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The study of antimutagen effect of the birch bark extract containing betuline

Global pollution of biosphere has an adverse effect on the hereditary mechanism of all alive. The quantity of mutagens - the chemical substances causing changes in the hereditary device, rates promptly in an environment: toxic compounds of industrial discharges and drains, insecticides in an agriculture, household chemical goods and perfumery, some medical products.

The remote consequences of genetic damage are perceived by the majority of the population not so intently, as really appreciable factors conducting to illnesses. But already now it is possible to allocate the certain groups of risk of people who need antimutagenic protection. They are persons with diseases with increased mutation (hereditary, autoimmune, infectious diseases), people who are permanently or periodically exposed to mutagen influence of the medicinal or industrial origin, smoking persons, persons, which work is accompanied by psychoemotional pressure. Immunodeficiency patients have increased sensitivity to mutagens which cause release of free radicals capable to destroy enzymes, cells, such important structures as DNA and RNA that can lead to malignancies formations.

The most effective method to prevent the induced mutagenesis is detection and elimination of genotoxicants from human habitat. However it is impossible to exclude completely contact of people with mutagens. For today a number of mutagenic compounds cannot be withdrawn from practice through their economical and medical virtue, for example indispensable medical products. By some estimation, a person receives 2-3 g of mutagenic compounds per day from food, water, atmosphere and as a result of application of medical preparations.

That is why these above-listed facts clarify survey of antimutagens and ways of their preventive application for a protection of the human heredity. Recently the company "Birch world" has developed a way of the birch bark extract getting commercially with the content of Betulin not less than 70 %; it is also shown to have a wide spectrum of biological properties (Registration number SEC № 77.99.03.936.B 000201.02.04). [2].

The study of activity of the birch bark extract in genotoxicological research was expedient both from the point of view of analysis of antimutagenic properties in experiments on mammals, and in frameworks of pre-clinical estimations of application safety. Thereupon, the purpose of the present research was to study the influence of the birch bark extract on spontaneous and induced by dioxidine and cyclophosphamide mutagenesis at mice.

The research was conducted in the Zakusov Research Institute of Pharmacology of RAMS under the direction of the Professor Durnev A.D.

The research was executed by a method of the registration of the bone marrow cells with chromosomal aberrations at mice C57Bl/6. The dry birch bark extract was administrated peroral in dozes 50, 150, 450 and 1500 mg/kg, mutagens - dioxidine (200 mg / kg) and cyclophosphamide (20 mg / kg) - by intraperitoneal injection.

Materials and methods

The estimation of mutagenic activity of the dry birch bark extract (150 and 1500 mg/kg, peroral) on mice has been executed according to methodical recommendations of Pharmacological committee of Ministry of Public Health of the Russian Federation [4].

Experiments were conducted in three variants. In the first variant the dry birch bark extract was administrated once simultaneously with a mutagen (acute experiment). In the second variant the dry birch bark extract was administrated daily for five days. The last intake of the birch bark extract was combined with an injection of mutagen (preconditioning). In the third variant the birch bark extract and mutagen were administrated once a day within five days (joint administration). In the latter case the slaughter of animals was carried out in 6 hours after the last administration, in other cases - in 24 hours.

Results and discussion

The cytogenetic analysis of the bone marrow cells was executed after the single and five-day administration of the birch bark extract (150 mg/kg) to male and female mice and after its single administration to male mice in a doze of 1500 mg/kg. It has not revealed mutagenic activity of the extract. The number of cells with chromosomal aberrations in experimental series of research almost did not differ from the values established at control animals.

The table №1 represents the results that describe the influence of the birch bark extract on the cytogenetic effect of dioxidine (DN) and cyclophosphamide (CP), in doses 200 and 20 mg/kg, accordingly.

Administration of DN in a single doze of 200 mg/kg resulted in increase of cells with chromosomal aberrations up to value 10.2 1.4 %, that is significantly higher than the value of the control (1.6 0.6 %).

