Application of totarol and pharmaceutical composition containing totarol
11534412 · 2022-12-27
Assignee
Inventors
Cpc classification
A61K9/0036
HUMAN NECESSITIES
A61K9/06
HUMAN NECESSITIES
International classification
Abstract
The invention relates to application of totarol for production of a preparation for treatment of vaginal mucous membrane inflammations of bacterial origin, for alleviation of symptoms in such a treatment, and for prophylaxis and prevention of relapses of such inflammations. The invention includes also a pharmaceutical composition for treatment of vaginal mucous membrane inflammations of bacterial origin, for alleviation of symptoms in such a treatment and for prophylaxis and prevention of relapses of such inflammations, the composition containing from 75 to 95 parts by wt. of a cellulose derivative, from 0.5 to 5 parts by wt. of lactic acid, from 0.5 to 5 parts by wt. of a basic polymer, wherein the stoichiometric ratio of lactic acid to the basic polymer is comprised in the range of 1:1 to 8:1, and comprising totarol in an amount of 0.001 to 5 parts by wt. as the active substance.
Claims
1. A method for treatment of vaginal membrane inflammations of bacterial origin, for alleviation of symptoms in such a treatment, for prophylaxis and prevention of relapses of such inflammations, for treatment of anaerobic bacterial vaginosis (BV) and aerobic vaginitis (AV) comprising administering a pharmaceutical composition comprising totarol in an amount of 0.001 parts by weight (wt.) to 5.0 parts by wt. as an active agent.
2. A method for treatment of anaerobic bacterial vaginosis (BV) and aerobic vaginitis (AV), comprising: administering a pharmaceutical composition comprising totarol to a vaginal mucous membrane, wherein administering a pharmaceutical composition comprising totarol comprises administering a pharmaceutical composition including totarol in an amount of 0.001 parts by weight (wt.) to 5.0 parts by wt. as an active substance.
3. The method according to claim 2, wherein administering the pharmaceutical composition comprising totarol treats vaginal mucous membrane inflammations of bacterial origin, alleviates symptoms in such a treatment and provides prophylaxis and prevention of relapses of such inflammations.
4. The method according to claim 2, wherein administering the pharmaceutical composition comprising totarol limits disturbances in a composition of vaginal bacterial flora.
5. The method according to claim 2, wherein the pharmaceutical composition further comprises: from 75 parts by weight (wt.) to 95 parts by wt. of a cellulose derivative; from 0.5 parts by wt. to 5 parts by wt. of lactic acid; and from 0.5 parts by wt. to 5 parts by wt. of a basic polymer, wherein a stoichiometric ratio of lactic acid to the basic polymer is 1:1 to 8:1.
6. The method according to claim 5, wherein the cellulose derivative comprises methylcellulose.
7. The method according to claim d, wherein the basic polymer comprises at least one of the following: an acrylic polymer, a copolymer of methacrylic acid and ethyl acrylate, chitosan, polyvinylpyrrolidone or any combination thereof.
8. The method according to claim 2, wherein the pharmaceutical composition is in a powder form, in a gel form, in a molded pessaries form, or in a tablet form.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The figures of the drawing show:
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DETAILED DESCRIPTION OF THE DRAWINGS
(6) The composition in accordance with the invention is intended for use in prevention and treatment of inflammations of vaginal mucous membrane. It is characterised by a high effectiveness and selectiveness of antibacterial activity, due to application of a strictly defined amount of totarol as the active substance. Additionally, the composition may contain substances exhibiting antifungal and antiprotozoan activity.
(7) The most important, unique advantage of the composition according to the invention in its described form is the fact that the highly selective antibacterial activity of totarol, directed against pathogenic bacteria, does not infract the proper biocenosis and does not destroy lactic acid bacilli of the Lactobacillus genus, being the most significant component of the physiological flora. An additional agent stimulating growth and restoration of correct vaginal flora is constituted by lactic acid, which, due to its regulating function of lowering the pH, forms a barrier against microbes sensitive to acidic environment and creates favourable conditions for growth of bacteria of the Lactobacillus genus.
