€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ *****JOURNAL OF BIOLOGICAL CHEMISTRY***** Stadler R Zenk MH The purification and characterization of a unique cytochrome P-450 enzyme from Berberis stolonifera plant cell cultures. In: J Biol Chem (1993 Jan 15) 268(2):823-31 A new cytochrome P-450 enzyme, isolated from Berberis stolonifera plant cell suspension cultures, has been purified to electrophoretic homogeneity. The purified hemoprotein migrated as a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis with a minimal M(r) = 46,000. The enzyme could be purified to a high specific content of P-450 (18.2 nmol/mg protein) after fast protein liquid chromatofocusing, displaying an isoelectric point of 6.05. Spectral analysis of the homogeneous enzyme showed that it is predominantly low spin in the oxidized state, with a slight red- shifted ferrous carbonyl complex that exhibits a maximum at 452 nm. The purified cytochrome P-450, successfully reconstituted with NADPH- cytochrome P-450 reductase, displayed a maximal turnover rate of 50 nmol of substrate/nmol of P-450/min. In the purified and reconstituted form, the enzyme catalyzed the oxidation of three different chiral benzyltetrahydroisoquinoline substrates, namely (S)- coclaurine, (R)-N-methylcoclaurine, and (S)-N-methylcoclaurine, leading to the formation of three distinct dimeric products, (R,S)- berbamunine, (R,S)-2'-norberbamunine, and (R,R)-guattegaumerine, that are also present in the plant cell cultures in vivo. This is the first report of a P-450 enzyme that mediates regio- and stereoselective intermolecular oxidative phenol coupling to furnish natural dimeric compounds. In this catalytic cycle cytochrome P-450 functions as an oxidant in a bisubstrate reaction without transfer of the activated oxygen atom to either of the two chiral substrates. €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ *****ARCHIVES OF MEDICAL RESEARCH***** Morales MA Gonzalez E Torres R Martinez JL Cardiodepressor effects of 7-O-demethylisothalicberine, bisbenzylisoquinoline alkaloid isolated from Berberis chilensis. In: Arch Med Res (1993 Summer) 24(2):177-81 7-O-demethylisothalicberine (7-O-DI), an alkaloid isolated from Berberis chilensis Gillies ex Hook, induces a negative inotropism and chronotropism on guinea pig isolated atria (IC50 = 1.5 x 10(-4) and 1.8 x 10(-4) M, respectively). As these effects were not antagonized by propranolol, 7-O-DI inhibition of isoproterenol-induced atrial activation seems to be attained through a mechanism distinct of beta- receptor blockade. A probable cholinergic mechanism of action was also ruled out. 7-O-DI atrial effects were completely reversed by a 50% increase of calcium concentration. The spontaneous frequency of rat sinus node was also deeply modified by 7-O-DI, in accordance with previous results obtained in frog cardiac pacemaker cells. It is concluded that 7-O-DI should perform its pharmacological action through calcium entry blockade. These results open expectations for future research about probable therapeutic use for 7-O-DI. €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ *****ARCHIVES ROUMAINES DE PATHOLOGIE EXPERIMENTALES ET DE MICROBIOLOGIE***** Gancevici GG Bioeffects of the plant Mahonia sempervirens. In: Arch Roum Pathol Exp Microbiol (1990 Apr-Jun) 49(2):183-90 Water extracts of mature fruits of Mahonia sempervirens possess four biological properties: hemagglutinin, hemolysin, complement antagonism and antistreptoccocal activity. Partial characterization of the crude extract was carried out by barbital-mediated spectrophotometry, polyacrylamide gel electrophoresis, thin layer chromatography and interaction chromatography. Fracctionation of the Mahonia extract by ionic exchange static adsorption resulted in the quantitative separation of the fruit red pigment with recovery of the entire anticomplementary activity and indicated that the complement inhibitor was different from the aggregating and lytic