After the administration of the combination of the birch bark extract in a doze of 50 mg/kg and DN 4.8 1.0 % of aberrant metaphases were detected. This value significantly differs from that of the positive control and corresponds to 53 % decrease of DN effect. Using the birch bark extract in a doze of 150 mg/kg increased its antimutagenic effect to 61 %: 4.0 0.9 % of damaged metaphases were registered. Further increasing the doze of the birch bark extract up to 450 mg/kg resulted in decrease of antimutagenic effect to 33 %.

Antimutagenic effect of the birch bark extract was confirmed further by using mutagen CP (table №1).

The same antimutagenic effect was registered when the birch bark extract was used in dozes of 50 and 150 mg/kg: the decrease of damaging effect of mutagen in both cases was 60 % (level of chromosomal aberrations was 3.5 ± 0.9 % against 8.8 ±1.4 % in the positive control). Further increasing the dose of the birch bark extract up to 450 mg/kg resulted in the absence of the antimutagenic effect revealed: the received value - 6.4 ± 1.1 % of aberrant metaphases did not differ significantly from the value of the positive control.

In the following part of experiments the influence of 5-day preconditioning with the birch bark extract on mutagenic effect of DN was investigated. The results of these experiments are summarized in the table №2.

In this case the antimutagenic effect of the birch bark extract in comparison with the single administration was greater. 49 % decrease in damaging action of mutagen in a doze of 50 mg/kg was observed. With increasing the doze of the birch bark extract up to 150 mg/kg its protective effect increased to 63 %. When using the extract in the doze of 450 mg/kg the maximal antimutagenic effect was registered - total suppression of mutagen's damaging effect. The received value - 2.6 ± 0.7 % of aberrant metaphases did not differ significantly from a spontaneous level of a mutation (1.6 0.6 %).

Antimutagenic effect of the birch bark extract at 5-day time preconditioning, in comparison with the single administration, increased also when using CP as a mutagen (table №2).

After the CP exposure to animals processed with the birch bark extract in a doze of 50 mg/kg it was registered 5.5 ± 1.1 % of damaged metaphases that corresponds to 54 % decrease of cytogenetic effect of the mutagen. With increasing the doze of the birch bark extract its antimutagenic effect achieved 65 % (4.2 ± 0.9 % of aberrant metaphases against 12.0 ± 1.5 % in the positive control). Contrary to the single administration, the birch bark extract showed antimutagenic effect also in the doze of 450 mg/kg. The received value, 4.4 ± 0.9 % of the damaged cells corresponded to 63 % reduction of the mutagen action.

In the following series of experiments the influence of the birch bark extract on mutagenic effects of DN and CP after the five-day concurrently administration was investigated. The received results are represented in the table №3.

After five-day treatment of animals with DN in a doze of 200 mg/kg chromosomal aberrations were registered in 11.6 1.4 % of the investigated metaphases. After the combined application of the birch bark extract in a doze of 50 mg/kg and DN it was revealed 6.6 1.2 % of damaged cells that corresponds to 43 % reduction of mutagen's effect.

After the combined application of the mutagen and the birch bark extract in dozes of 150 and 450 mg/kg it was observed 4.8 1.1 % and 5.8 1.0 % of aberrant metaphases, respectively. Paired comparison of the registered results with the data of the positive control revealed significantly distinctions: in a doze of 150 mg/kg the antimutagenic effect was 59 %, and in a doze of 450 mg/kg - 50 %.

When using CP the antimutagenic effect of the birch bark extract was revealed at all investigated dozes. After combined administration of the birch bark extract in a doze of 50 mg/kg and the mutagen, it was registered 3.8 ± 0.7 % of aberrant metaphases that corresponds to 54 % reduction of CP effect. At application of the birch bark extract in higher dozes: 150 and 450 mg/kg - its antimutagenic properties were similar. The received values 5.0 ± 1.0 and 5.2 ± 1.0 % of damaged metaphases correspond to 40 and 38 % of a reduction of damaging effect of the mutagen, respectively.

Thus, the results of conducted investigations demonstrate the antimutagenic activity of the birch bark extract. Based on the total received data the antimutagenic effect of the birch bark extract is most evident in the case of its preliminary administration in a doze of 150 mg/kg during many days. The comparative analysis of antimutagenic effect regarding to the damaging effect of the used mutagens shows that the birch bark extract is most effective when using DN as a mutagen for positive control.

