Coptis Japonica Research Articles

Effects of baicalein, berberine, curcumin and hesperidin on mucin release from airway goblet cells.

Baicalein, berberine, curcumin and hesperidin are the major components derived from Scutellaria baicalensis, Coptis japonica, Curcuma longa and Poncirus trifoliata, respectively. These plants have been used for the treatment of diverse chronic inflammatory diseases including respiratory disease in oriental medicine and their respective major components were reported to have various biological effects including anti-inflammatory activity. In the present study, we investigated whether these four natural products affect mucin release from airway goblet cells and compared the possible activities of these agents with the inhibitory action on mucin release by PLL and the stimulatory action by ATP. Confluent primary hamster tracheal surface epithelial (HTSE) cells were metabolically radiolabeled using 3H-glucosamine for 24 h and chased for 30 min in the presence of varying concentrations of each agent to assess the effects on 3H-mucin release. The results were as follows: (i) baicalein did not affect mucin release significantly; (ii) berberine, curcumin and hesperidin increased mucin release at the highest concentration (10 - 4 M); (iii) PLL inhibited and ATP increased mucin release. We conclude that berberine, curcumin and hesperidin can increase mucin release by directly acting on airway mucin-secreting cells and suggest that these agents be further studied for possible use as mild expectorants during the treatment of chronic airway diseases. Abbreviations. PLL:poly- L-lysine ATP:adenosine triphosphate HTSE:hamster tracheal surface epithelial DMSO:dimethylsulfoxide IL-12:interleukin-12 PBS:phosphate-buffered saline

Application of vanadate-induced nucleotide trapping to plant cells for detection of ABC proteins.

The vanadate-induced nucleotide trapping technique, which has been conventionally used to characterize mammalian ATP-binding cassette (ABC) proteins, was applied to berberine-producing plant cell cultures, Thalictrum minus and Coptis japonica. One membrane protein at ca. 180 kDa was photoaffinity-labeled with 8-azido-[alpha-(32)P]ATP in the T. minus cells in the presence of vanadate, which was specifically induced by the addition of benzyladenine in a similar manner as the induction of berberine biosynthesis in these cell cultures, whereas three bands were observed in the C. japonica cells in the size region between 120 and 150 kDa corresponding to full-sized ABC protein. The benzyladenine-induced band in T. minus showed properties similar to those of human MDR1, including the recognition of berberine, which suggests that the ABC protein detected in T. minus takes this endogenous alkaloid as a putative substrate for transport. This is the first application of this technique to plant cells.

Antimalarial Activity of Herbal Extracts Used in Traditional Medicine in Korea

Aqueous extracts of 6 traditional Korean medicines used to treat malaria were tested in vitro for their antimalarial activity against Plasmodium falciparum. The EC50 values for the herbal extracts were in the range 1.4-8.1 microg/ml. Significant antimalarial activity was observed with Coptis japonica (EC50=1.4 microg/ml), but it demonstrated no selective toxicity (selectivity=1). In contrast, Kalopanax pictus showed antimalarial activity (EC50=4.6 microg/ml) and higher selective toxicity (>4). This indicated that K. pictus may be potent for a new antimalarial agent.

Molecular cloning of columbamine O-methyltransferase from cultured Coptis japonica cells.

To identify all of the O-methyltransferase genes involved in isoquinoline alkaloid biosynthesis in Coptis japonica cells, we sequenced 1014 cDNA clones isolated from high-alkaloid-producing cultured cells of C. japonica. Among them, we found all three reported O-methyltransferases and an O-methyltransferase-like cDNA clone (CJEST64). This cDNA was quite similar to S-adenosyl-l-methionine:coclaurine 6-O-methyltransferase and S-adenosyl-l-methionine:isoflavone 7-O-methyltransferase. As S-adenosyl-l-methionine:columbamine O-methyltransferase, which catalyzes the conversion of columbamine to palmatine, is one of the remaining unelucidated components in isoquinoline alkaloid biosynthesis in C. japonica, we heterologously expressed the protein in Escherichia coli and examined the activity of columbamine O-methyltransferase. The recombinant protein clearly showed O-methylation activity using columbamine, as well as (S)-tetrahydrocolumbamine, (S)-, (R,S)-scoulerine and (R,S)-2,3,9,10-tetrahydroxyprotoberberine as substrates. This result clearly indicated that EST analysis was useful for isolating the candidate gene in a relatively well-characterized biosynthetic pathway. The relationship between the structure and substrate recognition of the O-methyltransferases involved in isoquinoline alkaloid biosynthesis, and a reconsideration of the biosynthetic pathway to palmatine are discussed.

