RO125279B1 - Process for preparing bioactive complexes of hedera helix herba and medicine product to be used in anti-tumour therapy - Google Patents

Description

The invention relates to a technological process applicable at the industrial level by which the glycosidated triterpenic components of Hedera helix grass are fully utilized, which involves the extraction, separation and purification of pharmaceutically and cosmetically active substances.

It is known that all anti-tumor drugs obtained by chemical synthesis or biosynthesis, regardless of their chemical structure and mode of action, have a multitude of serious adverse effects, which limit their therapeutic use, sometimes imposing the interruption of cytostatic treatment in order to protect the patient's life.

For these reasons, it is of particular therapeutic interest the possibility of obtaining new antitumor drugs based on natural compounds of plant origin, which have therapeutic qualities (selectivity, specificity, safety) superior to cytostatic chemotherapeutic drugs.

The nomenclatures of pharmaceutical products at national and international level contain besides the cytostatic synthetic drugs and a limited number of antitumor products of plant origin, the most used being:

- ISCADOR, HELIXOR containing lectures from Viscum album-,

- VINBLASTIN - vinblastine sulphate from Vinca rosea-,

- VINCRISTIN - vincristine sulphate from Vinca roșea-,

- VINDESIN - vindesine sulphate from Vinca rosea-,

- TAXOL - paclitaxel from Taxus species.

These products are commonly used in oncology clinics, although they have some adverse effects, which are of a lower gravity than the chemotherapy cytostatic ones.

Worldwide, intensive phytochemical and pharmacological research is being carried out, in order to obtain bioactive substances and products with antitumor activity that are free from adverse effects and can be used as such or in concomitant administration with synthetic cytostatic agents, in order to obtain it enhances their activity and reduces their toxicity [1,2, 3, 4, 5],

From a phytochemical point of view, the bioactive compounds of plant origin most studied for this purpose belong to the class of pentacyclic oxygenated triterpenoids with chemical structure type ursan and oleanan, in free or glycosidated state, in the form of triterpenic saponins complex, named after the name of the triterpenic aglicon of the plant from which they come:

- oleanozides - aglicon oleanolic acid from Calendula off .;

- aralozide - aglicon oleanolic acid from Aralia species;

- ginsenozide - aglycone oleanolic acid + protopanaxadiol + protopanaxatriol from Panax ginseng;

- hederasaponine-aglycone oleanolic acid and hederagenin from Hedera sp.

The complex of triterpenic saponins existing in the plant Hedera helix (ivy) family

Araliaceae has predominantly hederagenin aglycone, together with oleanolic acid in smaller quantities, constituting the specific bioactive components of this plant. The analytical studies of comparative evaluation of the composition of the triterpenic saponins complex in Hedera helix led to the qualitative and quantitative highlighting of the structural groups of triterpenic glycosidated compounds, the most important being:

The name of the triterpenic saponin aglycone structural Carbohydrate chain 3 C28 to hederin Hederagenina arabinose-rhamnose - β- Hederine Oleanolic acid arabinose-rhamnose -

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Table (continued) 1

The name of the triterpenic saponin aglycone structural Carbohydrate chain 3 C28 Hederacozide B Oleanolic acid arabinose-rhamnose (glucose) _2- brnose Hederacozide C Hederagenina arabinose-rhamnose (glucose) _2- brnose

depending on the geographical area of the plant, the harvesting period, the plant part of 7 that is collected - leaves from flowering branches, young leaves, old leaves, the content in the main triterpenic glycosides varies [6], the presence and other 9 triterpenic saponins in smaller quantities: δ-Hederine, Hederasaponins D, E, F, G, Η, I, etc.

Hydroalcoholic extracts from Hedera helix are the basic component of only 11 red anti-cough pharmaceutical products provided in the European drug nomenclature, [7] and glycolic extracts are used to obtain anti-cellulite and antilipemic dermatological cosmetics.

Due to the special properties of water-soluble preparations of CuNa-chlorophyll obtained from 15 plants such as lucerne, grass, nettle, algae, pelin, namely: tincture properties, structural similarity with hemoglobin, the practical applications of soluble chlorophyll salts 17 are found in industry 17 cosmetics, food and nutritional supplements [8; 9; 10; 11;

12; 13; 14; 15], 19 in the last period, studies have appeared in the specialized magazines on the antioxidant and antimutagenic effects of the soluble chlorophyll salts (16; 17; 18; 19; 20; 21; 22; 21

2. 3; 24).

The following are the patents of the invention and patent applications 23 which present processes for obtaining bioactive products applicable in therapeutics, based on triterpenic glycosidated compounds with hederagenin structure, obtained from plant 25 Hedera helix.

- French patent FR 1.531.621 / 24.05.1967 (25), Process for preparing the triterpene complexes in plants, shows the obtaining of four strains of saponins from Hedera helix, three in the form of oily liquids and a of a yellow 29 brown powder that were characterized by thin layer chromatography highlighting the presence of

2-10 spots of saponins in each sample. The process for obtaining this involves the extraction of 31 plant raw materials with 60% ethanol and the processing of the total extract obtained in powder form by successive extraction with 96% ethanol, acetone, petroleum ether, methanol, and the purification step 33 is carried out by adsorption on activated carbon. in the ratio 1/4 extract / adsorbent and subsequent desorption with different solvents: methanol, chloroform, ethyl acetate, benzene, pyridine. The 35 products obtained which are complexes of saponins are not analytically characterized by their content in active substance, but only by spot detection methods by thin layer chromatography 37. The process is laborious with many extraction and separation stages, it uses various slightly flammable extraction and purification solvents, carcinogenic (benzene, pyridine, methanol, 39 chloroform), uneconomical obtaining low extraction yields even when complexes are obtained - mixtures of triterpenic compounds (from 100 kg plant between 4 and 450 g 41 oily liquid extracts).

- Patent application US 20060210660 / 21.09.2006 / A1, entitled: "Processes for the preparation of an extract of ivy leaves, presents a process for the preparation of an extract of ivy leaves, consisting of a mixture of 2 extracts processed 45 separately for enrichment in hederacozide C and α-hederin. The enriched extract in

RO 125279 Β1 α-hederin is obtained by the enzymatic fermentation of the ivy leaves with water, at 30 ° C, in order to convert hederacozide C into α-hederin which is extracted with ethyl alcohol 30% hydroalcoholic extracts being processed as sicc extract in the form of powder, with an enriched content in α-hederin: at least 5%, due to the total conversion of the C hederacozide existing in the plant. The extract enriched in hederacozide C is obtained by treating the plant with superheated water vapor at 120 ° C, in order to inactivate the enzymes that convert hederacozide C to α-hederin, after which extraction with 30% ethyl alcohol is carried out, the hydroalcoholic extracts tend to be processed sicc with high content of hederacozide C: minimum 10%. In order to be used in therapeutics, a mixture of the 2 extracts mentioned above is prepared, obtaining a special final extract with a content of

5 ... 8%) C hederacozide and 3 ... 5% α-hederin. Regarding the pharmacological activity of these 2 bioactive extracts, mention is made of the respiratory tract, mumps, expectorant and antiobstructive effects, which justify their use in the treatment of respiratory disorders.

- GB patent 1106133 / 13.03.1968 [26] shows the obtaining of extracts from Hedera helix: saponin, sapogenin and magnesium salt of sapogenin, by extraction with 70% ethyl alcohol, precipitation of the saponin complex with ammonium sulphate, purification with 93% ethyl alcohol and 98% methanol, and for the removal of pigments, resins and oils, acetone, ethyl acetate, methyl ethyl ketone, ethylene trichloride, chloroform are dissolved by dissolving concentrated sapogenin extracts as a dry residue, followed by filtration, for the removal of organic solvents. In this way saponin is obtained, in the form of an amorphous powder, of a white-cream color, slightly soluble in water, methanol, ethanol, but insoluble in acetone and ethyl acetate, having a melting point between 195 and 200 ° C, and sapogenin. as a white powder, insoluble in water at acid pH, soluble in methanol, ethanol isopropanol. Saponin, sapogenin and magnesium salt obtained according to the invention are inhibitors of cell division and growth having no mitoclastic effect.

- Chinese patent CN 200710034341 / 29.01.2007 [27] shows the preparation of a 98.2% Hederacozide C preparation, determined by HPLC, by hydroalcoholic extraction 30 ..95% at reflux, obtaining the crude extract, dissolving it in water in an ultrasound generator and subsequent extraction with ether and ethyl acetate to remove ballast substances, which impurify the extract. The extract thus obtained is extracted with n-butanol, and purification is continued with 95% ethanol, ethyl acetate (to dissolve the insoluble part) methanol, and column chromatography using chloroform as the eluent: methanol 65:35. The process is uneconomical, it cannot be applied on an industrial scale, using expensive installations (ultrasound generator, chromatographic columns), solvent mixtures that are difficult to recover and used and the product obtained is obtained with extremely low yields for applications in the pharmaceutical industry 0, 21 g / 100 g vegetable raw material.

- Patent GB 2051575 / 08.01.1980 [28], entitled: "Pharmaceutical compositions containing dandruff ivy extracts, presently obtain pharmaceutical compositions based on ivy extracts (Hedera helix) containing: Hederasaponin C (Hederacozide C 60) ... 90% or α-hederin. The process of obtaining the extracts provides for the successive extraction of the plant with acetone and methanol, followed by precipitation of hederasaponne C from the methanolic solution by treatment with ethyl ether. The result is hederasaponin C, with a content of 60%, which is purified by column chromatography of AI ₂ O ₃ eluted with methanol, from which the purified hederasaponine C, with a content of 90%. The transformation of C-90% hederasaponin into α-hederin is also presented, by alkalizing with NaOH or 2N KOH, resulting in purified α-hederin. It is mentioned the possibility of using ivy extracts, obtained according to the invention and conditioned in the form of tablets, solution for injection, ointment, as drugs with antifungal and antiparasitic activity, in human and veterinary therapeutics.

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- US Patent Application 20060057236 / 16.03.2006 / A1 [29], entitled: 1 "Process for producing an extract of ivy leaves, presents a process for obtaining a leaf extract of Hedera helix, containing α- ivy, which provides 3 fermentation of ivy leaves, with water at 30 ° C, when hederacoside C is enzymatically transformed into α-ivydine which is extracted from the plant with hydroalcoholic mixtures (ethanol) 5 obtaining a powdered sico extract . It is mentioned that by the total enzymatic transformation of hederacoside C from the plant into α-hederin, the sico extract obtained according to the 7 invention is enriched in α-hederine from 0.53% to 4.74% and can be conditioned as pharmaceuticals. internal and external use, applicable in the treatment of respiratory disorders 9, due to the bronchospasmolytic effects of α-hederin.

- Patent CN 1245693 / 01.03.2000 [31] presents a medicinal powder for the treatment of various forms of leukemia, which contains as main material the ivy powder, together with other pulverized vegetable products, the therapeutic effect having a 13 rate of 96% cure with no toxic side effects.

