SUBSTANCE IN THE FORM OF AN AQUEOUS EXTRACT OF VEGETAL RAW MATERIAL FOR TREATING ONCOLOGICAL DISEASES, AND METHOD FOR PRODUCING THE SAME

Abstract: The present invention relates to a substance in the form of an aqueous extract of vegetal raw material which is used for treating oncological diseases, as well as a method for producing said substance. The substance comprises the aqueous extracts of plants from the genus Geranium and Plantago as well as from the species Calendula officinialis, wherein said extracts are obtained from the following amounts in grams of said plants and water : from 10 to 60 of dried Geranium or from 300 to 360 of fresh Geranium ; from 10 to 60 of dried Plantago ; from 10 to 60 of dried Calendula officinialis : and 300 grams of water. This substance may also contain an extract of a plant from the species Pentaphilloides fruticosa obtained from an amount of 10 to 60 grams of dried plant as well as an extract of a plant from the genus Angelica obtained from an amount of 10 to 60 grams of dried plant. The method for producing said aqueous extracts comprises macerating the plants in water before press-filtering the mixture and submitting it to a centrifugal treatment. This substance may be used in oncological practice as part of a cancer therapeutic program.

Description: The present invention relates to medicine, particularly to phytotherapy, and still more particularly to a composition for treating oncological diseases, said composition comprising an aqueous extract of vegetable stock, and to a process for preparing thereof. An anticancer preparation is known in the art, comprising as one of components an extract of a plant of the genus Geranium. The formulation of this preparation comprises also sulfuric acid, boric acid, an alcohol, and glycerol [1]. A disadvantage of this composition is that it can be recommended for external application only. A composition is known, recommended as an auxiliary means which enhances the antitumoric activity, whose formulation, among other aqueous or aqueous-organic extracts of plants, comprises extracts of Astragalis, Pasonia, and Angelica [2]. Also known in the art is a multicomponent composition for antitumoric use, comprising plants of the genera Plantago and Angelica as components [3]. These compositions are disadvantageous in being multicomponent. The present invention was directed to the provision of a composition for treating oncological diseases in the form of aqueous extracts of vegetable stock, said stock featuring a sufficiently simple composition, and to the provision of a process for preparing thereof. For solving the posed problem, a composition is proposed, comprising aqueous extracts of plants of the genera Geranium, Plantago, plant of the genus Calendula officinalis, prepared with the following ratio of water and plants, g: Geranium 300-360 fresh or 10-60 dry Plantago 10-60 dry Calendula officinalis 10-60 dry Water 3000 and Pentaphylloides fruticosa in an amount of 10-60 g (dry). Besides Pentaphylloides fruticosa, a plant of the genus Angelica in an amount of 10-60 g (dry) can be added. The use of separate components of the proposed composition in amounts smaller than the lower levels of said ranges lowers the therapeutic effect, whereas the upper levels of said ranges are limited by the volume of water, required for preparing said aqueous extracts. In the proposed composition the plant of the genus Geranium can be represented by the species sibiricum, pratense, and robertianum; Plantago can be represented by the species lanceolata and media; Angelica can be represented by the species sylvestris and decurrens. A process is known for preparing an extract of plants by treating them with water or with an aqueous-organic solvent [4]. A process of preparing a composition of vegetable origin is also known, which comprises extraction of comminuted grass with water by maceration, followed by filtration and re-maceration of the vegetable stock [5]. This process appears to be technologically complicated, since it involves a two-stage maceration. It is an object of the proposed invention to provide a simple process for preparing an aqueous extract of plants for