properties. €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ *****CHUNG-KUO CHUNG YAO TSA CHIH CHINA JOURNAL OF CHINESE MATERIA MEDICA***** Jiang YL Wang XL Xue SG [Extraction of berberine hydrochloride from Mahonia bealei (Fort.) Carr] In: Chung Kuo Chung Yao Tsa Chih (1993 Jun) 18(6):347-8, 382 (Published in Chinese) The article reports the processing methods for extracting berberine hydrochloride from Mahonia bealei by means of regressive test. The result shows that A2B3C1D2E3F2 is the best way, namely, extractive solvent--0.5% sulfuric acid, salting out--24% salt, soaking temperature--25 degrees C, soaking time--48 hours, pH = 1.5, precipitation time--24 hours. The recovery is 1.45%-1.47%. €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ *****EUROPEAN JOURNAL OF BIOCHEMISTRY***** Amann M Nagakura N Zenk MH Purification and properties of (S)-tetrahydroprotoberberine oxidase from suspension-cultured cells of Berberis wilsoniae. In: Eur J Biochem (1988 Jul 15) 175(1):17-25 A novel oxidase, catalyzing in the presence of oxygen the removal of four hydrogen atoms from a number of tetrahydroprotoberberines with simultaneous production of 1 mol H2O2 and H2O each, has been discovered and purified to homogeneity from Berberis wilsoniae cell cultures. This enzyme, (S)-tetrahydroprotoberberine oxidase, exhibited strict specificity for the (S)-enantiomer of tetrahydroprotoberberines and 1-benzylisoquinoline alkaloids, a pH optimum at 8.9, a molecular mass of 105 kDa and consisted of two subunits each of 53 kDa and covalently bound flavin. The Km values for (S)-scoulerine and (S)-norreticuline were 25 microM and 150 microM respectively. Concentration of the end-products, either protoberberines or H2O2, greater than 0.5 mM caused severe enzyme inhibition. This catalyst was responsible for the conversion of (S)- tetrahydrocolumbamine to the key intermediate, columbamine, in the metabolic pathway leading to berberine, jatrorrhizine and palmatine. €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ *****GENERAL PHARMACOLOGY***** Morales MA Gallardo LR Martinez JL Puebla RS Hernandez DA Effects of 7-O-demethylisothalicberine, a bisbenzylisoquinoline alkaloid of Berberis chilensis, on electrical activity of frog cardiac pacemaker cells. In: Gen Pharmacol (1989) 20(5):621-5 1. In spontaneously beating preparations of sinus venosus of the chilean frog Caudiverbera caudiverbera, the electrophysiological effects of 7-O-demethylisothalicberine (7-O-DI) on transitional pacemaker cells were investigated. 2. 7-O-DI in concentration 1 x 10(- 4) M blocked the action potential of transitional cells. This blockade was preceded by subthreshold oscillations and depolarization of membrane potential. 3. Lower concentration of the drug to induce complete blockade (5 x 10(-5) M), allowed to observe a great depression of bioelectric cell characteristics in transitional fibres. 4. 7-O-DI induced blockade of transitional cells action potential was preceded by the appearance of a notch in their upstroke and the persistence of a fast depolarizing activity that remained unblocked. This 7-O-DI resistant fast component of the upstroke was blocked by tetrodotoxin. 5. Transitional cells completely blocked by 7-O-DI were depolarized to about 40 mV. 6. The results indicate a close similarity between 7-O-DI and verapamil effects on action potential configuration. €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ Sotnikova R Kost'alova D Vaverkova S Effect of bisbenzylisoquinoline alkaloids from Mahonia aquifolium on the isolated rat aorta. In: Gen Pharmacol (1994 Nov) 25(7):1405-10 1. The extract from roots of Mahonia aquifolium as well as berbamine and oxyacanthine relaxed K(+)-precontracted rat aortal rings with IC50 values of about 20 mumol/l. 2. Relaxation was not inhibited by denudation of the endothelium or by premedication of the aortas with indomethacin, methylene blue or propranolol. 