- Patent RU 2127605 / 20.03.1999 [32] presents a method of stimulating cellular immunity 15 using triterpenic glycosides isolated from different plant organs of the Hedera genus as an adjuvant bioactive product, the method being applicable to vaccine preparation. 17 regarding the use of the Hedera helix plant in therapeutics, it is clear from the knowledge in the prior art that no medicinal products have been made from this plant 19 applicable in antitumor therapy, based on an activity to influence the evolution of the tumors or to counteract the adverse effects. of cytostatic drugs. 21

Due to the high social incidence of neoplastic diseases and a constantly increasing mortality rate, the chances of mitigating tumor evolution and extending life with the help of chemotherapy cytostatic drugs are still limited, hence the special therapeutic importance, given with priority research to obtain new anti-tumor drugs, with safety and therapeutic efficacy superior to those used in the current stage in oncology. 27

The technical problem that the present invention aims to solve is to obtain the entire glycosylated triterpenic complex from the Hedera Helix herb plant. 29

The process according to the invention solves the technical problem of obtaining the whole triterpenic glycosidated complex from Hedera helix grass. 31

The solution proposed according to the invention consists of a process for separating and purifying the entire triterpenic glycosidated complex of hederagenin and oleanolic acid from grass 33.

Hedera helix.

The main object of the present invention is to obtain 4 bioactive complexes 35 glycosides, one of which in the form of an aqueous CuNa-chlorophyll extract, from the plant Hedera helix. 37

Another object of the invention is a process for the preparation of bioactive glycosidated complexes and drug products containing the bioactive complex 39 glycosidate.

Accordingly, the invention relates to a process for obtaining bioactive complexes 41 from Hedera helix grass, used in antitumor therapy, wherein, in a first step, the dried Hedera helix grass is subjected to dynamic extraction by ethyl alcohol of 43 80% concentration in a ratio 1:15, at ambient temperature, for 12 hours, followed by the concentration of the alcoholic extract solution, the concentrated extract is then subjected to the liquid-liquid extraction with ethyl acetate in a ratio of 1: 3, which results an aqueous extract and a phase of ethyl acetate, which is further separated, filtered and concentrated in vacuo to a ratio 47 1:30, suspended under stirring in acetone, resulting in a first crude bioactive complex which

RO 125279 Β1 is subjected to purification by dissolving in ethyl alcohol 95% bleaching with activated carbon and concentration, which leads to an acetonic filtrate, and a crystallized one which is purified, resulting in a first purified bioactive complex, and then in a second step , the aforementioned aqueous extract is subjected to a liquid-liquid separation, which results in an aqueous phase and an n-butanolic phase, which is subsequently subjected to concentration under vacuum, resulting in a second crude bioactive complex, which is suspended in acetone, and the precipitate separated by filtration and purified by dissolving with 70% ethyl alcohol, bleaching with coal and concentration, resulting in a second purified bioactive complex, then, in a third step, the acetonic filtrate is concentrated in vacuo and combined with the aqueous phase from the liquid-liquid extraction of the previous step, it is concentrated and conditioned in propylene glycol, which results in a third bioactive complex, and finally, the acetonic purification filtrate from step 1 is concentrated and suspended in the alcoholic solution at pH 8.5 ... 12.5, separated by filtration and suspended in 10% copper sulphate solution in the environment acid, followed by solubilization of Cu-chlorophyll in an aqueous sodium hydroxide solution at pH 8.5 ... 12.5, resulting in an aqueous extract of Cu Nachlorophyll.

In a preferred embodiment of the invention, in the process defined above, the purified bioactive complex of step 1 is a white crystalline powder, having a minimum content of 95% total monodesmozide triterpenic saponins with aglycone hederagenin, of which 70 ... 80% of -Hederină.

In another preferred embodiment of the invention, in the process defined above, the purified bioactive complex of step 2 is a yellowish-white powder, having a content of at least 90% total bisdesmozide triterpenic saponins with hederagenin aglycone, of which 50 ... 75 % Hederacozide C.

In another preferred embodiment of the invention, in the process defined above, the bioactive bioactive complex in step 3 is in the form of a reddish-brown liquid extract, having 1-3 g / 100 ml of bisdesmozide saponins with oleanolic acid aglycone and 1.5 - 5 g / 100 ml polyphenolcarboxylic acids In another preferred embodiment of the invention, in the process defined above, the aqueous CuNa-chlorophyll extract has 0.2 ... 1 g / 100 ml contained in monodesmozide saponins with oleanolic acid aglycone and 0 , 1 ... 0.5 g / 100ml total chlorophyll extract.

The invention also relates to a medicinal product, consisting of 1 ... 2% of a peppermint bioactive complex by the above conditioned process in the form of an injectable or buccal solution, gel, cream or suppositories.

The invention also relates to a medicinal product consisting of 100 mg of a bioactive complex prepared by the process defined above, conditioned in capsule form.

The therapeutic products obtained according to the invention are:

- a glycosidated bioactive complex in the form of purified crystallized white powder with a content of total monodesmozidic triterpenic saponins with aglicon hederagenin of at least 95%, of which α-hederin between 70 and 80%, called GSO-1;

- a purified crystallized white powdered glycoside bioactive complex with a content of total bisdesmozide triterpenic saponins with a minimum 90% aglycone hederagenin, of which Hederacozide C between 50 and 75%, called GSO-2;

- a glycosidated bioactive complex in the form of a reddish-brown liquid extract characterized by a content of bisdesmozide saponins with oleanolic acid aglycone between 1 and 3 g / 100 ml and polyphenolcarboxylic acids between 1.5 and 5 g / 100 ml conditioned in a solvent cosmetically acceptable (propylene glycol, butylene glycol, glycerin), termed GSO-3;

RO 125279 Β1

- an aqueous CuNa-chlorophyll extract usable in the cosmetic industry, containing 1 in monodesmozide saponins with oleanolic acid aglycone between 0.2 and 1 g / 100 ml and total chlorophyll between 0.1 and 0.5 g / 100 ml extract. 3

The invention presents the conditioning processes of triterpenic glycosidated compounds in the form of medicinal products for human and veterinary use, highlighting in 5 in vitro and in vivo studies of the antitumor, antiangiogenic, hepatoprotective and cosmetic products, developed based on the dyeing, antibacterial, anti-bacterial, anti-bacterial properties. antelopes - 7 thoracic and anti-cellulite.

From the data presented regarding the state of the art in the field of obtaining 9 substances or bioactive products from the Hedera helix plant, the following results:

• compared to the prior art, the novelty elements according to the invention are represented by:

- the different percentages of active substances in each bioactive complex; 13

- the presented procedure consists of a certain different sequence of stages and parameters corresponding to each stage; 15

- the medicinal products obtained contain a different proportion of the active principles used in tumor disorders; 17 • according to the prior art, no procedures have been performed for the therapeutic purpose of obtaining the whole triterpenic glycosidated complex having aglycone hederagenin and oleanolic acid 19 existing in the plant Hedera helix;

• The processes presented above are different from the present invention, having 21 other steps, solvents and parameters;

• the processes present a series of technological disadvantages, such as: 23

- they are laborious having many extraction and separation stages, they use various slightly flammable extraction and purification solvents, carcinogens (benzene, pyridine, methanol, 25 chloroform), they are uneconomical obtaining low extraction yields for applications in the pharmaceutical industry 0, 21 g / 100 g vegetable raw material for purified products, 27 and for complexes - mixtures of triterpenic compounds of 100 kg plant between 4 and 450 g oily liquid extracts) [25]; 29

- cannot be applied on an industrial scale using expensive installations (ultrasonic generator, chromatographic columns, expensive adsorbents AI ₂ O ₃ ), solvent mixtures 31 difficult to recover and used [27];

- uses highly flammable and explosive solvents - ethyl ether - to obtain hederacozide 33 C 60% by precipitation from the methanolic solution, resulting in a precipitation medium consisting of ethyl ether-methanol, difficult to distill and separated at industrial level for recovery and reuse 35 solvents [28];

- I suppose the processing of the primary extractive solutions in the form of sicc extracts, with a 37 content low in α-hederin (3 ... 5%) and hederacozide C (5 ... 8%) [29, 30], which decreases them the chance of application in therapeutics, conditioned as medicines, being usable as nutritional supplements;

- determines the production of bioactive products containing only 1 ... 2 hederasapo- 41 nine, with aglycone hederagenin (α-hederin and hederacozide C), without the technological and therapeutic use of hederacozide B - with oleanolic acid aglycone [29, 30] , 43

Regarding the use of the Hedera helix plant in therapeutics, the patents studied and presented in the prior art state the following: 45

- no medicinal products from this plant were applied in antitumor therapy, based on an activity to influence the evolution of tumors or to counteract 47 the adverse effects of cytostatic drugs;

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- The prior art documents present pharmaceutical compositions containing different proportions from the present invention, of the active principles used: hederacoside C of purity 60 .. .90%, conditioned as a drug with antifungal and antiparasitic activity [28]; sicc extract with 4.74% α-hederin, conditioned in the form of pharmaceuticals applicable in the treatment of respiratory disorders [29]; sicc extract containing 5 ... 8% hederacozide C and

3 ... 5% α-hederin, applicable in the treatment of respiratory disorders [30];

- most of the patents issued in the state of the art regarding Hedera helix show the use of glycolic extracts, hydrogens and colts, and hydroalcoholics in the conditioning of pharmaceutical and cosmetic products in the form of gel, cream, balsam, with anti-inflammatory, antipruritic, antilipemic effects and anti-cellulites [33, 41],

According to the proposed process, 4 different preparations representing bioactive intermediates for the pharmaceutical, cosmetic, food and nutritional supplements are obtained, as follows: the plant raw material grass Hedera helix dried and milled is extracted with ethyl alcohol for pharmaceuticals with a concentration of 50 .. .80%, followed by the concentration of the extracted solutions obtained and the recovery of the solvent; the concentrated aqueous extract obtained is subjected to a first separation step by liquid / liquid extraction with ethyl acetate, at which time the monodesmoside glycosides selectively pass from the aqueous phase to the organic phase, from which they are separated after concentration in vacuo, by suspension in acetone and filtration. , in the form of a green powder with a content of total monodesmozide triterpenic saponins with aglycone hederagenin in the range of 50-60%, of which α-Hederine 30-40%, which represents the GSO-1-crude preparation.

For GSO-1-crude purification, it is subjected to a recrystallization with 95% ethyl alcohol, obtaining the final product with a content of total monodesmozide triterpenic saponins with at least 95% aglycone hederagenin, of which α-Hederine between 70 and 80% determined by HPLC called GSO-1.

The second step consists in obtaining the GSO-2 preparation from the residual aqueous extract which is subjected to liquid / liquid extraction with n-butanol, vacuum concentration and suspension in acetone, at which time hederasaponins precipitate as a sandy powder separable by filtration. Thus, a crude preparation of GSO-2 with a total bisdesmozide triterpenic saponins content of 50-60% aglycone hederagenin is obtained, of which Hederacozide C between 30 and 40%.

