RU2533228C2 - Method for preparing agent possessing antiulcerogenic action - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the pharmaceutical industry, particularly to a method for preparing an agent possessing an antiulcerogenic action. A method for preparing the agent possessing the antiulcerogenic action by extracting the aspen bark in 40% ethanol by the multi-step reverse-flow extraction followed by purification, vacuum concentration and drying in a vacuum chamber; the prepared dry extract is added with the Ludipress extract in the specific environment and used to fill solid gelatine capsules.

EFFECT: preparing the agent possessing the antiulcerogenic action.

15 dwg

Description

The invention relates to the field of pharmacy and relates to a method for producing funds with antiulcer action.

One of the urgent problems of modern gastroenterology is the search and study of new highly effective means of treating diseases of the gastrointestinal tract. Despite the significant successes achieved in the field of synthetic antiulcer drugs, plants continue to be one of the promising sources of new biologically active compounds. For example, herbal preparations are widely used, such as Vikair, Vikalin, Befungin, etc., which have anti-ulcer action (Mashkovsky M.D. Medicines. 15th ed., Revised, corrected and add. M .: LLC "Publishing House New Wave", 2005. 1200 p .: ill.).

Among plant raw materials containing biologically active anti-ulcer biologically active substances (BAS), one should single out the bark of the common aspen (Populus tremula L.), the family of willow (Salicaceae), the genus poplar (Populus), which contains significant amounts of various phenolic compounds (phenological glycosides phenolic acids, flavonoids) (Turetskova V.F. Theoretical and experimental substantiation of the rational use of bark and shoots of sea buckthorn and buckthorn buckthorn bark and aspen bark: abstract of diss. ... Dr. Pharm. Sciences. Perm, 2001. 49 p.).

There is a method of producing an anti-ulcer agent from willow leaves in the form of an infusion, which is an aqueous extract from dried ground willow leaves, obtained by extracting raw materials with boiling water at a ratio of 1: 9, infusion time 30 minutes, cooling 45 minutes (Patent No. 2187322).

The disadvantages of this method are:

- the inability to use in an industrial environment due to the chemical and microbiological instability of aqueous extracts during storage (shelf life of not more than 1 day);

- the use of boiling water in the manufacturing process, which can lead to the destruction of the main biologically active substances (phenological glycosides) of this type of raw material.

The closest to the technical result achieved, the prototype is a method of obtaining funds with anti-ulcer action, the essence of which is that extracted with 40 ° ethanol, crushed to 5-10 mm aspen bark with a complete or unfinished cycle in a battery of 5-6 diffusers at 18-25 ° C and with a ratio of raw materials: extractant 1: 2.3-3.0 for 8-16 hours once, stand for 3 days at a temperature of 6-8 ° C, thicken for 18 hours at 32 ° C under vacuum and then dried in a vacuum oven at 40 ° C for After 3-6 hours or a 40-degree extraction, spray-dried is used, having previously driven off 1/2 of the extractant under vacuum or diluting the 40-degree extraction 2 times with water, at an inlet temperature of 120 ° C, and at the outlet 65 ° C for 2-3 s (Patent No. 2086251).

The disadvantages of this method are:

- the inability to ensure high accuracy of dosing, since it is not a metered drug;

- the impossibility of masking a bitter taste;

- the ability to reduce stability due to dampness of the extract during storage.

The technical result of the claimed invention is to increase the accuracy of dosing and the effectiveness of the extract, as well as reduce the wetness and increase the convenience of its use by introducing an adjuvant of ludipress and / or using an encapsulated dosage form.

The technical result is achieved by the fact that ludipress is added to dry aspen bark extract in a mass ratio of 35:13, respectively, followed by filling of hard gelatin capsules of 0.48 g of the resulting mass.

The authors propose a method of obtaining funds with anti-ulcer action, which allows to increase gastroprotective activity of dry aspen bark extract, as well as to eliminate its dampness while correcting organoleptic properties.

The method is as follows

The method is illustrated in figures 1-15.

Figure 1 presents data on the study of the technological properties of dry aspen bark extract.

