RU2804299C1 - Method of obtaining oreganol a, having nephrotropic and neurotropic activity - Google Patents

Abstract

FIELD: chemical; pharmaceutical industry.

SUBSTANCE: invention relates to a method of producing oreganol A, which has nephrotropic and neurotropic activity. A method of producing oreganol A, which has nephrotropic and neurotropic activity, with the preliminary production of aqueous-alcoholic extract from medicinal plant raw materials using the sorbent chromatography method, according to which the extract is obtained from the oregano herb by ordinary extraction with 70% ethyl alcohol in the raw material-extractant ratio of 1:5, extraction is carried out by fractional maceration until the raw material is completely depleted, the resulting combined aqueous-alcoholic extract is evaporated to a thick extract, which is then mixed with silica gel L 40/100, dried and applied to a column filled with silica gel in the form of a suspension in chloroform, the target substance is eluted a mixture of chloroform:ethyl alcohol 96% in a ratio of 70:30 using column chromatography on silica gel, further purification is carried out by reprecipitation in a mixture of acetone and chloroform with a yield of 1.05% and a degree of purity of 99.5%.

EFFECT: above method allows to obtain oreganol A, which has nephrotropic and neurotropic activity with a substance yield of 1.05% and a purity of 99.5%.

1 cl, 4 dwg, 1 ex

Description

The invention relates to the chemical and pharmaceutical industry, in particular to the production of medicines in the form of substances and individual biologically active compounds (BACs) and concerns a method for producing 4'-O-β-D-glucopyranoside 3',4'-dihydroxybenzylprotocatechuic acid (oreganol A) from the herb oregano ( Origanum vulgare L.), which has nephrotropic and neurotropic activity. This BAS can be used as a diuretic and a treatment for anxiety.

Oregano herb is a promising plant species due to its rich chemical composition. Oregano raw materials contain essential oil (the leading group of biologically active substances), flavonoids, simple phenols and phenylpropanoids. Oregano herb is known to have expectorant, antibacterial, antifungal, antiviral, and antioxidant effects (1-5). The raw material is included in the multicomponent “Breast collection No. 1” and “Sedative collection No. 3”, as well as the herbal medicine “Urolesan” (1, 6).

There is a known method for isolating 4'-O-β-D-glucopyranoside 3',4'-dihydroxybenzylprotocatechuic acid from the leaves of oregano ( Origanum vulgare L.) (4). According to this method: raw oregano (100 g) is extracted with 70% methanol (about 3 l), filtered and concentrated in vacuum to about 1 liter. The resulting extract is divided between ethyl acetate and water. The water-soluble phase is concentrated under reduced pressure. An 80% ethanol solution (1 l) is added to the resulting concentrated extract and allowed to infuse at 25°C for 24 hours. The supernatant and precipitate are separated with filter paper, and the supernatant solution is further purified. The supernatant solution is concentrated in vacuo, and the resulting extract is subjected to column chromatography on Diaion HP-20 adsorbent resin (Mitsubishi Chem., 200 g). The adsorbent is washed successively with water and 70% methanol. The extract from 70% methanol was purified using Sephadex LH-20 by column chromatography. The column is treated with 70% methanol, and the resulting fractions are purified by liquid chromatography at medium pressure. The system (Tokyo Rikakiki) for this purification consisted of an EFC-1000 pump (stroke length 50; stroke frequency 70), a hand injector (SV-606010S) with a 2.0 ml sampling loop and a UV-D2 detector (A 254nm) . The isocratic mobile phase was methanol:water:acetic acid (50:50:0.1). The resulting fractions are further purified by HPLC (moving phase: acetic acid: methanol (1:4); flow rate: 5 ml/min, A: 254 nm). The result was a compound (270 mg) having a relative retention time of 12.4 minutes. This compound is the 4'-O-β-D-glucopyranoside of 3',4'-dihydroxybenzylprotocatechuic acid (2).

We took the above method as a prototype.

