Accumulation Of Berberine Research Articles

The interaction of alkaloids in Coptis chinensis Franch -Tetradium ruticarpum (A. Juss.) T.G. Hartley with hOCT1 and hOCT2

Ethnopharmacological relevanceZuojin Pill, a traditional poly-herbal drug, comprises Coptis chinensis Franch - Tetradium ruticarpum (A. Juss.) T.G. Hartley (6:1). The significant quantity of alkaloids found in the participating herbs is a key aspect of the Zuojin Pill. According to traditional Chinese medicine (TCM), these numerous alkaloidal compounds within Zuojin Pill have various essential therapeutic effects. Aim of the studyThe alkaloids in Tetradium are mainly indole alkaloids, while the alkaloids in Coptis are mostly isoquinoline alkaloids with low bioavailability. Alkaloids and their metabolites are nitrogen-containing compounds or weakly alkaline substances that can be partially ionized under physiological pH conditions. Fortunately, organic cation transporters (OCTs) play a crucial role in the cellular uptake of weakly alkaline compounds. Therefore, we speculated that the alkaloidal compounds might interact with liver cation transporters hOCT1 and kidney cation transporters hOCT2 to alter cell drug disposal. In order to clarify our hypothesis, a series of alkaloids-OCTs interaction experiments were conducted. Materials and methodsHEK293 cells stably expressing hOCT1 and hOCT2 were modeled and evaluated. Afterward, high-content screening (HCS) was conducted to analyze whether the main alkaloids and their metabolites of Coptis - Tetradium were inhibitors of hOCT1 and hOCT2 transporters. Meanwhile, LC-MS/MS was used to investigate whether the alkaloidal compounds were substrates of hOCT1 and hOCT2 transporters. Finally, drug interactions at the cellular level were assessed by LC-MS/MS after co-administration of berberine and rutacorine. ResultsBerberine, jateorhizine, coptisine, epiberberine, columbamine, demethyleneberberine, and berberrubine could significantly inhibit hOCT1 and hOCT2 activity. Isoquinoline alkaloids, including berberine, jateorhizine, coptisine, epiberberine, columbamine, and palmatine, were substrates of hOCT1 and hOCT2, but not the indole alkaloids evodiamine and rutaecarpine. Furthermore, evodiamine at a concentration of 20 μmol/L had a trivial effect on berberine accumulation in HEK293-hOCT2 cells. ConclusionsThese results support the idea that alkaloidal compounds within Coptis and Tetradium have hOCT1 and hOCT2 inhibitory activity or be their substrates, and the increased oral bioavailability of berberine in vivo was closely related to the potential interactions of small molecules in Coptis- Tetradium. Overall, our study provides a framework for investigating the potential interactions of small molecules in Coptis- Tetradium.

Alkaloid Biosynthesis in the Early Stages of the Germination of Argemone mexicana L. (Papaveraceae).

The synthesis of the benzylisoquinoline alkaloids, sanguinarine and berberine, was monitored in Argemone mexicana L. (Papaveracea) throughout the early stages of its hypocotyl and seedling development. Sanguinarine was detected in the cotyledons right after hypocotyl emergence, and it increased continuously until the apical hook unbent, prior to the cotyledonary leaves unfolding, when it abruptly fell. In the cotyledonary leaves, it also remained at low levels. Throughout development, berberine accumulation required the formation of cotyledonary leaves, whereas it was quickly detected in the hypocotyl from the time it emerged. Interestingly, the alkaloids detected in the cotyledons could have been imported from hypocotyls, because no transcriptional activity was detected in there. However, after turning into cotyledonary leaves, important levels of gene expression were noted. Taken together, these results suggest that the patterns of alkaloid tissue distribution are established from very early development, and might require transport systems.

