other useful nutrients, beetroot supplementation may provide a holistic means to prevent cancer and manage undesired effects associated with chemotherapy. The main aim of this narrative review is to discuss beetroot's nutrient composition, current studies on its potential utility in chemoprevention and cancer-related fatigue or treatment-related side effects such as cardiotoxicity. This review aims to provide the current status of knowledge and to identify the related research gaps in this area. The flavonoids and polyphenolic components present in abundance in beetroot support its significant antioxidant and anti-inflammatory capacities. Most in vitro and in vivo studies have shown promising results; however, the molecular mechanisms underlying chemopreventive and chemoprotective effects of beetroot have not been completely elucidated. Although recent clinical trials have shown that beetroot supplementation improves human performance, translational studies on beetroot and its functional benefits in managing fatigue or other symptoms in patients with cancer are still lacking.

Andrographolide is neither a human organic anion transporter 1 (hOAT1) substrate nor inhibitor.

Andrographolide, a major bioactive compound isolated from Andrographis paniculata (Burm. F.) Nees, was evaluated for its effects on the hOAT1 membrane transporter. Substrate determination and inhibition of hOAT1-mediated uptake transport assay was carried out using recombinant CHO-hOAT1 cells. The results showed that the uptake ratio of andrographolide was less than 2.0 at all concentrations tested, indicating that andrographolide is not a hOAT1 substrate. Andrographolide has no significant effects on the p-aminohippuric acid uptake and on the mRNA and protein expression of hOAT1. In conclusion, andrographolide may not pose a drug-herb interaction risk related to hOAT1.

The inhibitory effects of mitragynine on P-glycoprotein in vitro.

Mitragynine is a major component isolated from Mitragyna speciosa Korth or kratom, a medicinal plant known for its opiate-like and euphoric properties. Multiple toxicity and fatal cases involving mitragynine or kratom have been reported but the underlying causes remain unclear. P-glycoprotein (P-gp) is a multidrug transporter which modulates the pharmacokinetics of xenobiotics and plays a key role in mediating drug-drug interactions. This study investigated the effects of mitragynine on P-gp transport activity, mRNA, and protein expression in Caco-2 cells using molecular docking, bidirectional assay, RT-qPCR, Western blot analysis, and immunocytochemistry techniques, respectively. Molecular docking simulation revealed that mitragynine interacts with important residues at the nucleotide binding domain (NBD) site of the P-gp structure but not with the residues from the substrate binding site. This was consistent with subsequent experimental work as mitragynine exhibited low permeability across the cell monolayer but inhibited digoxin transport at 10 µM, similar to quinidine. The reduction of P-gp activity in vitro was further contributed by the downregulation of mRNA and protein expression of P-gp. In summary, mitragynine is likely a P-gp inhibitor in vitro but not a substrate. Hence, concurrent administration of mitragynine-containing kratom products with psychoactive drugs which are P-gp substrates may lead to clinically significant toxicity. Further clinical study to prove this point is needed.

The effects of deoxyelephantopin on the cardiac delayed rectifier potassium channel current (IKr) and human ether-a-go-go-related gene (hERG) expression.

Elephantopus scaber Linn and its major bioactive component, deoxyelephantopin are known for their medicinal properties and are often reported to have various cytotoxic and antitumor activities. This plant is widely used as folk medicine for a plethora of indications although its safety profile remains unknown. Human ether-a-go-go-related gene (hERG) encodes the cardiac IKr current which is a determinant of the duration of ventricular action potentials and QT interval. The hERG potassium channel is an important antitarget in cardiotoxicity evaluation. This study investigated the effects of deoxyelephantopin on the current, mRNA and protein expression of hERG channel in hERG-transfected HEK293 cells. The hERG tail currents following depolarization pulses were insignificantly affected by deoxyelephantopin in the transfected cell line. Current reduction was less than 40% as compared with baseline at the highest concentration of 50 µM. The results were consistent with the molecular docking simulation and hERG surface protein expression. Interestingly, it does not affect the hERG expression at both transcriptional and translational level at most concentrations, although higher concentration at 10 µM caused protein accumulation. In conclusion, deoxyelephantopin is unlikely a clinically significant hERG channel and Ikr blocker.