Figure 2 proposed four prescriptions, which included various amounts of excipients that allow you to adjust the technological properties of the extract of dry aspen bark.

- среднее значение; $$\Delta \overline{x}$$

- доверительный интервал.Figure 3 presents data on the study of humidity of the studied compounds obtained from various prescriptions, where $$\overline{X}$$

- average value; $$\Delta \overline{x}$$

- confidence interval.

- среднее значение; $$\Delta \overline{x}$$

- доверительный интервал.Figure 4 is a table for the study of the wetability of the studied compounds, where $$\overline{X}$$

- average value; $$\Delta \overline{x}$$

- confidence interval.

- среднее значение; $$\Delta \overline{x}$$

- доверительный интервал.Figure 5 presents data on the study of the hygroscopicity of the studied compositions, where $$\overline{X}$$

- average value; $$\Delta \overline{x}$$

- confidence interval.

- среднее значение; $$\Delta \overline{x}$$

- доверительный интервал.Figure 6 presents data on the study of flowability and angle of repose of the investigated compounds, where $$\overline{X}$$

- average value; $$\Delta \overline{x}$$

- confidence interval.

- среднее значение; $$\Delta \overline{x}$$

- доверительный интервал.Figure 7 presents a table for studying the indicator of bulk volume of the studied compounds, where $$\overline{X}$$

- average value; $$\Delta \overline{x}$$

- confidence interval.

On Fig given the calculated data on the selection of the size of hard gelatin capsules.

- среднее значение; $$\Delta \overline{x}$$

- доверительный интервал.Figure 9 gives a table of analysis of the obtained capsules according to the indicators "Description", "Humidity" and "Microbiological frequency" during the year of storage, where $$\overline{X}$$

- average value; $$\Delta \overline{x}$$

- confidence interval.

Figure 10 presents the average weight of the capsules, the deviation of the mass of each capsule and the mass of the contents of each capsule during one year of storage.

- среднее значение; $$\Delta \overline{x}$$

- доверительный интервал.Figure 11 presents data on the quantitative content of the main groups of biologically active substances during one year of storage, where $$\overline{X}$$

- average value; $$\Delta \overline{x}$$

- confidence interval.

- среднее значение; $$\Delta \overline{x}$$

- доверительный интервал.On Fig presents the main indicators of the tests "Disintegration" and "Dissolution" during one year of storage, where $$\overline{X}$$

- average value; $$\Delta \overline{x}$$

- confidence interval.

On Fig presents the effect of course administration of aspen bark extract, tabletted and encapsulated mixtures, on the development of neurogenic ulceration in the gastric mucosa in female mice CD ₁ (X ± m) (in this table and 2, 3 the numbers of comparable numbers are indicated before the significance level P groups).

On Fig presents the effect of course administration of aspen bark extract, tabletted and encapsulated mixtures, on the development of indomethacin ulceration in the gastric mucosa in female rats CD ₁ (X ± m).

On Fig presents the effect of the course of the aspen bark extract, tabletted and encapsulated mixtures, on the development of indomethacin ulceration in the gastric mucosa in female rats CD ₁ (X ± m).

An example of the method

350 g of crushed extract of dried aspen bark, obtained using multistage countercurrent extraction with 40% ethyl alcohol, followed by purification, concentration under vacuum at a temperature of 40-50 ° C and drying in a vacuum oven at a temperature of 40-50 ° C, are thoroughly mixed with 130 g ludipress (0-BASF, Germany), the resulting mixture is filled into hard gelatin capsules of cylindrical shape with a smooth surface 0 numbers of 0.48 g each on a machine (or semi-automatic machine) for filling capsules.

The obtained capsules have an average weight of 0.546 g, a deviation from the average weight of 0.05-4.12%, an average capsule content of 0.484 g, a deviation from the average capsule content of 0.10-4.96%; the content of phenological glycosides 0.0832 ± 0.0037 g; salicin content 0.0202 ± 0.0020 g; the content of phenolic acids is 0.0253 ± 0.0010 g; disintegration is 9.01 min; phenol glycoside dissolution 93.14 ± 3.94%; phenol acid dissolution 97.85 ± 0.25%; according to microbiological purity correspond to category 3B.