For the purification of the crude preparation of GSO-2, the technology according to the invention proposes differently from the data in the literature [27; 28] dissolving the crude preparation of hederacoside C in ethyl alcohol 60-70%, bleaching with carbon, concentrating the extract to the aqueous phase which is subjected to liquid / liquid extraction with n-butanol, followed by crystallization from the organic phase. Thus, the purified preparation of GSO-2 with a content of total bisdesmozide triterpenic saponins with aglycone hederagenin of at least 90% is obtained, of which Hederacozide C between 50 and 75%, determined by HPLC.

In order to obtain the liquid bioactive extract characterized in bisdesmozide glycoside compounds with oleanolic acid aglycone, the acetonic filtrate obtained after the separation of the crude preparation from GSO-2 is concentrated in vacuo to recover the solvent, it is combined with the aqueous extract remaining (from the extraction of hederacoside C ) rich in polyphenolic compounds and conditioned in an acceptable cosmetic solvent (butylene glycol, glycerin, propylene glycol) resulting in the preparation GSO-3 with a content of bisdesmozide saponins with oleanolic acid aglycone between 1 and 3 g / 100 ml and polyphenolcarboxylic acids comprising , 5 and 5 g / 100 ml, called GSO-3.

To obtain the water-soluble CuNa-chlorophyll extract, the residual acetonic extract containing the monodesmozidic triterpene glycosides of oleanolic acid as well as the chlorophyll pigment complex obtained after the separation of the crude GSO-1 preparation - is concentrated,

RO 125279 Β1 is alkalized, suspended in the solution of copper sulphate in acid medium, followed by 1 solubilization in alkaline medium of Cu-chlorophyll obtained in NaOH solution, at which time the water-soluble salt of chlorophyll pigments is formed, in the form a 3 times aqueous solution of intense green color, stable, characterized by a content of monodesmozide saponins with oleanolic acid aglycone between 0.2 and 1 g / 100 ml and of total chlorophyll between 0.1 and 5 0.5 g / 100 ml extract.

The advantages of the invention with respect to the prior art are the following: 7

- presents a technological process, applicable at industrial level, for the full utilization of the triterpenic glycosidated potential of the plant raw material Hedera helix by obtaining 4 preparations, namely:

- the glycosidated bioactive complex in the form of purified crystallized white powder with a content of total monodesmozidic triterpenic saponins with a minimum of 95% aglycone hederagenin, of which α-Hederine between 70 and 80%, called GSO-1; 13

- the glycosidated bioactive complex as a yellowish-white powder crystallized purified with a content of total bisdesmozide triterpenic saponins with 15 hederagenin aglicon of at least 90%, of which Hederacozide C between 50 and 75%, called GSO-2;

- the glycosidated bioactive complex in the form of a reddish-brown liquid extract, characterized by a content of bisdesmozide saponins with oleanolic acid aglycone between 1 and 3 g / 100 ml and polyphenolcarboxylic acids between 1.5 and 5 g / 100 ml, called GSO -3, conditioned in a cosmetically acceptable solvent 19 (propylene glycol, butylene glycol, glycerine);

- aqueous CuNa-chlorophyll extract, usable in the cosmetic industry, having a content of 21 in monodesmozide saponins with oleanolic acid aglicon between 0.2 and 1 g / 100 ml and of total chlorophyll between 0.1 and 0.5 g / 100 ml extract. 2. 3

- as opposed to the processes mentioned in the prior art, which show the obtaining of sicc extracts with a low content of α-Hederine and Hederacozide C of 3 ... 5%, 25 respectively, of 5 ... 8% [29; 30], the process according to the invention leads to the obtaining of glycosidated bioactive complexes with a content of monodesmozidic triterpenic saponins and 27 total bisdesmozides with aglycone hederagenin of minimum 90 ... 95%, of which a-Hederine of 70 ... 80% and respectively , Hederacozide C between 50 and 70%. 29

- Ecological judicious exploitation of the full potential of plant raw material

Hedera helix-, by applying this process, through the same technological flow after the primary hydro-alcoholic extraction 31, two liquid / liquid extraction stages are carried out with two different types of solvents; thus from the primary extract it is separated with the mixture of water: ethyl acetate, firstly 33 monodesmozide triterpenic glycosidated compounds and then by liquid / liquid water extraction: nbutanol, bisdesmozide glycosidic triterpenic compounds; 35

- the use of the liquid / liquid separation method in the technology of separating the triterpenic components instead of the chromatographic processes on aluminum oxide columns [28] is a great economic advantage, executing simply, after a non-laborious methodology, with a easily accessible equipment and in a short time; 39

- By applying this process according to the present invention, the purification step of Hederine and hederacozide C uses non-toxic solvents, easy to handle at industrial level 41 - ethyl alcohol of 50% and 96% concentration, n-butanol with high boiling point and easily recovered for reuse 43

- the use of ethyl acetate in the first stage of the liquid / liquid extraction is an advantage, because due to its selectivity, it leads to the separation in a single stage, with yields between 80 and 90% of the monodesmozide triterpenic components;

- the technology recovers the solvents used and makes use of the separate filtrates in the 47 purification stages, by obtaining the bioactive glycosidated complex, characterized by a content of bisdesmozide saponins with oleanolic acid aglicon between 1 and 3 g / 100 ml and polyphenolcarboxylic acids 49

EN 125279 Β1 between 1.5 and 5 g / 100 ml, termed GSO-3, and of the aqueous CuNa-chlorophyll extract, containing monodesmozide saponins with oleanolic acid aglicon between 0.2 and 1 g / 100 ml and total chlorophyll between 0.1 and 0.5 g / 100 ml extract.

- the phytotherapeutic products, obtained according to the present invention, have pharmacological effects different from those presented in the prior art: antitumor activity of inhibiting and slowing down the cancerous evolution, especially of mammary tumors, but also for those located in the lower abdominal basin: ovary, cervix and prostate, with antioxidant, hepatoprotective and immunoprotective activity to counteract the immunosuppressive and hepatotoxic adverse effects that occurred during and after aggressive cytostatic treatment, being applicable in human and veterinary therapy;

- compared with the bioproducts made according to the mentioned procedures [7,28,29], in which the antitussive, antifungal and antiparasitic effects of hederasaponin C and α-hederin were shown, in the phytotherapeutic preparations according to the invention, have been demonstrated by preclinical studies , in vitro and in vivo, performed on mitogenic stimulation model of normal lymphocytes, as well as on specific standardized tumor lines, the presence of antitumor activity (especially in mammary tumors, but also in cervical and prostate tumors), evidenced by:

- inhibition of the DNA synthesis phase from cell cycle sequencing;

- inhibition of tumor cell proliferation;

- induction of the phenomenon of early / late apoptosis;

- antiangiogenetic activity by which the irrigation of the tumor is reduced / inhibited;

- prophylactic effect on systemic tumor toxicity and diffusion.

In the context of tumor evolution and independent of it, the preparations according to the invention have other pharmacotoxicological qualities of therapeutic interest:

- reduced toxicity and absence of adverse effects, which gives them a higher efficacy / toxicity ratio than other existing anti-tumor drugs, which have a high degree of toxicity, expressed by a low therapeutic index, because the therapeutic doses are very close to the toxic doses;

- potential antioxidant and hepatoprotective relevant in oncological and human therapeutics, to counteract the immunosuppressive and hepatotoxic adverse effects induced by antineoplastic treatments;

- the selectivity of the cytotoxic effect, which contributes to maintaining the normal functionality of the main vital organs, to regulating the physiological parameters in the therapies associated with cytostatics.

based on these pharmacological effects, the phytotherapeutic preparations, obtained according to the invention, have been conditioned in the form of new medicinal and cosmetic products, for human and / or veterinary use such as: capsules, injectable solutions, mouthwashes and for external use, gel, cream, suppositories. , intended for new therapeutic applications, compared to those currently known.

The process according to the invention consists in the complete recovery at industrial level of the triterpenicyclic glycoside components of the plant raw material, the Hedera helix herb in dry and ground state, which is extracted with hydroethanol solution of 50 .... 80%, at the temperature of 18 ... 25 ° C, for 12 ... 18 h.

The obtained extraction solution is subjected to the vacuum concentration operation at 40 ° C, by obtaining an aqueous concentrated extract which is subjected to liquid / liquid extraction in two successive stages, as follows:

- In order to obtain crude GSO-1, in the first step, the concentrated aqueous extract is extracted with ethyl acetate in ratio 1: 1 - 1: 4 aqueous extract: ethyl acetate, the organic phase is collected and further processed by, concentration below vacuum in 1/30 ratio for the recovery of ethyl acetate and the suspension in acetone of the concentrated extract obtained, at which point

RO 125279 Β1 monodesmozide glycosides precipitate and separate by filtration as green powder 1 with total monodesmozide triterpene saponins containing hederagenin aglycone.

50 ... 60%, of which α-hederin 30 ... 40% representing GSO-1-crude preparation; 3

- for the purification of the crude preparation of GSO-1, it is subjected to a recrystallization with ethyl alcohol 95%, by dissolution, discoloration with coal and concentration, obtaining the final product 5 as a white crystalline powder, with a content of total monodesmozide triterpenic saponins. with aglycone hederagenin of at least 95%, of which it has between 7 70 and 80%;

- the remaining aqueous extract is subjected to the second stage of liquid / liquid separation with n-butanol 9 in ratio 1: 1 ... 1: 3 aqueous extract: n-butanol for the separation of bisdesmozide hederacosides in the organic phase, which is further processed by vacuum concentration for butanol recovery, acetone suspension of the concentrated extract obtained, at which time the hederasaponins precipitate and separate by filtration in the form of sandy yellow-brown powder 13 with a total bisdesmozideic triterpenic saponin content of hederagenic aglycone 50 ... 60%, of which Hederacozide C between 30 and 40%, thus obtaining 15 crude GSO-2 preparation;

- for the purification of the crude GSO-2 preparation, the technology according to the invention proposes 17 its dissolution in ethyl alcohol 60 ... 70% in 1/100 ratio, discoloration with carbon, concentration of the extract until the aqueous phase, liquid / liquid extraction with n-butane in ratio 1: 3, 19 followed by concentration and crystallization from the organic phase. Thus, the purified preparation of GSO-2 is obtained in the form of a crystalline white-yellow powder, with a total triterpene saponin content of 21 bisdesmozides total agglonin hederagenin of at least 90%, of which Hederacozide C between 50 and 75%; 2. 3

- to obtain the liquid bioactive complex, characterized in bisdesmozide glycoside compounds with oleanolic acid aglicon, the acetonic filtrate obtained after the filtration separation of the crude GSO-2 is concentrated in vacuo to recover the solvent, meets the remaining aqueous extract (from the extraction with hederacid). n-butanol), rich in polyphenolic compounds, 27 are concentrated and thus GSO-3 is obtained with a content of bisdesmozide saponins with oleanolic acid aglycone between 1 and 3 g / 100 ml and polyphenolcarboxylic acids between 1.5 and 5 g / 100 ml, 29 which is conditioned in an acceptable cosmetic solvent (butylene glycol, glycerin, propylene glycol);

