RU2189832C1 - Agent with antimetastatic activity - Google Patents

Abstract

FIELD: medicine, oncology, phytotherapy. SUBSTANCE: invention proposes an antimetastatic agent comprising extract from the above-ground part (herb) of Baikal aconite (Aconitum baikalense Steinb.), in part, a liquid extract from the dried milled above-ground part (herb) of Baikal aconite with 40% ethyl alcohol in the ratio of vegetable raw to extractant = 1:50 and a liquid extract from the dried and milled above-ground part (herb) of Baikal aconite with 30% ethyl alcohol in the ratio of vegetable raw to extractant = 1: 20 and a liquid extract from the dried milled above-ground part (herb) of Baikal aconite with 40% ethyl alcohol in the ratio of vegetable raw to extractant = 1:1 and a dry extract from the dried and milled above- ground part (herb) of Baikal aconite with 40% ethyl alcohol in the ratio of vegetable raw to extractant = 1:1. Invention proposes also an agent comprising the dried extract of total alkaloids isolated from the dried and milled above-ground part (herb) of Baikal aconite. Proposed agents show the sufficiently strong an antimetastatic effect and can be used for treatment of patients with metastasing tumors. EFFECT: enhanced effectiveness of agent, valuable antitumor effect. 7 cl, 3 tbl

Description

 The invention relates to the pharmaceutical industry and medicine, relates to methods for producing a medicinal product from plant materials and can be used to treat patients with metastatic tumors.

 The closest (prototype) is befungin, used in medicine in the treatment of cancer patients - befungin (chaga extract) [1]. Its antimetastatic properties are known [2, 3].

 However, at present, the issue of expanding the arsenal of drugs with antimetastatic action from natural raw materials is extremely urgent.

 The objective of the invention is to expand the arsenal of funds with antimetastatic activity from natural raw materials.

 This object is achieved in that the antimetastatic agent contains an alcohol extract from the aboveground part (grass) of Baikal aconite (Aconihim baikalense Turcz. Ex Rapaics), in particular a liquid extract from dried ground part (grass) of Baikal aconite 40% ethyl alcohol in the ratio of plant materials and extractant 1:50, and a liquid extract from the dried and crushed above-ground part (grass) of Baikal aconite with 30% ethanol with a ratio of plant materials and extractant 1: 20, and a liquid extract from dried and ground ground part (grass) of Baikal aconite 40% ethyl alcohol with a ratio of plant materials and extractant 1: 1, and a dry extract from dried and ground ground part (grass) of Baikal aconite 40% ethyl alcohol with a ratio of plant materials and extractant 1: 1 , as well as a dry extract of the amount of alkaloids isolated from the dried and crushed aboveground part (grass) of Baikal aconite.

 Means with antimetastatic activity are alcohol extracts from Baikal aconite containing a complex of active and related substances extracted from crushed grass of Baikal aconite 30% and 40% ethyl alcohol followed by settling and filtration, as well as an alkaloid extract extracted with chloroform and containing the amount of alkaloids.

 2% liquid plant anti-metastatic agent is a clear liquid of light brown color, a peculiar smell.

 10% liquid plant anti-metastatic agent is a clear brownish-brown liquid, a peculiar smell, burning taste.

 50% liquid plant anti-metastatic agent is a dark brown liquid, a peculiar smell and a burning taste.

 50% dry plant anti-metastatic agent is a brown powder, a peculiar smell and a burning taste.

 The standard liquid plant anti-metastatic agent is a brown liquid, a peculiar smell and a burning taste, containing 0.01-0.007% alkaloid.

 The herbal antimetastatic agent containing the amount of alkaloids is a white powder of a peculiar smell.

 The authors did not find these distinguishing features in the literature they analyzed. This set of distinctive features does not explicitly follow for a specialist. The proposed funds can be used in medical practice.

 Thus, the proposed solution meets the criteria of the invention - "novelty", "inventive step", "industrially applicable".