Comparative transcriptome analysis provides novel insights into the molecular mechanism of berberine biosynthesis in Coptis chinensis

Berberine is the primary medicinal component of Coptis chinensis, a perennial herb in Ranunculaceae. It has pharmacological effects, such as clearing away heat and dampness, purging fire and detoxifying, and resisting oxidation, and is widely used in clinical treatment. Anatomical analysis of the organs showed that berberine is mainly deposited in the xylem of the adventitious roots and rhizomes, as well as in thick-walled tissue layers containing vascular bundles in the leaves and petioles of C. chinensis. The content of total alkaloids in C. chinensis was highest in the rhizomes, among which berberine (54.44%) and coptisine (20.62%) were the main alkaloids. Furthermore, comparative transcriptome analysis of the leaves, petioles, rhizomes, and adventitious roots of C. chinensis was used to reveal the molecular regulation of alkaloid biosynthesis. A total 38 differentially expressed structural genes (DEGs) and 22 differentially expressed transcription factors were related to berberine biosynthesis in C. chinensis. Among these DEGs, the expression level of 13 genes was positively correlated with berberine content, including candidate structural genes (TYR, TYDC, TH, and PPO) and transcription factors (bHLH and WRKY), which may play key roles in berberine accumulation in C. chinensis. The results provide resources and technical support for improving the content of berberine in C. chinensis by genetic engineering, thus providing a theoretical foundation for enhancing the medicinal value of C. chinensis.

In vitro synthesis of berberine, a pharmacologically important isoquinoline alkaloid in callus cultures of Tinospora cordifolia

Callus cultures were raised from young green leaf segments of the medicinal climber Tinospora cordifolia in Murashige and Skoog’s media containing different growth regulator regime. The in vitro callus cultures were analysed for the accumulation of active principle through high-performance liquid chromatography. Quantitative analysis of berberine was achieved by thin-layer chromatography and UV-absorbance method. Callus cultures accumulated an appreciable amount of berberine. A maximum of 355 μg/g dry wt accumulation of berberine was noted when the callus was grown in the presence of 2 mg/l 2, 4-D and 0.1 mg/l BA. However, maximum callus growth was achieved in the presence of 0.5 mg/l BA and 2 mg/l 2,4-D. Maximum tissue growth didn't favour an increased accumulation of the berberine. Enhanced accumulation of berberine was found when callus growth was less and therefore may be associated with the stress faced by in vitro tissues. The experimental results indicate that it is desirable to adopt two different growth regulator regime - one for accelerated callus growth and another for enhanced in vitro synthesis of berberine. The in vitro cultures of T. cordfiolia also offers an ideal system for understanding the studies on elucidating alternate routes of berberine biosynthetic pathway as well as possible elite cells for scaling up of berberine.

Berberine-induced nucleolar stress response in a human breast cancer cell line

We investigated the novel molecular mechanisms of the antitumor effect of berberine. In this study, two different human cell lines (breast cancer MCF7 cells and non-tumorigenic epithelial MCF12A cells) were treated with various concentrations of berberine. Treatment with 1 and 10 μM berberine inhibited proliferation with G0/G1 cell cycle arrest in both cell lines, and treatment with 100 μM berberine triggered a marked level of cell death in MCF7 cells but not in MCF12A cells. Berberine increased the level of p53 protein and of its target p21 both time- and dose-dependently in MCF7 cells. At any concentration of berberine, immediate uptake (within 15 min) followed by predominantly mitochondrial accumulation were observed by confocal microscopy in both cell lines. At high concentrations (10 or 100 μM), accumulation in the nucleolus became prominent after the transition to the nucleoplasm, especially remarkable in MCF7 cells. Therefore, we evaluated the possibility of berberine-induced nucleolar stress and observed the disappearance of ribosomal protein (RP)L5 from the nucleolus and accumulation of p53 protein in the nucleus after treatment with 10 or 100 μM berberine in MCF7 cells. We also detected the accumulation of RPL5 and RPL11 in the nucleoplasm fraction where they bind to Mdm2. Moreover, downregulation of RPL5 inhibited berberine-driven induction of p53 and p21 and cell death in MCF7 cells. Whereas, in MCF12A cells, down-regulation of RPL5 had little effect on the growth inhibitory effect of high concentration of berberine. These results indicated that cell growth inhibition and cell death induced by higher doses (>10 μM) of berberine in MCF7 cells were due to the upregulation of p53 under the nucleolar stress response caused by a significant accumulation of berberine in the nucleoli.

