Antimicro bial Activity of some 13-Alkyl Substituted Proto berberinium salts Kinuko Iwasa, Miyoko Kamigauchi, Makiko Sugiura, and Hiroaki Nanba Abstract: Several 13-alkyl substituted analogs of berberine and palmatine were found to be highly active against two types of Staphylococcus aureus(SI and S2)of different origin. The most active 13-hexylberberine was 8 times more active(against SI)and the same order active(against S2)as kanamycin sulfate. 13-Hexylpatmatine displayed an activety against S aureus(S1)4 times greater than that of kanamycin sulfate. The activities of 13-hexylberberine against two types of S aureus were 64 and 128 times greater than those of the clinically used alkaloid berberine Additionally two hexyl derivatives possessed antifungal activity Key words: 13-Alkylrotoberinium salts, antimicrobial activity, antifungal activity. Introduction The quaternary is quinoline alkaloid berberine(1), isolated from a variety of plants, is widely used in Asia as a drug due to its antimicrobial activity. In Japan, berberine and the extracts of the dried rhizomes of Coptis japonica Makino( Ranunculaceae) and the dried bark of phellodendron amurense Ruprecht ( Rutaceae )are widely used as a stomach tonic. In a previous paper the antibacterial activity of berberine analogs has been reported (1). In brief, th derivative(2), the 13-ethyl analog(3), and the 13-ethyl-g-ethoxy homolog( 4 )displayed an increase in antibacterial activity against Staphylococcus aureus by factors of 2, 4, and 8, respectively, over the parent salt berberine. These results strongly suggested that the bulk of the alkoxy- and alkyl-substituents at C-9 and C-13 had a direct bearing upon the activity Results and discussion In the present work, the effects of variations in the length of the alkyl sidechain at C-13 on the antimicrobial activity were examined in both berberine(1)and palmatine( 8 )derivatives. Some known and some new substances(1-4, 8, 9, and5-7, 10-13)were tested against representative strains of Gram-positive, Gram-negative and fungal microorganisms by the 2-fold dilution techniques Two types of S aureus(SI and S2), as well as Escherichia coli, and Candida albicans were used The propyl-, butyl-, and hexyl-substituted derivatives at C-13 of berberine( 1)or palmatine(8)and 13-ethylpalmatine were prepared by treatment of the parent alkaloid with propionaldehyde, n-butylaldehyde, n-hexylaldehyde, and acetaldehyde, respectively e structures of these compounds(5-7 and 10-13)were confirmed by means of 1H-and C-NMR data including NOESY, DEPT, COSY, HMBC, and HMQC spectral data. These data enabled us also to assign the H-and C-NMr spectra unambiguously as shown in Tables I and 2. The mass spectra of all the new compounds were consistent with the assigned structures(Table 3).The retention times of the 13-alkyl derivatives in HPLC increased with increasing length of the alkyl sidechain at C-13(Table 3)
Antimicrobial Activity of Some 13-Alkyl Substituted Protoberberinium Salts Kinuko Iwasa,Miyoko Kamigauchi,Makiko Sugiura,and Hiroaki Nanba Abstract: Several 13-alkyl substituted analogs of berberine and palmatine were found to be highly active against two types of Staphylococcus aureus (S1 and S2)of different origin. The most active 13-hexylberberine was 8 times more active (against S1)and the same order active (against S2)as kanamycin sulfate.13-Hexylpatmatine displayed an activety against S.aureus(S1)4 times greater than that of kanamycin sulfate. The activities of 13-hexylberberine against two types of S.aureus were 64 and 128 times greater than those of the clinically used alkaloid berberine. Additionally two hexyl derivatives possessed antifungal activity. Key words:13-Alkylrotoberinium salts, antimicrobial activity, antifungal activity. Introduction The quaternary is oquinoline alkaloid berberine(1),isolated from a variety of plants, is widely used in Asia as a drug due to its antimicrobial activity. In Japan, berberine and the extracts of the dried rhizomes of Coptis japonica Makino (Ranunculaceae) and the dried bark of phellodendron amurense Ruprecht (Rutaceae)are widely used as a stomach tonic. In a previous paper the antibacterial activity of berberine analogs has been reported (1).In brief, the 13-methyl derivative(2) ,the 13-ethyl analog(3),and the 13-ethyl-9-ethoxy homolog(4) displayed an increase in antibacterial activity against Staphylococcus aureus by factors of 2,4,and 8,respectively,over the parent salt berberine. These results strongly suggested that the bulk of the alkoxy- and alkyl-substituents at C-9 and C-13 had a direct bearing upon the activity. Results and Discussion In the present work, the effects of variations in the length of the alkyl sidechain at C-13 on the antimicrobial activity were examined in both berberine(1) and palmatine (8)derivatives. Some known and some new substances (1-4,8,9,and5-7,10-13)were