This article has Open Peer Review reports available.
Potential antimutagenic activity of berberine, a constituent of Mahonia aquifolium
© Čerňáková et al; licensee BioMed Central Ltd. 2002
Received: 17 December 2001
Accepted: 19 February 2002
Published: 19 February 2002
As part of a study aimed at developing new pharmaceutical products from natural resources, the purpose of this research was twofold: (1) to fractionate crude extracts from the bark of Mahonia aquifolium and (2) to evaluate the strength of the antimutagenic activity of the separate components against one of the common direct-acting chemical mutagens.
The antimutagenic potency was evaluated against acridine orange (AO) by using Euglena gracilis as an eukaryotic test model, based on the ability of the test compound/fraction to prevent the mutagen-induced damage of chloroplast DNA.
It was found that the antimutagenicity of the crude Mahonia extract resides in both bis-benzylisoquinoline (BBI) and protoberberine alkaloid fractions but only the protoberberine derivatives, jatrorrhizine and berberine, showed significant concentration-dependent inhibitory effect against the AO-induced chloroplast mutagenesis of E. gracilis. Especially berberine elicited, at a very low dose, remarkable suppression of the AO-induced mutagenicity, its antimutagenic potency being almost three orders of magnitude higher when compared to its close analogue, jatrorrhizine. Possible mechanisms of the antimutagenic action are discussed in terms of recent literature data. While the potent antimutagenic activity of the protoberberines most likely results from the inhibition of DNA topoisomerase I, the actual mechanism(s) for the BBI alkaloids is hard to be identified.
Taken together, the results indicate that berberine possesses promising antimutagenic/anticarcinogenic potential that is worth to be investigated further.
Traditional Chinese medicine has been practised by Chinese communities world-wide for many generations, and there is a wealth of literature information available related to the therapeutic use of this type of medicine. In recent years, there has been a global surge in the popularity of herbal/traditional medicine, and currently there is enormous interest in developing new pharmaceutical products from such resources.
All the above findings raise the possibility that protoberberines may be effective in deactivation of carcinogens and tumour promoters. For in vitro screening of antimutagens/anticarcinogens a variety of prokaryotic and eukaryotic micro-organisms have been used. In the present study, a new eukaryotic model, viz. green unicellular flagellate Euglena gracilis was chosen . This micro-organism possesses a multigenomic system with nuclear, mitochondrial and chloroplast DNAs. The detection ability of this model is based on the preferential and selective sensitivity of the chloroplast genetic apparatus to xenobiotics resulting in elimination of the functional chloroplast from the cells. Antichloroplastic activity of mutagens is macroscopically manifested by an irreversible loss of the capability of cells to form green colonies (bleaching effect). Compounds with antimutagenic properties prevent such a bleaching of E. gracilis cells by mutagens / carcinogens. Previous studies on several standard mutagens and antimutagens revealed a high sensitivity and reliability of the model [16–19].
Thus, the aim of the present work was to study the possible antimutagenic effect of berberine and jatrorrhizine as well as of the BBI alkaloids fraction and of the overall M. aquifolium crude extract, on acridine orange (AO) – induced mutagenicity in the E. gracilis assay.
Materials and methods
The stem bark of Mahonia aquifolium (Pursh) Nutt. (Berberidaceae) was collected in October 1998 in the Arboretum Tesárske Mlyòany, Slovakia. Voucher specimens are deposited at the Herbarium of the Faculty of Pharmacy, Comenius University, Bratislava (No. Ma-108/8). Dried and powdered stem bark was macerated at room temperature with 62% aqueous EtOH (1:10 w/v) for 5 days and then filtered. The filtrate (crude M. aquifolium extract) was stored in a refrigerator until use in the biological assay. The BBI and the protoberberine alkaloid fractions were separated and purified using standard procedures as described previously .
Berberine [CAS 2086-83-1] and jatrorrhizine [CAS 3621-38-3], the main quaternary protoberberine alkaloids were isolated as iodides from the protoberberine fraction and identified by direct comparison of their physical and spectral properties with the literature data .
HPLC analysis of the BBI alkaloids was performed according to the procedure reported earlier . Totally 6 tertiary BBI alkaloids (oxyacanthine, aromoline, baluchistine, berbamine, obamegine and aquifoline) were positively identified by referring to authentic compounds described earlier . The identification was made on the basis of their UV absorption spectra and retention time.
Euglena gracilis (strain Z) was obtained from S.H. Hutner, Haskins Laboratory, Pace University, New York, NY, USA and maintained on Cramer-Myers (CM) medium  under static conditions at 27°C and with permanent lighting (16.4 W/m2).