Growth inhibition of human liver carcinoma HepG2 cells and α-glucosidase inhibitory activity of Murdannia bracteata (C.B. Clarke) Kuntze ex J.K. Morton extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: The juice of the entire fresh herb and infusion of dried sample of Murdannia bracteata are consumed to treat liver cancer and diabetes in Malaysia. However, no scientific evidence of these bioactivities has been reported. MATERIALS AND METHODS: To verify the therapeutic potentials of sequential extracts and infusion of this plant by determining its cytotoxicity against human liver carcinoma HepG2 cells and α-glucosidase inhibitory activity. The cytotoxic activities of the extracts against HepG2 were determined using a methylene blue assay, and an α-glucosidase inhibitory assay was used to assess anti-diabetic activity. The molecular basis of the anti-hepatocellular carcinoma activity of the most active extract was determined using RT-PCR. Chemical profiling of the most active extract was performed using GC-MS and UPLC analyses. RESULTS: The results obtained from the cytotoxic screening revealed the dose-dependent growth inhibition of the HepG2 cells by only the hexane extract, with an EC50 value of 37.17±1.00 µg/ml. The HepG2 cell death was found to be apoptotic in nature and based on the significant biphasic induction of caspase-3, suggesting that the extract inhibited cell growth through a caspase-3-dependent pathway. The hexane extract also displayed α-glucosidase inhibitory activity, with an EC50 of 117.04±2.34 µg/ml. GC-MS analysis revealed that α-tocopherol was the major volatile compound in the hexane extract, and two phenolics (apigenin and caffeic acid derivatives) were detected using UPLC. CONCLUSIONS: Based on various published reports, it could be suggested that α-tocopherol and apigenin derivatives might be involved in the apoptosis-based cytotoxicity of the active extract of this plant against HepG2 carcinoma cells. The effects of this plant in the treatment of diabetes can be related to the presence of α-glucosidase inhibitors, such as the caffeic acid derivative identified in the active extract.

The effects of Andrographis paniculata (Burm.f.) Nees extract and diterpenoids on the CYP450 isoforms' activities, a review of possible herb-drug interaction risks.

Andrographis paniculata (Burm.f.) Nees is a popular medicinal plant and its components are used in various traditional product preparations. However, its herb-drug interactions risks remain unclear. This review specifically discusses the various published studies carried out to evaluate the effects of Andrographis paniculata (Burm.f.) Nees plant extracts and diterpenoids on the CYP450 metabolic enzyme and if the plant components pose a possible herb-drug interaction risk. Unfortunately, the current data are insufficient to indicate if the extracts or diterpenoids can be labeled as in vitro CYP1A2, CYP2C9 or CYP3A4 inhibitors. A complete CYP inhibition assay utilizing human liver microsomes and the derivation of relevant parameters to predict herb-drug interaction risks may be necessary for these isoforms. However, based on the current studies, none of the extracts and diterpenoids exhibited CYP450 induction activity in human hepatocytes or human-derived cell lines. It is crucial that a well-defined experimental design is needed to make a meaningful herb-drug interaction prediction.

Evaluation of the cardiotoxicity of mitragynine and its analogues using human induced pluripotent stem cell-derived cardiomyocytes.

INTRODUCTION: Mitragynine is a major bioactive compound of Kratom, which is derived from the leave extracts of Mitragyna speciosa Korth or Mitragyna speciosa (M. speciosa), a medicinal plant from South East Asia used legally in many countries as stimulant with opioid-like effects for the treatment of chronic pain and opioid-withdrawal symptoms. Fatal incidents with Mitragynine have been associated with cardiac arrest. In this study, we determined the cardiotoxicity of Mitragynine and other chemical constituents isolated using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). METHODS AND RESULTS: The rapid delayed rectifier potassium current (IKr), L-type Ca2+ current (ICa,L) and action potential duration (APD) were measured by whole cell patch-clamp. The expression of KCNH2 and cytotoxicity was determined by real-time PCR and Caspase activity measurements. After significant IKr suppression by Mitragynine (10 µM) was confirmed in hERG-HEK cells, we systematically examined the effects of Mitragynine and other chemical constituents in hiPSC-CMs. Mitragynine, Paynantheine, Speciogynine and Speciociliatine, dosage-dependently (0.1∼100 µM) suppressed IKr in hiPSC-CMs by 67%∼84% with IC50 ranged from 0.91 to 2.47 µM. Moreover, Mitragynine (10 µM) significantly prolonged APD at 50 and 90% repolarization (APD50 and APD90) (439.0±11.6 vs. 585.2±45.5 ms and 536.0±22.6 vs. 705.9±46.1 ms, respectively) and induced arrhythmia, without altering the L-type Ca2+ current. Neither the expression, and intracellular distribution of KCNH2/Kv11.1, nor the Caspase 3 activity were significantly affected by Mitragynine. CONCLUSIONS: Our study indicates that Mitragynine and its analogues may potentiate Torsade de Pointes through inhibition of IKr in human cardiomyocytes.

Cytochrome P450 induction properties of food and herbal-derived compounds using a novel multiplex RT-qPCR in vitro assay, a drug-food interaction prediction tool.

A multiplex RT-qPCR was developed to examine CYP1A2, CYP2D6, and CYP3A4 induction properties of compounds from food and herbal sources. The induction of drug metabolizing enzymes is an important pharmacokinetic interaction with unique features in comparison with inhibition of metabolizing enzymes. Cytochrome induction can lead to serious drug-drug or drug-food interactions, especially if the coadministered drug plasma level is critical as it can reduce therapeutic effects and cause complications. Using this optimized multiplex RT-qPCR, cytochrome induction properties of andrographolide, curcumin, lycopene, bergamottin, and resveratrol were determined. Andrographolide, curcumin, and lycopene produced no significant induction effects on CYP1A2, CYP2D6, and CYP3A4. However, bergamottin appeared to be a significant in vitro CYP1A2 inducer starting from 5 to 50 µmol/L with induction ranging from 60 to 100-fold changes. On the other hand, resveratrol is a weak in vitro CYP1A2 inducer. Examining the cytochrome induction properties of food and herbal compounds help complement CYP inhibition studies and provide labeling and safety caution for such products.