The inventive method of obtaining funds with antiulcer action with an extract of aspen bark dry has the following advantages over the well-known: increasing the accuracy of dosing and the effectiveness of the extract, as well as reducing the wettability and increasing the convenience of its use due to the scientifically based choice of pharmaceutical factors (administration of an auxiliary substance ludipress and / or use of the encapsulated dosage form).

From the point of view of biopharmacy, the effectiveness of a drug depends to a large extent on the correct choice of pharmaceutical factors used in the development and production of drugs, and, first of all, on the excipients used. From modern positions, there are no indifferent and universal excipients, in connection with which the composition of each particular drug should be scientifically substantiated [Tentsova AI, 1971; Demina NB, 2013].

The choice of excipient and dosage form based on the following pharmacotechnological experiments.

The results of studies on the technological properties of the extract showed that it has a satisfactory flowability, large bulk volume, low humidity, dampness and a sufficiently high hygroscopicity (figure 1).

An analysis of the technological properties of dry aspen bark extract suggested the possibility of making hard gelatin capsules with a lid with the addition of auxiliary substances that improve flowability and reduce hygroscopicity. In this connection, we developed and received 4 prescriptions, which included various amounts of excipients that allow you to adjust the above indicators and study their technological properties (figure 2).

An analysis of the results showed that the moisture content (Fig. 3) and wetness (Fig. 4) of the dry extract reduced only ludipress (copy No. 2), all other excipients, on the contrary, increased these indicators. The hygroscopicity of prescription No. 2 with ludipress compared with the extract decreased from 20.43 ± 0.56 to 11.27 ± 0.47% (Fig. 5).

Therefore, further studies to determine the technological properties were carried out with all the above mixtures.

The research results indicate that the encapsulated mass prepared according to the recipe No. 2 has the best flowability (Fig.6). All prescriptions have high indicators of bulk volume (from 803.86 ± 0.24 to 909.5 5 ± 0.46 kg / m ³ ) (Fig.7).

A comparison of the obtained data on technological indicators allowed us to conclude that it is most rational to make capsules according to prescription No. 2, so this recipe provides both good flowability, as well as lower humidity, dampness and hygroscopicity of the encapsulated mass.

The selection of the size of hard gelatin capsules for the selected recipe is carried out based on the average capacity of the capsule according to the requirements of the Global Fund XI, v.2., P.144, article "Capsules".

Capsule capacity is calculated by the formula:

,

,

where V is the average capacity of the capsule, ml;

m is the mass of dry aspen bark extract;

p is the bulk volume, g / cm ³ .

Calculations showed that for encapsulation of a therapeutic dose of dry aspen bark extract according to prescriptions No. 2, it is necessary to use capsules 0 numbers (Fig. 8).

The study of the stability of the obtained encapsulated preparation according to the indicators regulated by the requirements of the Global Fund XI ed., General Pharmacopoeia Monograph “Capsules” and OSTU 91500.05.001-00 “Standards for the quality of medicines. The main provisions ", as well as the moisture content of the capsules during natural storage at a relative humidity of 40% and a temperature of 15-25 ° C for one year are presented in the tables (Fig.9-12).

From the data presented in (Fig.9), it follows that the indicators description, moisture content of the capsules and microbiological purity meet the requirements and do not change for one year.

Determination of the average weight of the capsules, the deviation of the mass of each capsule and the mass of the contents showed that all series correspond to the requirements (figure 10).

The study of the stability of the encapsulated preparation according to the content of two groups of biologically active substances (phenolic acids and phenological glycosides by the SPS method), as well as the main phenol glycoside of salicin by HPLC, showed that the content of active substances practically does not change during one year of storage (Fig. 11).

High rates of tests "Disintegration" and "Dissolution", corresponding to the requirements of the Global Fund XI ed. and OFS 42-0003-00 “Dissolution” (Fig. 12) of the test experimental preparation confirm the rationality of the choice of capsules as the dosage form.

