RU2827099C1 - Method of producing herbal agent having anti-inflammatory action - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry, namely to a method for producing an herbal agent having anti-inflammatory action. Method for producing a herbal agent having anti-inflammatory action by extracting the plant material with a water-alcohol solution by fractional maceration while stirring, removing the extractant and obtaining a dry residue differs by the fact that the herb of Erodium cicutarium is used as the plant material with the raw material particle size of 3 mm; extraction is carried out in three stages with 50% ethanol at the ratio of raw material : extractant of 1:14 and temperature of +60 °C, contact time of the phases: the first one – 90 minutes, the second one – 60 minutes, the third one – 30 minutes, settling of the combined aqueous-alcoholic extract is carried out at a temperature of +6 °C for 3 days, dry residue is milled and sieved; the dry residue contains the sum of flavonoids of not less than 5%, the sum of phenolcarboxylic acids of not less than 6%.

EFFECT: said method simplifies the method by using one type of medicinal plant material and one extractant, increase content of biologically active substances in end product and its output, increase the anti-inflammatory activity of the agent by a greater anti-exudative effect, manifesting a proliferative action, reducing a degree of tissue damage.

1 cl, 4 tbl, 1 ex

Description

The invention relates to the pharmaceutical industry, namely, to the production of a product from plant materials that has anti-inflammatory activity.

Medicinal plant materials and preparations based on them continue to play an important role in the pharmacotherapy of many chronic and sluggish human diseases. As studies have shown, herbal preparations make up about 20-25% of the total number of drugs registered in the Russian Federation [1, 2].

The main advantages of herbal preparations over synthetic ones include a broad spectrum of action, low toxicity, minimization of side effects, the possibility of long-term use and affordability. In addition, these advantages explain the loyalty of elderly people suffering from chronic diseases to herbal preparations [1].

Inflammation is a fundamental pathological process that forms the pathophysiological basis of many human diseases [7]. The social significance of inflammatory diseases is constantly growing all over the world. This determines the need to develop new anti-inflammatory drugs. The complexity of developing effective and at the same time safe means for the treatment of inflammation lies in the fact that this is a polyvalent process with many alternative and intersecting pathways that exist both at the level of intracellular interactions of signaling cascades and at the level of regulation of the production of inflammatory mediators. Accordingly, the effect on only one target of pathogenesis is either not accompanied by a sufficient pharmacological effect or causes a number of side effects. Thus, there is a need to create new drugs that can regulate the functional activity of not one, but many molecules that affect all stages of inflammation [6]. These include herbal preparations, the action of which is based on the many biologically active substances included in them.

Herbal-based medications have long been used as effective anti-inflammatory agents.

So, the official types of raw materials have an anti-inflammatory effect - chamomile flowers, calendula flowers, linden flowers, harvested from the plants of small-leaved linden and broad-leaved linden, buds of Scots pine, grass and flowers of common yarrow, three-part Bidens grass, leaves of medicinal sage, leaves of rod-shaped eucalyptus. The main biologically active substances of these plants, providing an anti-inflammatory effect, are phenolic compounds (flavonoids, phenolic carboxylic acids, tannins), essential oils.

A "Method for obtaining a product possessing anti-inflammatory activity" is known [Patent of the Russian Federation No. 2637414. Authors: Pupykina K.A., Kazeeva A.R., et al.]. The method consists of the following: the herb of the medicinal burnet, crushed to a particle size of 2.0 mm, is extracted three times at 90°C with 70% ethyl alcohol, the resulting extract is filtered, evaporated, concentrated and dried in a vacuum drying cabinet. The product exhibits anti-inflammatory activity due to tannins, which, when interacting with the inflamed surface, provide its compaction and an active reduction in the area of the inflammatory process.

The anti-inflammatory effect of the multicomponent product "Medicine with anti-inflammatory action" is demonstrated [Patent RU No. 2636818, authors Karnopoltseva T.V., Botoeva E.A., Aseeva T.A.]. The medicinal product is a polyextract consisting of dry extracts of sea buckthorn fruits, long pepper fruits, Ural licorice roots, broadleaf eryngium roots, and apple fruits, taken in a ratio of 2:2:1:1:1, mass parts, obtained in a specific way. This medicinal product is obtained by mixing pre-obtained dry extracts from the listed types of plant materials. In this case, there is a high probability of developing side effects in the form of allergic reactions due to intolerance to the components, as well as taking into account contraindications when taking licorice root preparations, sea buckthorn fruits in the presence of diseases such as chronic hepatitis, liver disease with cholestasis, liver cirrhosis, as well as in childhood [Vidal Handbook. Medicines, 2020].