(8) In the solutions used currently, the therapeutic substance in the form of an antibiotic destructs the pathogenic flora, simultaneously destroying the physiological flora and the lactic acid bacilli being the main component of the latter. The pharmaceutical composition according to the invention, due to the presence of an antibacterially active totarol concentration, determined experimentally, acts in a specific and selective way, only on pathogenic bacteria being the cause of the inflammation resulting from BV or AV. This concentration was determined as a result of a research process, carried out by the present inventors for the first time. Additional presence of lactic acid in the composition, buffered with a basic polymer, acidifies the environment and support growth and development of existing Lactobacillus bacteria. Due to this fact, the composition exhibits a very high effectiveness with a simultaneous lack of adverse impacts on the physiological biocenosis, which limits disease relapses recorded with the majority of patients after BV or AV, occurring in the case of application of classic therapies. Simultaneously, the known possibility for exact pH adjustment by changing the ratio of lactic acid to the basic polymer may be utilised in the composition. The composition adheres well to the whole surface of the mucous membranes, which extends its residence time inside the vagina and provides a uniform contact with the antibacterial agent in the form of totarol.
(9) The composition to be applied in the case of mixed infections may be obtained by supplementing the basic constitution of the composition with substances having profiled antifungal or antiparasitic activity, while selecting a pH proper for the treatment. The composition may be used also for prophylactic purposes or as a product for daily intimate hygiene. The composition in the form of a loose powder may be used for direct gynaecological treatments as a dusting powder or for making preparations in other forms, such as gels or vaginal solutions, after adding a properly selected amount of purified water, or for production of tablets after mixing with known substances which enable pelletizing, or pessaries. The target constitution of all mentioned final preparation may be supplemented with substances exhibiting antifungal or antiprotozoan activity.
(10) By the method according to the invention, a composition with a selective antibacterial activity directed only against pathogenic bacteria is obtained. Due to the uniform application of totarol on particles of a chemically modified cellulose derivative, an effect of development of surface of the contact of the active substance with the mucous membrane in the whole volume of the used preparation is achieved. Also, this solution allowed for significant limiting, and in some cases—eliminating of the necessity to use of large amounts of water-miscible organic solvents in the final products, in order to dissolve the hydrophobic totarol. Simultaneously, as a result of buffering effect of the basic polymer used, an effect of gradual and prolonged release of the active substance together with the lactic acid adjusting the vaginal pH is achieved. The described activity was not possible to obtain using the complex known from the patent descriptions Nos. pl 166898, 194437, 201868, and 201869. The composition produced by the method according to the invention provides its good adhesion to the whole surface of the mucous membranes, which extends its residence time inside the vagina and provides a uniform contact with the antibacterial agent in the form of totarol.
(11) The invention includes also use of the composition defined above for production of a preparation for supportive treatment and alleviation of symptoms in treatment of vaginal mucous membranes' inflammations of bacterial origin. The composition is particularly useful in the case of relapsing infections of the vaginal mucous membrane.
(12) The invention is presented in more detail in the examples.
Example 1
(13) Test of antibacterial activity of totarol using DMI (dimethylisosorbide) as a solvent.
(14) A suspension of 0.5 McF in physiological saline (suspension density 10.sup.8 cfu/ml) was prepared from a 24-hour culture of a standard strain. 1 ml of the suspension was collected to 9 ml of a TSB broth (dilution 10.sup.−). Suspensions with a dilution of 10.sup.−6 were prepared in the described way. A suspension with a cell density of 10.sup.5 cfu/ml was selected for further investigations. For each strain, 2 96-well plates were prepared: the first one was intended for the OD measurement, the second one—for quantitative culture on solid media. A/ to wells B1-D1, B2-D2 and B-D 4-11, 100 pl of the broth were added; B/ to wells B3-D3, 200 pl of the broth were added; C/ to wells B1-D1, 1 pl of stock I was added; D/ to wells B2-D2, 1 pl of stock II was added; E/ to wells B3-D3, 2 pl of stock III were added.