treating oncological diseases. To solve this problem, it is proposed to macerate plants (grass, leaves or roots) in water, with subsequent straining and centrifugation. The biological activity of the plant extracts was assayed by determining the antioxidant and cytotoxic activity in vitro, as well as the toxicity and antitumoric activity in vivo. The antioxidant activity of the extracts was determined from the ability of a sample to inhibit the lipid peroxidation processes (LPP) in murine liver homogenate [6]. The cytotoxic activity of the extracts in vitro was determined with the help of a biological test based on inhibition of the proliferation of the inoculated cell culture of human pulmonary adenocarcinoma A-549 [7]. The antitumoric activity of the extracts was assessed on mice BDF 1 suffering from leukemia, using different doses of the extracts. The assessment criteria were inhibition of the tumor growth and an increase in the lifetime of the animals. In addition, the effect of the extracts on the therapeutic activity of the cytostatic cis-dichlorodiamine platinum (DDP) [8] was estimated. The proposed invention is illustrated by the Examples presented hereinbelow. Comparative Example 1 360 g of comminuted fresh grass Geranium sibiricum, 25 g of dry leaves of Plantago lanceolata, and 25 g of dry leaves of Calendula officinalis are soaked in 3000 g of water at 25°C for 8 days. On completion of the maceration procedure, the extract is strained and centrifugated under standard conditions (20°C, 3000 rpm, 40 min). The precipitate is discarded, and the extract is used. Comparative Examples 1a-1e Extracts of plants are prepared by following the procedure of Example 1, but the quantity of the mixture components and the maceration conditions are varied. The formulations are specified in Table 1. Examples 2-2h Extracts of plants are prepared by following the procedure of Example 1, but comminuted dry sprouts of the plant Pentaphylloides fruticosa are added to the formulation of the mixture being soaked. The formulations of the vegetable stock and the maceration conditions are specified in Table 1. Examples 3-3f Extracts of plants are prepared by following the procedure of Example 1, but, in addition to Pentaphylloides fruticosa, comminuted underground organs of the plant Angelica (species sylvestris and decurrens) are introduced into the formulation of the mixture being soaked. The formulations of the vegetable stock and the maceration conditions are specified in Table 1. Ex. No. Description of Stock Consistence of stock Weight (g) Temp. (°C) Time (days) 1 Grass Geranium sibiricum Fresh 360 28 8 Leaf of Calendula officinalis Dry 25 Leaf of Plantago lanceolata Dry 25 1a Grass Geranium pratense Dry 60 26 7 Leaf of Calendula officinalis Dry 10 Leaf of Plantago lanceolata Dry 10 1b Grass Geranium pratense Dry 25 25 8 Leaf of Calendula officinalis Dry 25 Leaf of Plantago lanceolata Dry 25 1c Grass Geranium sibiricum Fresh 300 28 7 Leaf of Calendula officinalis Dry 60 Leaf of Plantago lanceoloata Dry 10 1d Grass Geranium sibiricum Fresh 320 27 6 Leaf of Calendula officinalis Dry 10 Leaf of Plantago lanceoloata Dry 60 1e Grass Geranium sibiricum Dry 10 28 7 Leaf of Calendula officinalis Dry 60 Leaf of Plantago media 25 2 Grass Geranium sibiricum Fresh 360 26 8 Leaf of Calendula officinalis Dry 25 Leaf of Plantago lanceolata Dry 25 2a Sprouts of Pentaphylloides fruticosa Dry 25 Grass Geranium sibiricum Fresh 360 25 6 Leaf of Calendula officinalis Dry 25 Leaf of Plantago lanceolata Dry 25 Sprouts of Pentaphylloides fruticosa Dry 10 2b Grass Geranium sibiricum Fresh 360 25 6 Leaf of Calendula officinalis Dry 25 Leaf of Plantago lanceolata Dry 25 Sprouts of Pentaphylloides fruticosa Dry 10 2c Grass Geranium robertianum Dry 60 28 7 Leaf of Calendula officinalis Dry 25 Leaf of Plantago lanceolata Dry 25 Sprouts of Pentaphylloides fruticosa Dry 10 2d Grass Geranium robertianum Dry 10 28 7 Leaf of Calendula officinalis Dry 25 Leaf of Plantago lanceolata Dry 25 Sprouts of Pentaphylloides fruticosa Dry 60 2e Grass Geranium sibiricum Fresh 360 28 8 Leaf of Calendula officinalis Dry 10 Leaf of Plantago lanceolata Dry 10 Sprouts of Pentaphylloides