RU2244553C1 - Hepatoprotective agent - Google Patents

Abstract

FIELD: medicine, pharmaceutical industry.

SUBSTANCE: invention relates to agents used for treatment of chronic hepatitis. The hepatoprotective agent representing extract prepared from biomass of Maackia amurensis Rupr. et Maxim and obtained by the callus culture method comprises polyphenolic complex consisting of daidzein, retusin, genistein, formononetin, maackianin and medicarpin. The hepatoprotective agent promotes to effective treatment of chronic hepatitis.

EFFECT: valuable medicinal properties of agent.

2 tbl

Description

The invention relates to medicine and pharmacology, and relates to agents that can be used to treat chronic hepatitis.

Known hepatoprotectors of plant origin, such as LIV-52, Katergen, Silibor. Currently, the most widely used drug is Carsil (synonyms: Legalon, Silymarin, Silibinin), obtained from the seeds of the plant of milk thistle, which is an analogue of the domestic preparation of Silibor [1].

However, in the treatment of these drugs, in some cases, low therapeutic efficacy, as well as the presence of various kinds of side effects in these drugs [2, 3].

Closest to the proposed hepatoprotective agent is the hepatoprotector Maksar, which is a dry extract from the Amurian maakia sound wood. It is more effective in the treatment of chronic hepatitis compared with the drug Karsil and the absence of side effects [4].

However, Maksar is obtained from the wood of the plant Maakia Amur (Maackia amurensis Rupr. Et Maxim), which is a rare species. Resource studies show that intensive harvesting of wild medicinal plants leads to widespread depletion of their reserves, the disappearance of populations and certain plant species, so the search for alternative raw materials is an urgent problem. Currently, the development of biotechnology (culturing cells to obtain biologically active substances) allows us to solve this problem.

The objective of the invention is the expansion of the arsenal of hepatoprotective agents.

The problem was solved by creating a new hepatoprotective agent based on the extract of Maackia amurensis and is a biomass extract of Maackia amurensis Rupr. et Maxim obtained by the method of callus culture, and contains a polyphenolic complex consisting of daidzein, retuzin, genistein, formononetin, maakiain, medicarpin.

Callus culture of Maackia amurensis Rupr. et Maxim is prepared as follows: explants of various parts of the plant Maackia amurensis Rupr. et Maxim is placed in 230 ml Erlenmeyer flasks containing 50 ml of agarized growth medium of the following composition, mg / l of water:

NH ₄ NO ₃ 350–450

KNO ₃ 1500-2300

K ₂ HPO ₄ 150-190

CaCl ₂ × 6H ₂ O 600–730

MgSO ₄ × 7H ₂ O 350-390

H ₃ BO ₄ 4.2-8.0

MnSO ₄ × 4H ₂ O 15.6-26.7

CuSO ₄ × 5H ₂ O 0.02-0.03

CoCl ₂ × 2H ₂ O 0.02-0.03

ZnSO ₄ × 7H ₂ O 6.0-10.3

Na ₂ MoO ₄ × 2H ₂ O 0.2-0.3

KI 0.52-0.90

FeSO ₄ × 7H ₂ O 25.0-30.6

Na ₂ EDTA × 2H ₂ O 33.6-41.0

Mesoinositis 80-120

Thiamine hydrochloride 0.15-0.25

Nicotinic acid 0.4-0.6

Pyridoxine hydrochloride 0.4-0.6

6-Benzylaminopurine 0.45-0.55

α-Naphthylacetic acid 1.5-2.5

Casein Hydrolyzate 40-60

Sucrose 24000-26000

Agar 5800-6200

Water up to 1 L

pH of the medium 5.6-5.8 before autoclaving

Explants are incubated for 30 days in the dark at 25 ° C. Calluses formed on the explants are separated and cultured on medium of the same composition for ten passages at monthly intervals to stabilize the callus biosynthetic activity. Then calluses are removed from the culture vessels and dried in a stream of hot air at 50-55 ° C.

The dried calluses are treated with 95% ethanol at 50-55 ° C in the ratio of raw materials: extractant - 1: 5 in a percolator battery, followed by evaporation and drying of the alcoholic extracts under vacuum. The obtained powder of dark brown color is a preparation containing a complex of polyphenols, the composition of which was analyzed by high performance liquid chromatography.

Chemical and pharmacological studies of the drug from calli of Amur maakia were carried out in comparison with the drug Maksar from Amur maakia wood (PKM-NDR).

A chemical study of the polyphenolic complex contained in calluses of Amur maakia (PKM-KL) showed that it is substantially different in composition from the polyphenol complex contained in the extract from Amur maakia sound wood. It has been established that the culture of callus of Maakia Amur does not contain, unlike wood, a plant of stilbenes, namely piceatannol and resveratrol. In addition, in contrast to the native plant, the obtained callus culture does not produce stilbenes and dimeric stilbenes, while the polar fractions of extracts of Amurian maakia wood contain up to 30% oligomeric stilbenes (scirpusin A, scirpusin B, maakin, maakin A) [5-7 ], as well as stilbenolignan maakolin [7] and isoflavonostilbene maakiazine [8]. However, calli synthesize a larger amount of isoflavones (retuzin, genistein, and formomonetin) and, especially, pterocarpans (maakiain and medicarpin) than in the plant (Table 1).

