RU2306945C1 - Prophylaxis composition based on phenolic substances and phospholipids - Google Patents

Abstract

FIELD: pharmacology, medicine, in particular new complex preparation in liposomal form.

SUBSTANCE: claim composition useful in prevention or treatment of pathological processes and maintaining of high levels of organism functional activity includes combination of one ore more extracts from medicative herms, containing phenolic substances and phospholipids., wherein phenolic substances are incapsulated into phospholipid capsule. Said phospholipid is selected from group containing phospholipids from bovine or porcine brain or skin, phosphatidylcholine, egg lecitine etc., and capsule is made in form liposome containing 11-40 % of cholesterol. Said phenolic substances are selected from group containing 5,6,7,4'-tetraoxyflavon, 5,6,7-trioxyflavon, gesperidine, etc. Claimed agent is capable of stable aqueous microdispersion formation useful in patentheral administering.

EFFECT: composition of high hydrophilic properties and bioavailability and improved therapeutic activity.

3 tbl, 1 dwg, 2 cl, 3 ex

Description

The invention relates to the field of pharmacology and medicine, specifically to a new complex preparation in liposome form, characterized by high hydrophilicity, improved bioavailability and therapeutic properties compared to free bioflafonoids and other substances of a phenolic nature.

The flavonoid family, widely distributed among medicinal plants, includes an extensive collective class of polyphenols. Flavonoid compounds are divided into the main groups of flavones, flavonols, flavanones, flavanonols, catechins and anthocyanins. Flavonoids have a wide spectrum of biological and antioxidant activity. Currently, pharmacological properties of flavonoids, such as antioxidant, peripheral, vasodilatory activity, vasoprotective activity (effect on the fragility and permeability of capillaries), anti-inflammatory, antispasmodic, antihistamine and antitumor effects, have been well studied and documented.

Despite the promising prospects for the clinical use of flavonoids, their therapeutic efficacy is often insufficient when applied topically and orally. With oral administration, there is a weak absorption of flavonoids in the intestine, due to bacterial degradation and the formation of complexes of flavonoids not absorbed in the intestine with other substances of the digestive tract. Since flavonoids are practically insoluble in water, their parenteral administration is also difficult. For the purposes of practical medicine, the search for ways to increase the bioavailability and therapeutic effectiveness of drugs based on flavonoids is of undoubted relevance. One such route is the use of lipophilic complexes of flavonoids with phospholipids developed by the authors of US patent No. 5043323.

Although the authors' unconditional merit is a slight increase in the bioavailability of flavonoids by improving their absorption in the intestine when used per os, the high lipophilicity of the complexes is a serious obstacle to the parenteral use of drugs, which is necessary for more radical therapy of a number of pathologies.

The RF patent No. 2141488 describes the use of lipophilic complexes of flavolignans, which are a mixture of silibinin, silidianin and silicristin with natural or synthetic phospholipids for the treatment of tumors of the female genital area. The authors managed to achieve a pronounced therapeutic effect with the oral administration of drugs, however, a further increase in the therapeutic efficacy of flavolignans in this case is also limited by the high lipophilicity of the drugs, which creates obstacles to their parenteral use.

The closest technical solution is a prophylactic composition used to prevent or treat pathological processes and maintain a high level of functional activity of the body, including combinations of one or more extracts of medicinal plants containing phenolic substances and phospholipid (US Pat. RF №2252029). Soybean phospholipid complex was used as a phospholipid.

Thus, the authors of the above works partially solved the problem of increasing the bioavailability of flavonoids by increasing their absorption in the intestine in the form of complexes with phospholipids, however, the problem of increasing the solubility of flavolignans in aqueous solutions in order to create injection forms remains unresolved.

An inventive task is to create a hydrophilic, liposome composition with increased bioavailability, with higher therapeutic activity, the ability to form stable aqueous microdispersions, which, unlike the previously proposed lipophilic preparations, can be used for parenteral administration and has a higher therapeutic activity.

The inventive problem is solved by the fact that a prophylactic composition is proposed that is used to prevent or treat pathological processes and maintain a high level of functional activity of the body, including combinations of one or more extracts of medicinal plants containing phenolic substances, and phospholipid, phenolic substances are enclosed in a shell of phospholipid selected from the group: bovine or porcine brain phospholipids or skin, phosphatidylcholine, phosphatidylserine, phosphatidyla anolamin containing the same or different acyl groups, egg lecithin, wherein the shell is made in the form of liposomes containing cholesterol in an amount of 11-40%.

In this case, phenolic substances are selected from the group: 5,6,7,4'-tetraoxyflavone, 5,6,7-trioxyflavone, g-schisandrine, apigenin, astragaline, aureuzidine, visnagine, visnadine, hesperidin, hyperoside, deoxyschisandrine, dihydrosamidine, digidrosilibin, izolikviritigenin, izoramnitin, inokosterone, quercetin, quercitrin, kvinkvelozid, Kellin, kaempferol, leptozidin, likviritigenin, luteolin, myricetin, naringenin, piranokumarin, rosavin, rutin, salidroside, silidianin, silibinin, silychristin, scopoletin, sulfuretin, schizandrin, skhizandrol, tyliroside, tricin , umbelliferone, 6-methoxy-7,8-dioxicoumarin, furocoumarin, ecdysterone, integrasterones, 6,7-dioxicoumarin and their derivatives.