The closest method for obtaining a plant-based multicomponent agent with anti-inflammatory activity consists of black bergenia leaves, chamomile flowers, mint leaves and yarrow herb [RU Patent No. 2331429. Authors: Lubsandorzhieva P.-N. B., Azhunova T. A. et al.]. The agent is a dry extract obtained from plant materials of black bergenia leaves, chamomile flowers, mint leaves and yarrow herb (in a ratio of 3:2.8:2.4:1.8). The resulting dry extract contains polyphenols (9.77%), carotenoids (16.29 mg%), ascorbic acid (6.58 mg%), flavonoids (0.14%), and anthocyanins (1.35%). The disadvantages of this method are: the need to use four different types of plant materials, while the physicochemical properties of biological compounds of these plants can interfere with the isolation of the main group of biologically active substances that provide an anti-inflammatory effect (flavonoids and tannins); the use of step-by-step extraction with a change of extractant - initially 80% ethyl alcohol is used at room temperature with subsequent extraction for 2 hours twice, then - at a temperature of 90 ± 5 ° C 40% ethyl alcohol, the extraction of raw materials is carried out for an hour twice. At the first stage, in the case of using 80% ethyl alcohol, mainly lyophobic components - carotenoids and chlorophyll - are released. Chlorophyll is contained in mint leaves, yarrow herb and is well extracted with 80% ethyl alcohol. In the process of obtaining a dry extract, the presence of chlorophyll complicates the purification and drying stage. At the extraction stage at high temperature, a significant decrease in the content of thermolabile biologically active substances is observed, as well as a change in their chemical structure. The use of these methods reduces the efficiency of extraction and the yield of active substances in the finished product.

Dry extract is one of the dosage forms, which is characterized by a high content of biologically active substances. Dry extract is a concentrated extract from plant raw materials in the form of a powder with good flowability, with a residual moisture content of no more than 5%. Dry extracts have advantageous distinctive features in comparison with other dosage forms - tinctures, liquid extracts and water extracts. Dry extracts do not contain alcohol, which allows to significantly expand the scope of application. In addition, they contain more biologically active substances and a minimum amount of ballast, are convenient in dosing and use, have a long shelf life due to low contamination.

The objective of the present invention is to expand the range of anti-inflammatory drugs based on medicinal plants with a high content of biologically active substances.

The technical result is a simplification of the method due to the use of one type of medicinal plant material and one extractant, an increase in the content of biologically active substances in the target product and an increase in its yield, an increase in anti-inflammatory activity due to a greater anti-exudative effect, the manifestation of a proliferative effect, and a decrease in the degree of tissue damage.

The technical result is achieved by three-stage extraction with 50% ethyl alcohol of one type of plant material, at a temperature of +60°C, with a phase contact time of 90 minutes at the first extraction stage, 60 minutes at the second, and 30 minutes at the third extraction stage, carrying out a stage of purification of the combined aqueous-alcoholic extracts, concentrating the purified extract and obtaining a dry residue.

The new technical result is that the above-ground part is used as plant material - the grass of the hemlock (Erodium cicutarium). The hemlock is an annual herbaceous plant, has leafy stems up to 20 cm high, usually with several non-glandular and small glandular hairs. The leaves are petiolate, almost twice pinnate. The flowers consist of 5 petals, the color from pink to burgundy, collected in an umbel inflorescence. Characteristic are the fruits with a bordered pit at the top and a short (2.5-4.5 cm) nose. The hemlock is widespread throughout almost the entire territory of Russia, as well as in Eurasia and North Africa. According to our data, the above-ground organs of the hemlock herb contain flavonoids - rutin, hyperoside, cynaroside, kaempferol, luteolin, quercetin, phenolic acids - chlorogenic, caffeic, neochlorogenic. In the herb of the hemlock herb, the quantitative content of biologically active compounds extracted with 50% ethyl alcohol is: the sum of flavonoids (in terms of rutin) - 2.16%, phenolic acids (in terms of chlorogenic acid) - 3.16%, carotenoids (in terms of β-carotene) - 0.34%, ascorbic acid - 0.4%.