The disadvantages of the prototype are the low yield of the target substance - 4'-O-β-D-glucopyranoside 3',4'-dihydroxybenzylprotocatechuic acid, the use of large quantities of extremely dangerous methanol (poison) not only as an extractant, but also during column chromatography, multi-stage and duration of obtaining the target substance.

Thus, the purpose of the invention is to develop a method for producing a substance with nephrotropic and neurotropic activity.

The technical result is the creation of a method for producing 4'-O-β-D-glucopyranoside 3',4'-dihydroxybenzylprotocatechuic acid (oreganol A) from oregano herb using column chromatography.

The technical result is achieved by the fact that the extract is obtained from the oregano herb by ordinary extraction with 70% ethyl alcohol in a raw material-extractant ratio of 1:5, the target substance is eluted with a mixture of chloroform:ethyl alcohol 96% in a ratio of 70:30 using column chromatography on silica gel, and further purification is carried out by reprecipitation in a mixture of acetone and chloroform.

The method is implemented as follows.

Crushed air-dried raw materials of oregano herb (100.0 g) are extracted with 70% ethyl alcohol in a ratio of 1:5 until the raw materials are completely depleted by fractional maceration. The resulting combined aqueous-alcoholic extract is evaporated on a rotary vacuum unit to a thick extract, which is then mixed with 70 g of silica gel L 40/100, dried and applied to a column (8 x 10 cm) filled with silica gel in the form of a suspension in chloroform. The chromatographic column is washed with horoform (0.25 l) and a mixture of chloroform and ethyl alcohol in a ratio of 80:20 (1.0 l). The target substance is eluted with a mixture of chloroform and ethyl alcohol in a ratio of 70:30 (1 l). The separation of substances is monitored by chromatography on Sorbfil PTSKH-AF-A-UF-254 plates in a solvent system: chloroform-ethyl alcohol-water (26:16:3). Eluates containing 3',4'-dihydroxybenzylprotocatechuic acid 4'-O-β-D-glucopyranoside are combined for subsequent reprecipitation in a mixture of acetone and chloroform. The resulting amorphous substance is the finished product (4'-O-β-D-glucopyranoside 3',4' dihydroxybenzylprotocatechuic acid) having a purity of 99.5%.

The proposed method is illustrated by the following example.

Example 1.

Crushed air-dried raw materials of oregano herb (100.0 g) are extracted with 70% ethyl alcohol in a ratio of 1:5 until the raw materials are completely depleted by fractional maceration. The resulting combined aqueous-alcoholic extract is evaporated on a rotary vacuum unit to a thick extract, which is then mixed with 70 g of silica gel L 40/100, dried and applied to a column (8 x 10 cm) filled with silica gel in the form of a suspension in chloroform. The chromatographic column is washed with horoform (0.25 l) and a mixture of chloroform and ethyl alcohol in a ratio of 80:20 (1.0 l). The target substance is eluted with a mixture of chloroform and ethyl alcohol in a ratio of 70:30 (1 l). The separation of substances is monitored by chromatography on Sorbfil PTSKH-AF-A-UF-254 plates in a solvent system: chloroform-ethyl alcohol-water (26:16:3). Eluates containing 3',4'-dihydroxybenzylprotocatechuic acid 4'-O-β-D-glucopyranoside are combined for subsequent reprecipitation in a mixture of acetone and chloroform. 1.05 g of the substance is obtained with a yield of 1.05% by weight of the air-dried raw material and a purity of 99.5%.

The chemical structure of 3',4'-dihydroxybenzylprotocatechuic acid 4'-O-β-D-glucopyranoside is shown in FIG. 1.

Spectral and physicochemical properties of 4'-O-β-D-glucopyranoside 3',4'-dihydroxybenzylprotocatechuic acid. Amorphous substance of light yellow color with composition C ₂₁ H ₂₄ O ₁₁ . UV spectrum (EtOH, λ _max , nm): 220, 263.