fruticosa Dry 10 2f Grass Geranium sibiricum Fresh 360 28 8 Leaf of Calendula officinalis Dry 60 Leaf of Plantago lanceolata Dry 10 Sprouts of Pentaphylloides fruticosa Dry 10 2h Grass Geranium sibiricum Fresh 360 28 8 Leaf of Calendula officinalis Dry 10 Leaf of Plantago lanceolata Dry 60 Sprouts of Pentaphylloides fruticosa Dry 10 3 Grass Geranium pratense Dry 10 28 6 Leaf of Calendula officinalis Dry 25 Leaf of Plantago media Dry 25 Sprouts of Pentaphylloides fruticosa Dry 25 Root of Angelica silvestris Dry 60 3a Grass Geranium pratense Dry 10 28 6 Leaf of Calendula officinalis Dry 25 Leaf of Plantago media Dry 25 Sprouts of Pentaphylloides fruticosa Dry 10 Root of Angelica silvestris Dry 60 3b Grass Geranium pratense Dry 60 27 7 Leaf of Calendula officinalis Dry 25 Leaf of Plantago media Dry 25 Sprouts of Pentaphylloides fruticosa Dry 10 Root of Angelica decurrens Dry 10 3c Grass Geranium pratense Dry 40 27 7 Leaf of Calendula officinalis Dry 25 Leaf of Plantago media Dry 25 Sprouts of Pentaphylloides fruticosa Dry 10 Root of Angelica decurrens Dry 10 3d Grass Geranium pratense Dry 60 27 8 Leaf of Calendula officinalis Dry 25 Leaf of Plantago lanceolata Dry 25 Sprouts of Pentaphylloides fruticosa Dry 25 Root of Angelica decurrens Dry 60 3e Grass Geranium pratense Dry 60 28 8 Leaf of Calendula officinalis Dry 25 Leaf of Plantago lanceolata Dry 25 Sprouts of Pentaphylloides fruticosa Dry 40 Root of Angelica silvestris Dry 30 3f Grass Geranium pratense Dry 60 26 7 Leaf of Calendula officinalis Dry 10 Leaf of Plantago lanceolata Dry 10 Sprouts of Pentaphylloides fruticosa Dry 10 Root of Angelica silvestris Dry 60 Determination of the Antioxidant Activity of vegetable Extracts The antioxidant activity was used by using a method based on the ability of the sample being investigated to inhibit LPP processes in murine liver homogenate [6]. The liver of BDF-1 (nondescript) mice was homogenized in a glass homogenizer and then centrifugated. The liver homogenate was introduced in an amount of 5-6 mg of protein per assay. The volume of the assay was brought to 0.5 ml with a solution of the sample being tested for the antioxidant activity. The assays were incubated for 3 h at 37°C. The concentration of the LPP products was determined from the color reaction with thiobarbituric acid. The quantity of the antioxidant required for the 50% inhibition of LPP was adopted as a conventional unit of the sample under investigation. The results are presented in Tables 2 and 2a. Comparison of the antioxidant activity of samples of vegetable extracts with the activity of prior art antioxidant preparations Nos. Preparation Antioxidant activity, conv. units (1/mg) 1 1 333 2 2 333 3 3 333 4 Retinol acetate (vitamin A) 83 5 Ascorbic acid (vitamin C) 12 6 β-Carotene 6 Comparison of the antioxidant activity of samples of vegetable extracts under storage Ex. No. Quantity of sample, required for attaining 50% inhibition of LPP, mg/ml of assay 2 days after preparation 16 days after preparation 30 days after preparation 1 3.0 x 10 -3 9.0 x 10 -3 9.2 x 10 3 1b 7 x 10 -3 1.2 x 10 -2 1.3 x 10 -2 1c 3.2 x 10 -3 9.1 x 10 -3 9.0 x 10 3 2 3.4 x 10 -3 7.0 x 10 -3 6.9 x 10 3 2d 6.8 x 10 -3 6.9 x 10 -3 7.0 x 10 3 2h 3.8 x 10 -3 6.9 x 10 -3 6.8 x 10 3 3 3.8 x 10 -3 6.0 x 10 -3 6.3 x 10 3 3b 7.1 x 10 -3 1.5 x 10 -2 1.5 x 10 -2 3c 9.2 x 10 -3 1.9 x 10 -2 2.0 x 10 -2 As can be seen from Table 2, extracts 1, 2, and 3 have a high antioxidant activity in the test in vitro, which exceeds appreciably the antioxidant activity of the known antioxidative vitamins: A, C and β-carotene (4.28 and 55-fold, respectively). On storage of the vegetable extracts (Table 2a) for 16-30 days, their antioxidant activity becomes lower by a factor of 1.5-2 due to the formation of a precipitate, into which active components are involved as well. Nevertheless, the antioxidant activity remains appreciably higher than the activity of vitamin preparations. Determination of the Cytotoxic Activity of Extract Samples in vitro When carrying out the cytotoxic biological test in vitro, each well of a flat-bottomed 96-well microtiter (Sarstedt, USA) was charged with 10 µl of a suspension of A549-line