Using a multichannel pipette, 100 pl of the sample were collected from each of the B3-D3 wells and placed in wells B4-D4 (mixing by sucking the content in and letting it out for 5 times). The pipette tips were replaced and 100 pl of the content were collected from of the B4-D4 wells, and placed in wells B5-D5, mixing as previously. These operations were repeated through the wells B11-D11. The content in the B11-D11 wells was mixed, and 100 pl of the fluid were collected and discarded. This way, a series of dilutions of totarol samples in DMI in the range of 0.32% to 0.0004% was obtained. Then, 100 pl of a corresponding bacterial culture were added to the wells B, C, D 1-11. In consequence, a change in the final concentration range occurred, to 0.16%-0.0002%.
Control Preparation: Negative control: 100 pl of the broth+1 pl DMI were added to the Al well. Positive control (I): 100 pl of the broth with the bacterial culture were added to the wells E1-G1. Diluent positive control (II): 100 pl of the broth with the bacterial culture+1 pl DMI were added to the wells E2-G2.
The OD value in the individual wells was read at the wavelength of 620 nm (read time 0). From the second 96-well plate, a culture was inoculated by a quantitative method from every dilution onto a well with a proper solid medium, and incubated for 24 h at 37° C. under aerobic or anaerobic conditions, depending on the strain. The plates were incubated under aerobic conditions or in an atmosphere of an elevated CO.sub.2 concentration (for the strains requiring it) in an incubator at 37° C. The OD was read after 8 h, 16 h, and 24 h, respectively. Before every OD measurement, the contents of the wells were mixed delicately. In parallel to the OD readout, cultures we inoculated from the second 96-well plate, from every dilution, into plates with a proper solid medium, and incubated for 24 h at 37° C., under aerobic or anaerobic conditions. For the analysis of the experimental results, an average value of the OD measurement obtained from three repetitions in the individual times (0, 8, 16, and 24 h) for the individual microbes (Gardnerella vaginalis, Streptococcus agalactiae, Enterococcus faecalis, Lactobacillus gasseri/plantarum) was taken. Additionally, the number of bacteria grown on solid media was taken into account. The results are presented in Tables 1, 2, 3, 4, and 5. The results of the tests confirm the specific, concentration-dependent selectiveness, and the high antibacterial effectiveness of totarol against the pathogenic microorganisms: Gardnerella vaginalis, Streptococcus agalactiae, Enterococcus faecalis at low MIC values safe for bacteria of the Lactobacillus genus.
Example 2
(15) Test of totarol impact on the human vaginal epithelial cells of the A-431 cell line (ATCC® The study was carried out using the following test: Annexin-V-Fluos (Roche, Mannheim, Germany), during incubation for 2, 8 and 24 hours, under in vitro conditions, according to the instructions of the test's manufacturer. The totarol sample was prepared by dissolution of 3 mg of totarol in 3 ml of 100% DMI.
(16) The cultivation time for the A-431 cell line was 20 days. The culture was carried out at a temperature of 37° C. in an atmosphere containing 10% CO.sub.2, on a DMEM culture medium (Institute of Immunology and Experimental Therapy PAS [IITD PAN], Wroclaw), with an addition of 10% of foetal bovine serum (FBS, Sigma-Aldrich). The culture fluid were being replaced regularly every 48 hours. After achieving a decantable growth or the so-called “monolayer”, the cells were passaged using (for approx. 10 min) 0.25% trypsin (Sigma-Aldrich). Then, the obtained tissue line was passed to 24-well plates (TPP), adjusting their densities to the value of 5×10.sup.5 per well. The tissue culture of the studied lines was carried out for 3 next days on the surface of sterile microscope slides placed on the bottom of a 24-well plate, until a decantable growth was obtained on the surface of the microscope slides. After obtaining the so-called “monolayer”, the cells were washed with PBS without Ca.sup.2+ and Mg.sup.2+ ions (IITD PAN, Wroclaw), and poured with 700 pl of fresh DMEM medium with 10% FBS, together with 300 μl of properly prepared totarol. Negative control: 1000 μl of fresh DMEM medium with 10% FBS. Solvent control: 700 μl of fresh DMEM medium with 10% FBS+300 μl of DMI solvent. Necrosis positive control: 900 μl of fresh DMEM medium with 10% FBS+100 μl H.sub.2O.sub.2 (30%).