tested against representative strains of Gram-positive, Gram-negative and fungal microorganisms by the 2-fold dilution techniques. Two types of S.aureus(S1 and S2),as well as Escherichia coli, and Candida albicans were used. The propyl-, butyl-,and hexyl-substituted derivatives at C-13 of berberine(1) or palmatine(8) and 13-ethylpalmatine were prepared by treatment of the parent alkaloid with propionaldehyde, n-butylaldehyde, n-hexylaldehyde, and acetaldehyde, respectively. The structures of these compounds(5-7 and 10-13)were confirmed by means of 1H-and 13C-NMR data including NOESY, DEPT, COSY, HMBC, and HMQC spectral data. These data enabled us also to assign the 1H-and 13C-NMR spectra unambiguously as shown in Tables 1 and 2.The mass spectra of all the new compounds were consistent with the assigned structures (Table 3).The retention times of the 13-alkyl derivatives in HPLC increased with increasing length of the alkyl sidechain at C-13(Table 3)
Activity against the Gram-positive bacterium S aureus proved to be much higher than against the Gram-negative bacterium and fungus(Table 4), Activity against S aureus for the 3-propyl-, 13-butyl-, and 13-hexylberberine (5, 6, and 7)increased as the length of the alkyl chain increased. This is in line with what was previously observed for 13-methyl-, and 13-ethylberberine(2 and 3 )(1). In like fashion, in palmatine analogs tested for the first time, activity against S aureus increased as the length of alkyl chain rose in the order methyl, ethyl, propyl, butyl and hexyl groups. 13-Alkyl derivatives of the berberine-type, with methoxy groups at the same positions (palmatine-type). Activity against S, aureus ATCC 3061(S2) was generally lower than against S aureus X(SI) isolated from hospitalized patients Among the 13-alkyl substituted analogs, 13-hexyl derivatives(7 and 13) displayed the highest activity against all strains. 13-Hexylberberine()showed the best activity against SI and S2, with Impressive 128-fold over paren berberine( 1). Similarly, 13-hexyl palmatine( 13)demonstrated stronger activity against SI and S2 than the parent compound by factors of 128 and 32, respectively Among the 13-alkyl substituted analogs, 13-hexyl derivatives(7 and 13)displayed the highest activity against all strains. 13-Hexylberberine(7)showed the best activity against SI and S2, with an impressive 64- and 128-fold increase over the parent berberine( 1). Similarly, 3-hexylpalmatine (13) compound by factors of 128 and 32, respectively. The activity of 13-hexylberberine against S2 was, in fact, comparable to that of kanamycin ulfate. 13-Hexylberberine(7)and 13-hexylpalmatine(13)showed potent activity against Sl, and proved to be 8 and 4 times, respectively superior to kanamycin sulfate. 13-Hexylberberine(7)and 13-hexylpalmatine(13)showed potent activity against Sl, and proved to be 8 and 4 times, respectively, superior to kanamycin sulfate. 13-Hexylberberine( 7)and 13-hexylpalmatine(13)also showed activity against Candida albicans IFO 1061(MIC 15.6 H g/ml) although kanamycin sulfate was inactive(MIC>2000 g/ml) Only 13-hexylberberine( 7)demonstrated a relatively high activity(MIC250 u g/ml) against E coli IFO 026, even though the activity was only one-eighth of that of kanamycin sulfate. The other 13-alkyl derivatives, except for 13-hexylpalmatine( 13), exhibited weak activity(MIC>1000 H g/ml). 13-Butylberberine(6) and 13-butylpalmatine (12) possessed significant activity against n conclusion, among the tested compounds, 13-hexylberberine(7) and 13-hexylpalmatine(13) were found to be 8 and 4 times, respectively, more active against S aureus(S1)than kanamycin ulfate. 13-Hexylberberine(7)showed high activity against S aureus(S2) comparable to that of kanamycin sulfate. 13-Hexylberberine(7)and 13-hexylpalmatine(13) had very strong activity against Saureus compared with the clinically used alkaloid berberine (1). The 13-hexyl derivatives of berberine and palmatine are highly selective in inhbiting the growth of Gram-positive, S aureus, while they were less potent against the fungus Candida albicans. Their activities were also very low against Gram-negative E coli Further evaluations are required in