- the chlorophyll acetonic filtrate, obtained after the crude GSO-1 separation, is concentrated, 31 is alkalized at pH = 8.5 ... 12.5, suspended in 10% copper sulphate solution, in acidic medium at pH = 2 . ..4.5, for a minimum of 30 minutes, at ambient temperature, followed by solubilization 33 Cu-chlorophyll obtained in alkaline medium in NaOH solution at pH = 8.5 ... 12.5, at which time the salt is formed soluble in water of chlorophyll pigments, in the form of an aqueous solution of 35 intense green color, stable over time, having a content of monodesmozide saponins with oleanolic acid aglicon between 0.2 and 1 g / 100 ml and of total chlorophyll between 0.1 and 0 , 5 g / 100 ml extract. 37 below, are described 10 embodiments of the invention, which refer to the process of obtaining the herbal products from the Hedera helix plant, to the tests performed 39 in order to highlight the specific activity and to evaluate the applications in therapeutics, as well as the conditioned medicinal and cosmetic products. . 41

Example 1. Process for obtaining phytotherapeutic preparations from the plant Hedera helix kg plant raw material grass Hedera helix dried and ground is introduced into 43 extraction vessel type 300 L stainless steel percolator, extracted by dynamic percolation with ethyl alcohol for pharmaceutical use with an 80% concentration, in relation to 1:15 plant raw material 45: solvent, at ambient temperature, for 12 hours, followed by the concentration of the extractive solution obtained with solvent recovery. 47

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Obtaining the GSO-1 preparation

The concentrated extract obtained in the amount of 50 L is subjected to liquid / liquid extraction, with 150 L of ethyl acetate. The separate ethyl acetate phase is filtered and concentrated in vacuo, until a 1/30 ratio is suspended under stirring in 10 L acetone when precipitation of the ahederin occurs, which is separated by vacuum filtration and drying at 65 ° C. The powder obtained 700 g of green color representing the crude GSO-1 preparation has a content of total monodesmozide triterpenic saponins with 50% aglycone hederagenin, of which 35% a-hederine.

To obtain a substance with an advanced purity the crude product of GSO-1 is subjected to a purification operation by dissolving in 40 L ethyl alcohol 95% discoloration using activated carbon and concentration with solvent recovery and crystallization, after filtering and drying 200 white powder are obtained. crystalline content of total monodesmozide triterpenic saponins with 95% aglycone hederagenin, of which 75% α-hederin, which represents the phytotherapeutic preparation GSO-1.

Obtaining the GSO-2 preparation

The remaining aqueous extract is subjected to the second stage of liquid / liquid separation with 75 L n-butanol for the separation of bisdesmozide hederacosides in the organic phase. The n-butanolic phase in the amount of 75 L is collected and further processed by vacuum concentration, to recover the butanol, suspension of the concentrated extract obtained in 60 L acetone, at which time the hederacoside C precipitates and is separated by filtration. Thus, the crude preparation of GSO-2 is obtained in an amount of 1.2 kg as a brownish sandy powder, with a total bisdesmozide triterpene saponins content of 58% aglycone hederagenin, of which 32% Hederacozide C. To obtain a substance with an advanced purity, the crude product GSO-2,1,2 kg is dissolved in 120 L 70% ethyl alcohol, discolored with activated carbon, and after the concentration of the extract to the aqueous phase, it is subjected to liquid / liquid with 12 L n-butanol, followed by concentration under vacuum and crystallization from the organic phase. Thus, the purified preparation of GSO-2 is obtained in the amount of 440 g, as a white-yellow powder with a total bisdesmozide triterpene saponins content of 90% hederagenin aglycone, of which 65% Hederacozide C, which represents the phytotherapeutic preparation of GSO-2. 2.

Obtaining liquid bioactive extract GSO-3

In order to obtain the liquid bioactive extract characterized in bisdesmozide glycoside compounds with oleanolic acid aglycone, the acetonic filtrate obtained after separation of the GSO-2 crude preparation in 60 L quantity is concentrated in vacuo, to recover the solvent, it is combined with the remaining 50 L aqueous extract (from extraction of hederacozide C with n-butanol) rich in polyphenolic compounds and conditioned by concentration in 5 L propylene glycol. The standardized liquid bioactive extract is thus obtained in bisdesmozide triterpene glycosidated compounds with oleanolic acid aglycone of 1.2 g / 100 ml and polyphenolcarboxylic acids of 1.7 g / 100 ml.

Obtaining the preparation of CuNa-chlorophyll

The acetonic filtrate -10 L- obtained after the separation by filtration of the crude GSO-1 powder, which contains monodesmozidic triterpenic glycosidated compounds with oleanolic acid aglycone and the chlorophyll pigment complex, is concentrated to a ratio of 1/20, suspended in 25 L alkaline aqueous solution at pH = 8.5 ... 12.5, separated by filtration and then suspended in 10 L copper sulphate solution 10%, in acidic medium at pH = 2.4.5 for at least 30 minutes, at ambient temperature, followed by solubilization of Cu-chlorophyll obtained in an aqueous alkaline solution, of NaOH at pH = 8.5 ... 12.5, when the water-soluble sodium salt of chlorophyll pigments is formed , conditioned in the form of an aqueous solution of intense green color, stable in time, in the amount of 15 L, standardized in monodesmozidic saponins with aglicon oleanolic acid of 0,42 g / 100 ml and of 0,35 g total chlorophyll / 100 ml extract .

Based on the results of the in vitro / in vitro and in vivo tests, the herbal preparations of plant 1 Hedera helix, obtained according to the invention, can be conditioned in the form of medicinal products for internal and external use, intended for the treatment of benign and malignant disorders, 3 in particular mammary neoplasia therapy for human and veterinary use.

The drug products, conditioned according to the invention, can be administered 5 parenterally, orally or topically, for 1 ... 12 months, depending on the evolution of the disease, the following formulation examples not excluding the possibility of developing other 7-conditioning formulas, having in view of the diversity and multitude of excipients known and currently used in the pharmaceutical industry. 9

In vitro and in vivo tests on the antitumor activity of the phytotherapeutic preparation with GSO1 and GSO2 11

The special advances made in the last decade in the field of biochemistry and molecular biology have decisively influenced the pharmacological researches carried out in order to deepen the physiological processes that take place in the human body, as well as to highlight the biochemical, histological and enzymatic transformations that occur under different pathological conditions. . In this field, the extent of in vitro research in cellular and cellular systems, which precede in vivo tests, is mentioned as complementing their results with new experimental data, which elucidates new mechanisms of action of hormones and enzymes or deepens the biotransformations that occurred in the course of metabolization of new drugs. 19

For in vitro testing of bioactive complexes, normal cells (normal human lymphocytes, unstimulated and mitogenic stimulated) and relevant tumor cells were used in the target neoplastic pathology 21 to determine cytotoxicity and specific action (HeLa - adherent cells of cervical carcinoma epithelial, Hep-2 - adherent cells of cervical carcinoma epithelial type 23 (derived from HeLa), MCF-7 - adherent cells of breast adenocarcinoma, DU 145 adherent cells of prostate carcinoma epithelium). 25

1. Antitumor activity in the preliminary pharmacological screening for investigating the antitumor activity 27 of the phytotherapeutic preparations, in vitro experimental studies were performed on the cytotoxicity and the specific action exerted on some relevant parameters in the proliferative cell status 29 on the induction of early and late apoptosis. , cell cycle sequencing and cell proliferation. The influence of biocomplexes in the angiogenesis process was investigated as an alternative mechanism to block tumor progression. In the same vein, the in vitro model of inducing mitogenic stimulation of separate lymphocytes from human peripheral blood 33 has been experimented to extrapolate phenomena in vivo, mimicking a polyclonal immune response mediated by T. lymphocytes. toxic and specific action of GSO1 bioactive complexes (purified crystallized white powder 37 glycosidated bioactive complex with a content of total monodesmozide triterpenic saponins with aglicon hederagenin of at least 95% standardized in α-hederin of 70 ... 80%) and GSO2 39 (purified crystallized white crystalline powdered glycoside bioactive complex with a content of total bisdesmozide triterpenic saponins with a minimum of 90% aglycone hederagenin, 41 standardized in 60 ... 75% Hederacozide C). Experimental data were reported as appropriate, to culture and solvent controls. 43

1. Activity on cytotoxic status and metabolic activity - performed by standardized tests to determine the reduction of MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxy-45 methoxyphenyl) -2-H-tetrazolium ) and LDH (lactate dehydrogenase) using ELISA technique. The effects of biocomplexes on cell viability and cell proliferation were tracked (test values 47

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MTS) correlated with the activity on the cytotoxic potential, expressed by the values obtained in the LDH test. This demonstrates the cytotoxic and / or cytostatic action of bioactive complexes.

Table 1

Cytotoxic / cytostatic effect of GSO1 biocomplex on standardized tumor cells. The IC ^ - statistical significance p <0.05 was determined by the one-way ANOVA algorithm

The line cell / Under the- die MCF7 HeLa Hep MTS IC50 (μΜ) LDH IC50 (μΜ) MTS IC50 (PM) LDH IC50 (PM) MTS IC50 (PM) LDH IC50 (PM) MC 18.8 ± 0.075 19.6 ± 0,201 18.2 ± 0.086 17.6 ± 0.193 9.2 ± 0.126 10.6 ± 0.279 THX 16.5 ± 0.145 17.4 ± 0.089 16.9 ± 0.106 15.9 ± 0.073 8.4 ± 0.084 9.5 ± 0.203 GSO1 14.7 ± 0.057 14.5 ± 0.142 12.3 ± 0,312 12.7 ± 0.105 5.1 ± 0.135 6.8 ± 0.079 a- hederin standard 16.2 ± 0.137 20.2 ± 0.069 14.7 ± 0,157 18.3 ± 0.099 8.6 ± 0.163 11.7 ± 0.109

MC = cell control MS = solvent control

From the analysis of the data presented in table 1, it is highlighted that the GSO1 biocomplex exerts on the mammary (MCF) and cervical (He and HeLa) tumor cells a moderate cytotoxic action and a significant cytostatic effect by decreasing the MTS IC ₅₀ and LDH IC ₅₀ values, indicating a slight decrease of cell viability and inhibition of cell multiplication. The cytotoxic action of the standard substance compared to GSO1 and MS is noted, by decreasing the MTS IC ₅₀ value and increasing the LDH IC ₅₀ value, indicating decreased cell viability and multiplication by increasing LDH release.

The GSO1 product has a statistically significant cytostatic effect, at ranges of concentrations specific to each cell line tested compared to the heavy cytotoxic action of the standard substance (purity> 95% HPLC).