Acridine orange [CAS 65-61-2] was purchased from Merck, Darmstadt, Germany. Stock solutions of AO, berberine, jatrorrhizine, and BBI alkaloids were prepared by dissolving them in distilled water.
E. gracilis cells diluted to concentration 8 × 105 cells/ml CM medium were used in the experiments. Aliquots (0.2 ml) of the cell suspension were dispensed into test tubes, along with 2.3, 11.4 or 22.8 μM AO, indicated concentrations of berberine (0.03, 0.06, 0.15, 0.30, 0.45 μg/ml), jatrorrhizine (0.72, 1.44, 14.4, 28.8, 43.2 μg/ml), BBI alkaloids (0.72, 1.44, 14.4, 28.8,43.2 μg/ml) or the M. aquifolium extract (5.5, 11.0, 110,220,330 μg/ml), and at last the content of the incubation mixture was completed to a final volume (5 ml) by the addition of CM medium. Following a 24-h co-treatment, the cells were centrifuged at 3000 rpm for 20 min, the resultant pellet suspended in fresh CM medium and again centrifuged. After the pellet was resuspended in fresh CM medium, the cells were finally cultivated 14 days at 27°C under permanent illumination (16.4 W/m2). The experiments were repeated in three independent series. Just before the counting, the movement of E. gracilis was stopped by adding a drop of EtOH and the counting of green and white (mutant) colonies was carried out in a Bürker chamber under a microscope. The viability of the Euglena cells was estimated by counting the total white mutants in the presence of mutagen (positive control) compared to the number of the spontaneous white mutants in the absence of mutagen (negative control). We defined the relative decrease of the bleaching as the antimutagenic potency (AP) which was calculated by the formula
where B0 is the AO-induced E. gracilis bleaching (%) and Br is the AO-induced and antimutagen-reduced E. gracilis bleaching (%).
The statistical significance of all the calculated values were determined by paired Student's t-test. The values represent the means ± standard deviation (SD).
Results and discussion
The mutagenic effect of AO on E. gracilis was tested in three concentrations, 2.3, 11.4 and 22.8 μM, which induced, respectively, 43 ± 2 %, 58 ± 2 % and 65 ± 3 % of white mutant cells. The AO concentrations were chosen so as to ensure no significant change in the viability of cells compared to the negative controls (Euglena cells in the absence of the mutagen). Similarly, no spontaneous white mutants were found in any sets of positive controls.
Antimutagenic potency (AP) [%] of berberine in reducing the acridine orange (AO)-induced bleaching of Euglena gracilis cells.
concentration of berberine [μg/ml]
89 ± 2.2
88 ± 5.3
90 ± 6.2
86 ± 1.7
78 ± 6.5
80 ± 3.0
86 ± 4.2
84 ± 1.6
83 ± 1.1
82 ± 3.6
54 ± 3.5
54 ± 1.8
49 ± 4.1
38 ± 2.3
36 ± 1.7
Although the BBI and protoberberine alkaloids studied in this laboratory exhibit a broad spectrum of biological activities, for most of them the molecular mechanism of action is still unclear. In the case of the BBI alkaloids, one possible mechanism of their antimutagenic activity could be based on the reports that tetrandrine and other BBI alkaloids are excellent scavangers of reactive oxygen species (ROS) such as singlet oxygen and/or superoxide anion radical  since it is well documented that that ROS play a central role in multistage mutagenesis and carcinogenesis [23, 24]. However, the potential mechanism based on the ability of BBIs to prevent oxidative damage of DNA by ROS is unlikely since the ability of AO to produce ROS in a redox system has never been published. Instead, the antimutagenic potency of the BBI fraction is more likely linked to modulation of DNA at the transcriptional level. Recently, the role of eukaryotic transcription factors, NF-κB and AP-1, has been highlighted in mutagenesis and carcinogenesis [25, 26] and tetrandrine is known to inhibit the activation of NF-κB in the alveolar macrophage . Nevertheless, the identification of the ultimate mechanism of the antimutagenic effect of the BBI alkaloids awaits further study.