^1H NMR spectrum (399.78 MHz, DMSO-d ₆ , δ, ppm, J/Hz): 9.41 (1H, br.s, 3'-OH group), 8.67 (1H, s, 3'- OH-group), 7.41 (1Н, dd, 2.0 and 9.0 Hz, Н-6), 7.34 (1Н, d, 2 Hz, Н-2), 7.07 (1Н, d, 9 Hz, Н-5'), 6.97 (1Н, d, 9 Hz, Н-5), 6.85 (1Н, d, 2 Hz, Н-2'), 6.77 (1Н, d, 2 and 9 Hz, Н-6'), 5.11 (2Н, c, H-7'), 4.66 (d, J = 7.0, H-1'' glucopyranose), 3.78 (3H, s, CH ₃ O), 3.22-3.66 (m, 6H glucopyranose).

¹³ C NMR spectrum (100.52 MHz, DMSO-d ₆ , δ _C , ppm): 165.91 (C-7), 152.54 (C-4'), 147.27 (C-4), 146.82 (C-3' ), 145.75 (C-3), 132.01 (C-2), 131.54 (C-6), 131.45 (C-5'), 122.45 (C-1), 122.13 (C-1'), 117.13 (C- 5), 116.31 (C-2'), 116.22 (C-6'), 102.73 (C-1'' glucose), 77.74 (C-5'), 76.49 (C-3'''), 73.83 (C -2''), 70.34 (C-4''), 66.10 (C-7), 61.29 (C-6''), 56.17 (CH ₃ O).

HR-ESI-MS, 180°С, m/z : 456.1500 [M+NH ₄ ] ⁺ , 461.1054 [M+Na] ⁺ , 477.0794 [M+K] ⁺ .

4'-O-β-D-glucopyranoside 3',4'-dihydroxybenzylprotocatechuic acid, obtained by the developed method, was studied for the presence of nephrotropic and neutrotropic activity.

The study of nephrotropic and neutrotropic activity was carried out on outbred white rats of both sexes weighing 200-220 g. The animals were kept in a vivarium on a normal diet with free access to water. Each group consisted of ten animals. The study drug was administered intragastrically through a tube at a dose of 0.5 mg/kg. Purified water served as a control in the study of neurotropic activity. The drug was administered once against the background of a similar water load, the experiment was carried out 2 hours after administration of the drug (7). The study of neutrotropic activity was carried out using the Porsolt test (8). At the same time, the individual time of the animals’ active attempts to get out of the water was recorded for five minutes. The obtained data were processed statistically using the Mann-Whitney test. The results of the study of the effect on the motor activity of rats using the Porsolt test method are presented in Table 1 - Fig. 2.

Based on the results obtained, we can conclude that 4'-O-β-D-glucopyranoside 3',4' dihydroxybenzylprotocatechuic acid at a dose of 0.5 mg/kg with a single intragastric administration contributed to a significant decrease in the motor activity of animals in the experimental group by 47% in relation to water control.

The study of nephrotropic (diuretic) activity was carried out as follows: the control group of animals received a 3% water load intragastrically, the experimental animals received the study drug at a dose of 0.5 mg/kg against the background of a similar water load. After all manipulations, the animals were placed in exchange cages for 24 hours. Urine samples were collected after 4 and 24 hours of the experiment. Sample volume (diuresis) and creatinine concentration were determined. Creatininuresis was determined by colorimetry using CPK-3. Statistical processing of the obtained results was carried out using the Mann Whitney method with Bonferroni correction.

The results of the study of the effect of oreganol A on the excretory function of the kidneys are presented in table 2 - Fig. 3 and table 3- Fig. 4.

Based on the results obtained, we can conclude that 4'-O-β-D-glucopyranoside 3',4' dihydroxybenzylprotocatechuic acid at a dose of 0.5 mg/kg with a single intragastric administration contributed to a significant increase in diuresis (by 73% and 42%) for 4 and 24 hours of experiment, respectively, as well as creatinine reduction (by 47%) for 24 hours of experiment in relation to the water control.