Table 1
The composition of the polyphenols of wood and callus of Maakia Amur (mg / g dry weight) Components Piceatannol Resveratrol Daidzein Retuzin Genistein Formononetin Maakiain Medicarpine Wood 6.32 ±
0.52 6.10 ±
0.18 traces 1.15 ±
0.06 1.92 ±
0.23 1.15 ±
0.19 0.35 ±
0.06 0.28 ±
0.05 Calli - - 0.52 ±
0.18 0.91 ±
0.34 2.53 ±
0.42 4.23 ±
1.01 5.78 ±
1.94 2.68 ±
0.45

The total content of polyphenols in wood and calluses was 17.3 ± 0.18 and 16.7 ± 0.27 mg / g dry weight, respectively. Thus, maakia calli accumulate the amount of polyphenols comparable to Amur maakia wood. But at the same time, there are significant differences both in the composition and in the ratio of polyphenols synthesized by calluses of Amur maakia and wood of this plant. Therefore, it was not obvious that the polyphenolic complex of calli Maackia amurensis Rupr. et Maxim will have hepatoprotective activity, similar to the polyphenol complex of wood Maackia amurensis Rupr. et Maxim.

Studies of the hepatoprotective effect of the preparation of callus maakia were carried out on a group of laboratory mice in which artificial hepatitis was induced by the action of carbon tetrachloride (CCl ₄ ) (Table 2).

table 2
Hepatoprotective activity of the maakia polyphenolic complex obtained from callus culture compared to the complex isolated from sound wood with CCl ₄ hepatitis in mice Experienced groups
Indicators Intact animals CCl _{4 -} hepatitis CCl ₄ hepatitis + (PCM-NUCLEUS) CCl _4- hepatitis + (PKM-KL) Mortality,% 0 33 12.5 0 Change in body weight, g + 0.95 ± 0.24 -1.63 ± 0.97 ^a -1.15 ± 0.75 ^a -1.13 ± 0.64 ^a The specific gravity of the liver, mg 60.6 ± 3.3 78.3 ± 3.9 ^a 66.0 ± 5.7 70.6 ± 2.6 ^a The duration of hexenal sleep, min 18.5 ± 0.9 70.2 ± 2.2 ^a 40.5 ± 2.4 ^{a, b} 45.3 ± 3.0 ^{a, b} Fatty liver, points 0 ± 0 3.0 ± 0 ^a 2.1 ± 0.33 ^{a, b} 1.9 ± 0.22 ^{a, b} Note: (M ± m, averages from 6-8 observations), p <0.05 with respect to: ^a - to intact animals; ^b - to CCl ₄ hepatitis

As a result of these studies, it was found that on the seventh day of the experiment, the animals developed severe CCl ₄ hepatitis, which was characterized by a four-fold increase in the duration of hexenal sleep, indicating inhibition of the activity of microsomal liver enzymes. Against the background of a decrease in body weight, the specific gravity of the liver increased by 30% by the end of the experiment. The degree of hepatocyte fatty infiltration corresponded to a 3-point scale on a five-point scale. The death in the group of mice with induced hepatitis was 33%. Mortality in the group of mice treated with the drug from sound wood was 12.5%. In the group of animals treated with the drug from the callus culture, mortality was not observed. Both drugs significantly (more than 50%) reduced the duration of hexenal sleep and the degree of obesity of hepatocytes (to 1.9-2.1 points), more moderate values of the specific gravity of the liver were noted. Thus, the results obtained indicate the presence of pronounced hepatoprotective properties of the polyphenolic complex isolated from the Amak maia culture. At the same time, the polyphenolic complex from the culture of calluses of Maakia Amur has a more pronounced pharmacological activity.

At the same time, pharmacological studies were performed in rats with induced CCl ₄ hepatitis. As a result, it was found that a preparation of sound wood at a dose of 150 mg / kg (the previously established optimal effective dose) prevents hyperlipidemia and hyperbilirubinemia, reduces the activity of alanine and aspartate aminotransferases, as well as alkaline phosphatase. A preparation from callus culture has an equal effect with a preparation from sound wood when it is used in a lower dose, 100 mg / kg. A drug from callus culture has a tendency towards a more pronounced normalizing effect on the concentration of lipids in blood serum increased by a toxicant. Therefore, the effective dose of the drug from callus culture is 1.5 times lower compared to the effective dose of the drug from sound wood, which is obviously due to the higher content of biologically active substances in the preparation obtained from callus culture.

Thus, pharmacological studies of the callus preparation Maackia amurensis have shown that it exhibits a significant hepatoprotective effect.

LITERATURE

1. Yakovenko EP et al. The effectiveness of therapy in patients with chronic hepatitis with hepatoprotectors // Materials of the final scientific-practical conference “Clinical Medicine Issues”, MH RSFSR, M., 1989, p.228-231.

2. Grigoriev P.Ya. and others. “Diagnosis and treatment of chronic hepatitis and cirrhosis of the liver” // Soviet medicine, 1985, No. 12, pp. 61-67.

3. Blinkov I.L. and others. The experience of clinical use of the hepatoprotective agent legalon // Symposium “Clinical significance of the drug legalon”, M., 1981, p.113-123.

4. RF patent No. 2175237, A 61 K 35/78, A 61 P 1/16, 2001.

5. Chem. prir. Comp., 1988, No. 6, pp. 80-804.

6. Chem. prir. Comp., 1992, No. 5, p. 468-476.

7. Phytochemistry, 1995, vol. 40, No. 3, p. 1001-1003.

8. Chem. prir. Comp., 1986, No. 1, pp. 39-42.

Claims (1)

A hepatoprotective agent based on an extract of Maackia amurensis, characterized in that it is an extract of the biomass of Maackia amurensis Rupr. et Maxim, obtained by the method of callus culture, and contains a polyphenolic complex consisting of daidzein, retuzin, genistein, formononetin, maakiain, medicarpine.