As described above, the significant antimutagenic potency against AO was observed for the two protoberberines studied herein. The potency of jatrorrhizine alone is comparable to that found for the overall BBI fraction and the potency of berberine is even by more than 2 orders of magnitude higher. To our knowledge, the ability of protoberberines to reduce the mutagenicity of environmental chemicals has not yet been reported so that the mechanism of their antigenotoxic action was not discussed. However, the cytotoxic activity of berberine and its analogues has been attributed to DNA topoisomerase I (TOPO-I) and II (TOPO-II) poisoning [11–13]; recently, a mechanistic model  for TOPO-I poisoning by protoberberines was developed according to which berberine binds to DNA and TOPO-I at the interface of the binary complex DNA – TOPO-I in such a manner that rings C and D (Fig. 1) intercalate into the DNA helix, while rings A and B protrude out of the helix interior into the minor groove, where they are accessible to interact with specific functional groups on the enzyme. Thus, both drug-DNA and drug-enzyme interactions contribute to the overall affinity (potency) of the drug. In this light it is reasonable to suggest that the antimutagenicity of berberine against the genotoxic effect of AO on plastid DNA is exerted by a similar, if not identical, mechanism. A comparison of the antimutagenic potency of the two protoberberines tested here supports this notion. The only difference between berberine and jatrorrhizine (Fig. 1) is restricted to the substitution on the A ring (2,3-methylenedioxy in berberine vs. 2-methoxy-3-hydroxy substitution in jatrorrhizine), i.e. to the portion of the molecule which is involved in interactions with TOPO-I. Thus, insertion of the protoberberine molecule between the base pairs of DNA prevents the interaction of the AO mutagen at the same binding site as AO is known as a frameshift mutagen due to strong intercalative binding. A similar detrimental effect of the free hydroxy group on the TOPO-II poisoning activity has also been observed for berberrubine . In this context it is important to note that camptothecin, a natural product currently in clinical use for the treatment of a variety of cancers [12, 25], has a pharmacological profile very similar to that of protoberberines; its antineoplastic and antimutagenic activity was demonstrated against many organic mutagens and also the DNA-TOPO-II complex was identified as its molecular target . It is therefore reasonable to consider that the drug-induced poisoning of TOPO enzymes may be of general importance for anticancer and antimutagenic drugs.
In summary, the data presented here showed that the BBI alkaloid fraction and especially the protoberberines, jatrorrhizine and berberine, inhibited the AO-induced mutagenicity in the E. gracilis assay. Berberine demonstrated significant antimutagenic activity against the chloroplast damaging effects of AO with a high potency. The protective effects of the latter compound against the chemical carcinogen suggest its potential chemopreventive ability, which is under investigation using other bacterial tests as well as animal tumour models.
This research was supported by the Grant Agency of the Slovak Republic, Projects No. 1/7359/20, 1/8216/01, 1/8215/01 and 2005.
- Amin AH, Subbaiah V, Abbasi KM: Berberine sulphate: antimicrobial activity, bioassay, and mode of action. Canad J Microbiol. 1969, 15: 1067-1076.View ArticleGoogle Scholar
- Okunade AL, Hufford CD, Richardson MD, Petterson JR, Clark AM: Antimicrobial properties of alkaloids from Xanthoriza simplicissima. J Pharm Sci. 1994, 83: 404-406.View ArticlePubMedGoogle Scholar
- Iwasa K, Kamigauchi M, Sugiura M, Nanba H: Antimicrobial activity of some 13-alkyl substituted protoberberinium salts. Planta Med. 1997, 63: 196-198.View ArticlePubMedGoogle Scholar
- Sarma BK, Pandey VB, Mishra GD, Singh UP: Antifungal activity of berberine iodide, a constituent of Fumaria indica. Folia Microbiol. 1999, 44: 164-166.View ArticleGoogle Scholar
- Kuo CL, Chou CC, Yung BYM: Berberine complexes with DNA in the berberine-induced apoptosis in human leukemic HL-60 cells. Cancer Letters. 1995, 93: 193-200. 10.1016/0304-3835(95)03809-B.View ArticlePubMedGoogle Scholar