Rat lens aldose reductase inhibitory activities of Coptis japonica root-derived isoquinoline alkaloids.

The inhibitory activity of Coptis japonica root-derived materials was evaluated against lens aldose reductase isolated from male Sprague-Dawley rats and compared to that of three commercially available isoquinoline alkaloids (berberine sulfate, berberine iodide, and palmatine chloride), as well as quercitrin as aldose reductase inhibitor. The biologically active constituents of C. japonica extract were characterized as the isoquinoline alkaloids, berberine chloride and palmatine iodide, by spectral analysis. The inhibitory effects varied with both chemical and concentration used. The IC(50) values of berberine chloride and palmatine iodide are 13.98 and 13.45 nM, respectively. Among three berberines and two palmatines, the inhibitory activity was much greater for the choridated and sulfated analogues than for those with iodide. Quercitrin was a much more potent inhibitor than berberines and palmatines. Nonetheless, berberines and palmatines may be useful as lead compounds and new agents for aldose reductase inhibition.

Characterization of berberine transport into Coptis japonica cells and the involvement of ABC protein.

Cultured Coptis japonica cells are able to take up berberine, a benzylisoquinoline alkaloid, from the medium and transport it exclusively into the vacuoles. Uptake activity depends on the growth phase of the cultured cells whereas the culture medium had no effect on uptake. Treatment with several inhibitors suggested that berberine uptake depended on the ATP level. Some inhibitors of P-glycoprotein, an ABC transporter involved in multiple drug resistance in cancer cells, strongly inhibited berberine uptake, whereas a specific inhibitor for glutathione biosynthesis and vacuolar ATPase, bafilomycin A1, had little effect. Vanadate-induced ATP trap experiments to detect ABC proteins expressed in C. japonica cells showed that three membrane proteins of between 120 and 150 kDa were photolabelled with 8-azido-[alpha-32P] ATP. Two revealed the same photoaffinity-labelling pattern as P-glycoprotein, and the interaction of these proteins with berberine was also demonstrated. These results suggest that ABC proteins of the MDR-type are involved in the uptake of berberine from the medium.

CULTIVATION OF MEDICINAL AND AROMATIC PLANTS IN INDIA - A COMMERCIAL APPROACH

CULTIVATION OF MEDICINAL AND AROMATIC PLANTS IN INDIA - A COMMERCIAL APPROACH

Molecular Cloning and Characterization of CoclaurineN-Methyltransferase from Cultured Cells of Coptis japonica

S-adenosyl-L-methionine:coclaurine N-methyltransferase (CNMT) converts coclaurine to N-methylcoclaurine in isoquinoline alkaloid biosynthesis. The N-terminal amino acid sequence of Coptis CNMT was used to amplify the corresponding cDNA fragment and later to isolate full-length cDNA using 5'- and 3'-rapid amplification of cDNA ends (RACE). The nucleotide sequence and predicted amino acid sequence showed that the cDNA encoded 358 amino acids, which contained a putative S-adenosyl-L-methionine binding domain and showed relatively high homology to tomato phosphoethanolamine-N-methyltransferase. A recombinant protein was expressed in Escherichia coli, and its CNMT activity was confirmed. Recombinant CNMT was purified to homogeneity, and enzymological characterization confirmed that Coptis CNMT has quite broad substrate specificity, i.e. not only for 6-O-methylnorlaudanosoline and norreticuline but also for 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline. The evolution of N-methyltransferases in secondary metabolism is discussed based on sequence similarity.

A novel Coptis japonica multidrug-resistant protein preferentially expressed in the alkaloid-accumulating rhizome.

A full-length cDNA, Cjmdr1, which belongs to the multidrug-resistant (mdr) gene family, was isolated by nested RT-PCR from alkaloid-producing cultured cells of Coptis japonica. The cDNA is 4192 nucleotides long and has an ORF of 1289 amino acids. Northern analysis of the intact plant showed a clear preference in its expression in the rhizome, where alkaloids are highly accumulated compared to other organs.