The proper examination using the Annexin-V-Fluos test causes green staining of apoptotic cells. For staining of necrotic cells, propidium iodide (PI) is used. This dye has an ability to penetrate the cell's interior freely only through a damaged cellular membrane, staining the interior red. In the case of a lack of damages of the cellular membrane, propidium iodide remains on its surface, forming a characteristic red “halo” around the cell. After a corresponding study time, i.e. 2, 8, and 24 hours, the culture fluid was removed, the content was washed with PBS (pre-heated to a temperature of 37° C.) twice. Then, 100 μl of the fluorescent stains mixture including in the Annexin-V-Fluos (Roche) kit were applied on the surface of each microscope slide (placed in a culture well) and additionally, Hoechst stain was used (cellular DNA staining). The staining procedure was carried out according to the manufacturer's recommendations. The culture together with the stains applied onto it were incubated at room temperature for 20 min. The intensity of light emitted by the cells stained with the individual stains was observed using a fluorescent microscope at the wavelength of 494-520 nm, using 400× magnification. The cells were counted in five visual fields, and the results were averaged. The reliability of the tests carried out is confirmed by hydrogen peroxide used as a positive control, causing a distinct necrosis process already after 2 hours of incubation. Data analyses were carried out on the basis of the average number of cells from five visual fields in the individual times (2, 8, and 24 h). The results are presented in Table 6 and in the form of photographic documentation—
Example 3
Preparation of the Composition in the Form of Powder
(17) Composition Ingredients:
(18) TABLE-US-00002 1. Totarol 0.1 parts by wt. 2. Lactic acid 1.0 parts by wt. (2 moles) 3. Eudragit E-100 1.5 parts by wt. (1 mole) 4. Methylcellulose 97.4 parts by wt.
The composition is prepared by dissolving Eudragit in lactic acid in a stoichiometric ratio mentioned in the composition's constitution, in a mixer equipped with a mechanical stirrer, mixing for 20 minutes, then leaving the whole mixture for 24 hours. After this time, the obtained component is dissolved in 95% ethyl alcohol, using 10 parts by wt. of the alcohol and 0.1 parts by wt. of totarol per total amount of ingredients. The whole mixture is mixed until the component is dissolved, then the obtained solution is sprayed through a nozzle onto methylcellulose placed in a mixer of a nozzle sprayer, and mixed until the methylcellulose is wetted uniformly, but not shorter than for 10 minutes. The wet product is subjected to drying at a temperature of 25° C. in a dryer with forced air circulation and solvent condenser. During the drying, ethyl alcohol is removed. A loose powder is obtained, which is mixed to a complete homogenisation and dosed to plastic or glass containers.
Example 4
Preparation of the Composition in the Form of Gel
(19) Composition Iingredients:
(20) TABLE-US-00003 1. Totarol 0.5 parts by wt. 2. Lactic acid 0.5 parts by wt. (1 mole) 3. Chitosan 0.83 parts by wt. (1 mole) 4. Methylcellulose 98.2 parts by wt. 5. Purified water 25 parts by wt. per total amount of ingredients
The composition is prepared similarly as in Example 3, but water is added with mixing to the obtained dry product in the form of powder, and then, after obtaining a homogeneous mixture, it is left for 30 min without mixing. The obtained gel is dosed into plastic containers with applicators.