Alkaloid synthesis is coupled to shoot morphogenesis in Argemone mexicana L. (Papaveraceae) in vitro cultures

During the induction process of an in vitro callus culture of Argemone mexicana L. (Papaveraceae), the levels of two benzylisoquinoline alkaloids known as berberine and sanguinarine displayed opposing trends. While the berberine levels steadily decreased from the initial explant stage up to the early proliferation of unorganized parenchymatous cell masses, the sanguinarine content increased. Once the callus culture was established, sanguinarine was the primary alkaloid present and berberine could no longer be detected. However, upon shoot regeneration, the berberine accumulation recovered, but sanguinarine was found in the newly formed leafy tissue. After root formation, sanguinarine was relocated to this organ, whereas berberine was evenly distributed between both tissues. Explants from stem internodes did not form callus, and berberine—plus sanguinarine—containing axillary shoots emerged from lateral buds in the induction medium. In contrast to callus-derived shoots, no root formation was observed. Therefore, alkaloid synthesis in A. mexicana in vitro cultures is related to the level of tissue organization in different ways, and while berberine accumulation seems to require the presence of differentiated organs, this is not the case for sanguinarine. Moreover, leafy parts of rootless shoots acquired the capacity to accumulate sanguinarine, which is usually absent in aerial tissues of mature plants. However, when these shoots were rooted, sanguinarine was mainly located in the newly formed roots, while berberine was detected in the shoots at similar levels found in the roots.

In Vitro Anti-Helicobacter pylori Activity of Berberine and Barberry Extracts: A Preliminary Report

The berberine accumulation in Berberis vulgaris and antibacterial activity of plant extracts against Helicobacter pylori were examined. The antibacterial activities of berberine, the main alkaloid of B. vulgaris, and standard antimicrobials metronidazole and tetracycline were also determined. Berberine content was investigated using high-performance liquid chromatography. The antibacterial activity of plant extracts, berberine and reference antibiotics was tested by the broth microdilution method. The highest amount of berberine was found in the root bark (3.99 g/kg, dry wt). The plant extracts tested exhibited in vitro antibacterial activity against H. pylori, but the activities were lower than those of berberine.

Mitochondrial membrane potential played crucial roles in the accumulation of berberine in HepG2 cells.

Berberine is a natural alkaloid that has antineoplastic effects. However, in hepatoma cells like HepG2, the expressions of uptake transporters are minimal but efflux transporters are relatively high. Hence, how berberine enters and reaches a cytocidal concentration remains to be elucidated. In the present study, we revealed the accumulation mechanism of berberine in HepG2 cells. Cell organelles were isolated based on differential centrifugation; berberine concentration was measured using a liquid chromatography-tandem mass chromatography method or flow cytometry. Subcellular distribution of berberine was observed using a laser scanning confocal microscopy. The results showed that berberine was concentration-, temperature-, and time-dependently taken up and accumulated in HepG2 cells. Membrane drug transporters and cell membrane potential had limited effects in berberine uptake. However, qualitative and quantitative studies showed that berberine was enriched in the mitochondria; inhibition of mitochondrial membrane potential (MMP) by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) significantly decreased the intracellular berberine by up to 70%. More importantly, MMP not only significantly enhanced berberine uptake driven by cell membrane potential (P<0.01) but also inhibited p-glycoprotein (P-gp)-mediated berberine efflux (P<0.01). In brief, our results for the first time showed that MMP played crucial roles in berberine accumulation in HepG2 cells.

Renal vectorial transport of berberine mediated by organic cation transporter 2 (OCT2) and multidrug and toxin extrusion proteins 1 (MATE1) in rats.