Purification and characterization of coclaurine N-methyltransferase from cultured Coptis japonica cells

S-Adenosyl- l-methionine (SAM): coclaurine N-methyltransferase (CNMT), which catalyzes the transfer of a methyl group from S-adenosyl- l-methionine to the amino group of the tetrahydrobenzylisoquinoline alkaloid coclaurine, was purified 340-fold from Coptis japonica cells in 1% yield to give an almost homogeneous protein. The purified enzyme, which occurred as a homotetramer with a native M r of 160 kDa (gel-filtration chromatography) and a subunit M r of 45 kDa (SDS-polyacrylamide gel electrophoresis), had an optimum pH of 7.0 and a pI of 4.2. Whereas ( R)-coclaurine was the best substrate for enzyme activity, Coptis CNMT had broad substrate specificity and no stereospecificity; CNMT methylated norlaudanosoline, 6,7-dimethoxyl-1,2,3,4-tetrahydroisoquinoline and 1-methyl-6,7-dihydroxy-1,2,3,4,-tetrahydroisoquinoline. The enzyme did not require any metal ion. p-Chloromercuribenzoate and iodoacetamide did not inhibit CNMT activity, but the addition of Co 2+, Cu 2+ or Mn 2+ at 5 mM severely inhibited such activity by 75, 47 and 57%, respectively. The substrate-saturation kinetics of CNMT for norreticuline and SAM were of the typical Michaelis–Menten-type with respective K m values of 0.38 and 0.65 mM.

Lignans from the rhizomes of Coptis japonica differentially act as anti-inflammatory principles.

Coptis japonica Makino (Ranunculaceae) is known to possess several biological activities such as anti-inflammatory effects. In this study, five lignans, isolariciresinol (1), lariciresinol glycoside (2), pinoresinol (3), pinoresinol glycoside (4) and syringaresinol glycoside (5), isolated from the rhizomes of C. japonica were tested to evaluate their in vitro anti-inflammatory effects. Pinoresinol and isolariciresinol showed higher inhibitory effects on TNF-alpha production, whereas syringaresinol glycoside strongly suppressed lymphocyte proliferation. The results indicate that the lignans may differentially modulate inflammatory cell responses, suggesting that these compounds may participate in anti-inflammatory processes by C. japonica.

Inaugural Article: Metabolic engineering of plant alkaloid biosynthesis

Plant alkaloids, one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the biosynthetic pathways for scopolamine, nicotine, and berberine have been cloned, making the metabolic engineering of these alkaloids possible. Expression of two branching-point enzymes was engineered: putrescine N-methyltransferase (PMT) in transgenic plants of Atropa belladonna and Nicotiana sylvestris and (S)-scoulerine 9-O-methyltransferase (SMT) in cultured cells of Coptis japonica and Eschscholzia californica. Overexpression of PMT increased the nicotine content in N. sylvestris, whereas suppression of endogenous PMT activity severely decreased the nicotine content and induced abnormal morphologies. Ectopic expression of SMT caused the accumulation of benzylisoquinoline alkaloids in E. californica. The prospects and limitations of engineering plant alkaloid metabolism are discussed.

In vitro antiinflammatory effects of neolignan woorenosides from the rhizomes of Coptis japonica.

Five dihydrobenzofuran neolignans, woorenosides I (1), II (2), III (3), IV (4), and V (5), isolated from Coptis japonica (Ranunculaceae), suppressed tumor necrosis factor (TNF)-alpha and nitric oxide (NuOmicron) production, as well as lymphocyte proliferation triggered by inflammatory signals such as various mitogens, in a dose-dependent manner. The results indicate that the woorenosides strongly inhibit the mitogenic response by activated macrophage and lymphocytes and suggest that these compounds may participate in regulating inflammatory processes.

Molecular Characterization of the S-Adenosyl-l-methionine:3′-Hydroxy-N-methylcoclaurine 4′-O-Methyltransferase Involved in Isoquinoline Alkaloid Biosynthesis in Coptis japonica