Activity against the Gram-positive bacterium S.aureus proved to be much higher than against the Gram-negative bacterium and fungus(Table 4),Activity against S.aureus for the 13-propyl-,13-butyl-,and 13-hexylberberine (5,6,and 7)increased as the length of the alkyl chain increased.This is in line with what was previously observed for 13-methyl-,and 13-ethylberberine(2 and 3)(1).In like fashion, in palmatine analogs tested for the first time ,activity against S.aureus increased as the length of alkyl chain rose in the order methyl, ethyl, propyl, butyl, and hexyl groups.13-Alkyl derivatives of the berberine-type, with methoxy groups at the same positions (palmatine-type).Activity against S,aureus ATCC 3061(S2) was generally lower than against S.aureus X(S1) isolated from hospitalized patients. Among the 13-alkyl substituted analogs, 13-hexyl derivatives (7 and 13) displayed the highest activity against all strains.13-Hexylberberine(7) showed the best activity against S1 and S2,with an impressive 64- and 128-fold increase over the parent berberine(1).Similarly,13-hexylpalmatine(13) demonstrated stronger activity against S1 and S2 than the parent compound by factors of 128 and 32,respectively. Among the 13-alkyl substituted analogs, 13-hexyl derivatives (7 and 13) displayed the highest activity against all strains.13-Hexylberberine (7) showed the best activity against S1 and S2,with an impressive 64- and 128-fold increase over the parent berberine(1). Similarly, 13-hexylpalmatine (13) demonstrated stronger activity against S1 and S2 than the parent compound by factors of 128 and 32,respectively. The activity of 13-hexylberberine against S2 was, in fact, comparable to that of kanamycin sulfate.13-Hexylberberine (7)and 13-hexylpalmatine (13) showed potent activity against S1,and proved to be 8 and 4 times, respectively ,superior to kanamycin sulfate .13-Hexylberberine(7) and 13-hexylpalmatine (13) showed potent activity against S1,and proved to be 8 and 4 times, respectively, superior to kanamycin sulfate.13-Hexylberberine(7) and 13-hexylpalmatine (13) also showed activity against Candida albicans IFO 1061 (MIC 15.6μg/ml) although kanamycin sulfate was inactive (MIC>2000Μg/ml). Only 13-hexylberberine(7) demonstrated a relatively high activity (MIC250μg/ml) against E.coli IFO 026,even though the activity was only one-eighth of that of kanamycin sulfate. The other 13-alkyl derivatives, except for 13-hexylpalmatine(13), exhibited weak activity (MIC>1000μ g/ml).13-Butylberberine (6) and 13-butylpalmatine (12) possessed significant activity against S.aureus. In conclusion, among the tested compounds,13-hexylberberine(7) and 13-hexylpalmatine (13) were found to be 8 and 4 times, respectively, more active against S.aureus (S1) than kanamycin sulfate.13-Hexylberberine (7) showed high activity against S.aureus (S2) comparable to that of kanamycin sulfate.13-Hexylberberine(7) and 13-hexylpalmatine (13) had very strong activity against S.aureus compared with the clinically used alkaloid berberine (1). The 13-hexyl derivatives of berberine and palmatine are highly selective in inhbiting the growth of Gram-positive, S.aureus, while they were less potent against the fungus Candida albicans .Their activities were also very low against Gram-negative E.coli. Further evaluations are required in
order to establish the in vivo efficacy of the most active derivatives Materials and Methods Berberine (1)and palmatine(8)are commercially available. 13-Methylberberine (2)(2) 3-methylpalmatine(9)(2), and 9-O-ethyl-13-ethylberberrubine(4)(1)were previously prepared Preparation of 13-alkyl substituted berberine and palmatine(5-7 and 10-13) A mixture of the dihydro derivative(3)of berberine or palmatine and the corresponding aldehyde in 20%CH3 COOH was placed in a glass-stoppered bottle and heated for 24-48 h for 105-120C. After the mixture cooled, the solution was acidified with HCL. The resulting crystals were filtered to give the berberine or palmatine chloride. The filtrate was cooled and the resulting crystals were collected to supply the mixture of the corresp 13-alkyl derivative and berberine or palmatine. The crystalline mixture was recrystallized repeatedly to give the 13-alkyl The antimicrobial activity was measured according to the method previously described( 1) using the strains: S aureus(ATCC 3061), E coli (IFo 026), Candida albicans(IFO 1061)and S aureus(X) isolated from hospitalized patient
order to establish the in vivo efficacy of the most active derivatives. Materials and Methods Berberine (1) and palmatine (8) are commercially available. 13-Methylberberine(2)(2), 13-methylpalmatine(9)(2),and 9-O-ethyl-13-ethylberberrubine (4)(1) were previously prepared. Preparation of 13-alkyl substituted berberine and palmatine (5-7 and 10-13) A mixture of the dihydro derivative (3) of berberine or palmatine and the corresponding aldehyde in 20%CH3COOH was placed in a glass-stoppered bottle and heated for 24-48 h for 105-120℃.After the mixture cooled, the solution was acidified with HCl. The resulting crystals were filtered to give the berberine or palmatine chloride. The filtrate was cooled and the resulting crystals were collected to supply the mixture of the corresponding 13-alkyl derivative and berberine or palmatine. The crystalline mixture was recrystallized repeatedly to give the 13-alkyl derivative. The antimicrobial activity was measured according to the method previously described(1) using the strains: S.aureus (ATCC 3061), E.coli (IFO 026),Candida albicans (IFO 1061) and S.aureus(X) isolated from hospitalized patients