These compositions can be obtained by ultrasonic treatment, extrusion, injection of ether or alcohol solutions of mixtures containing components in the claimed proportions.

Upon receipt of an aqueous solution of the proposed composition, a stable dispersion of microvesicles is formed, which can be used to obtain injection or infusion dosage forms.

The invention is based on a new liposome composition, which may contain one or more substances of a phenolic nature or one or more extracts of medicinal plants and phospholipids containing these substances, characterized by the ability to form stable aqueous microdispersions, which, unlike the previously proposed lipophilic preparations, can be used for parenteral administration and has a higher therapeutic activity.

The compositions of the proposed liposome composition are summarized in table 1.

Table 1 №№ The ratio of active substance / phospholipid Active substance Phospholipid Shell containing cholesterol,% one 1:10 quercetin phosphatidylcholine 10 2 1: 5 hyperoside phosphatidylcholine / phosphatidylserine mixture 38 3 1: 0.5 silymarin soya lecithin eleven four 1: 1 Milk Thistle Extract soya lecithin fifteen 5 1:20 extracts of milk thistle and echinacea phosphatidylcholine 12 6 1:50 routine bovine brain phospholipid 40 7 1:30 5,6,7-trioxy flavone porcine brain phospholipid 35 8 1:15 g-schizandrin skin phospholipid 32 9 1:25 apigenin phosphatidylserine twenty 10 1:40 astragaline phosphatidylethanolamine thirty eleven 1:10 Aureuzidine egg lecithin 16

Similarly, compositions with the rest of the phenolic substances mentioned above were obtained. The inventive preparation, including complexes of starting materials of phenolic nature or plant extracts and phospholipids containing them, is characterized by its ability to form stable dispersions of microvesicles in aqueous solutions. The stability and hydrophilicity of the claimed drug is confirmed by the absence of sediment during long-term storage, as well as microscopy data (drawing).

Example 1. Antioxidant activity of the obtained compositions.

The antioxidant activity of the compositions was evaluated by their effect on the generation of reactive oxygen species in macrophages caused by chrysotile asbestos.

(0% - отсутствие антиоксидантной активности). В качестве препаратов сравнения использовали смеси фосфолипидов с веществами фенольной природы или содержащими их растительными экстрактами.0.85 ml of medium containing 110 mM NaCl, 10 mM Tris-HCl, pH 7.4, 5 mM glucose, 2.5 mM MgCl ₂ , 0.65 mM luminol, pH 7.4 was placed in a measuring cell of a chemiluminometer. Then, 0.1 ml of a macrophage suspension (5 × 10 ⁶ cells / ml) was added to the cuvette and the chemiluminescence intensity of the cells was recorded at 37 ° C with constant stirring. After 5 min, the level of spontaneous chemiluminescence of macrophages was measured. Then, 0.05 ml of the studied complexes were injected into the cuvette (the concentration of a phenolic substance in all cases was 0.1 mg / ml), and after 3 minutes 0.05 ml of phys. a solution containing 0.3 mg of chrysotile asbestos. After 5 min, the maximum macrophage luminescence level (CL _OD ) was measured. As a control sample used a similar system that does not contain the studied drugs (CL _K ). The antioxidant activity of the studied complexes was evaluated in percent by the formula:

(0% - lack of antioxidant activity). Mixtures of phospholipids with phenolic substances or plant extracts containing them were used as comparison preparations.

The results of the study are shown in table 2.

table 2 Liposome Compositions Antioxidant activity,% Complex drug Mixture quercetin / phosphatidylcholine 46.8 30.6 hyperoside / phosphatidylcholine / phosphatidylserine 38,0 27.9 silymarin / soya lecithin 51.9 35.8 Milk Thistle Extract / Echinacea Extract / Phosphatidylcholine 58.4 41.3

As can be seen from table 2, the antioxidant activity of substances of phenolic nature or extracts containing them can be significantly increased when they are included in the inventive complex preparation. Moreover, the antioxidant activity of the claimed complex preparations significantly exceeds the activity of simple mixtures of phenolic substances with phospholipids.

Similar results were obtained in the study of the antioxidant activity of the other phenolic substances listed above, which are part of complex preparations with phospholipids.

Example 2. Specific hepatoprotective activity of a liposome composition comprising silibinin and phosphatidylcholine.