 Distinctive features showed new properties in the claimed combination. It was found for the first time that products containing extracts from dry ground ground part of the grass of Baikal aconite 30%, 40% ethanol in the proposed ratio, as well as the amount of alkaloids have high antimetastatic activity.

 Examples of specific obtaining funds with antimetastatic activity.

 Example 1. Extraction is carried out by maceration. The collected air-dried grass of Baikal aconite is crushed to 3 mm, 100 g of raw material is poured into 1000 l of 40% ethyl alcohol and insisted in a closed vessel for 7 days at room temperature, stirring occasionally. Filter through filter paper and measure the recovery volume. It is equal to 860 ml. The raw materials are squeezed well, the meal is poured with the missing amount of extractant (140 ml) and squeezed again, the extraction is brought to the original volume (1000 ml) with 40% ethanol. Received 2% herbal remedy - 2% tincture of Baikal aconite.

 Example 2. Extraction is carried out by the method of fractional maceration. 100 g of air-dried crushed grass of Baikal aconite is placed in a percolator equipped with a false bottom, covered with a layer of filter material (calico). The raw materials are compacted and placed on top of the clamping lattice. 0.54 l of 30% ethyl alcohol is fed into the percolator, the percolator is sealed and left to infuse for 24 hours.

 On the second day, 0.25 L of extraction is poured from the percolator into the collection, while simultaneously feeding 0.25 L of extractant (30% ethyl alcohol) into the percolator, and left for 2 hours to infuse.

 After 2 hours, 0.25 L of extraction is poured from the percolator into the collection, while simultaneously feeding 0.25 L of extractant into the percolator, and left to infuse for 2 hours.

 After 2 hours, 0.25 L of extract is drained from the percolator, while simultaneously feeding 0.20 L of extractant into the percolator, and left to infuse for 2 hours.

 After 2 hours, the extract is completely drained from the percolator into the collection tank. Extract from the collection pumped into the sump and left for 5 days at room temperature or in a cool place.

 From the sump, the extraction merges in layers, without stirring up sediment, and is filtered through a triple layer of calico. The yield of the finished product should be at least 1 liter. Received 10% herbal remedy - 10% tincture of Baikal aconite.

 Example 3. 200 g of air-dried crushed grass of Baikal aconite are loaded into five extractors and the first of them is fed from above with 40% ethanol in an amount that creates a layer over the raw material of 5-7 cm and left for 17 hours. After this period, the liquid from the first extractor is poured into the second, and 40% ethanol is fed into the first extractor until the initial level is obtained. After 17 hours, successive drains are made from the 2nd extractor to the 3rd, from the 1st to the 2nd, and fresh solvent is fed into the first. Every 17 hours, these operations are repeated until the 5th extractor is filled. Then, 200 ml of extract is obtained from the 5th extractor, while simultaneously producing successive plums from the 1st to the 2nd, from the 2nd to the 3rd, from the 3rd to the 4th, and from the 4th to the 5th suspended extractor. After an interval of 17 hours, from the 5th extractor with a similar sequence of moving the liquid, we obtain a liquid extract in 200 ml portions four more times. The resulting extracts are combined (1 L). Received 50% liquid herbal remedy - akonite extract of Baikal liquid.

 Example 4. 200 g of air-dried crushed grass of Baikal aconite are loaded into five extractors and the first of them is fed from above with 40% ethanol in an amount that creates a layer over the raw material of 5-7 cm and leave it for 17 hours. After this period, the liquid from the first extractor is poured into the second, and 40% ethanol is fed into the first extractor until the initial level is obtained. After 17 hours, successive drains are made from the 2nd extractor to the 3rd, from the 1st to the 2nd, and fresh solvent is fed into the first. Every 17 hours, repeat these operations until the 5th extractor is filled. Then, 200 ml of extract is obtained from the 5th extractor, while simultaneously producing successive plums from the 1st to the 2nd, from the 2nd to the 3rd, from the 3rd to the 4th, and from the 4th to the 5th suspended extractor. After an interval of 17 hours, from the 5th extractor with a similar sequence of fluid movement, a 200 ml liquid extract is obtained in portions of 200 ml. The resulting extracts are combined (1 L). The liquid extract is evaporated under vacuum, followed by grinding the resulting residue to a powder state. The yield of the target product is 12.5% by weight of air-dried raw materials. Received 50% dry herbal remedy - dry Baikal aconite extract.