Table 2

Cytotoxic / cytostatic effect of GSO2 biocomplex on standardized tumor cells. The IC ^ - statistical significance p <0.05 was determined by the one-way ANOVA algorithm

Cell line DU 145 Substance MTS IC ₅₀ (μΜ) LDH IC ₅₀ (μΜ) MC 19.3 ± 0.096 19.7 ± 0.153 GSO2 16.2 ± 0.209 * ^a 16.7 ± 0.359 * ^a Hederacozide C standard 17.3 ± 0.072 20.9 ± 0,173

MC = cell control

The statistically significant differences of the proliferation index (P <0.05) are indicated by:

*** - extremely significant P <0.001

RO 125279 Β1 ** - very significant * - significant non - insignificant a - vs MC

0.001 <P <0.01

0.01 <P <0.05 P> 0.05

Experimental data indicate for the GSO2 complex (purity> 60% HPLC) a statistically moderate moderate cytostatic effect higher than the action of the standard substance (purity> 95% 7).

HPLC) on cells derived from prostate cancer.

In conclusion, the biocomplexes tested obtained within Biotechnos have a low cytotoxic profile 9 and statistically significant cytostatic effects higher than the action of the standard substances.

2. Activity on apoptosis - evaluated based on the evidence of phosphatidyl-serine translocation, 11 early event of apoptosis induction simultaneously with nuclear fragmentation and permeabilization of the plasma membrane characteristic of late apoptosis (double fluorescence marking with Annexin 13-FITC, respectively, prop.

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Table 3

Effect of the 6S01 biocomplex on the induction of apoptotic versus necrotic processes in cervical tumor cells. Determined / C _w - statistical significance p <0.05 by the 'one-way ANQVA' algorithm

Test compound (μΜ) Hep 2 cell line HeLa cell line % live cells % apoptosis % necrosis % live cells % apoptosis % necrosis THX 93.3 + 0.374 1 +0.195 5.6 + 0.097 81.6 + 0.642 14.9 + 0.187 3.5 + 0.043 GSO1 [8pm] 631 + 0.815 "'" ⁵ 30.9 + 0.426 ^ 6 + 0.383 38.5 + 0.226 '' a, '' b 60.7 + 0.543 "^ 0.8 + 0.0003 * ' GSO1 [10 pM] 70.2 + 0.515 ^ 21.1 + 0.912 " 8.7 + 0.296 ' 54.3 + 0.426 '' to 42.2 + 0.274 ' 3.6 + 0.009 GSO1 [14pM] 79.6 + 0.671 ' 14.1 + 0.613 '' 6.3 + 0.520 57.9 + 0.698 '' to 39.2 + 0.140 '' 2.9 + 0.019

MS = solvent control

The statistically significant differences of the proliferation index (P <0.05) are indicated by: extremely significant '' 'P <0.001' '- very significant 0.001 <P <0.01' significant 0.01 <P <0.05 ns -significant P> 0.05 a- vsMCS; b- vsGSO114 pM;

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Table 4 1

Effect of GS01 biocomplex on the induction of apoptotic versus necrotic processes in mammary tumor cells. IC ^ - statistical significance p <0.05 3 was determined using the one-way AN OVA algorithm

Test compound (pM) IVICF7 cell line % live cells % apoptosis % not necrosis MS [8 pM] 77.9 ± 0.357 12.82 ± 0.395 9.2 ± 0.123 MS [10 pM] 78.9 ± 0.182 14.1 ± 0.091 8 ± 0.195 MS [14 pM] 80.77 ± 0.097 11.50 ± 0.106 7.73 ± 0.274 GSO1 [8 μΜ] 64.5 ± 0.241 ** ^a 24.30 ± 0.126 ** ^a 11.14 ± 0.085 GSO1 [10 μΜ] 57.5 ± 0.075 *** ^b 31.5 ± 0.221 *** ^b 11.1 ± 0.175 GSO1 [14 μΜ] 54.1 ± 0.395 “* ^c 36.55 ± 0.113 * “ ^c 9.4 ± 0.048

MS = solvent control 13

The statistically significant differences of the proliferation index (P <0.05) are indicated by: 15 *** - extremely significant P <0.001 ** - very significant 0.001 <P <0.01 17 * - significant 0.01 <P <0.05 ns - insignificant P> 0.05 19 a - vs MS 8 μΜ; b - vs MS 10 pM; c - MS 14 pM

Table 5 21

Effect of GSO2 biocomplex on the induction of apoptotic versus necrotic processes in prostate cancer cells. IC ^ - significance 23 statistical p <0.05 was determined by the one-way algorithm AN OVA statistic p <0.05 by the one-way algorithm ANOVA

Test compound (pM) Cell line DU 145 % live cells % apoptosis % necrosis MC 85.3 ± 0,395 11.3 ± 0,395 3.4 ± 0.014 GSO2 [10 μΜ] 5.4 ± 0.0935 *** ^a 93.2 ± 0.326 *** ^a 1.4 ± 0.009 * ^a GSO2 [14 μΜ] 5.4 ± 0.019 *** ^a 93.7 ± 0.157 *** ^a 0.9 ± 0.003 *** ^a GSO2 [24.56 μΜ] 33.3 ± 0.085 *** ^a 62.2 ± 0.301 * of 4.5 ± 0.026

MS = solvent control 31

The statistically significant differences of the proliferation index (P <0.05) are indicated by: 33 *** - extremely significant P <0.001 ** - very significant 0.001 <P <0.01 35 * - significant 0.01 <P <0.05 ns - insignificant P> 0.05 37 a - vs MC, b - vs GSO2 24.56 uM

After receiving apoptotic signals from antitumor agents, cells enter early apoptosis, followed by late apoptosis / secondary necrosis. Bioactive complexes act 41 by inducing apoptotic cell death with specific kinetics through nuclear fragmentation, caused by endonuclear cleavage of genomic DNA. 43

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Table 4 shows the selective action of GSO1 for the progressive induction of apoptosis in MCF7 cells (breast adenocarcinoma), exerted in dose-effect manner.

The experimental data summarized in table 3 show that at the level of cervical carcinoma, in both Hep2 and HeLa type cells, an increase in the number of apoptotic cells is observed as an effect of the presence of GSO1 in the extracellular environment inversely proportional to concentration of bioactive complex.

On the other hand, it is noted that GSO2 (6-12 μΜ) acts selectively in prostate carcinoma (DU line 145), inducing processes characteristic of apoptosis - see Table 5.

The increase of the apoptotic cell fraction is doubled by the presence of a percentage of necrotic cells compared to the culture control / solvent control, which means a convenient toxic effect of the product.

3. Activity on cell cycle sequencing revealed by determining the percentage of cells in the cell cycle phases, Go / G1, S (DNA synthesis) and G2M by flow cytometry and fluorescence marking of cell nuclei with propidium iodide linked to DNA stoichiometry.

Table 6

Percentage variation of cells in the DNA synthesis phase of the cell cycle as an effect of GSO1 biocomplex action on MCF 7 HeLa and Hep 2 cells. IC ^ was determined - statistical significance p <0.05 by one-way ANOVA

Cell line / Biocomplex % S phase of DNA synthesis MCF 7 HeLa Hep MS [8.5 pM] 86.56 ± 1.018 35.57 ± 1.741 30.10 ± 1.484 MS [10.6 pM] 83,2511,009 31.54 ± 1.091 33.08 ± 0.878 MS [14.1 pM] 85.25 ± 1.097 24.08 ± 1.568 34.09 ± 0.358 GSO1 [8.5 pM] 56.37 ± 0.815 ** ^a 24.46 ± 0.279 “ ^a 7,820 ± 0.716 * “ ^a GSO1 [10.6 pM] 53.08 ± 0.215 ** ^b 24.44 ± 0.859 * ^b 12.00 ± 0.732 * "b GSO1 [14.1 pM] 33.72 ± 0.317 *** ^c ** ^d 23.33 ± 0.930 ^nsc 15.09 ± 0.273 *** ^b

MS = solvent control

The statistically significant differences of the proliferation index (P <0.05) are indicated by: *** - extremely significant P <0.001 ** - very significant 0.001 <P <0.01 * - significant 0.01 <P <0 , 05 ns - insignificant P> 0.05 a - vs MS 8.5 μΜ; b - vs MS 10.6 pM; c - vs MS 14.1 pM; d - vs GSO1 8.5 pM

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Table 7 1

The variation of the percentage of allied cells takes the GO / Gt phase within the cell cycle as an effect of the action of the GSO1 pheocomplex on the IWCF cells

7, HeLa and Hep 2. IC _S was determined - statistical significance p <0.05 by the one-way algorithm ANO VA 3

Cell line / Biocomplex % GO / G1 phase of the cell cycle % G2 / M phase of the cell cycle MCF7 HeLa Hep MCF7 HeLa Hep MS [8,5uM] 12.41 + 0.364 54.46 + 0.148 65.75 + 0.245 1.03 + 0.106 10.06 + 0.317 4.15 + 0.554 MS [10.6 μΜ] 15.21 + 0.354 56.36 + 0.465 60.39 + 0.843 1 36 + 0.222 12.05 + 0.614 6.53 + 0.34 MS [14.1 μΜ] 13.22 + 0.225 60.54 + 0.786 62.4 + 0.124 0.98 + 0.144 11 + 0.247 5.43 + 0.443 GSO1 [8,5pM] 42.75 + 0.412 '“ ^d 66.2 + 0.317 '' 82.33 + 0.348 ' 1.88 + 0.096 9.5 + 0.156 9.85 + 0.113 ' GSO1 [10,6pM] 4544 + 0.365 '” ^d 61.74 + 0.235 ^b 75.04 + 0.454 “ ^b 4.17 + 0.088 ' 13.82 + 0.456 12.95 + 0765 " GSO1 [14.1 μΜ] 63.02 + 0.554 ' 65.3 + 0.287 74.27 + 0.113 ' 3.26 + 0.012 ' 11.54 + 0.273 10.65 + 036 '

MS = solvent control 13 a vs MS 3.5 μΜ, b - vs MS 10.6 μΜ, c - vs MS 14.1 μΜ, d - vs GSO114.1 μΜ

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The data summarized in Table 6 show that mammary and cervical tumor cells are particularly responsive to GSO1 action in terms of the mechanisms of DNA synthesis, so that significant inhibition of replicative cells is found. The cell cycle analysis by the breast and cervical tumor cells under the influence of GSO1 shows that the cell cycle sequencing is modulated and in terms of the number of cells in the other phases - table 7. The mechanisms of cell growth and multiplication at the level of cell cycle initiation are influenced, at the interface with the possibility of cellular blocking at the Go point.

4. Activity on cell proliferation - evaluated by flow cytometry and fluorescence labeling with carboxy fluorescein diacetate succinimidyl ester.