Example 5
Preparation of the Composition in the Form of Tablets
(21) Composition Ingredients:
(22) TABLE-US-00004 1. Totarol 0.08 parts by wt. 2. Lactic acid 0.5 parts by wt. (1 mole) 3. Polyvinylpyrrolidone-90 5.0 parts by wt. (1 mole) 4. Methylcellulose 94.4 parts by wt.
The composition is prepared similarly as in Example 3, but known substances used in pelletizing are added to the obtained dry product in the form of powder if necessary, and then, the product is pelletized by a known method into proper therapeutic doses.
Example 6
Preparation of the Composition in the Form of Moulded Pessaries
(23) Composition Ingredients:
(24) TABLE-US-00005 1. Totarol 0.5 parts by wt. 2. Lactic acid 4.0 parts by wt. (8 moles) 3. Chitosan 0.83 parts by wt. (1 mole) 4. Methylcellulose 94.4 parts by wt.
(25) Additives per total amount of composition ingredients:
(26) TABLE-US-00006 1. Gelatine 16.5 parts by wt. 2. Purified water 83.5 parts by wt.
The composition is prepared similarly as in Example 3, but the obtained dry product in the form of powder is dissolved in part of the water, giving a gel, to which an aqueous gelatine solution (prepared from the rest of the water), heated to a temperature of 90° C., is added. Then, the product is mixed and poured into moulds, obtaining moulded pessaries.
Appendix
(27) TABLE-US-00007 TABLE 1 Determination of the MIC value of totarol against the Enterococcus faecalis strain. Enterococcus faecalis - clinical strain (initial working density 7.0 × 10.sup.5 cfu/ml) 0 h 8 h 16 h 24 h Totarol OD, OD, OD, OD, concen- Inoculation average of 3 Inoculation average of 3 Inoculation average of 3 Inoculation average of 3 tration cfu/ml measurements cfu/ml measurements cfu/ml measurements cfu/ml measurements Stock I 0.16% 6 × 10.sup.5 0.638 0 0.651 0 0.510 0 0.369 Stock II 0.08% 3.2 × 10.sup.5 0.448 0 0.539 0 0.499 0 0.491 Stock III 0.04% 2.6 × 10.sup.5 0.552 0 0.667 0 0.555 0 0.538 0.02% 3.1 × 10.sup.5 0.322 0 0.384 0 0.374 0 0.371 0.01% 4.1 × 10.sup.5 0.302 0 0.419 0 0.368 0 0.306 0.005% 6 × 10.sup.5 0.302 0 0.349 0 0.320 0 0.296 0.0025% 5 × 10.sup.5 0.284 0 0.297 0 0.295 0 0.302 0.0013% 4 × 10.sup.5 0.293 .sup. 6 × 10.sup.4 0.297 .sup. 6 × 10.sup.4 0.399 .sup. 1 × 10.sup.7 0.549 0.0007% 2.5 × 10.sup.5 0.288 3.1 × 10.sup.5 0.291 3.8 × 10.sup.5 0.445 2.1 × 10.sup.7 0.602 0.0004% 3 × 10.sup.5 0.298 2.1 × 10.sup.5 0.303 1.1 × 10.sup.6 0.491 1.9 × 10.sup.7 0.677 0.0002% 4 × 10.sup.5 0.292 4.1 × 10.sup.5 0.295 2.1 × 10.sup.6 0.490 1.6 × 10.sup.7 0.681 Bacterial Positive 7 × 10.sup.5 0.238 6.2 × 10.sup.5 0.381 6.2 × 10.sup.6 0.531 3.1 × 10.sup.7 0.659 broth control (I) Bacterial Positive 8 × 10.sup.5 0.206 .sup. 6 × 10.sup.5 0.313 4.9 × 10.sup.6 0.470 2.1 × 10.sup.7 0.648 broth + control (II) DMI Broth + Negative 0 0.248 0 0.249 0 0.240 0 0.238 DMI control