Berberine, a well-known plant alkaloid derived from Rhizoma coptidis, has potential applications as a therapeutic drug for diabetic nephropathy. However, the transporter-mediated renal transport of berberine remains largely unclear. This study aimed to investigate the renal transport mechanism of berberine using transfected cells, kidney slices and animal experiments. In Madin-Darby canine kidney (MDCK) cells stably expressing rat OCT2 (MDCK-rOCT2) and kidney slices, saturable and non-saturable uptake of berberine was observed, and corticosterone could inhibit the uptake of berberine, with IC50 values of 0.1μm and 147.9μm, respectively. In double-transfected cells, the cellular accumulation of berberine into MDCK-rOCT2 and MDCK-rOCT2-rMATE1 (MDCK cells stably expressing rOCT2 and rMATE1) cells was significantly higher than the uptake into MDCK cells. Meanwhile, berberine transcellular transport was considerably higher in double-transfected MDCK-rOCT2-rMATE1 cells than in MDCK and MDCK-rOCT2 cells. Corticosterone for MDCK-rMATE1 and MDCK-MDR1 and pyrimethamine for MDCK-rMATE1 at high concentrations could inhibit the efflux of berberine. In animal experiments, compared with the berberine alone group, the cumulative urinary excretion of berberine significantly decreased in the corticosterone or pyrimethamine pretreatment groups. In the rat kidney, pyrimethamine increased, and a low dose of corticosterone (5mg/kg) decreased, the berberine concentration. However, there was no apparent change in the renal concentration of berberine in rats pretreated with corticosterone (10 or 20mg/kg). Thus, berberine is not only a substrate of OCT2 and P-glycoprotein, but is also a substrate of MATE1. Both OCT2 and MATE1 mediate the renal vectorial transport of berberine.

A multidrug and toxic compound extrusion transporter mediates berberine accumulation into vacuoles in Coptis japonica

Plants produce a large variety of alkaloids, which have diverse chemical structures and biological activities. Many of these alkaloids accumulate in vacuoles. Although some membrane proteins on tonoplasts have been identified as alkaloid uptake transporters, few have been characterized to date, and relatively little is known about the mechanisms underlying alkaloid transport and accumulation in plant cells. Berberine is a model alkaloid. Although all genes involved in berberine biosynthesis, as well as the master regulator, have been identified, the gene responsible for the final accumulation of berberine at tonoplasts has not been determined. This study showed that a multidrug and toxic compound extrusion protein 1 (CjMATE1) may act as a berberine transporter in cultured Coptis japonica cells. CjMATE1 was found to localize at tonoplasts in C. japonica cells and, in intact plants, to be expressed preferentially in rhizomes, the site of abundant berberine accumulation. Cellular transport analysis using a yeast expression system showed that CjMATE1 could transport berberine. Expression analysis showed that RNAi suppression of CjbHLH1, a master transcription factor of the berberine biosynthetic pathway, markedly reduced the expression of CjMATE1 in a manner similar to the suppression of berberine biosynthetic genes. These results strongly suggest that CjMATE1 is the transporter that mediates berberine accumulation in vacuoles.

Uptake of and Resistance to the Antibiotic Berberine by Individual Dormant, Germinating and Outgrowing Bacillus Spores as Monitored by Laser Tweezers Raman Spectroscopy.

Berberine, an alkaloid originally extracted from the plant Coptis chinensis and other herb plants, has been used as a pharmacological substance for many years. The therapeutic effect of berberine has been attributed to its interaction with nucleic acids and blocking cell division. However, levels of berberine entering individual microbial cells minimal for growth inhibition and its effects on bacterial spores have not been determined. In this work the kinetics and levels of berberine accumulation by individual dormant and germinated spores were measured by laser tweezers Raman spectroscopy and differential interference and fluorescence microscopy, and effects of berberine on spore germination and outgrowth and spore and growing cell viability were determined. The major conclusions from this work are that: (1) colony formation from B. subtilis spores was blocked ~ 99% by 25 μg/mL berberine plus 20 μg/mL INF55 (a multidrug resistance pump inhibitor); (2) 200 μg/mL berberine had no effect on B. subtilis spore germination with L-valine, but spore outgrowth was completely blocked; (3) berberine levels accumulated in single spores germinating with ≥ 25 μg/mL berberine were > 10 mg/mL; (4) fluorescence microscopy showed that germinated spores accumulated high-levels of berberine primarily in the spore core, while dormant spores accumulated very low berberine levels primarily in spore coats; and (5) during germination, uptake of berberine began at the time of commitment (T1) and reached a maximum after the completion of CaDPA release (Trelease) and spore cortex lysis (Tlysis).