A study of the specific hepatoprotective activity of the compositions was carried out on a model of acute toxic hepatitis in mice. White outbred mice in adulthood were used in the experiments. An aqueous solution of the drug was administered intragastrically using a probe at a dose of 100 mg / kg for silibinin. Paracetamol (a model of drug hepatitis) was used as a hepatotropic agent. Animals were injected with the drug for the first time 1 hour before the introduction of paracetamol, and then another 5 times daily. As a comparison drug, a simple mixture of silibinin and phosphatidylcholine in the form of an aqueous suspension was used, which was administered according to the corresponding scheme at a dose of 100 mg / kg for silibinin. Control animals were administered only paracetamol. A day after the last injection of the animals, the animals were decapitated and an autopsy and a study of blood biochemical parameters were performed. The activity of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) was determined by the Wrightman-Frenkel method. Total protein was determined using a biuret reagent. Relative liver mass in control animals with hepatitis was increased by 45.6% compared with healthy intact animals; in experimental animals, this indicator was 23.1% (30.2% when taking a simple mixture). Thus, the severity of inflammatory processes in animals treated with the claimed liposome complex preparation, significantly decreased. A study of the activity of transaminases in blood serum showed that in control animals with hepatitis, the levels of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) were increased in comparison with intact animals by 432.9% and 211.5%; in mice treated with the inventive complex preparation, these indicators decreased to 208.6% and 52.3%, respectively (267.3% and 76.0% when taking a simple mixture). The level of total protein in the blood serum of patients with hepatitis control mice decreased to 63.2% (100% in intact healthy animals), while the use of the claimed drug increased this indicator to 89.7%. When using a simple mixture, the level of total protein was only 79.1% of that for healthy control animals.

Thus, the use of the proposed drug had a positive effect on detoxification functions of the liver of animals with acute drug hepatitis. Moreover, the effect of the complex preparation exceeded the effect of the comparison drug (a simple mixture of silibinin and phosphatidylcholine).

Example 3. Antitumor activity of a liposome composition comprising extracts of milk thistle and echinacea and soya lecithin.

, где а - короткий, b - длинный диаметр опухоли. Относительный размер опухолей (ОРО) определяли по формуле:

, где Р_оп - средний размер опухолей в опытной группе, Р_к - средний размер опухолей у контрольных животных. Увеличение продолжительности жизни леченых животных по сравнению с контролем определяли по формуле:

, где Т - СПЖ леченых животных, дни, С - СПЖ контрольных животных, дни.The study of the antitumor activity of the compositions was carried out on a model of melanoma line B16 in C57BI / 6 mice. To induce solid tumors, the mice were injected subcutaneously with a cell suspension (2 × 10 ⁵ cells in 0.1 ml of physiological saline). The experimental animals were injected intravenously with the inventive complex preparation in physiological saline. An aqueous suspension of a simple mixture containing extracts of milk thistle and echinacea and soya lecithin in the same amounts, administered intragastrically through a tube, was used as a comparison drug. The drugs were administered once a week, only three doses, starting from 3 days after tumor inoculation. Each experimental group consisted of six animals, the control - of ten. The size of solid tumors was measured once every 2-3 days. Tumor volume was calculated by the formula

where a is short, b is the long diameter of the tumor. The relative size of the tumors (ORO) was determined by the formula:

where P _op - the average size of the tumors in the experimental group, P _to - the average size of the tumors in control animals. The increase in the life expectancy of the treated animals compared with the control was determined by the formula:

where T is the LSS of treated animals, days, C is the LSS of control animals, days.

The results of the study are shown in table 3. As can be seen from the table, the antitumor effect of the use of a simple mixture was mild. At the same time, the use of the inventive liposome complex preparation had a significant inhibitory effect on tumor growth, and also led to a significant increase in the life expectancy of animals compared to the control.

Table 3 A drug ORO,% USPZH,% Fiz. solution (control) one hundred - Mixture [milk thistle extract / echinacea extract / soya lecithin], aqueous suspension intragastrically 76,2 20.3 Liposomal composition, the same components, aqueous solution intravenously 51.9 52.0

Thus, the use of phenolic substances that are part of the inventive complex preparation, significantly increases their antitumor effect due to increased bioavailability and use in the form of an injection.

Claims (2)

1. Preventive composition used to prevent or treat pathological processes and maintain a high level of functional activity of the body, including combinations of one or more extracts of medicinal plants containing phenolic substances and phospholipid, characterized in that the phenolic substances are enclosed in a shell of phospholipid, selected from the group: bovine or porcine brain or skin phospholipids, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine containing the same or various acyl groups, egg lecithin, while the shell is made in the form of liposomes containing cholesterol in an amount of 11-40%. 2. The prophylactic composition according to claim 1, characterized in that the phenolic substances are selected from the group: 5,6,7,4′-tetraoxyflavone, 5,6,7-trioxyflavone, g-schisandrin, apigenin, astragaline, aureuzidine, visnagin , visnadine, hesperidin, hyperoside, deoxyschisandrine, dihydrosamidine, dihydrosilibine, isoquiritigenin, isoramnitine, inocosterone, quercetin, quercetrin, quinqueloside, kellin, campferol, leptosidine, liquirithininininirininidinininirinininirinininirinininirinininirinininirinininirinininirinininirinininirinininirinininirinininirininininirininininirininininirinininirininininirinininirininininirininininirinininirinininirinininirininininirinininirininininirininininininininininerininininininininininininininininin , silikristin, scoproletin, sul furetin, schizandrin, schizandrol, tyliroside, tricin, umbelliferone, 6-methoxy-7,8-dioxicoumarin, furocoumarin, ecdysterone, integrasterones, 6,7-dioxicoumarin and their derivatives.

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