Optimal parameters for the process of maximum yield of biologically active substances from the herb of hemlock that determine anti-inflammatory activity - flavonoids and phenolic acids - have been experimentally established. Extraction agent - 50% ethyl alcohol, which allows extracting glycosides and aglycones of flavonoids. The particle size of the raw material is 3 mm. The maximum yield of flavonoids, phenolic acids and extractive substances occurs when using a raw material-extractant ratio of 1:14. The yield of biologically active substances and the quality of the finished product are significantly affected by the temperature and dynamic processes in the extraction mixture. The optimal temperature of the extraction process is +60°C, which ensures the highest yield and preservation of biologically active substances. The maximum yield of flavonoids, phenolic acids and extractive substances from the plant material of the hemlock occurs during the first phase contact of 90 minutes, the second - 60 minutes, the third - 30 minutes (the extraction process is carried out with stirring) (Table 1).

The increased content of the main biologically active substances in the dry extract of hemlock is influenced by the stage of purification from ballast substances by settling the combined alcohol-water extract at a temperature of +6°C for three days, followed by filtration.

Obtaining dry extract from the herb of hemlock.

Example.

Extraction of the raw material was carried out by maceration under dynamic conditions with three-fold phase contact at a temperature of +60°C. During the first phase contact, 140 ml of 50% ethyl alcohol was poured into 10.0 g of pre-crushed raw material to a particle size of 3.0 mm. Extraction was carried out for 90 minutes. The resulting aqueous-alcoholic extract was poured through a filter into a collector in a volume of 110 ml, after which the loss of the extractant was replenished in the same volume (110 ml) of 50% ethyl alcohol and transferred to the extractor. Extraction during the second phase contact was carried out under similar conditions for 60 minutes. The resulting aqueous-alcoholic extract was poured through a filter in a volume of 86 ml and transferred to the collector. During the third phase contact, the loss of the solvent was replenished in a volume of 86 ml of 50% ethyl alcohol and transferred to the extractor, extraction was continued for another 30 minutes. The aqueous-alcoholic extract was drained in a volume of 83 ml, squeezing out the raw material. The combined aqueous-alcoholic extracts were kept for 3 days at a temperature of +6°C. Then they were filtered through a pressure filter, evaporated in a vacuum evaporator, and separated. Drying of the resulting combined extract was carried out at a temperature of +50°C for 8 hours. The dry residue contained a residual moisture of 4.8%, it was crushed, sifted to a homogeneous free-flowing state.

The dry extract of hemlock is a light-brown powder, the quantitative content of the sum of flavonoids in terms of rutin is 5.52%, the sum of phenolic carboxylic acids in terms of chlorogenic acid is 6.57%, carotenoids in terms of β-carotene are 0.51%, and ascorbic acid is 0.77%. The yield of dry extract from raw materials is 37.88±1.50%. According to this technological scheme, three additional series of dry extract were obtained from 3 batches of hemlock raw materials with the following content of total flavonoids: 5.47%, 5.71%, 5.59%; phenolic carboxylic acids - 6.46%, 6.54%, 6.91%; carotenoids - 0.47%, 0.41%, 0.48%; ascorbic acid - 0.74%, 0.72%, 0.77%. The recommended minimum value of biologically active substances that provide anti-inflammatory activity of the dry extract of hemlock: the amount of flavonoids is not less than 5%, phenolic carboxylic acids are not less than 6%.

Pharmacological studies of the anti-inflammatory activity of the obtained dry extract of hemlock were conducted in accordance with the documents “On approval of the rules of laboratory practice” (Ministry of Health of the Russian Federation, order of June 19, 2003, No. 267), Good Laboratory Practice for Nonclinical Laboratory Studies (FDA, 21 CFR Part 58, December 22, 1978), OECD Principles on Good Laboratory Practice (OECD, ENV/ MC/ CHEM (98) 17, 1977). The results were processed statistically and presented as “mean value (M) ± standard error (m)”. Reliability was established according to Student’s t-test [3].

The experiments were conducted on 90 white Wistar rats of both sexes weighing 180-200 g. For the experiments, animals with average motor activity, prevailing in the population, showing the most typical reactions to any impact were selected. The animals were kept in vivarium conditions (with natural lighting; at a temperature of 22-24°C), as well as free access to water and food. Before the experiment, the animals were quarantined for 10-14 days [8].