Organic anion-transporting polypeptides contribute to the hepatic uptake of berberine

1. The purpose of this study was to investigate the mechanism of hepatic uptake of berberine. Berberine accumulation in hepatocytes was found to be highly dependent on active uptake, which could not be explained by liver organic cation transporter (OCT) alone.2. Our studies indicated that berberine uptake was significantly suppressed by rifampicin, cyclosporine A and glycyrrhizic acid, which act as specific inhibitors of different Oatp isoforms (Oatp1a1, Oatp1a4 and Oatp1b2) in rat hepatocytes. The combination of OCT and OATP inhibitors further reduced berberine accumulation in both rat and human hepatocytes. The uptake of berberine could be increased in human HEK293-OATP1B3 but not in OATP1B1-transfected HEK 293 cells.3. Rifampicin could reduce the berberine liver extraction ratio (ER) and double its concentration in the effluent in isolated rat livers. Further in vivo study indicated that berberine plasma exposure could be significantly increased by co-administration of the OATP inhibitor rifampicin or the substrate rosuvastatin.4. In conclusion, this study demonstrated that both OCT and OATP contribute to the accumulation of berberine in the liver. OATPs may have important roles in berberine liver disposition and potential clinically relevant drug--drug interactions.

Quantitative evaluation of berberine subcellular distribution and cellular accumulation in non-small cell lung cancer cells by UPLC–MS/MS

Berberine, an isoquinoline alkaloid, has been demonstrated to be a safe anti-cancer agent with multiple effects on mitochondria. Intracellular concentration and distribution around the targeting sites are determinants of efficacy, but subcellular distribution of berberine has not been fully elucidated yet, which relies on the sensitive and robustness assay. In this study, a sensitive and robust UPLC–MS/MS method has been developed and validated with optimized extraction solvents and detection conditions. Key factors such as the purity and integrity of isolated organelle fractions, and the effects of isolation procedures on the subcellular concentration of berberine were systemically evaluated. With the developed assay, we found that the intracellular accumulations of berberine in two gefitinib resistant NSCLC cell lines H1650 and H1975 were 2–3 folds higher than that of normal epithelial cells BEAS-2B. Moreover, significantly different subcellular distribution profiles in NSCLC cancer cells from that of BEAS-2B cells with a striking increase in content in most organelles may contribute to its selective cytotoxicity to cancer cells. Furthermore, a predominant accumulation of berberine was observed for the first time in microsomal fraction for all three cell lines. Therefore, this method could be used for quantitative evaluation of subcellular distribution and cellular accumulation of berberine and for further evaluation of the concentration–effects relationship.

Inhibition of P-glycoprotein by HIV protease inhibitors increases intracellular accumulation of berberine in murine and human macrophages.

BackgroundHIV protease inhibitor (PI)-induced inflammatory response in macrophages is a major risk factor for cardiovascular diseases. We have previously reported that berberine (BBR), a traditional herbal medicine, prevents HIV PI-induced inflammatory response through inhibiting endoplasmic reticulum (ER) stress in macrophages. We also found that HIV PIs significantly increased the intracellular concentrations of BBR in macrophages. However, the underlying mechanisms of HIV PI-induced BBR accumulation are unknown. This study examined the role of P-glycoprotein (P-gp) in HIV PI-mediated accumulation of BBR in macrophages.Methodology and Principal FindingsCultured mouse RAW264.7 macrophages, human THP-1-derived macrophages, Wild type MDCK (MDCK/WT) and human P-gp transfected (MDCK/P-gp) cells were used in this study. The intracellular concentration of BBR was determined by HPLC. The activity of P-gp was assessed by measuring digoxin and rhodamine 123 (Rh123) efflux. The interaction between P-gp and BBR or HIV PIs was predicated by Glide docking using Schrodinger program. The results indicate that P-gp contributed to the efflux of BBR in macrophages. HIV PIs significantly increased BBR concentrations in macrophages; however, BBR did not alter cellular HIV PI concentrations. Although HIV PIs did not affect P-gp expression, P-gp transport activities were significantly inhibited in HIV PI-treated macrophages. Furthermore, the molecular docking study suggests that both HIV PIs and BBR fit the binding pocket of P-gp, and HIV PIs may compete with BBR to bind P-gp.Conclusion and SignificanceHIV PIs increase the concentration of BBR by modulating the transport activity of P-gp in macrophages. Understanding the cellular mechanisms of potential drug-drug interactions is critical prior to applying successful combinational therapy in the clinic.