3. Berbamine and oxyacanthine inhibited contractions induced by noradrenaline, phenylephrine and serotonin in a non-competitive manner. Further, they competitively blocked calcium-induced contraction in Ca(2+)-free depolarizing PSS. 4. The blockade of calcium entry appears to be the main mechanism of the dilatory effect of the drugs tested, but their interaction with alpha-adrenoceptors could not be excluded. €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ *****PLANTA MEDICA***** Muller K Ziereis K Gawlik I The antipsoriatic Mahonia aquifolium and its active constituents; II. Antiproliferative activity against cell growth of human keratinocytes [letter] In: Planta Med (1995 Feb) 61(1):74-5 The extract of the bark of Mahonia aquifolium is an inhibitor of keratinocyte growth with an IC50 of 35 microM. Of its main alkaloids tested, berberine inhibited cell growth to the same extent as did the Mahonia extract, while the benzylisoquinoline alkaloids berbamine and oxyacanthine were more potent inhibitors by a factor of three. €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ Muller K Ziereis K The antipsoriatic Mahonia aquifolium and its active constituents; I. Pro- and antioxidant properties and inhibition of 5-lipoxygenase. In: Planta Med (1994 Oct) 60(5):421-4 The effects of the extract of the bark of Mahonia aquifolium and its main constituents (berberine, berbamine, oxyacanthine) on 5- lipoxygenase, lipid peroxidation in phospholipid liposomes induced by 2,2'-azo-(bis-2-amidinopropane), deoxyribose degradation, and their reactivities against the free radical 2,2-diphenyl-1-picrylhydrazyl have been studied. The extract of M. aquifolium inhibits 5-LO with an IC50 value of 50 microM, whereas no appreciable effects were observed by its constituent alkaloids. Reactivity against DPPH increased in the following order: berberine < M. aquifolium < oxyacanthine, berbamine. Pro-oxidant effects by M. aquifolium or its constituents can be excluded, since deoxyribose degradation was not influenced as determined by the release of malondialdehyde. The most prominent feature of M. aquifolium is its efficacy in inhibition of lipid peroxidation (IC50 = 5 microM) which was not mediated by the alkaloids berberine, berbamine, and oxyacanthine. €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ ****PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES** Kraus PF Kutchan TM Molecular cloning and heterologous expression of a cDNA encoding berbamunine synthase, a C--O phenol-coupling cytochrome P450 from the higher plant Berberis stolonifera. In: Proc Natl Acad Sci U S A (1995 Mar 14) 92(6):2071-5 A cDNA encoding a cytochrome P450-dependent oxidase, berbamunine synthase (EC 1.1.3.34; CYP80), from cell suspension cultures of the higher plant Berberis stolonifera Koehne and Wolf (barberry) has been isolated and heterologously expressed in functional form in insect cell culture using a baculovirus-based expression system. This cytochrome P450-dependent enzyme is unusual in that it catalyzes the regio- and stereoselective formation of a C--O phenol couple in bisbenzylisoquinoline alkaloid biosynthesis without concomitant incorporation of activated oxygen into the product. Consistent with the function of an oxidase rather than a monooxygenase, an essential glycine residue in the distal helix, which forms the oxygen-binding pocket in the well-studied bacterial enzyme P-450cam, is replaced by proline at the equivalent position in berbamunine synthase. This oxidase was accumulated in an active form in insect cell microsomes and accepted electrons from the endogenous NADPH-cytochrome P450 reductase. The heterologously expressed enzyme oxidatively couples either two molecules of (R)-N-methylcoclaurine to form the (R,R) dimer guattegaumerine or one molecule each of (R)- and (S)-N- methylcoclaurine to form the (R,S) dimer berbamunine. The ratio of the two bisbenzylisoquinolines formed could be altered by reductase source or by varying the enantiomer composition of the substrates. €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€