Appendix
Appendix
(28) TABLE-US-00008 TABLE 2 Determination of the MIC value of totarol against the Streptococcus agalactiae strain. Streptococcus agalactiae - clinical strain (initial suspension density 1.0 × 10.sup.5 cfu/ml) 0 h 8 h 16 h Totarol OD, OD, OD, 24 h concen- Inoculation average of 3 Inoculation average of 3 Inoculation average of 3 Inoculation tration cfu/ml measurements cfu/ml measurements cfu/ml measurements cfu/ml OD Stock I 0.16% .sup. 2 × 10.sup.5 0.512 0 0.605 0 0.569 0 0.615 Stock II 0.08% .sup. 5 × 10.sup.5 0.563 0 0.564 0 0.540 0 0.430 Stock III 0.04% .sup. 3 × 10.sup.5 0.406 0 0.535 0 0.489 0 0.405 0.02% 10.sup.5 0.318 0 0.323 0 0.324 0 0.324 0.01% 10.sup.5 0.297 0 0.295 0 0.301 0 0.307 0.005% 1.5 × 10.sup.5 0.288 0 0.292 0 0.296 0 0.300 0.0025% 2.6 × 10.sup.5 0.289 0 0.290 0 0.291 0 0.291 0.0013% 3.3 × 10.sup.5 0.299 0 0.302 0 0.302 0 0.302 0.0007% 4.5 × 10.sup.5 0.297 6 × 10.sup.4 0.319 3.5 × 10.sup.6 0.630 6 × 10.sup.7 0.941 0.0004% .sup. 6 × 10.sup.5 0.305 6 × 10.sup.6 0.318 9.1 × 10.sup.5 0.493 2 × 10.sup.7 0.668 0.0002% 3.4 × 10.sup.5 0.289 4 × 10.sup.6 0.314 1.1 × 10.sup.6 0.498 1 × 10.sup.7 0.682 Bacterial Positive 1.0 × 10.sup.5 0.299 3 × 10.sup.7 0.322 .sup. 8 × 10.sup.7 0.501 8 × 10.sup.7 0.748 broth control (I) Bacterial Positive .sup. 3 × 10.sup.5 0.277 8 × 10.sup.7 0.299 .sup. 6 × 10.sup.7 0.501 6 × 10.sup.7 0.747 broth + control (II) DMI Broth + Negative 0 0.333 0 0.344 0 0.330 0 0.343 DMI control
Appendix
Appendix
(29) TABLE-US-00009 TABLE 3 Determination of the MIC value of totarol against the Gardnerella vaginalis strain. Gardnerella vaginalis - clinical strain (initial suspension density 2.8 × 10.sup.5 cfu/ml) 0 h 8 h 16 h 24 h Totarol OD, OD, OD, OD, concen- Inoculation average of 3 Inoculation average of 3 Inoculation average of 3 Inoculation average of 3 tration cfu/ml measurements cfu/ml measurements cfu/ml measurements cfu/ml measurements Stock I 0.16% 1.4 × 10.sup.5 0.822 0 0.564 0 0.451 0 0.438 Stock II 0.08% 6.1 × 10.sup.5 0.740 0 0.523 0 0.455 0 0.441 Stock III 0.04% 3.7 × 10.sup.5 0.460 0 0.416 0 0.411 0 0.379 0.02% 2.1 × 10.sup.5 0.344 0 0.332 0 0.333 0 0.334 0.01% 1.6 × 10.sup.5 0.319 0 