- Anis KV, Kuttan G, Kuttan R: Role of berberine as an adjuvant response modifier during tumour therapy in mice. Pharm Pharmacol Commun. 1999, 5: 697-700.View ArticleGoogle Scholar
- Anis KV, Rajeshkumar NV, Kuttan R: Inhibition of chemical carcinogenesis by berberine in rats and mice. J Pharm Pharmacol. 2001, 53: 763-768. 10.1211/0022357011775901.View ArticlePubMedGoogle Scholar
- Yang IW, Chou CC, Yung BYM: Dose-dependent effects of berberine on cell cycle pause and apoptosis in Balb/c 3T3 cells. Naunyn-Schmiedeberg's Arch Pharmacol. 1996, 354: 102-106.View ArticleGoogle Scholar
- Orfila L, Rodriguez M, Colman T, Hasegawa M, Merentes E, Arvelo F: Structural modification of berberine alkaloids in relation to cytotoxic activity in vitro. J Ethnopharmacol. 2000, 71: 449-456. 10.1016/S0378-8741(00)00177-X.View ArticlePubMedGoogle Scholar
- Sanders MM, Liu AA, Li TK, Wu HY, Desai SD, Mao Y, Rubin EH, LaVoie EJ, Makhey D, Liu LF: Selective cytotoxicity of topoisomerase-directed protoberberines against glioblastoma cells. Biochem Pharmacol. 1998, 56: 1157-1166. 10.1016/S0006-2952(98)00243-3.View ArticlePubMedGoogle Scholar
- Kobayashi Y, Yamashita Y, Fujii N, Takaboshi K, Kawakami T, Kawamura M, Mizukami T, Nakano H: Inhibitors of DNA topoisomerase I and II from the Coptis Rhizomes. Planta Med. 1995, 61: 414-418.View ArticlePubMedGoogle Scholar
- Li TK, Bathory E, LaVoie EJ, Srinivasan AR, Olson WK, Sauers RR, Liu LF, Pilch DS: Human topoisomerase I poisoning by protoberberines: Potential roles for both drug-DNA and drug-enzyme interactions. Biochemistry. 2000, 39: 7107-7116. 10.1021/bi000171g.View ArticlePubMedGoogle Scholar
- Krishnan P, Bastow KF: The 9-position in berberine analogs is an important determinant of DNA topoisomerase II inhibition. Anti-Cancer Drug Des. 2000, 15: 255-264.Google Scholar
- Li WY, Lu H, Xu CX, Zhang JB, Lu ZH: Spectroscopic and binding properties of berberine berberine to DNA and its application to DNA detection. Spectrosc Lett. 1998, 31: 1287-1298.View ArticleGoogle Scholar
- Ebringer L: Interaction of drugs with extranuclear genetic elements and its consequences. Teratogen Carcinogen Mutagen. 1990, 10: 477-501.View ArticleGoogle Scholar
- Foltínová P, Grones J: Euglena gracilis as an eukaryotic test organism for detecting mutagens and antimutagens. Mutation Res. 1997, 393: 1-6. 10.1016/S1383-5718(96)00071-X.View ArticlePubMedGoogle Scholar
- Križková L, Lopes MH, Polónyi J, Belicová A, Dobias J, Ebringer L: Antimutagenicity of a suberin extract from Quercus suber cork. Mutation Res. 1999, 446: 225-230.View ArticlePubMedGoogle Scholar
- Križková L, Nagy M, Polónyi J, Dobias J, Belicová A, Grančai D, Krajčovič J: Phenolic acids inhibit chloroplast mutagenesis in Euglena gracilis. Mutation Res. 2000, 469: 107-114.View ArticlePubMedGoogle Scholar
- Križková L, Polónyi J, Košíková B, Dobias J, Belicová A, Krajčovič J, Ebringer L: Lignin reduces of loxacin-induced mutagenicity in Euglena assay. Anticancer Res. 2000, 20: 833-836.PubMedGoogle Scholar
- Košt'álová D, Uhrín D, Hrochová V, Tomko J: A new bisbenzylisoquinoline alkaloid from Mahonia aquifolium (Pursh) Nutt. Collect Czech Chem Commun. 1987, 52: 242-246.View ArticleGoogle Scholar
- Cramer M, Myers J: Growth and photosynthetic characteristic of Euglena gracilis. Arch Microbiol. 1952, 17: 384-402.Google Scholar
- Chen F, Sun S, Kuhn DC, Lu Y, Gaydos LJ, Shi X, Demers LM: Tetrandrine inhibits signal-induced NF-κB activation in rat alveolar macrophages. Biochem Biophys Res Commun. 1997, 231: 99-102. 10.1006/bbrc.1997.6057.View ArticlePubMedGoogle Scholar
- Ames BN: Endogeneous oxidative DNA damage, aging and cancer. Free Radical Res Commun. 1989, 7: 121-128.View ArticleGoogle Scholar
- Lunec J: Free radicals: Their involvement in disease processes. Ann Clin Biochem. 1990, 27: 173-182.View ArticlePubMedGoogle Scholar
- Pantazis P, Giovanella B, Rotenberg ML: The camptothecins: From discovery to the patient. New York Academy of Sciences, New York. 1996Google Scholar
- Keskin O, Bahar I, Jernigan BL, Beutler JA, Shoemaker RH, Sausville EA, Covell DG: Characterization of anticancer agents by their growth inhibitory activity and relationships to mechanism of action and structure. Anti-Cancer Drug Des. 2000, 15: 79-98.Google Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/2/2/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.