cells in a concentration of 1x10 5 cell/ml; 24 h later series dilutions of the vegetable preparations in the range of 1:4-1:64 were added, this corresponding to 3.0-0.1 mg/ml in the volume of 100 µl/well. The cytotoxic activity of the extracts and DDP was estimated colorimetrically [7]. The results were expressed in the percentage of inhibition of the A-549 test-culture proliferation. The cytotoxic activity of the sample was adopted to be the maximum titer of the sample, at which the biologically significant (50%) level of inhibition of the A-549 test-culture proliferation was observed. The results are presented in Table 3. Cytotoxic activity of samples of vegetable extracts in terms of inhibition of the proliferation of cell culture of human pulmonary adenocarcinoma A-549 Example No. Maximum titer Inhibition, % 1 1/8 (1.2 mg/ml) 50 1a 1/8 (1.2 mg/ml) 58 1b 1/8 (1.2 mg/ml) 60 1c 1/8 (1.2 mg/ml) 59 2 1/64 (0.1 mg/ml) 66 2a 1/64 (0.1 mg/ml) 80 2b 1/64 (0.1 mg/ml) 72 2d 1/64 (0.1 mg/ml) 78 3 1/4 (3.0 mg/ml) 79 3a 1/4 (3.0 mg/ml) 83 3c 1/4 (3.0 mg/ml) 80 DDP 10 µg/ml 68 As can be seen from Table 3, the investigated samples of the vegetable extracts have a cytotoxic activity in the experiments in vitro. The maximum ability to inhibit proliferation of the cell culture A-549 was observed in sample 2 (66-80% at the concentration of 0.1 mg/ml), but it was one tenth that of DDP (68% at the concentration of 0.01 mg/ml), i.e., of the preparation conventionally used for induced cytotoxicity control. Investigation of Tolerance to Vegetable Extract Samples The tolerance to and harmlessness of the samples were investigated on animals with L-1210 inoculated subcutaneously. Samples of the extracts were administered through a probe many times throughout the lifetime of the animals, starting with the first day of the tumor growth, in single doses of 5.4 and 10.8 ml/kg in the volume of 0.2 ml or intraperitoneally in the volume of 1 ml. Control mice were administered a 0.9% NaCl solution instead of the vegetable extracts. The toxic effect was estimated from the change in the body weight of mice and also from their external appearance (behavior, the appearance of the hair-cover, their motor activity). For a quantitative estimation of changes in the motor activity, an integral characteristic was introduced: (a/b)x100%, where a is the total number of mice with a lowered motor activity from the 1 st to the 6 th day of the tumor growth, b is the total number of observations from the 1 st to the 6 th day of the tumor growth (1 observation - 1 living mouse per day). The results of the experiments showed that the vegetable extracts in the single dose of 5.4 ml/kg do not affect the body weight, behavior, the external appearance of the hair-cover or the mobility of the mice. The administration of the vegetable extracts of some of the series in the single dose of 10.8 ml/kg brought about a weak toxic effect manifested in a reduction of the motor activity of the test animals in 5-10% of the observations (Fig. 1). An increase of the single dose of the vegetable extracts to 1 ml (maximum permissible volume) in the case of single intraperitoneal administration of the preparations of series b, c, d did not lead to the development of visible disturbances in the behavior and external appearance of the animals or cause their death. This suggests that the LD 10 , LD 50 , and LD 100 values cannot be determined for these particular vegetable extracts. Determination of the Antitumoric Activity of Vegetable Extract Samples Lymphoid leukemia L1210 was inoculated to BDF 1 males in a sterile physiologic salt solution, 3x10 6 cells in 0.2 ml volume. The day of the inoculation was regarded as the zero day of tumor growth. The extract samples and DDP were administered in the doses and under the conditions specified in Tables 4, 5. Treatment of the animals was started 24 hours after the tumor inoculation, control animals were administered a physiologic salt solution instead of the vegetable extracts. The antitumoric effect was estimated from the tumor growth inhibition (TGI) calculated from theformula: TGI = [(Vcontrol - Vexperiment)/Vcontrol]x100%; mean lifetime (ML), lifetime