Table 8

Variation of cell proliferation index in MCF7, HeLa and Hep2 lines in the presence of GSO1 in the culture medium. IC ^ - statistical significance p <0.05 was determined by one-way ANOVA algorithm

Cell line / Biocomplex Proliferation Index (IP) MCF7 HeLa HepG2 MC 7.76 ± 0.42 1.470 ± 0.069 4.150 ± 0.213 GSO1 [8.5 μΜ] 5.24 ± 0.083 1,720 ± 0.27 * ^a 2 ± 0.127 *** ^a GSO1 [10.6 μΜ] 5.02 ± 0.116 1.673 ± 0.034 1.97 ± 0.029 *** ^a GSO1 [12 μΜ] 4.91 ± 0.231 ** ^a 1,026 ± 0.018 * ^a 1.83 ± 0.102 *** ^a GSO1 [14.1µΜ] 3.56 ± 0.198 *** ^a 1.00 ± 0.008 * ^b 1.4 ± 0.2 * ^a GSO1 [16 μΜ] 1.45 ± 0.242 *** ^a “* ^b 0.876 ± 0.054 * ^a 1 ± 0.078 *** ^a ** ^b MTX [5 ηΜ] 3.77 ± 0.046 * “ ^a 0.398 ± 0.008 *** ^a 1.675 ± 0.067 * “ ^a

MC = cell control MTX = metrotexate

The statistically significant differences of the proliferation index (P <0.05) are indicated by:

*** - extremely significant P <0.001 ** - very significant 0.001 <P <0.01 * - significant 0.01 <P <0.05 ns - insignificant P> 0.05 at -vs MC; b - vs MTX 5 nM.

Table 8 shows the marked inhibition in the dose-effect manner of the proliferative capacity of Hep2-like mammary and cervical adenocarcinoma cells, as well as the induction of a slight anti-proliferative effect on HeLa cervical tumors.

GSO1 reduces the number of generations in the proliferative processes in the brain cells studied compared to the positive control.

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Table 9 1

Variation of the cell proliferation index in the DU 145 cell line in the presence of GSO2 in the culture medium. IC ^ - statistical significance p <0.05 was determined by the one-way algorithm 3

ANOVA

Cell line OF 145 biocomplex Proliferation Index (IP) MC 4.150 ± 0.213 GSO2 [10 μΜ] 2 ± o, 127 *** ^a * ^b GSO2 [14 μΜ] 1,750 ± 0.28 *** ^a GSO2 [24.56 μΜ] 1.4 ± 0.2 *** ^a MTX 5 nM 1.67 ± 0.0,067 *** ^a

MC 13

The statistically significant differences of the proliferation index (P <0.05) are indicated by:

*** - extremely significant P <0.001 ** - very significant 0.001 <P <0.01 17 * - significant 0.01 <P <0.05 ns - insignificant P> 0.05 19 a - vs. MC, b - vs MTX

GSO2 (10-24.56 μΜ) reduces, in dose-effect manner, the number of generations in the proliferative processes of DU 145 cells, therefore the biocompound exerts a specific antiproliferative effect 23 on prostate cancer cells.

5. Activity on the processes of angiogenesis, evaluated by the Ribatti method, on the 25 chorioalantoic embryonic membrane of chickens (43). The antiangiogenic action of the GSO1 compound was determined, by which it is shown that the antitumor effect is manifested by 27 alternative mechanisms of blocking the tumor progression due to the processes of reduction or inhibition of the tumor irrigation. 29

GSO1 exerts a strong healing effect evidenced both by the test on the embryo egg (blood vessel restoration) and in preclinical experiments on the B6D2F1 mouse, in which 31 it was noted the rapid recovery of some induced lesions. The action of GSO1 induces hemolysis, then scarring and survival of the embryo through mechanisms of vascularization reduction 33, which highlights the antiangiogenic character of the biocomplex.

6. Activity on modulating the DNA synthesis phase of normal lymphocytes, separated 35 from human peripheral blood, mitogenic stimulated with phytohemagglutinin (PHA) 20 µg / ml, by which the in vivo phenomena regarding the response of lymphocytes to the intervention of a proliferation growth factor 37 are extrapolated. (eg stimulated multiplication in tumor processes).

The tests applied to highlight the influence of bioactive complexes on the DNA sequence of the specific cell cycle of the stimulated mononuclear cells in vitro consisted of monitoring the DNA synthesis phase and the induced effect on the proliferative capacity of 41 non-stimulated lymphocytes, under the presence of GSO1 in the environment, respectively. , GSO2.

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Table 10

The effect of GSO1 and GSO2 on mitogenic stimulated lymphocytes and on basal proliferative capacity, expressed by the percentages of unstimulated lymphocytes that go through the DNA synthesis phase under the conditions of culture maintenance 48 h

% S phase of DNA synthesis Non-stimulated lymphocytes Lymphocytes stimulated with PHA 48 h MC 0.15 ± 0.0001 21.55 ± 10.59 GSO1 [8 μΜ] 0 16.5 ± 0.54 “* ^b GSO1 [12 μΜ] 0.0410,0009 *** ^a 14.03 ± 0.01 *** ^b GSO2 [32 μ Μ] 0 21.7 ± 0.38 ^nsb GSO2 [50 μ Μ] 0 23.6 ± 0.41 ^nsb

MC = cell control a - vs unstimulated MC; b - vs MC stimulated with PHA

Table 10 shows that the biocomplexes do not influence the basal proliferative capacity of the lymphocytes expressed by the number of unstimulated cells present in the DNA synthesis phase, which shows that the presence of GSO1 and GSO2 does not affect the normal physiological status in vitro. However, there is a significant inhibitory effect, in dose-effect manner, of GSO1 on mitogen-stimulated cells that are in synthesis phase S. Also, GSO2 action is present in the experimental system, indicating a decrease in the percentage of replicative mononuclear cells directly proportional to the growth. biocomplex concentration. The realization of the in vitro model of inducing mitogenic stimulation of separate lymphocytes from human peripheral blood for extrapolation of in vivo phenomena has shown that the intervention of bioactive complexes induces a polyclonal immune response mediated by the T lymphocytes favoring the antiproliferative action imposed in the uncontrolled growth conditions of the tumors.

From the analysis and corroboration of the presented experimental data, it can be concluded that the 2 glycosidated bioactive complexes in the form of purified crystallized powder, one containing total monodesmozide triterpenic saponins standardized in 70 -80% hederin and the other with a saponin content. Total bisdesmozide triterpenes standardized in Hederacozide C of 60 ... 75% induce moderate cytotoxicity and a significant cytostatic effect compared to the existing standards of α-hederin (minimum 95%) and Hederacozide C (minimum 95%) and show remarkable biological activity in the status tumor proliferative with proven in vitro antitumor effects.

II. In vitro antioxidant and regenerative activity

The analytical model applied to demonstrate the antioxidant potential of the GSO1 structural group used the indirect spectrophotometric method with 1,1-diphenyl-2-picryl hydrazide (DPPH) as a stable free radical generating system DPPH.

Table 11

Reductive capacity of extracts on the free radical DPPH

Concentration/ Subata lnhibiţie% 10 μg / ml 20 μg / ml 40 μg / ml 50 μg / ml 80 μg / ml GSO1 13.02 18.82 27.94 31.47 45.73

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Table 11 (continued) 1

Concentration/ Subata lnhibiţie% 10 pg / ml 20 pg / ml 40 pg / ml 50 pg / ml 80 pg / ml GSO2 6.9 13.66 24.55 26.74 49.91 Trolux 21 27 32 40 52

Significant antioxidant capacity, manifested in the free radicals by 7 GSO1, is highlighted against Trolux, considered a positive control, protecting the cells from the attack of reactive oxygen species (SRO). 9 ///. In vivo hepatoprotective activity of GSO1 and GSO2, in the context of their efficacy in antitumor activity 11

The hepatoprotective action of the bioactive complexes, expressed by determining the total antioxidant status, the evaluation of the enzymatic activity of the catalase and the dosing of 13 MDA malondialdehyde (specific indicator for lipid peroxidation) was shown on an experimental model of induction of liver damage in Wistar rats with CCI ₄ . 15

There were 3 studies:

I - the animals were treated for 7 days with GSO1, on day 5 hepatotoxic was given 17;

II - the animals were treated for 14 days with GSO2, on the 10th day 19 hepatotoxic was given;

III - the animals received for 7 days physiological serum, then were treated for 7 days 21 with GSO1, respectively, GSO2, on day 7 hepatotoxic was given.

following the poisoning of animals with 1 ml / kg gc CCI ₄ : sunflower oil, 1: 1, v / v, the concentration of free radicals in plasma increased 23 (intoxicated control - 0.054 mmol / l compared to non-toxic control - 0.018 mmol / l) for the purpose of determining the systemic response to GSO1 and 25 GSO2 action.

Table 12

Effect of GS01 on total antioxidant status at plasma level 29

Parameter / Lot Total antioxidant status without KICs ₄ with KIC ₄ Study I LMV (n = 6) 0.018 ± 0.001 0.052 ± 0.013 LGSO1_1 (n = 6) 0.016 ± 0.003 0.026 ± 0.009 ** ^a LGSO1 2 (n = 6) 0.014 ± 0.0016 0.020 ± 0.005 ** ^a

LMV = vehicle control lot (oil)

LGSO1_2 = batch treated with GSO1 75 mg / kg. g.c, 37

LGSO1_2 = batch treated with GSO1 150 mg / kg. G.C.

The statistically significant differences of the proliferation index (P <0.05) are indicated by:

*** - extremely significant P <0.001 41 ** - very significant 0.001 <P <0.01 * - significant 0.01 <P <0.05 43 a - vs control

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According to the results obtained from the statistical processing (table 12), it was observed that the pretreatment of the animals with GSO1 (75 mg / kg gc and 150 mg / kg gc) decreased the level of free radicals existing in the plasma, an effect evidenced in the case of the hepatotoxic intoxicated animals. . In the case of lot LGSO1_2, the concentration of free radicals in plasma (0.020 mmol / l) was similar to that of the non-toxic control (0.018 mmol / l), therefore the influence of GSO1 maintained the normal physiological value of the oxidative potential of plasma. Based on the reducing action of GSO1, it significantly counteracts the negative effects of reactive oxygen species, thus manifesting a remarkable systemic antioxidant effect.

Table 13

Effect of GSO1 on plasma lipid peroxidation

plasma

Parameter / Lot Lipid peroxidation without KICs ₄ with KIC ₄ Study I LMV (n = 6) 0.483 ± 0.054 1.458 ± 0.269 LGSO1 1 (n = 6) 0.475 ± 0.094 1.127 ± 0.103 LGSO1 2 (n = 6) 0.442 ± 0.055 1.128 ± 0.087

LMV = vehicle control lot (oil)

LGSO1_2 = batch treated with GSO1 75 mg / kg. G. C,

LGS01_2 = batch treated with GSO1 150 mg / kg. G. C,

The statistically significant differences of the proliferation index (P <0.05) are indicated by: *** - extremely significant P <0.001 ** - very significant 0.001 <P <0.01 * - significant 0.01 <P <0 , 05

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Table 14 1

The effect of GSO1 and GSO2 on the oxidative system expressed by the degree of lipid peroxidation and the activity of hepatic homogenate catalase.