 Example 5. Act as in example 2. The resulting tincture is analyzed for alkaloids according to [4, 5] and diluted with 30% ethyl alcohol as calculated to a concentration of 0.01-0.007%.

 Example 6. 100 g of dried and ground raw materials are moistened with a 5% soda solution and loaded into a percolator. 2 hours after loading, 2000 ml of chloroform is poured into it. Extraction with chloroform is carried out in a continuous laboratory apparatus consisting of a percolator, a film evaporator and a direct refrigerator. Continuous circulation of the solvent is carried out at a speed of 2 l / h. Duration of exhaustive extraction is 5 days. The concentrated chloroform extract is drained through an evaporator tap. The last portion of the chloroform extract from the percolator is drained through the bottom valve of the percolator and concentrated in the evaporator. The obtained chloroform extract of alkaloids is purified from non-alkaloid compounds by extraction with an aqueous solution of sulfuric acid. Further purification of the amount of alkaloids is carried out by repeated chloroform extraction from the sulfate recovery after neutralization and alkalization to pH = 10-12. In order to get rid of traces of alkali that could pass into the chloroform layer, it is washed with 50 ml of water. The chloroform extract is then filtered through a desiccant (15 g of anhydrous sodium sulfate). At the last stage, the resulting amount of alkaloids is evaporated to dryness. The yield of 0.23%. Received herbal remedy containing the amount of alkaloids. It has been established that the composition of the alkaloids includes the known diterpenic alkaloids: napellin, zongorin, mesaconitin, hypaconitin and the new alkaloid N-oxide 12-epinapellia [6].

 To select the most effective dosage form of Baikal aconite, 2% herbal remedy (2% tincture), 10% herbal remedy (10% tincture), 50% liquid and dry herbal remedies (liquid and dry extracts), herbal remedy containing the amount were used: alkaloids.

 Comparison drugs were: 5-fluorouracil - an antimetabolite from the fluoropyrimidine group. This is a basic drug that has a wide spectrum of antitumor activity and is most widely used in oncological practice [7] and the prototype is befungin, a semi-thick extract from chaga (birch mushroom). Clinical studies of the drug from chaga showed a number of positive effects. There is literature data on the successful treatment of breast cancer [8], inoperable forms of cancer of the female genital area [9], radiation leukopenia [10], etc. In experimental medicine, the use of chaga preparations delays the development of transplanted tumors, and often their resorption [ eleven].

 There is a method of producing an antitumor agent of befungin (chaga extract), which is a semi-thick extract obtained from mushroom growths (birch fungus fungi) formed on birches by the phytopathogenic parasite Inonotus obliguus [12].

 A study of the effect of herbal remedies from Baikal aconite on the growth and dissemination of metastases was carried out in mice with Lewis lung carcinoma, B-16 melanoma. The experiments were conducted on non-inbred mice of both sexes weighing 18-25 g, obtained from the collection fund of the Research Laboratory of Experimental Biomedical Modeling of the Siberian Branch of the Russian Academy of Medical Sciences (certificate is available). Tumors were inoculated using standard well-known methods [13].

1 - R=OH мезаконитин
2 - R=H гипаконигин
Мезаконитин (1) - C₃₃H₄₅NO₁₁, М⁺ 631, т. пл. 195,5-196^oС (метанол), [α] $\genfrac{}{}{0ex}{}{\text{20}}{\text{546}}$ +32,75 (С 3,47; хлороформ).