increase (LI) calculated from the formula: LI = [(MLexperiment - MLcontrol)/Mlcontrol]x100%. TGI > 50%, LI > 50% were considered to be biologically significant effects. The obtained data were processed in accordance with the Fisher-Student procedure, using statistical programs. The differences were regarded confident at p < 0.05. The results of action of the vegetable extracts are presented in Table 4, from which it is seen that preparations 2 and 3 are able to inhibit the growth of L1210 in vivo. The results of action of the vegetable extracts on the curative effect of DDP are presented in Table 5. As can be seen from Table 5, preparations 2 and 3 increase the therapeutic effect of the cytostatic, bringing about an increase of the TGI value by the 6 th , 8 th days of the tumor growth. Influence of vegetable extract samples on L-1210 growth Ex. No. Dose, ml/kg TGI, % days LI, % 4 6 8 2 5.4 26 46 36 24 2d 5.4 28 48 39 10 2f 5.4 20 44 35 12 2 10.8 41 38 37 2 2d 10.8 41 36 37 8 2f 10.8 40 37 34 5 3 5.4 56 24 25 -7 3c 5.4 50 25 24 2 3f 5.4 57 25 24 -1 3 10.8 65 27 22 -7 3c 10.8 67 27 23 4 3f 10.8 65 30 21 0 Influence of vegetable extract samples on curative effect of DDP Action Dose, ml/kg TGI, % days LI, % 4 6 8 DDP 9 mg/kg 100 93 33 41 DDP +2 10.8 100 92 43 37 DDP +2d 10.8 100 90 39 40 DDP +2f 10.8 100 90 38 35 DDP +3 10.8 100 92 69 30 DDP +2b 5.4 100 40 47 11 DDR +2e 5.4 100 41 40 8 DDP +3 5.4 100 63 41 23 DDP +3c 5.4 100 65 44 20 DDP +3e 5.4 100 66 44 24 DDP 10 mg/kg 100 87 65 32 DDP +2 5.4 100 100 88 33 DDP +2c 5.4 100 98 87 29 DDP +2e 5.4 100 100 90 32 DDP +3 5.4 100 95 74 31 DDP +3d 5.4 100 91 71 32 DDP +3e 5.4 100 97 79 30 As can be seen from Tables 4 and 5, preparations 2 and 3 do not reduce the therapeutic effect of DDP, and under definite administration conditions they even enhance it. Preparation 3 has its own weakly pronounced antitumoric effect, causing a biologically significant inhibition of tumor growth in the early periods of the tumor development. Hence, the vegetable extracts may be promising for use in oncological practice, when included into antitumoric therapy procedures.

Claims: A composition for treating oncological diseases in the form of an aqueous extract of vegetable stock, comprising extracts of plants of the genera Geranium, Plantago and a plant of the species Calendula officinalis as well as a plant of the species Pentaphylloides fruticosia, prepared with the following ratio of plants and water, g: Geranium 10-60 dry or 300-360 fresh Plantago 10-60 dry Calendula officinalis 10-60 dry Pentaphylloides fruticosa 10-60 dry Water 3000 Composition according to Claim 1, characterized in that it additionally comprises a plant of the species Angelica, in an amount of 10-60 g dry. A process for preparing an aqueous extract from vegetable stock for treating oncological diseases, characterized in that plants of the genera Geranium, Plantago and a plant of the species Calendula officinalis and a plant of the species Pentaphylloides fruticosa are macerated in water, this being followed by straining and centrifugation. A process according to Claim 3, characterized in that the plant Angelica is additionally introduced into the composition of vegetable stock for maceration.

Inventors: CHISSOV VALERY IVANOVICH, GAVRILJUK OLGA ALEXEEVNA, KAZACHKINA NATALYA IVANOVNA, LUZHKOV JURY MIKHAILOVICH, NEMTSOVA ELENA ROMANOVNA, SHULTS ELVIRA EDUARDOVNA, TOLSTIKOV GENRIKH ALEXANDROVIC, VINOKUROVA ELENA JURIEVNA, VOROZHTSOV GEORGY NIKOLAEVICH, YAKUBOVSKAYA RAISA IVANOVNA, CHISSOV, VALERY IVANOVICH, GAVRILJUK, OLGA ALEXEEVNA, KAZACHKINA, NATALYA IVANOVNA, LUZHKOV, JURY MIKHAILOVICH, NEMTSOVA, ELENA ROMANOVNA, SHULTS, ELVIRA EDUARDOVNA, TOLSTIKOV, GENRIKH ALEXANDROVICH, VINOKUROVA, ELENA JURIEVNA, VOROZHTSOV, GEORGY NIKOLAEVICH, YAKUBOVSKAYA, RAISA IVANOVNA
Applicants: G NAUCHNY TS ROSSIISKOI FEDERA, MO NI ONKOLOGICHES INST, NOVOSIB I ORCH KHIM, GOSUDARSTVENNY NAUCHNY TSENTR ROSSIISKOI FEDERATSII "NIOPIK" (GNTS RF "NIOPIK"), MOSKOVSKY NAUCHNO-ISSLEDOVATELSKY ONKOLOGICHESKY INSTITUT IMENI P.A. GERTSENA, NOVOSIBIRSKY INSTITUT ORGANICHESKOI KHIMII
Application year: 1997
Main classification: A61K35
IPC codes: A61K9, A61K36, A61P35