Thus, the proposed method for obtaining 4'-O-β-D-glucopyranoside 3',4'-dihydroxybenzylprotocatechuic acid from oregano herb using column chromatography was developed for the first time for this type of raw material and has the following advantages:

1. The developed method makes it possible to obtain a higher yield of the substance 4'-O-β-D-glucopyranoside 3',4'-dihydroxybenzylprotocatechuic acid - 1.05% instead of 0.27% for the prototype (4).

2. The developed method is less labor-intensive, since it includes three stages of obtaining the target substance: extraction of raw materials with ethanol, evaporation, chromatographic purification on silica gel using an eluent, which is a mixture of two solvents (chloroform and ethyl alcohol), instead of seven stages in the prototype using various methods (column chromatography and HPLC), sorbents (Diaion HP-20 adsorbent resin, Sephadex LH-20) and toxic eluent - methanol (4).

3. Extraction of oregano from raw materials was obtained with the consumption of a small amount of non-toxic extractant (1:5), compared to the prototype, where the raw material-extractant ratio is 1:30, and toxic methanol is used as an extractant (4).

4. The target product oreganol A, obtained by the developed method, has nephrotropic and neutrotropic activity.

INFORMATION SOURCES:

1. Kurkin V.A. Pharmacognosy: A Textbook for Pharmacy Students. universities - Ed. 4th, revised and additional - Samara: Ofort LLC, Federal State Budgetary Educational Institution of Higher Education Samara State Medical University of the Ministry of Health of Russia. 2019. - 1278 p.

2. Mirovich V. M. Pharmacognostic study of representatives of the genera Origanum L. and Rhododendron L. of the flora of Eastern Siberia: abstract. dis. ... d. pharmacist. date: 04/14/02/ Mirovich Vera Mikhailovna. - Ulan-Ude, 2010. - 41 p.

3. Knez Hrnčič M., Cör D., Simonovska J. et al. Extraction techniques and analytical methods for characterization of active compounds in Origanum species. Molecules, 2020; 25 (20): 4735.

4. Matsura H., Chiji H., Asakawa C. et al.. DPPH radical scavengers from dried leaves of Oregano ( Origanum vulgare ). Bioscience, Biotechnology, and Biochemistry, 2003; 67 (11): 2311-2316.

5. Fujie A., Yoshida K., Ôba K. et al. Antioxidative phenolic acids from Oregano ( Origanum vulgare L.) leaves. Nippon Shokuhin Kagaku Kogaku Kaishi, 2003 50(9):404-410. DOI:10.3136/nskkk.50.404

6. Mashkovsky M.D. Medicines. 16th ed., revised, corrected. and additional New wave, 2020. - 1216 p.

7. Zaitseva E.N., Zaitsev A.R., Dubishchev A.V. Device for introducing water load to laboratory animals. Patent for PM 115651 Ros. Federation. No. 2011138631/13; application 20.09.11; publ. 05/10/12, Bulletin. No. 13. 2 s.

8. Khabriev R.U. Guide to experimental (preclinical) study of new pharmacological substances / ed. RU. Khabrieva. - 2nd ed., revised. and additional M.: OJSC Publishing House "Medicine", 2005. - 832 p.

Claims (1)

A method for producing oreganol A, which has nephrotropic and neurotropic activity, with the preliminary production of aqueous-alcoholic extract from medicinal plant raw materials using the chromatography method on sorbents, characterized in that the extract is obtained from oregano herb by ordinary extraction with 70% ethyl alcohol in the raw material-extractant ratio » 1:5, extraction is carried out by fractional maceration until the raw material is completely depleted, the resulting combined aqueous-alcoholic extract is evaporated to a thick extract, which is then mixed with silica gel L 40/100, dried and added to a column filled with silica gel in the form of a suspension in chloroform, target the substance is eluted with a mixture of chloroform:ethyl alcohol 96% in a ratio of 70:30 using column chromatography on silica gel, further purification is carried out by reprecipitation in a mixture of acetone and chloroform with a yield of the substance of 1.05% and a purity of 99.5%.