0.323 0 0.325 0 0.328 0.005% 3.1 × 10.sup.5 0.318 0 0.318 0 0.323 0 0.327 0.0025% 1.3 × 10.sup.5 0.312 5 × 10.sup.4 0.311 5.2 × 10.sup.4 0.314 2.3 × 10.sup.4 0.317 0.0013% .sup. 1 × 10.sup.5 0.321 2.7 × 10.sup.4 0.314 6.2 × 10.sup.5 0.318 1.2 × 10.sup.5 0.322 0.0007% .sup. 9 × 10.sup.4 0.315 6 × 10.sup.4 0.307 2.1 × 10.sup.5 0.326 3.8 × 10.sup.5 0.346 0.0004% 2.2 × 10.sup.5 0.326 2 × 10.sup.5 0.334 4.9 × 10.sup.5 0.362 3.9 × 10.sup.5 0.390 0.0002% 1.5 × 10.sup.5 0.313 2 × 10.sup.5 0.328 .sup. 2 × 10.sup.5 0.364 2.2 × 10.sup.5 0.398 Bacterial Positive .sup. 3 × 10.sup.5 0.293 7 × 10.sup.5 0.312 6.7 × 10.sup.6 0.312 .sup. 8 × 10.sup.6 0.429 broth control (I) Bacterial Positive .sup. 3 × 10.sup.5 0.321 6 × 10.sup.5 0.336 7.3 × 10.sup.6 0.336 1.1 × 10.sup.7 0.516 broth + control (II) DMI Broth + Negative 0 0.342 0 0.333 0 0.312 0 0.323 DMI control
Appendix
Appendix
(30) TABLE-US-00010 TABLE 4 Determination of the MIC value of totarol against the Lactobacillus plantarum strain. Lactobacillus plantarum - clinical strain (initial suspension density 1.9 × 10.sup.5 cfu/ml) 0 h 8 h 16 h Totarol OD, OD, OD, 24 h concen- Inoculation average of 3 Inoculation average of 3 Inoculation average of 3 Inoculation tration cfu/ml measurements cfu/ml measurements cfu/ml measurements cfu/ml OD Stock I 0.16% 3.7 × 10.sup.5 0.472 .sup. 4 × 10.sup.4 0.320 7.1 × 10.sup.4 0.346 4.1 × 10.sup.4 0.371 Stock II 0.08% 3.2 × 10.sup.5 0.401 1.2 × 10.sup.4 0.413 3.5 × 10.sup.4 0.368 .sup. 5 × 10.sup.4 0.357 Stock III 0.04% 2.6 × 10.sup.5 0.398 1.6 × 10.sup.5 0.370 2.4 × 10.sup.5 0.379 2.4 × 10.sup.5 0.397 0.02% 7.5 × 10.sup.5 0.330 1.0 × 10.sup.5 0.299 5.1 × 10.sup.6 0.837 3.1 × 10.sup.7 1.375 0.01% 5.5 × 10.sup.5 0.295 .sup. 5 × 10.sup.5 0.298 6.4 × 10.sup.6 0.871 3.4 × 10.sup.7 1.445 0.005% 4.2 × 10.sup.5 0.288 1.2 × 10.sup.5 0.300 1.8 × 10.sup.6 0.895 3.8 × 10.sup.7 1.476 0.0025% 2.8 × 10.sup.5 0.284 1.0 × 10.sup.5 0.303 8.4 × 10.sup.6 0.914 1.4 × 10.sup.7 1.552 0.0013% 3.8 × 10.sup.5 0.293 3.2 × 10.sup.5 0.305 5.1 × 10.sup.6 0.913 4.1 × 10.sup.7 1.521 0.0007% 6.5 × 10.sup.5 0.292 4.5 × 10.sup.5 0.300 8.1 × 10.sup.6 0.902 5.1 × 10.sup.7 1.504 0.0004% 2.1 × 10.sup.5 0.304 .sup. 2 × 10.sup.5 0.309 3.9 × 10.sup.6 0.892 3.1 × 10.sup.7 1.508 0.0002% 3.9 × 10.sup.5 0.291 3.5 × 10.sup.5 0.305 4.2 × 10.sup.6 0.854 2.9 × 10.sup.7 1.469 Bacterial Positive 1.9 × 10.sup.5 0.324 6.1 × 10.sup.5 0.317 6.2 × 10.sup.6 0.939 3.2 × 10.sup.7 1.560 broth control (I) Bacterial Positive 1.7 × 10.sup.5 0.308 3.5 × 10.sup.5 0.303 2.9 × 10.sup.6 0.851 .sup. 2 × 10.sup.7 1.397 broth + control (II) DMI Broth + Negative 0 0.330 0 0.331 0 0.329 0 0.311 DMI control