Statistical processing of the experimental data was performed by applying the one-way ANOVA algorithm, followed by Dunnett's t-test 3

Homogenate liver Parameters / Lot Lipid peroxidation nmolMDA / g protein Catalase unit / mg protein without CCI ₄ cuCCI ₄ without CCI ₄ cuCCI ₄ Study LMV (n = 6) 0.8630 + 0.0086 2.15 + 0.109 30.10 + 1.091 10.71 + 1.137 LGSO1_1 (n = 6) 0.7735 + 0.0158 * ' 1.33 + 0.088 * ” ^a 31.13 + 2.414 12.01 + 1.018 LGSO1_2 (n = 6) 0.2992 + 0.005 "'" ^b 1.17 + 0.01 in ^b 47.44 + 1,441 * '*** ^b 22.49 + 1.076 '“ ^b Study LM (n = 6) 0.7700 + 0.06351 1.65 + 0.106 42.28 + 1.372 16.99 + 1.973 LGSO2 1 (n = 6) 0.2745 + 0.1155 * ' 1.25 + 0.125 * ^ 68.35 + 1,338 * ^a * ^c 62.67 + 4.425 = - LGSO2 2 (n = 6) 0.1140 + 0.0220 ** ' 0.29 + 0.095 * ' 80.68 + 2,612 '^{a; c} 94.18 + 6.175 ** ' LHep 0.1045 + 0.0055 ** ' 0.91 + 0.024 ^ 177.90 + 2.6862 ** ' 112.01 + 3.606 ' Study LMV (n = 6) - 1.63 + 0.211 - 4.95 + 1.432 LM (n = 6) - 1.65 + 0.185 - 15.02 + 1.973 LGSO1 2 (n = 6) - 0.76 + 0.107 * ' - 87.46 + 3.478 LGSO2 2 (n = 6) - 1.22 + 0.2133 = - 78.47 + 4.102

LMV = vehicle treated lot (oil) 21

LM = lot treated with physiological serum

LGSO1.1 = batch treated with GSO175 mg / kg. G.C. 2. 3

LGSO1 _2 = lot treated with GSO1150 mg / kg. G.C.

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LGSO2_1 = lot treated with GSO2 75 mg / kg. G. C,

LGSO2_2 = batch treated with GSO2 150 mg / kg. G.C.

LHep = batch treated with arginine for infusion (positive control for hepatoprotective activity)

The statistically significant differences of the proliferation index (P <0.05) are indicated by:

*** - extremely significant P <0.001 ** - very significant 0.001 <P <0.01 * - significant 0.01 <P <0.05 a - vs LMV, b - vs GSO1_1, c - vs LHep, d - vs LM

According to the obtained data, there is a significant increase of the MDA concentration in the liver tissue of Wistar rats after the administration of hepatotoxic, indicating the installation of lipid peroxidation, due to the microsomal metabolism of carbon tetrachloride. The highest level of peroxidation is reached, as expected, in the tetrachloride intoxicated group (2.15 nmolMDA / g protein), representing an increase of 59.6% compared to the non-intoxicated control. In the liver tissue, in the case of GSO1 administration to healthy laboratory animals, a decrease of the MDA concentration is observed depending on the dose administered (table 14 - Study I), the tendency highlighted also in the animals which were induced inflammation (group LGSO1_1 with 37 , 5%, respectively, the lot LGSO1_2 with 45.6%). In the case of plasma samples (table 13), a weak action of the bioactive compound is observed, exerted on the mechanisms of initiation and propagation of lipid peroxidation. Applying an animal model similar to that used in Study I, it was found that GSO2 administration in the absence of xenobiotic does not induce liver toxicity and has the same protective properties as GSO1 and arginine infusion used in medical practice.

If the bioactive complexes were administered to the subjects with liver injury due to hepatotoxic poisoning (Table 14 - Study III), GSO1 presented better protection than the GSO2-like substance, causing a decrease in the MDA concentration 53.24% as compared to 26.37%).

The catalase-specific enzyme activity is significantly lower in animals that were intoxicated with CCU, with the exception of the pretreated LGSO22 group, which maintained the increased enzyme level. The administration of the two bioactive complexes increased the concentration of catalase in the liver tissue in a dose-effect manner, due to the action of slowing / inhibiting the processes of radical formation. Interestingly, the difference between the catalase concentration observed in the control groups (30 units / mg protein) and LGSO1_1 (31 units / mg protein) are not very different, but between the two doses tested, the differences are statistically significant. When the analyzed structural groups were administered to subjects with potential hepatic parenchymal lesions (Table 14 Study III), a significant improvement of the enzymatic status was observed in both variants studied, but GSO1 (87.46 units / mg protein) showed a enhancement of enzyme activity of catalase much higher than GSO2 (78.47 units / mg protein).

Table 15

Effect of GS01 and GSO2 on hepatic protein metabolism

Parameters / Lot Liver protein mg / g tissue Difference from witness (%) without KICs ₄ with KIC ₄ without KICs ₄ with KIC ₄ The study I LMV (n = 6) 93.71 ± 6.156 84.29 ± 3.257 - - LGSO1_1 (n = 6) 6,450 ± 179 117.9 ± 6447 20 14.118 LGSO1 2 (n = 6) 142.6 ± 9164 140.1 ± 7.05 * ^a 52.172 66.212

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Table 15 (continued) 1

Parameters / Lot Liver protein mg / g tissue Difference from witness (%) without KICs ₄ with KIC ₄ without KICs ₄ with KIC ₄ The study II LM (n = 6) 43.44 ± 5.041 *** ^b 31.25 ± 1.902 - LGSO2_1 (n = 6) 116.9 ± 3.443 *** ^b 147.6 ± 9.616 *** ^b 169.172 372.320 LGSO2_2 (n = 6) 123.4 ± 6.882 * ^b 70.47 ± 1,288 * ^b ** ^c 184.070 125.504 LHep 140.8 ± 4.830 * ^b 59.65 ± 1,010 *** ^c 224.125 90.880 The study III LMV (n = 6) - 4540 ± 49.2 - LM (n = 6) - 31.25 ± 1.902 - LGSO1_2 (n = 6) - 80.89 ± 2.014 * ^a - 45.784 LGSO2-2 (n = 6) - 82.51 ± 2.482 * ^b - 164.032

LMV = vehicle treated lot (oil)

LM = lot treated with physiological serum 17

LGSO1_1 = lot treated with GSO1 75 mg / kg. G. C,

LGSO1_2 = batch treated with GSO1 150 mg / kg. G.C. 19

LGSO2_1 = lot treated with GSO2 75 mg / kg. G. C,

LGSO2_2 = batch treated with GSO2 150 mg / kg. G.C. 21

LHep = lot treated with arginine for infusion a - vs LMV, b - vs LM, c - vs LGSO2_1 23

Administration of KIC ₄ causes a decrease in hepatic proteins which is restored by treatment with GSO1 and GSO2 so that in both cases there is an increase of hepatic proteins by over 50%. 27 following these studies, it turned out that GSO1 and GSO2 do not induce hepatotoxic activity in the untreated groups with the hepatotoxic substance, and in the intoxicated groups with CCI ₄ - inducer of 29 liver dysfunctions - both biocomplexes determine the protection and restoration of the liver tissue, by suppressing the metabolic processes. developed by KIC _{4 the} chain of events that follows 31 after the induction of inflammation is interrupted.

In conclusion, the data of the corroborated experimental studies show that the first 2 phytotherapeutic preparations 33 according to the invention aim to obtain products with antitumor effects and a remarkable efficacy / toxicity ratio by exercising a concomitant action 35 antitumor, antioxidant and hepatoprotective, usable in both oncology and veterans. for human use. In particular, GSO1 is an active biocomplex with antitumor action in 37 in vitro complex, especially for breast adenocarcinoma and cervical carcinoma. GSO2 has a moderate antitumor effect in prostate cancer by inducing apoptosis and 39 antiangiogenic effect.

Example 2. Anti-tumor drug product - solution for injection containing 2% 41 phytotherapeutic product from Hedera helix

Conditioning formula: 43

- Phytotherapeutic preparation-GSO-1 ..................................... 22 g

- benzyl alcohol ............................................... 100 ml 45

- aqueous phosphate buffer pH = 8 .............................. 100 ml

- propylene glycol ............................................ ad. 1000 ml 47

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Method of preparation: the phytotherapeutic product is dissolved in hot (6O ... 7O ° C) in 200 ml mixture 1: 1 v / v benzyl alcohol: propylene glycol, the propylene glycol residue and the aqueous phosphate buffer solution are added with stirring with pH = 8, and the resulting homogeneous solution is filtered aseptic and conditioned in 1 ... 2 ml vials containing 20 mg / ml of herbal product from Hedera helix.

The product is administered parenterally, in doses of 1 ... 3 ampoules / day, for 1 ... 6 months, in the treatment of human and veterinary use of benign and malignant tumors, especially in the treatment of breast neoplasia, as well as immunoprotective for counteracting the adverse, immunosuppressive effects of chemotherapy with cytostatic synthetic drugs.

Example 3. Antitumour drug product - Oral solution containing 2% herbal product from Hedera helix Conditioning formula:

- herbal product .............................................. 22 g

- Glycerin ................................................ ... 200 ml

- propylene glycol ................................................ 200 ml

- ethyl alcohol 95 ° ............................................. 200 ml

- sorbitol ................................................ ...... 40 g

- Methyl p-hydroxybenzoate ......................................... 1 g

- propyl p-hydroxybenzoate ........................................ 2 g

- aqueous phosphate buffer pH = 8 .......................... ad. 1000 ml

Method of preparation: The phytotherapeutic product is dissolved in hot (6O ... 7O ° C) in 400 ml 1: 1 v / v mixture of ethyl alcohol: propylene glycol, followed by addition of the amount of glycerin and the aqueous solution of phosphate buffer with pH = 8, in which the two preservatives provided in the formula and sorbitol dissolved.

Stir for homogenization and filter, and the resulting clear solution is conditioned in vials of 100 ... 200 ml.

The product contains 20 mg / ml Hedera helix herbal product which is administered orally, in doses of 5 ... 10 ml / day, for 1 ... 6 months, in the treatment of human and veterinary use of benign and malignant tumors. , especially in mammary neoplasia therapy, as well as as an immunoprotective for counteracting the adverse, immunosuppressive and hepatotoxic effects of chemotherapy with cytostatic synthetic drugs.