Antimicrobial and Efflux Pump Inhibitory Activity of Caffeoylquinic Acids from Artemisia absinthium against Gram-Positive Pathogenic Bacteria

BackgroundTraditional antibiotics are increasingly suffering from the emergence of multidrug resistance amongst pathogenic bacteria leading to a range of novel approaches to control microbial infections being investigated as potential alternative treatments. One plausible antimicrobial alternative could be the combination of conventional antimicrobial agents/antibiotics with small molecules which block multidrug efflux systems known as efflux pump inhibitors. Bioassay-driven purification and structural determination of compounds from plant sources have yielded a number of pump inhibitors which acted against gram positive bacteria.Methodology/Principal FindingsIn this study we report the identification and characterization of 4′,5′-O-dicaffeoylquinic acid (4′,5′-ODCQA) from Artemisia absinthium as a pump inhibitor with a potential of targeting efflux systems in a wide panel of Gram-positive human pathogenic bacteria. Separation and identification of phenolic compounds (chlorogenic acid, 3′,5′-ODCQA, 4′,5′-ODCQA) was based on hyphenated chromatographic techniques such as liquid chromatography with post column solid-phase extraction coupled with nuclear magnetic resonance spectroscopy and mass spectroscopy. Microbial susceptibility testing and potentiation of well know pump substrates revealed at least two active compounds; chlorogenic acid with weak antimicrobial activity and 4′,5′-ODCQA with pump inhibitory activity whereas 3′,5′-ODCQA was ineffective. These intitial findings were further validated with checkerboard, berberine accumulation efflux assays using efflux-related phenotypes and clinical isolates as well as molecular modeling methodology.Conclusions/SignificanceThese techniques facilitated the direct analysis of the active components from plant extracts, as well as dramatically reduced the time needed to analyze the compounds, without the need for prior isolation. The calculated energetics of the docking poses supported the biological information for the inhibitory capabilities of 4′,5′-ODCQA and furthermore contributed evidence that CQAs show a preferential binding to Major Facilitator Super family efflux systems, a key multidrug resistance determinant in gram-positive bacteria.

Different concentrations of berberine result in distinct cellular localization patterns and cell cycle effects in a melanoma cell line

Natural products represent a rich reservoir of potential small molecule inhibitors exhibiting antiproliferative and tumoricidal properties. An example is the isoquinoline alkaloid berberine, which is found in plants such as goldenseal (Hydrastis canadensis). Studies have shown that berberine is able to trigger apoptosis in different malignant cell lines, and can also lead to cell cycle arrest at sub-apoptotic doses. A particularly interesting feature of berberine is the fact that it is a fluorescent molecule, and its uptake and distribution in cells can be studied by flow cytometry and epifluorescence microscopy. To test the relationships between berberine uptake, distribution and cellular effect in melanoma cells, K1735-M2 mouse and WM793 human melanoma cells were treated with different concentrations of berberine, and alterations in cell cycle progression, DNA synthesis, cell proliferation, and cell death measured. Cell proliferation was measured by sulforhodamine B assays, cell death by flow cytometry, berberine uptake and distribution by laser scanning confocal microscopy and flow cytometry, cell cycle progression by flow cytometry, and DNA synthesis, M-phase, and mitochondrial effects by immunolabeling and epifluorescence microscopy methods. In these melanoma cell lines, berberine at low doses (12.5-50 muM) is concentrated in mitochondria and promotes G1 arrest. In contrast, higher doses (over 50 muM) result in cytoplasmic and nuclear berberine accumulation, and G2 arrest. DNA synthesis is not markedly affected by low doses of berberine, but 100 muM is strongly inhibitory. Even at 100 muM, berberine inhibits cell growth with relatively little induction of apoptosis. Berberine displays multiphasic effects in these malignant cell lines, which are correlated with the concentration and intracellular distribution of this alkaloid. These results help explain some of the conflicting information in the literature regarding the effects of berberine, and suggest that its use in clinical development may be more as a cytostatic agent than a cytotoxic compound.