Considering the fact that excipients are one of the most significant factors influencing the effectiveness of drugs, a comparative assessment of the pharmacological activity of equal doses of aspen bark extract was carried out in the presence of the adjuvant ludipress selected above in technological studies for capsules and starch mixtures and talc, previously used in the preparation of tablets with dry aspen bark extract (Patent No. 2144899).

A comparative assessment of the pharmacological activity of the tabletted mixture of aspen bark extract and dry capsule mixture was carried out on models of neurogenic and indomethacin ulcerogenesis in two animal species (52 outbred female CD1 mice and 39 outbred female CD rats). The choice of these models of ulceration is due to the importance of the pathogenesis factors underlying these models in the initiation and chronicity of the gastric and duodenal ulcer and other acid-dependent gastrointestinal diseases.

Neurogenic ulcer. In the mechanism of the genesis of the stress ulcer model, the leading place is occupied by the neurohumoral factor, therefore this model is the most adequate among the rapidly reproducible acute experimental ulcers [Zavodskaya IS, Moreva EV, 1981]. The neurogenic damage model is used as a screening model to identify agents with antiulcer activity. The research methodology was as follows: mice were hung by forceps by the cutaneous neck fold, as a result of 24-hour partial immobilization, the animals developed ulcerative lesions of the gastric mucosa. The last injection of the drug occurred 1 hour before the onset of the action of the ulcer-forming factor (the course was 4 injections).

Indomethacin ulcer. The ulcerogenic effect of non-steroidal anti-inflammatory drugs (NSAIDs), used according to vital indications as part of cardiac and antirheumatic therapy, determines the relevance of the development of preventive and therapeutic gastroenterological measures. Among non-steroidal anti-inflammatory drugs, indomethacin has the most pronounced ulcerogenic effect, which served as the basis for choosing this drug as a damaging agent.

The model of indomethacin damage to the gastric mucosa of rats was reproduced by a single intragastric administration of indomethacin at a dose of 60 mg / kg in 1 ml of physiological solution at the end of the course appointment (4 days) of the studied substances.

The last injection of substances occurred 1 hour before the onset of the action of the ulcer-forming factor.

Performance criteria for antiulcer activity

At the end of the experiments, the mice were sacrificed by the method of craniocervical dislocation, the rats by inhalation of CO ₂ , the stomachs were removed, opened by small curvature, washed with cold physiological saline, and the number and area of destruction, which were differentiated by point (less than 1 mm), were determined using a magnifier under bright lighting. and large (more than 1 mm). The average number of ulcerations per animal in the group, the percentage of animals with ulcers [Hadzhai Ya.I. et al., 1962].

The Pauls Index (PI) was determined as an integral indicator of the number of destruction according to the formula:

Antiulcer activity (PA) of drugs was determined as the ratio of the Pauls index in the control group to the Pauls index in the experimental group. The test agent was considered active if the PA was 2 or more units [Pauls F., Wick A.M., Mac Key E.M. et al., 1947; Dobryakov Yu.I., 1978]. The degree of damage (SP) of the gastric mucosa and the severity of damage (TP) were characterized on the basis of calculations of the total length of all injuries of the gastric mucosa (in mm) in each rat and the percentage decrease in TP in experimental animals with respect to this indicator in control rats.

Neurogenic damage model

The following groups were formed on the model system of neurogenic damage to the gastric mucosa of mice.

1. Stress control (11) - mice received a solvent of distilled water in a volume of 0.4 ml intragastrically through a probe with a daily preventive course for 4 days, the last injection 1 hour before immobilization.

2. Aspen bark extract, tableted mixture, 100 mg / kg (10) - mice received the drug in a volume of 0.4 ml of distilled water intragastrically through a probe with a daily preventive course for 4 days, the last injection 1 hour before immobilization.

3. Aspen bark extract, tableted mixture, 200 mg / kg (10) - the mice received the drug in a volume of 0.4 ml of distilled water intragastrically through a probe with a daily preventive course for 4 days, the last injection 1 hour before immobilization.

4. Aspen bark extract, encapsulated mixture, 100 mg / kg (10) - mice received the drug in a volume of 0.4 ml of distilled water intragastrically through a probe with a daily preventive course for 4 days, the last injection 1 hour before immobilization.