Example 4. Anti-tumor drug product - Hard capsules containing 100 mg / capsule herbal product from Hedera helix

Conditioner / capsule formula:

- herbal product - minimum 90% .............................. 110 mg

- lactose monohydrate .......................................... 150 mg

- starch sodium glycolate ...................................... 9.00 mg

- polyvinylpyrrolidone K30 ....................................... 6.50 mg

- sodium lauryl sulfate ......................................... 2.00 mg

- aerosill ................................................ ... 1.47 mg

- talc ................................................ ...... 7.25 mg

- magnesium stearate ........................................ 2.90 mg

Method of preparation: the herbal product is placed on a number II screen with excipients: lactose monohydrate and aerosil; to the mixture obtained is added a solution made by dissolving the polyvinylpyrrolidone K30 in ethyl alcohol, homogenizing, resulting in granules, which are dried at 35 ... 45 ° C and sieved by sieve number V; add sodium starch glycolate, sodium lauryl sulphate, talc and magnesium stearate, homogenize, and the powder thus obtained is encapsulated in size 1 capsules.

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The product contains 100 mg / capsule phytotherapeutic product of Hedera helix and is administered orally, in doses of 2 ... 4 capsules / day, for 1 ... 12 months, in antitumor therapy for human or veterinary use, with especially in breast neoplasia, as well as immunoprotective for counteracting the immunosuppressive adverse effects of chemotherapy with cytostatic synthetic drugs. 5

Example 5. Antitumor drug product - soft capsules containing 100 mg / capsule herbal product from Hedera helix 7

Conditioning formula:

- herbal product - minimum 90% ................................ 22 g 9

- ethyl alcohol 95 ° ............................................. 250 ml

-glycerin ................................................ ... 250 ml 11

- seaweed oil ........................................... ad. 1000 ml

Method of preparation: The herbal product is dissolved in hot (6O ... 7O ° C) in 500 ml 13 mixture 1: 1 v / v ethyl alcohol 95 °: glycerin, resulting in a homogeneous solution, which is brought into the distillation flask. to a rotary evaporator, distill at 3O ... 35 ° C, 15 under vacuum, until the total removal of ethyl alcohol is added, then add the amount of seaweed oil and mix with stirring. A semi-colloidal solution 17 is obtained which contains 20 mg / ml of herbal product and is conditioned in soft gelatin capsules, each of 5 ml of product, which is administered orally, in doses of 2 ... 4 capsules / day, 19 for 1 ... 12 months, in antitumor therapy for human and veterinary use, especially in mammary tumors, as well as as an immunoprotective for counteracting the 21 immunosuppressive effects of chemotherapy with synthetic cytostatic drugs.

Example 6. Anti-tumor drug-gel product containing 1% herbal product 23 from Hedera helix

Packaging formula / capsule: 25

- herbal product - minimum 90% ................................ 11 g

- carbopol 940 ............................................... .. 20 g 27

- Glycerin ................................................ .... 50 g

- ethyl alcohol ............................................... ... 50 g 29

-trietanolamină ................................................ 15 g

- propylene glycol ................................................ 60 g 31

- Methyl p-hydroxybenzoate ........................................ 1 g

- propyl p-hydroxybenzoate ....................................... 2 g 33

- purified water ........................................... ad. 1000 g

Method of preparation: The gel mass is prepared by dispersing 20 g Carbopol 940 35 in the mixture of glycerin, ethyl alcohol and purified water, in which the mixture of preservatives provided in the formula is dissolved. Shake gently, then leave to stand for 24 hours at 37 ° C to 25 ° C, then add the mixture of the herbal powder, propylene glycol and the triethanolamine solution under slow stirring, adjusting to pH 39 = 7.0 and stirring continued to perfect the homogenization, followed by packing.

The product contains 1% phytotherapy product from Hedera helix and is given topically, in 41 doses of 3 ... 5 g / day, for 1 ... 6 months, in local antitumor therapy of mammary neoplasia or other benign and malignant tumors. , located externally, for human and veterinary use. 43

Example 7. Anti-tumor drug product - cream containing 1% herbal product from Hedera helix 45

Conditioning formula:

- herbal product - minimum 90% ................................ 11 g 47

- cetyl alcohol ............................................... 160 g

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- Vaseline ................................................ .... 200 g

- Glycerin ................................................ .... 107 g

- Tween 80 ............................................... ..... 70 g

- Methyl p-hydroxybenzoate ......................................... 1 g

- propyl p-hydroxybenzoate ........................................ 2 g

- purified water ............................................ ad. 1000 g

Method of preparation: the herbal product is dissolved in hot (6O ... 7O ° C) in the amount provided by glycerin, and the resulting solution is incorporated in the ointment mass, obtained by melting at 60 ° C of the cetyl alcohol and the grease, followed emulsification, by adding the quantities provided by Tween 80 and purified water, in which the 2 preservatives, homogenization and conditioning were dissolved previously.

The product contains 1% herbal product from Hedera helix and is given topically, in doses of 3 ... 5 g / day, for 1 ... 6 months, in local antitumor therapy of mammary neoplasia or other benign and malignant tumors, located externally, for human and veterinary use.

Example 8. Anti-tumor drug product - ointment containing 1% herbal product from Hedera helix

Conditioning formula:

- herbal product - minimum 90% ................................. 11 g

-propylene glycol ................................................ 100 g

- polyethylene glycol 4000 ........................................... 300 g

- polyethylene glycol 400 ........................................ ad 1000g

Method of preparation: the herbal product is dissolved in heat (6O ... 7O ° C) in the amount provided by propylene glycol, and the solution obtained is incorporated after cooling the mass of ointment resulting by melting at 65 ... 70 ° C of the mixture of polyethylene glycols 4000 and 400, followed by homogenization by shaking and conditioning.

The product contains 1% herbal product from Hedera helix and is given topically, in doses of 3 ... 5 g / day, for 1 ... 6 months, in local antitumor therapy of mammary neoplasia or other benign and malignant tumors, located externally, for human and veterinary use.

Example 9. Antitumor drug product - external use solution containing 1% herbal product from Hedera helix Conditioning formula:

- herbal product - minimum 90% ................................. 11 g

- ethyl alcohol 95 ° ............................................. .100 ml

- refined castor oil ........................................ ad. 1000 g

Method of preparation: the phytotherapeutic product is dissolved in hot (6O ... 7O ° C) in 200 ml mixture 1: 1 v / v ethyl alcohol 95 °: castor oil, resulting in a homogeneous solution, in which the rest of the oil is added of castor bean, homogenized by stirring, filtered and conditioned.

The product contains 10 mg / ml herbal product of Hedera helix and is given topically, in doses of 3 ... 5 g / day, for 1 ... 6 months, in local antitumor therapy of mammary neoplasia or other benign tumors and malignant, externally located, for human and veterinary use.

Example 10. Anti-tumor drug product - suppositories containing 1% herbal product from Hedera helix

Conditioning formula:

- herbal product - minimum 90% ................................. 11 g

- polyethylene glycol 400 ............................................ 100 g

- methyl parahydroxybenzoate ..................................... 300 g

- propyl parahydroxybenzoate ...................................... 2 g

- Supposition (semisynthetic triglycerides) ........................ ad. 1000 g

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Method of preparation: the herbal product is dissolved in heat (6O ... 7O ° C) in the quantity 1 provided by polyethylene glycol 400, and the resulting solution is incorporated in the Supocir table, previously heated to 40 ° C. 3

Add the mixture of preservatives, mix and condition as 2.5 g suppositories. 5

The product contains 25 mg / suppository herbal product from Hedera helix and is administered rectally, in doses of 2 ... 4 suppositories / day, for 1 ... 6 months, in the treatment of human 7 and veterinary use of benign tumors and malignancies, especially in mammary neoplasia therapy, as well as as immunosuppressants of chemotherapy with cytostatic synthetic drugs. 9

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Claims (7)

claims 1. Process for the preparation of bioactive complexes from Hedera helix grass, used 3 in antitumor therapy, characterized in that, in a first step, the dried Hedera helix grass is subjected to dynamic extraction with ethyl alcohol of 80% concentration 5 in -a ratio 1:15 at ambient temperature for 12 hours, followed by the concentration of the alcoholic extraction solution, the concentrated extract is then subjected to liquid-liquid extraction with 7 ethyl acetate in a ratio of 1: 3, resulting in an aqueous extract and a phase of ethyl acetate, which is further separated, filtered and concentrated in vacuo to a ratio of 1:30, suspended under 9 stirring in acetone, resulting in a first crude bioactive complex, which is subjected to dissolution by purification in 95% ethyl alcohol, bleaching with activated carbon and concentration, which leads to 11 an acetonic filtrate and a crystallized one which is purified, resulting in a first purified bioactive complex, after which, in a second step, the aforementioned aqueous extract is subjected to a liquid-liquid separation, which results in an aqueous phase and an n-butanolic phase which is subsequently subjected to concentration in vacuo, resulting in a second bioactive complex. crude, which is suspended in acetone, 15 and the precipitate is separated by filtration and purified by dissolution with 70% ethyl alcohol, bleaching with coal and concentration, resulting in a second purified bioactive complex, in 17 further, in a 3-. in step one, the acetonic filtrate is concentrated in vacuo and meets with the aqueous phase from the liquid-liquid extraction of the previous step, 19 is concentrated and conditioned in propylene glycol, resulting in a third bioactive complex, and finally, the acetonic filtrate for purification. from step 1 concentrate and suspend in the alcoholic solution at pH 8.5 ... 12.5, 21 separate by filtration and suspend in copper sulphate solution of 10% concentration in acidic environment, followed by solubilization of Cu-chlorophyll in an aqueous solution of sodium hydroxide 23 at pH 8.5 ... 12.5, resulting in an aqueous extract of Cu-Na-chlorophyll. Process according to claim 1, characterized in that the purified bioactive complex 25 of step 1 is a white crystalline powder, having a minimum content of 95% total monodesmozide triterpenic saponins with aglycone hederagenin, of which 70 ... 80% 27 a- hederin. Process according to claim 1, characterized in that the purified bioactive complex 29 of step 2 is a yellowish-white powder, having a content of at least 90% total bisdesmozide triterpenic saponins with hederagenin aglycone, of which 50 ... 75% 31 Hederacozide C. 4. Process according to claim 1, characterized in that the bioactive complex 33 of step 3 is in the form of a reddish-brown liquid extract, having 1 ... 3 g / 100 ml containing bisdesmozide saponins with oleanolic acid aglycone and 1.5. .5 g / 100 ml polyphenolcarboxylic acids 35 5. Process according to claim 1, characterized in that the aqueous CuNa-chlorophyll extract has 0.2 ... 1 g / 100 ml contained in monodesmozide saponins with 37 oleanolic acid aglycone and 0.1 ... 0.5 g / 100 ml total chlorophyll extract. Medicinal product, constituted dini ... 2% of a bioactive complex prepared by 39 the process defined in one of claims 2 ... 4, conditioned in the form of injectable or oral solution, gel, cream or suppositories, for use in antitumor therapy. 41 Medicinal product, consisting of 100 mg of a bioactive complex prepared by the process defined in one of claims 2 ... 4, conditioned in capsule form, 43 for use in antitumor therapy.