Betuline (74 % in investigated sample), is a part of the birch bark extract and have an antioxidant activity [6]. DN is the mutagen with a prooxidant effect; its action is effectively suppressed by the birch bark extract [1]. Therefore, the antimutagenic effect of the birch bark extract can be explained by its ability to suppress free radical oxidation.

The products of lipid peroxidation take part in the realization of the cytogenetic effect of CP. That is why suppression of the effect of this mutagen can also be explained by the antioxidant activity of the birch bark extract.

At the same time, CP is indirect mutagen; the main mechanism of its action is realized by its metabolites' (mainly acrolein) ability to alkylate a molecule of DNA [8]. This allows to assume that protective action of the birch bark extract can also be mediated by to suppression of activity of cytochrome P 450 participating in metabolism of CP.

We suggest that the mentioned above mechanisms do not exhaust all the possible ways of realization of antimutagenic effect of the birch bark extract, since previous experiments showed the ability of the birch bark extract to induce a production of interferons which are known to have positively effect on the DNA reparation[1].

The previous analysis [1] showed that the antimutagenic effect of natural compounds and their complexes generally does not exceed 50 %. Furthermore, along with a positive antimutagenic effect they frequently show undesirable comutagenic effect. The dry birch bark extract advantageously differs by the absence of comutagenic effect and efficiency of antimutagenic effect. Its protective effect in conditions of single and preliminary administration in most cases is not less than 60 %.

Thus, a set of the results received in experiments on mammals, allow concluding that the birch bark extract is characterized by the evident antimutagenic activity and has no undesirable mutagenic and comutagenic properties.

The company "Birch world" created some complex preparations on the basis of the dry birch bark extract - "Superantitox", "Betula-hit", "Betuline", "Betulanorm" and others, for the purpose of preventing the mutational damage of genome and, therefore, remote pathological consequences of induced mutagenesis; for increasing compensatory opportunities of human organism in the adverse conditions; for prolongation of human active lifetime. Medicinal and prophylactic administration of these preparations by the individuals with high professional risk of mutagenesis will reduce level of genetic damage of these people.

Literature

1. Durnev A. D., Seredenin S. B. "Mutagens: screening and pharmacological preventive measures of impacts"// Moscow, "Medicine", 1998, p. 326. (In Russian)
2. "The raw materials for the production of the biologically active additives to food "The dry birch bark extract". Registration number SEC 77.99.03.936.B. 000201.02.04 dated February 16, 2004. (In Russian)
3. Direction on the experimental (pre-clinical) study of a new pharmacological substances/Moscow, 2000. (In Russian)
4. Bochkov N. P., Durnev A. D., Zhurkov V. S. etc. System of searching and analysis of compounds with antimutagenic properties (Methodical recommendations)/ Khimiko-farmacevticheskiy zurnal , 1992, № 9 - 10, pp. 42 - 46, (In Russian)
5. Preston R.J., Dean B.J., Galloway S. at al. Mammalian in vivo cytogenetic assays. Analysis of chromosome aberrations in bone marrow cells// Mutat. Res., 1987, 189, 157-165.
6. Kahkonen M.P., Hopia A.I., Vuorela H.J., Rauha J.P., Pihlaja K., Kujala T.S. and Heinonen M. Antioxidant activity of plant extracts containing phenolic compounds// J Agric Food Chem, Oct 1999; 47(10): 3954-62.
7. Kanekal S., Kehrer J.P. Metabolism of cyclophosphamide by lypoxygenases.// Drug. Metab. Dispos.,1994, 22, 74-78.
8. Anderson D., Bishop J.B., Garner R.C. et al. Cyclophosphamide: review of its mutagenicity for an assessment of potential germ cell risk.// Mutat. Res., 1995, 330, 115-181.

Table №1.

Influence of the single administration of the dry birch bark extract (DBBE) on the induced by dioxidin (DN) and cyclophosphamide (CP) mutagenesis.

Table №2.

The influence of 5-day preliminary administration of the birch bark extract on the induced by DN and CP mutagenesis

Table № 3.

Influence of the birch bark extract on the induced by DN and CP mutagenesis in the case of 5-day combined administration.