S-adenosyl-L-methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'-OMT) catalyzes the conversion of 3'-hydroxy-N-methylcoclaurine to reticuline, an important intermediate in synthesizing isoquinoline alkaloids. In an earlier step in the biosynthetic pathway to reticuline, another O-methyltransferase, S-adenosyl-L-methionine:norcoclaurine 6-O-methyltransferase (6-OMT), catalyzes methylation of the 6-hydroxyl group of norcoclaurine. We isolated two kinds of cDNA clones that correspond to the internal amino acid sequences of a 6-OMT/4'-OMT preparation from cultured Coptis japonica cells. Heterologously expressed proteins had 6-OMT or 4'-OMT activities, indicative that each cDNA encodes a different enzyme. 4'-OMT was purified using recombinant protein, and its enzymological properties were characterized. It had enzymological characteristics similar to those of 6-OMT; the active enzyme was the dimer of the subunit, no divalent cations were required for activity, and there was inhibition by Fe(2+), Cu(2+), Co(2+), Zn(2+), or Ni(2+), but none by the SH reagent. 4'-OMT clearly had different substrate specificity. It methylated (R,S)-6-O-methylnorlaudanosoline, as well as (R, S)-laudanosoline and (R,S)-norlaudanosoline. Laudanosoline, an N-methylated substrate, was a much better substrate for 4'-OMT than norlaudanosoline. 6-OMT methylated norlaudanosoline and laudanosoline equally. Further characterization of the substrate saturation and product inhibition kinetics indicated that 4'-OMT follows an ordered Bi Bi mechanism, whereas 6-OMT follows a Ping-Pong Bi Bi mechanism. The molecular evolution of these two related O-methyltransferases is discussed.

Cordycepin: selective growth inhibitor derived from liquid culture of Cordyceps militaris against Clostridium spp.

The growth responses of nine human intestinal bacteria to liquid culture of Cordyceps militaris Link. Pt. (Ascomycotina: Clavicipitaceae) collected from a pupa of Bombyx mori L. (Lepidoptera: Bombycidae) were examined using spectrophotometric and impregnated paper disk methods and compared to those of tetracycline and chloramphenicol, as well as those of Coptis japonica root-derived berberine chloride. The biologically active constituent of the cultures was characterized as cordycepin (3'-deoxyadenosine) by spectroscopic analysis. This compound revealed potent growth-inhibiting activity toward Clostridium paraputrificum and Clostridium perfringens at 10 microgram/disk without adverse effects on the growth of Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium adolescentis, Lactobacillus acidophilus, and Lactobacillus casei, whereas tetracycline and chloramphenicol inhibited the growth of these lactic acid-producing bacteria, clostridia and Escherichia coli. However, C. militaris-derived materials revealed no growth stimulation on the bifidobacteria and lactobacilli. These results may be an indication of at least one of the pharmacological actions of C. militaris. As a naturally occurring antibacterial agent, cordycepin could be useful as a new preventive agent against various diseases caused by clostridia.

Antifeeding activity of isoquinoline alkaloids identified in Coptis japonica roots against Hyphantria cunea (Lepidoptera: Arctiidae) and Agelastica coerulea (Coleoptera: Galerucinae).

The anti-feeding activity of 3 isoquinoline alkaloids identified from roots of Coptis japonica Makino toward 4th-instar larvae of Hyphantria cunea Drury and adults of Agelastica coerulea Baly was examined using the leaf-dipping bioassay. The biologically active constituents of the Coptis roots were characterized as the isoquinoline alkaloids berberine, palmatine and coptisine by spectroscopic analysis. In a test with H. cunea larvae, the anti-feeding activity was much more pronounced in an application of a mixture of palmatine iodide and berberine chloride (1:1, wt:wt) at 250 ppm (82.3%) and 500 ppm (100%), compared with palmatine iodide (76.0%) and berberine chloride (75.4%) alone at 500 ppm. These results indicate a synergistic effect. With A. courulea adults, berberine chloride showed 57.5 and 91.1% anti-feeding activity at 125 and 250 ppm, respectively; whereas, weak activity was obtained in application of 500 ppm of palmatine iodide (41.4%) and coptisine chloride (52.4%) alone. The Coptis root-derived compounds merit further study as potential insect-control agents.

Antiphotooxidative activity of protoberberines derived from Coptis japonica makino in the chlorophyll-sensitized photooxidation of oil.