Appendix
Appendix
(31) TABLE-US-00011 TABLE 5 Determination of the MIC value of totarol against the Lactobacillus gasseri strain. Lactobacillus gasseri - clinical strain (initial suspension density 1.4 × 10.sup.5 cfu/ml) 0 h 8 h 16 h Totarol OD, OD, OD, 24 h concen- Inoculation average of 3 Inoculation average of 3 Inoculation average of 3 Inoculation tration cfu/ml measurements cfu/ml measurements cfu/ml measurements cfu/ml OD Stock I 0.16% 6.5 × 10.sup.5 0.427 2.5 × 10.sup.4 0.411 3.1 × 10.sup.1 0.409 .sup. 1 × 10.sup.1 0.406 Stock II 0.08% 2.9 × 10.sup.5 0.526 .sup. 1 × 10.sup.4 0.409 1.9 × 10.sup.2 0.463 1.2 × 10.sup.2 0.472 Stock III 0.04% 4.6 × 10.sup.5 0.352 .sup. 6 × 10.sup.4 0.335 4.5 × 10.sup.3 0.342 .sup. 5 × 10.sup.3 0.349 0.02% 1.4 × 10.sup.5 0.305 1.4 × 10.sup.5 0.299 2.1 × 10.sup.5 0.753 .sup. 1 × 10.sup.5 1.208 0.01% 3.9 × 10.sup.5 0.301 .sup. 7 × 10.sup.4 0.297 5.3 × 10.sup.6 0.799 1.3 × 10.sup.7 1.301 0.005% 1.2 × 10.sup.5 0.292 .sup. 2 × 10.sup.5 0.300 9.1 × 10.sup.6 0.843 2.1 × 10.sup.7 1.387 0.0025% 4.5 × 10.sup.5 0.285 1.5 × 10.sup.5 0.302 2.3 × 10.sup.6 0.880 2.3 × 10.sup.7 1.459 0.0013% 3.8 × 10.sup.5 0.298 1.8 × 10.sup.5 0.301 4.1 × 10.sup.6 0.879 2.1 × 10.sup.7 1.457 0.0007% 1.7 × 10.sup.5 0.289 1.9 × 10.sup.5 0.299 3.6 × 10.sup.6 0.879 .sup. 3 × 10.sup.7 1.458 0.0004% 5.1 × 10.sup.5 0.296 2.1 × 10.sup.5 0.306 3.9 × 10.sup.6 0.915 3.2 × 10.sup.7 1.484 0.0002% 3.1 × 10.sup.5 0.299 2.1 × 10.sup.5 0.302 5.9 × 10.sup.6 0.897 2.9 × 10.sup.7 1.492 Bacterial Positive 1.4 × 10.sup.5 0.293 .sup. 4 × 10.sup.5 0.307 .sup. 1 × 10.sup.7 0.885 .sup. 1 × 10.sup.8 1.463 broth control (I) Bacterial Positive .sup. 1 × 10.sup.5 0.297 1.4 × 10.sup.4 0.307 .sup. 9 × 10.sup.6 0.776 .sup. 9 × 10.sup.7 1.246 broth + control (II) DMI Broth + Negative 0 0.330 0 0.331 0 0.329 0 0.311 DMI control
Appendix
Appendix
(32) TABLE-US-00012 TABLE 6 Influence of the tested substance - totarol - on the apoptosis and necrosis phenomena of human cells of vaginal epithelium A-431. Human vaginal epithelium line A-431 2 h 8 h 24 h Alive Apopt. Necrot. Alive Apopt. Necrot. Alive Apopt. Necrot. Tested samples cells cells cells cells cells cells cells cells Cells Totarol 100 0 0 100 0 0 100 0 0 Negative 100 0 0 100 0 0 99 0 1 control Solvent 100 0 0 99 0 1 97 0 3 control Necrosis 0 0 100 0 0 100 0 0 100 positive control