1 - R = OH mesaconitin
2 - R = H hypaconigin
Mesaconitin (1) - C ₃₃ H ₄₅ NO ₁₁ , M ⁺ 631, mp 195.5-196 ^o With (methanol), [α] $\genfrac{}{}{0ex}{}{\text{20}}{\text{546}}$ +32.75 (C 3.47; chloroform).

3 - R₁=oh, R₂=H напеллин
4 - R₁+R₂=O зонгорин
Напеллин (3) - C₂₂H₃₃NO₃, M⁺ 359, т. пл. 156-157^oС (ацетон)
Зонгорин(4) - С₂₂Н₃₁NO₃, M⁺ 357, т.пл. 182-187^oС (эфир).Hypaconitin (2) - C ₃₃ H ₄₅ NO ₁₀ , M ⁺ 615, mp 179-181 ^o With (methanol)

3 - R ₁ = oh, R ₂ = H napellin
4 - R ₁ + R ₂ = O zongorin
Napellin (3) - C ₂₂ H ₃₃ NO ₃ , M ⁺ 359, mp 156-157 ^o With (acetone)
Zongorin (4) - C ₂₂ H ₃₁ NO ₃ , M ⁺ 357, mp 182-187 ^o C (ether).

N-окись 12-напеллнна
N-окись 12-напеллина (5) -C₃₃H₃₃NO₄, M⁺ 375
Выше приведена структура алкалоидов надземной части аконита байкальского.

N-oxide 12-napellnna
12-napellin N-oxide (5) -C ₃₃ H ₃₃ NO ₄ , M ⁺ 375
The structure of alkaloids of the aerial part of Baikal aconite is given above.

- индекс ингибирования метастазирования рассчитывали по формуле

где Ак - частота метастазирования в контрольной, А - в опытной группах, Вк - среднее число метастазов у животных контрольной, В - опытной группы.To simulate the metastatic process, treatment was started at a later date (10 days) after tumor inoculation. The most effective doses of 2% tincture of aconite for each type of tumor were selected empirically: for melanoma B-16 - a dose of 2.5 ml / kg, for Lewis lung carcinoma - 10 ml / kg. 10% tincture, liquid and dry extracts were administered to animals in doses equivalent to effective doses of 2% tincture of aconite in terms of dry raw materials. An alkaloid extract was introduced at a dose of 0.5 mg / kg. The most effective dose of befungin (1.0 ml / kg) was selected according to published data [14]. 5-fluorouracil was administered at a dose of 15.0 mg / kg [15]. All herbal remedies and the prototype drug befungin were administered intragastrically, 5-fluorouracil intramuscularly. Control animals received solvent in an equivalent volume. The antimetastatic effect of the drugs was determined by the frequency of tumor metastasis and the percentage of animals with metastases in relation to the total number of animals in the group, and also by the average number of lung metastases in mice per animal in the group. The criteria for evaluating the effectiveness of the antimetastatic action of drugs were also:
- the percentage of increase in life expectancy of animals, which was calculated by the formula

- the metastasis inhibition index was calculated by the formula

where Ak is the frequency of metastasis in the control, A is in the experimental groups, Bk is the average number of metastases in animals of the control, B is the experimental group.

и среднеквадратичной ошибки (m) О достоверности различий судили, используя параметрический (критерий Стьюдента) и непараметрический (критерий Вилкоксона-Манна-Уитни) методы. Для сравнения результатов экспериментов, где показатели выражались в долях, достоверность определяли с помощью углового преобразования Фишера [16].Statistical processing of the results was carried out by calculating the average

and standard error (m) The significance of differences was judged using the parametric (Student's test) and nonparametric (Wilcoxon-Mann-Whitney test) methods. To compare the experimental results, where the indices were expressed in fractions, the reliability was determined using the Fisher angular transformation [16].