Antiphotooxidative components were isolated from the methanolic extract of Coptis japonica Makino by liquid-liquid partitioning fractionation, subsequent column chromatography on Sephadex LH-20 and silica gel, and preparative silica gel TLC. The isolated compounds were identified as coptisine, jatrorrizhine, berberine, and magnoflorine by a combination of spectroscopic studies using UV-visible, IR, mass-spectrometry, and NMR. Coptisine, jatrorrizhine, and berberine isolated from Coptis japonica Makino showed strong antiphotooxidative activity in the chlorophyll-sensitized photooxidation of linoleic acid. However, these compounds did not show either inhibitory activity against lipid peroxidation in rat liver microsomes nor DPPH radical scavenging activity, indicating that their antiphotooxidative activity was not due to the radical chain reaction breaking ability but due to singlet oxygen quenching activity. Commercially available authentic protoberberines (berberine chloride and palmatine chloride) also showed strong antioxidative activity in the chlorophyll-sensitized photooxidation of linoleic acid. The antiphotooxidative activities of the berberine chloride and palmatine chloride were significantly higher than that of ascorbyl palmitate in the chlorophyll-sensitized photooxidation of linoleic acid. These results clearly showed for the first time the antiphotooxidative properties of protoberberines in chlorophyll-sensitized photooxidation of oil.

Methanolic extract of Coptis japonica Makino reduces photosensitized oxidation of oils

Antioxidative activities of methanolic extracts of 47 plants on the methylene blue-sensitized or chlorophyll-sensitized photooxidations of linoleic acid were studied to select the plant species which possess the strong antioxidative activity in sensitized photooxidation of oil. The methanol extract of Coptis japonica Makino showed the strongest antioxidative activity in both methylene blue-sensitized and chlorophyll-sensitized photooxidations of linoleic acid. The study on the extracting solvent effects indicated that the antiphotooxidative components in the Coptis japonica Makino possess strong polar property, and are easily extracted with highly polar solvent (methanol). The methanol extract of Coptis japonica Makino was fractionated into three fractions (ethyl ether fraction, ethyl acetate fraction and butanol fraction) by liquid–liquid partitioning fractionation. Among the tested fractions, the butanol fraction showed strongest antioxidative activity in both chlorophyll-sensitized and methylene blue-sensitized photooxidation of linoleic acid. The butanol fraction also significantly inhibited the photooxidation of model food emulsion (50% soybean oil emulsion). The treatment with 0.3% (w/w) butanol fraction resulted in 64.9% inhibition of photooxidation of the model food emulsion during 60 h fluorescent light illumination.

Growth-inhibiting effects of Coptis japonica root-derived isoquinoline alkaloids on human intestinal bacteria.

The growth-inhibiting activity of Coptis japonica (Makino) root-derived materials toward eight human intestinal bacteria was examined using an impregnated paper disk method and compared to that of four commercially available isoquinoline alkaloids [berberine sulfate (BS), berberine iodide (BI), palmatine chloride (PC), and palmatine sulfate(PS)], as well as that of Thea sinensis leaf-derived epigallocatechin gallate (EGCG). The biologically active constituents of the Coptis extract were characterized as the isoquinoline alkaloids berberine chloride (BC), palmatine iodide (PI), and coptisine chloride (CC) by spectral analysis. The growth responses varied with both chemical and bacterial strain used. In a test using 500 microg/disk, BC and PI produced a clear inhibitory effect against Bifidobacterium longum, Bifidobacterium bifidum, Clostridium perfringens, and Clostridium paraputrificum, whereas weak or no inhibition was observed in Bifidobacterium adolescentis, Lactobacillus acidophilus, Lactobacillus casei, and Escherichia coli. At 1000 microg/ disk, CC revealed weak or no growth inhibition toward all test bacteria, whereas EGCG exhibited weak growth inhibition against only C. perfringens and C. paraputrificum. Among various isoquinoline alkaloids, BC exhibited more potent inhibitory activity toward C. perfringens than BI and BS, whereas the inhibitory effect was more pronounced in PI compared to PC and PS. The Coptis root-derived materials did not promote growth of B. longum and C. perfringens.

Inhibitory effect of lignans from the rhizomes of Coptis japonica var. dissecta on tumor necrosis factor-alpha production in lipopolysaccharide-stimulated RAW264.7 cells.

The inhibitory effect of 10 lignan constituents isolated from the rhizomes of Coptis japonica var. dissecta on tumor necrosis factor (TNF)-alpha production in lipopolysaccharide (LPS)-stimulated macrophage cell line (RAW264.7 cells) has been studied. Among them, pinoresinol, woorenoside-V and lariciresinol glycoside showed significant inhibitory activities in the range from 37% to 55% at the concentration of 25 micrograms/ml. The results are first report that the lignans isolated from Coptis japonica inhibit TNF-alpha production, and suggest that the lignan components may partly participate in antiinflammatory and antiallergic effect of Coptis japonica through the inhibition of TNF-alpha production.