The mTOR signalling pathway in cancer and the potential mTOR inhibitory activities of natural phytochemicals.

The mammalian target of rapamycin (mTOR) kinase plays an important role in regulating cell growth and cell cycle progression in response to cellular signals. It is a key regulator of cell proliferation and many upstream activators and downstream effectors of mTOR are known to be deregulated in various types of cancers. Since the mTOR signalling pathway is commonly activated in human cancers, many researchers are actively developing inhibitors that target key components in the pathway and some of these drugs are already on the market. Numerous preclinical investigations have also suggested that some herbs and natural phytochemicals, such as curcumin, resveratrol, timosaponin III, gallic acid, diosgenin, pomegranate, epigallocatechin gallate (EGCC), genistein and 3,3'-diindolylmethane inhibit the mTOR pathway either directly or indirectly. Some of these natural compounds are also in the clinical trial stage. In this review, the potential anti-cancer and chemopreventive activities and the current status of clinical trials of these phytochemicals are discussed.

Identification of genes involved in the regulation of 14-deoxy-11,12-didehydroandrographolide-induced toxicity in T-47D mammary cells.

14-Deoxy-11,12-didehydroandrographolide is one of the principle compounds of the medicinal plant, Andrographis paniculata Nees. This study explored the mechanisms of 14-deoxy-11,12-didehydroandrographolide-induced toxicity and non-apoptotic cell death in T-47D breast carcinoma cells. Gene expression analysis revealed that 14-deoxy-11,12-didehydroandrographolide exerted its cytotoxic effects by regulating genes that inhibit the cell cycle or promote cell cycle arrest. This compound regulated genes that are known to reduce/inhibit cell proliferation, induce growth arrest and suppress cell growth. The growth suppression activities of this compound were demonstrated by a downregulation of several genes normally found to be over-expressed in cancers. Microscopic analysis revealed positive monodansylcadaverine (MDC) staining at 8h, indicating possible autophagosomes. TEM analysis revealed that the treated cells were highly vacuolated, thereby suggesting that 14-deoxy-11,12-didehydroandrographolide may cause autophagic morphology in these cells. This morphology may be correlated with the concurrent expression of genes known to affect lysosomal activity, ion transport, protein degradation and vesicle transport. Interestingly, some apoptotic-like bodies were found, and these bodies contained multiple large vacuoles, suggesting that this compound is capable of eliciting a combination of apoptotic and autophagic-like morphological characteristics.

Cytotoxic, apoptotic and anti-α-glucosidase activities of 3,4-di-O-caffeoyl quinic acid, an antioxidant isolated from the polyphenolic-rich extract of Elephantopus mollis Kunth.

ETHNOPHARMACOLOGICAL RELEVANCE: The decoction of the whole plant of Elephantopus mollis Kunth. is traditionally consumed to treat various free radical-mediated diseases including cancer and diabetes. AIM OF THE STUDY: This study was initiated to determine whether the most effective antioxidant compound isolated from the whole plant of Elephantopus mollis can also contribute to its claimed traditional values as anticancer and antidiabetes agents. MATERIALS AND METHODS: An active antiradical phenolic compound (3,4-di-O-caffeoyl quinic acid) was isolated from the methanol extract (with the highest in polyphenolic content) and their antioxidant activities were compared using four different assays, that are DPPH, FRAP, metal chelating, and ß-carotene bleaching tests. The compound was also evaluated for its cytotoxic activity, apoptotic induction and anti-glucosidase efficacies using methylene blue, DeadEnd™ assay and α-glucosidase assays, respectively. RESULTS: The compound acted as a greater primary antioxidant than its methanol extract, by having higher ferric reducing activity (EC(50) 2.18±0.05 µg/ml), ß-carotene bleaching activity (EC(50) 23.85±0.65 µg/ml) and DPPH scavenging activity (EC(50) 68.91±5.44µg/ml), whereas the methanol extract exhibited higher secondary antioxidant activity as a metal chelator with lower EC(50) value (49.39±3.68 µg/ml) than the compound. Cytotoxicity screening of this compound exhibited a remarkable dose-dependent inhibitory effect on NCI-H23 (human lung adenocarcinoma) cell lines (EC(50) 3.26±0.35 µg/ml) and was found to be apoptotic in nature based on a clear indication of DNA fragmentation. This compound also displayed a concentration-dependent α-glucosidase inhibition with EC(50) 241.80±14.29 µg/ml. CONCLUSIONS: The findings indicate the major role of 3,4-di-O-caffeoyl quinic acid to antioxidant capacities of Elephantopus mollis extracts. The compound also exerted apoptosis-mediated cytotoxicity and α-glucosidase inhibitory effects and is thus a promising non toxic agent in treating cancer and type 2 diabetes mellitus.