 The results of the study using melanoma B-16 are given in table. 1. F1 mice (CBA • C 57 DL6) with B-16 melanoma were treated on day 11. Comparison drug 5-fluorouracil on this model did not show significant antimetastatic activity, there was only a tendency to a decrease in the average number of metastases, the metastasis inhibition index was 13%. Under the influence of befungin, only 50% of the mice showed lung metastases (in control, 100%). Under the influence 2% aconite tinctures metastases were recorded only in 20% (78% in the control) The inhibition of metastasis growth (by their number) in the group treated with befungin was 12.9% (the difference with the control was not statistically significant), and in the group F, aconite tincture 97% (p <0.01). The differences between these effects are highly significant (p <0.01), which indicates a clear advantage of tincture. Under the influence of 2% tincture of aconite, metastases were recorded in 20% of animals (in the control in 78%), the average number of metastases was 0.2 ± 0.2 (in the control of 6.6 ± 3.4), the metastasis inhibition index was 1.8 times more than befungin (99 vs 56, respectively) Liquid and dry aconite extract significantly reduced the frequency of metastasis of B-16 melanoma. The index of inhibition of metastasis under the action of the dry extract exceeded that of the comparison drug befungin 1.3 times. This indicator was somewhat lower in the group where the animals received the liquid extract, nevertheless, it exceeded the comparison drug 5-fluorouracil by 3.2 times. Under the action of the alkaloid extract, 100% inhibition of metastasis was obtained, which significantly exceeded the effectiveness of the prototype drug befungin and, moreover, the comparison drug 5-fluorouracil.

 At the next stage, we conducted a comparative study of antimetastatic activity in another tumor model - Lewis lung carcinoma. The strain of this tumor is known for a high degree of lung damage with metastases. In the first series of experiments, mice with inoculated tumors were left for survival. The comparison drug 5-fluorouracil did not significantly affect the life expectancy of mice with this metastatic tumor (Table 2). 2% tincture of aconite, on the contrary, increased this indicator by 1.4 times, almost the same as 10% tincture and dry extract. Liquid extract led to a clear tendency to increase this parameter. A herbal remedy containing the amount of alkaloids increased the life expectancy of mice with a tumor by 1.7 times. In the second series of experiments, the effect of herbal remedies on the growth and dissemination of tumor cells was studied. The results of the experiments showed that the comparison drug 5-fluorouracil had a significant effect on the average number of metastases (1.6 times reduced this indicator compared to the control), but did not affect the frequency of metastasis (Table 3). The prototype drug befungin reduced the average number of metastases by 2.3 times and the frequency of metastasis by 2.5 times, which affected a high metastasis inhibition index - 83%. This indicator was slightly lower under the influence of 2% tincture of aconite, but the average number of metastases was 1.9 times less than the control. 10% tincture showed an even more significant anti-metastatic effect, as 2.5 times reduced the average number of metastases in the lungs and 2.2 times the frequency of metastasis. The iigibi index of 10% of tincture exceeded that of befungin. Liquid and dry extracts showed a clear tendency to decrease both the average number of metastases and the number of animals with metastases, and the metastasis igibi index exceeded 1.5 and 1.4 times, respectively, this indicator of the comparison drug 5-fluorouracil. An alkaloid extract reduced the average number of metastases by 5.4 times, only 82% of the animals showed dissemination of the tumor process, the inhibition index was 85%.

 Thus, as can be seen from experimental studies, all products containing extracts from the dried and crushed aerial parts of Baikal aconite have a fairly high antimetastatic effect with the highest activity in 10% of the Baikal aconite herbal product, 2% of the Baikal aconite herbal product and herbal product containing the amount of alkaloids.

Sources of information
1. Mashkovsky M.D. Medicines M., 1984. - 575 p.

 2. Rudakov V. F. The effect of chaga on metastasis of the Ehrlich tumor in mice / Higher mushrooms and their biologically active compounds - L .: Nauka, 1973. - P. 52-54.

 3. Zueva EP Preparations from plants of Siberia and the Far East in the complex therapy of malignant neoplasms (experimental study). Abstract diss ... doct. biol. Sciences, Tomsk, 1999.

 4. N. N. Pogodaeva, Ts. Zhapova, A.A. Semenov / Quantification of alkaloids in the aerial part of Aconitum baicalense Turcz. ex Rapaics // Rast. Resources, 1997, 1, pp. 87-88.