The control group of animals received water. The experimental group received dry extract at a dose of 150 mg/kg, which was dissolved in water before administration. Water and the test drug were administered intragastrically using a special probe in a volume of 2 ml. Studies of anti-inflammatory activity were reduced to assessing their effects on the exudative phase of inflammation according to Yu.E. Strelnikov [9] (first series of experiments), on the proliferation phase - according to F.P. Trinus [4] (second series of experiments) and on the alteration process - according to I.A. Oyvin [5] (third series of experiments).

In the first series of experiments, the antiexudative effect was studied on a model of acute inflammatory edema. Edema was caused by subplantar injection of 0.1 ml of a 2% aqueous formalin solution into the hind paw of rats. The extract and water under study were administered to the animals 2 hours before and 5 and 18 hours after the formalin was administered. Paw volume was measured using an oncometer. Activity was expressed as a percentage of edema suppression.

In the second series of experiments, the effect on proliferation processes was studied. For this purpose, sterile cotton balls weighing about 15 mg were implanted under the skin of rats in the back area under aseptic conditions. The studied extract and water were administered to rats once a day for 7 days. After 7 days, the granulomas were removed and the difference in weight before and after drying was determined. Weighing was performed on an analytical scale, the balls were dried at a temperature of 70°C for 24 hours (until constant weight).

In the third series of experiments, the effect on the processes of alteration and regeneration in the inflammation focus was studied. Inflammation was initiated by subcutaneous injection of 0.5 ml of 9% acetic acid solution into the back area of rats with pre-cut hair. Simultaneously, a dextran solution was administered intraperitoneally at a dose of 300 mg/kg. The first administration of the studied extract and water was carried out 1 hour before the administration of 9% acetic acid solution, then daily once a day for 25 days. On the 9th and 25th days of the experiment, the area of necrotic tissue was calculated by applying the necrosis contour to a transparent film.

As a result of the research, it was established that the dry extract of hemlock in the first series of experiments reduced formalin edema by 25% more effectively than in the control group, which indicates an antiexudative effect (Table 3).

In the second series of experiments, against the background of the introduction of the studied dry extract to the animals, delimitation of the focus of aseptic inflammation and stimulation of granulation tissue were observed on the 7th day. The mass of dry granuloma formed around the sterile ball was 15.2% greater than in the control group of animals (Table 3).

In the third series of experiments, it was found that the dry extract under study reduced the degree of tissue damage. In particular, on the 9th day, the area of necrotic tissue in rats that received the dry extract under study was 13% less than in the control group. The dry extract of hemlock stimulates regenerative processes, which resulted in a 26.5% decrease in the area of alteration on the 25th day of the experiment compared to the control (Table 4).

Thus, the studied dry extract of hemlock has anti-inflammatory activity.

Literature

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3. Medic V.A. Statistics in Medicine and Biology: A Manual in 2 Volumes / Medic V.A., Tokmachev M.S., Fishman B.B.; edited by Yu.M. Komarov; Moscow: Meditsina, 2000-2001. Vol. 1: Theoretical Statistics. - 455 p. Vol. 2: Applied Health Statistics. - 352 p.

4. Non-steroidal anti-inflammatory drugs: monograph / F.P. Trinus, N.A. Mokhort, B.M. Klebanov. - Kyiv: Zdorov'ya, 1975. - 240 p.

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6. Rydlovskaya A.V. Anti-inflammatory properties of the new herbal preparation artroflex and analysis of its mechanism of action: diss. - Institute of Toxicology, 2007.

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9. Strelnikov Yu.E. Comparative characteristics of the anti-inflammatory action of some pyrimidine derivatives // Pharmacology and toxicology - 1969. - No. 6. - P. 526-531.

Claims (1)

A method for obtaining a herbal remedy with an anti-inflammatory effect by extracting the plant material with a water-alcohol solution using fractional maceration with stirring, removing the extractant and obtaining a dry residue, characterized in that the herb used as the plant material is the hemlock grass with a raw material particle size of 3 mm, extraction is carried out in three stages with 50% ethyl alcohol at a raw material:extractant ratio of 1:14 and a temperature of +60°C, the phase contact time is: the first - 90 minutes, the second - 60 minutes, the third - 30 minutes, settling of the combined water-alcohol extract is carried out at a temperature of +6°C for 3 days, grinding and sifting the dry residue are carried out, the dry residue contains at least 5% of flavonoids, at least 6% of phenolic carboxylic acids.