Human MDR1 and MRP1 Recognize Berberine as Their Transport Substrate

To examine whether human ATP-binding cassette (ABC) transporters play a role in the detoxification of plant alkaloid berberine, we investigated berberine transport using multidrug resistance protein1 (MDR1) and multidrug resistance-associated protein1 (MRP1). Cells expressing MDR1 or MRP1 accumulated less berberine. Berberine accumulation depended on the cellular ATP level, and was reversed by typical inhibitors of MDR1, suggesting that human MDR1 and MRP1 directly efflux berberine as their substrate.

Molecular cloning and characterization of norcoclaurine synthase, an enzyme catalyzing the first committed step in benzylisoquinoline alkaloid biosynthesis.

(S)-Norcoclaurine synthase (NCS) (EC 4.2.1.78) catalyzes the condensation of 3,4-dihydroxyphenylethylamine (dopamine) and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in the biosynthesis of benzylisoquinoline alkaloids such as morphine, sanguinarine, and berberine, in plants. A molecular clone encoding NCS was isolated from a meadow rue (Thalictrum flavum ssp. glaucum) cell suspension culture cDNA library. Heterologous expression of the NCS cDNA, truncated to remove a putative signal peptide, produced a recombinant protein with NCS activity. Recombinant NCS showed sigmoidal saturation kinetics for dopamine (Hill coefficient=1.98), hyperbolic saturation kinetics for 4-HPAA (Km of 700 microm), and pH and temperature optima of 7.0 and 40 degrees C, respectively, all similar to the purified, plant-derived enzyme. NCS exhibits 28-38% identity, and putative structural homology, with the Bet v 1 allergen and pathogenesis-related (PR)10 protein families. NCS also displays 35% identity with the enzyme (HYP1) responsible for hypericin biosynthesis in St John's wort (Hypericum perforatum). The novel catalytic functions of NCS and HYP1 define a new class of plant secondary metabolic enzymes within the Bet v 1 and PR10 protein families. Weaker homology was also detected between NCS and proteins identified in the latex of Papaver somniferum (opium poppy), and in Arabidopsis thaliana. A family of three to five NCS genes is abundantly expressed in the rhizome, followed by petioles and roots of T. flavum. NCS transcripts were localized to the immature endodermis and pericycle in roots, and the protoderm of leaf primordia in rhizomes; thus, the sites of NCS gene expression and berberine accumulation are temporally and spatially separated in roots and rhizomes respectively.

Protoberberine Alkaloids From Callus and Cell Suspension Cultures of Tinospora cordifolia

Callus and cell suspension cultures were established from the stem explants of the plant Tinospora cordifolia. Accumulation of berberine and jatrorrhizine (protoberberine alkaloids) was observed in both callus and cell suspension cultures. The alkaloids were also exudated into the surrounding nutrient medium by non-immobilized and immobilized cultures. The root extracts of T. cordifolia showed higher levels of jatrorrhizine compared to the levels of berberine. An HPLC method was standardised for the separation, identification and quantification of these alkaloids.

Transfer of cationic antibacterial agents berberine, palmatine, and benzalkonium through bimolecular planar phospholipid film and Staphylococcus aureus membrane.

Some organic cations are known to be electrophoretically imported into bacterial cells and actively extruded from these cells by multidrug resistance (MDR) pumps. We have studied penetration of plant antimicrobial agents berberine and palmatine and synthetic antiseptic benzalkonium chloride through black planar phospholipid membrane (BLM) and membrane of Staphylococcus aureus cells. Gradients of these cations across BLM generated an electric potential difference. Penetrating anion tetraphenyl borate and phloretin (a plant substance decreasing membrane dipole potential) stimulated this effect. Under optimal conditions, the magnitude of the electric potential was close to theoretical, that is, 60 mV/10-fold cation gradient. Berberine accumulated in S. aureus cells as shown by direct measurement of berberine with a berberine-sensitive electrode. The berberine accumulation was prevented by protonophore CCCP and was stimulated by mutation in the MDR pump NorA. It is concluded that the plant alkaloids and benzalkonium are penetrating cations and substrates of an MDR pump.