5. Aspen bark extract, encapsulated mixture, 200 (11) - mice received the drug in a volume of 0.4 ml intragastrically through a probe with a daily preventive course for 4 days, the last injection 1 hour before immobilization.

Prophylactic course intragastric administration of an encapsulated mixture at doses of 100 and 200 mg / kg led to a significant increase in the resistance of the mucous membrane of the stomach of mice to the damaging effect of immobilization stress (Fig. 13). The gastroprotective effect of the new dosage form was manifested in a significant reduction in the number of animals with ulcers (P <0.01) in both study groups compared with the stress control index. It should be noted a significant decrease in the average number of ulcers in the capsule mixture prescribing groups at doses of 100 (P <0.05) and 200 (P <0.01) mg / kg due to a decrease in the number of point destruction by 2.6 and 5.1 times ( P <0.05), a decrease in the number of large ulcers by 9.1 (P <0.05) and 5.1 times, respectively, compared with similar indicators of the stress control group. The effectiveness of the new dosage form, based on the encapsulated mixture under study, is evidenced by the absence of strip-like ulcers when using a dose of 100 mg / kg and a significant decrease in this indicator by 1.7 times in the 200 mg / kg group. The revealed effect was dose-dependent in nature: an increase in the dose led to an increase in antiulcer activity (encapsulated mixture at a dose of 100 mg / kg - 6.33; at a dose of 200 mg / kg - 8.26).

Comparison drug - aspen bark extract (tableted mixture) showed pronounced antiulcer activity in the model of neurogenic damage. The average number of ulcers per animal, the number of large and pinpoint destruction did not significantly differ from those of the groups of destination of the encapsulated mixture. It should be noted a slight decrease in the number of animals with ulcers after applying the tablettable mixture in both doses compared to the same indicator in the stress control group (70% and 80% versus 90.9%). A higher anti-ulcerogenic effect when using a dose of 100 mg / kg (2.86 points) is due to a decrease in the average number of ulcers by 2.2 times (P <0.05), the number of point ulcers by 2.82 times (P <0.05) . The results obtained when using a tableted mixture in both doses (2.86 and 2.07 points) reproduced the data of previous experiments.

An analysis of the antiulcer efficacy indices of the studied dosage forms revealed a significant predominant specific activity of the encapsulated mixture. So, when using a tablettable mixture, the number of animals with ulcers did not have a statistically significant difference with that of the stress control indicator, whereas as a result of the administration of the capsule mixture in both doses, there was a decrease in the number of animals with ulcers 50% and 45.5% against 70% and 80 % A similar trend was observed in relation to other results of the studied mixture. If the values of strip-like ulceration of the comparison group did not practically differ from the control parameters, then in the case of using the encapsulated mixture, either the absence or decrease of 1.7 of this indicator was noted. A statistically significant decrease in the number of major ulcers was recorded when using the capsule mixture at a dose of 100 mg / kg, a significant decrease in this indicator (dose of 200 mg / kg) by 2.8 and 3.3 times relative to the effect of the tablet mixture (100 and 200 mg / kg) . As a result of the totality of the revealed changes, a statistically significant decrease in the average number of ulcers per 1 animal in both experimental groups was revealed, whereas when using a tableted mixture, when the optimal dose was 100 mg / kg. When comparing the antiulcer activity of the prototype and the encapsulated mixture, a significant advantage of the new substance was demonstrated, which was expressed in an increase in the effect by 2.2 and 4.0 times.

Thus, the results of the experiments suggest a significant gastroprotective effect - the encapsulated mixture used to obtain a new dosage form of dry aspen bark extract - on the model of neurogenic ulcer formation, which prevails compared to the prototype (tablet mixture).

Indomethacin ulcer model

The following groups were formed on the model system of indomethacin damage to the gastric mucosa of outbred female rats.

1. Control group (n = 10) - rats received distilled water in a volume of 0.5 ml intragastrically through a probe once daily with a prophylactic course for 4 days, the last injection occurred 1 hour before the use of indomethacin.