 5. Pogodaeva NN, Zhapova Ts., Vereshchagin A. L., Gorshkov A. G., Semenov A. A. / The study of the alkaloid composition of some species of Siberian aconites // Rast. resources, vol. 2, 2000, S.79-84.

 6. Zhapova C. The study of the chemical composition of the plant Aconitum baicalense. Abstract. diss ... cand. Chem. Sciences, Irkutsk, 1995.19 p.

 7. Siderman A.A. Fluoropyrimidines in tumor chemotherapy - Riga: Zinatne, 1982. - 173 p.

 8. Bielewski J. Z kasuistyki stoswania huby brozozy / Przegl. Lekar - 1958. - V. 14. - N.3, - S. 93-96.

 9. Glowaski H., Tomaszewski J. Proby stosowania wyciagow z czamej huby brzozowej (Poria obliqa Bres) w leczeniu nioperacynego raka narzadow rodnych and Kobiety / Ginecol. Polska. - 1962. - 33. - N.4 - S.445-450.

 10. Yakimov P.A. and other drug "Bin-chaga" / P.A. Yakimov, P.K. Bulatov, M.P. Berezina // News. USSR Academy of Sciences 4. - P. 88-91.

 11. Bogovsky P.A., Logna G.O. On the effect of chaga extract on the development of transplanted and induced tumors // Mater, confer. on the indirect effect on the tumor process - Tallinn, 1963. - P. 21-22.

 12. Muravyova I.A. Technology of drugs. T.1 "Medicine", 1980. 704 p.

 13. Experimental evaluation of antitumor drugs in the USSR and the USA / З. P. Sof'ina, A..B. Syrkin, A. Goldin, A. Kleinu. - M .: Medicine, 1979. - 296 p.

 14. Gribel N.V. - Features of the antitumor effect of a number of complex preparations of natural origin. Abstract. dis ... cand. biol. Sciences, Tomsk, 1987.18 s.

 15. Mandel H G et. al. Studies of antitumor action of 5-fluorouracil (5FU) / H.G. Mandel, Ph. Klubes, D.J. Fernandes // Bull. Cancer - 1979. - V.66. - N1. - P. 49-54.

 16. Gubler EV. Computational methods of analysis and recognition of pathological processes. - M .: Medicine, 1978.- 293 p.

Claims (7)

 1. The tool has an antimetastatic effect, containing an extract from plant materials with an extractant, characterized in that it contains an alcohol extract from the dried and crushed aboveground part (grass) of Baikal aconite.  2. The tool according to p. 1, characterized in that it contains a liquid extract from the dried and ground ground part (grass) of Baikal aconite with 40% alcohol at a ratio of plant material to extractant of 1: 50.  3. The tool according to p. 1, characterized in that it contains a liquid extract from the dried and ground ground part (grass) of Baikal aconite with 30% alcohol at a ratio of plant material to extractant of 1: 20.  4. The tool according to p. 1, characterized in that it contains a liquid extract from the dried and ground ground part (grass) of Baikal aconite with 40% alcohol with a ratio of plant material and extractant 1: 1.  5. The tool according to p. 1, characterized in that the tool contains a dry extract from the dried and ground ground part (grass) of Baikal aconite with 40% alcohol with a ratio of plant material and extractant 1: 1.  6. A tool with antimetastatic activity, containing a dry extract from the dried and crushed aboveground part (grass) of Baikal aconite, characterized in that it contains the amount of alkaloids. 7. The tool according to claim 6, characterized in that it contains mesaconitine (C ₃₃ H ₄₅ NO ₁₁ ), hypaconitin (C ₃₃ H ₄₅ NO ₁₀ ), napellin (C ₂₂ H ₃₃ NO ₃ ), zgorin (C) as alkaloids ₂₂ H ₃₁ NO ₃ ) and N-oxide 12-napellin (C ₂₂ H ₃₃ NO ₄ ).

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