2. Aspen bark extract, tableted mixture, 100 mg / kg (10) - rats received the drug in a volume of 0.5 ml of distilled water intragastrically with a prophylactic course for 4 days, the last injection occurred 1 hour before the use of indomethacin.

3. Aspen bark extract, encapsulated mixture, 50 mg / kg (10) - rats received the drug in a volume of 0.5 ml of distilled water intragastrically with a prophylactic course for 4 days, the last injection occurred 1 hour before the use of indomethacin.

4. Aspen bark extract, encapsulated mixture, 100 (9) - rats received the drug in a volume of 0.5 ml intragastrically daily with a prophylactic course for 4 days, the last injection occurred 1 hour before the use of indomethacin.

A new dosage form of aspen bark extract - an encapsulated mixture, administered prophylactically in doses of 50 and 100 mg / kg intragastrically daily for 4 days, limited the formation of all types of ulcerative destruction that occurs after exposure to indomethacin. So, the use of a substance at a dose of 50 mg / kg led to a decrease in the number of point ones by 1.9 times, large by 2.2 times (P <0.01) compared with the corresponding indices of the group of untreated animals (Fig. 14). As a result, the average number of destruction is statistically significantly decreased by 1.6 times (P <0.01). At the same time, there is a decrease in the area of all ulcerative destruction (Fig. 15): large - 1.9 times (P <0.01), strip-shaped - 1.3 times, point - 1.9 times (P <0.05). The revealed positive changes were reflected in a decrease in the degree of damage by 1.5 times (P <0.05) and the severity of the lesion to 64.8 against 100% of the control group.

Unidirectional changes are observed when using an encapsulated mixture at a dose of 100 mg / kg (FIGS. 14 and 15). There was a significant decrease in the number of strip-like ulcers by 1.4 times (P <0.05), large ulcers - 1.8 times (P <0.05), point ulcers - 1.3 times, as a result of which the average number of ulcers decreased by 1 4 times (P <0.05). The above and other significant positive changes identified with a dose of 100 mg / kg were reflected in the form of a decrease in the area of large ulcers by 1.9 times (P <0.01), strip-shaped - by 1.8 times (P <0.05), as a result, the degree of lesion decreased by 1.6 times (P <0.05), and the severity of the lesion to 60.7% versus 100% of the control group.

When analyzing the effectiveness of the compared tabletting mixture at a dose of 100 mg / kg, it should be noted that the course application of the substance led to a statistically significant decrease in the number of large defects by 1.7 times, point destruction - by 1.5 times, the average number of ulcers - by 1.4 (P <0.05) times (FIG. 14). The gastroprotective effect was manifested in a decrease in such indicators of the ulcer process as the area of large ulcers by 1.5 times (P <0.05), point ulcers - by 1.5 times (P <0.05), the degree of damage - 1.2 times , the severity of the lesion - up to 82.8% against 100% of the control group.

Thus, in the model of indomethacin damage to the gastric mucosa in rats, a pronounced antiulcer effect was revealed when using an encapsulated mixture of aspen bark extract in doses of 50 and 100 mg / kg, which exceeded the similar activity of the prototype tablet mixture (64.8 and 60.7% versus 82 , 8%, respectively).

Summarizing the foregoing, we can conclude in favor of the prevailing antiulcer activity - an encapsulated mixture, and, therefore, a new dosage form of aspen bark extract on dry models of experimental ulcerogenesis in mice and rats. The effect varied depending on the genesis of ulcerative destruction. In the case of neurogenic ulcer formation, the effectiveness of the new agent increased by 2.2–4.0 times, in the indomethacin ulcer model by 18.0–22.1% compared with the activity of the prototype.

Claims (1)

The method of obtaining funds with antiulcer action by extraction of aspen bark with 40% ethyl alcohol using multi-stage countercurrent extraction followed by purification, concentration under vacuum at a temperature of 40-50 ° C and drying in a vacuum oven at a temperature of 40-50 ° C, adding to the resulting dry extract of ludipress in a mass ratio of 35:13, respectively, followed by the filling of hard gelatin capsules of 0.48 g of the resulting mass.

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