CHEMICAL COMPOSITION AND BIOLOGICAL ACTIVITY OF Epilobium hirsutum L.

ABSTRACT

As a result of experiments to study the anti-inflammatory effect on the generally accepted model of formalin inflammation, it was established that the claimed total extract of fireweed has a pronounced anti-inflammatory effect and is not inferior in activity to the reference drug Ketoprofen.

АННОТАЦИЯ

В ходе экспериментов по изучению противовоспалительного действия на общепринятой модели формалинового воспаления установлено, что заявляемый суммарный экстракт кипрея оказывает выраженное противовоспалительное действие и по активности не уступает препарату сравнения Кетопрофен.

Keywords: ketoprofen, panadol, kiprey, HPLC-MC, anti-inflammatory, analgesic, antipyretic, Epilobium hirsutum L.

Ключевые слова: кетопрофен, панадол, кипрей, ВЭЖХ-МС, противовоспалительный, анальгетическую активность, жаропонижающий, Epilobium hirsutum L.

Introduction. Currently, research is being conducted around the world aimed at finding and introducing medicines based on plant metabolites. Interest in physiologically active substances obtained from plant materials is growing every year.

Non-narcotic analgesics are widely used in practical medicine. They are widely used for headaches, neurology, rheumatoid inflammation, other inflammatory processes, etc. They usually not only relieve pain, but also reduce body temperature (paracetamol, analgin, baralgin, pentalgin, trimol, etc.), and have anti-inflammatory activity (aspirin, ibuprofen, ortofen, butadione, indomethacin, etc.). However, all drugs used in medical practice as an analgesic that simultaneously exhibit anti-inflammatory and antipyretic properties have a significant number of disadvantages, namely, they inhibit hematopoiesis, cause an allergic reaction, bronchospasm, have hepatotoxic, nephrotoxic, ulcerogenic and other side effects, which limit scope of their application [1-2].

The study of the analgesic and anti-inflammatory activity of the compounds was carried out in comparison with a close analogue, ketoprofen [3].

Ketoprofen is a non-steroidal anti-inflammatory drug. It has anti-inflammatory, analgesic and antipyretic effects. Ketoprofen blocks the action of the enzymes COG-1 and COG-2 and, partially, lipoxygenase, which leads to suppression of prostaglandin synthesis (including in the central nervous system, most likely in the hypothalamus).

Ketoprofen does not have a negative effect on the condition of articular cartilage. However, when using ketoprofen, a number of side effects may occur, namely from the hematopoietic system: rarely - hemorrhagic anemia, hemolytic anemia, leukopenia; frequency unknown - agranulocytosis, thrombocytopenia, bone marrow dysfunction.

Experimental

Dried grinded aerial part (1 kg) of the Epilobium hirsutum L. (Onagraceae Jus.) (fireweed), was harvested in June 2020 in the Tashkent region. Extraction was carried out with methyl alcohol (4 l) at room temperature 25°C for 24 hours with periodic shaking (5 times). The extract was concentrated under reduced pressure and a temperature of 40-50°C to the consistency of a thick resinous mass. Water (300 ml) was added to the initial resinous mass obtained after primary (methanol) extraction (35 g) and with vigorous stirring a colored homogeneous solution was obtained. Next, the aqueous solution was extracted with 600 ml of chloroform. After evaporation of chloroform, the remainder of the extract was extracted with butanol. The butanol extract was evaporated to dryness. We obtained 20 g of butanol extract. The butanol extract was subjected to TLC (system: chloroform-methanol-water-acetic acid 9:3:0.5:0.5; chloroform-ethyl acetate 9:1; chromatographic: ammonia vapor and vanillin-sulfuric acid). Upon comparison by authentic compounds, phenolcarboxylic acids (gallic acid and its derivatives), flavonoids (quercetin and its glycosides) had been discovered.

HPLC-MS conditions of butanol fraction: Agilent 1260 Infinity II instrument equipped with a qD Acquity mass detector (Waters), ESI+ conditions in the mass range of 150-1250 Da. Substances of a phenolic nature were detected. HPLC conditions: for separation, water (A) was used as solvents: acetonitrile (B) in a gradient (00 min. 100% (A), 0% (B); 1.30 min. 96.1% (A); 3.9% (B); 2.50 min. 80% (A), 20% (B); 5 min. 55% (A), 45% (B); 10 min. 2% (A), 98% (B) HPLC column: Poroshell 120 EC18, 4 μl, 4.5x150 mm, injection volume: 10 mL, flow rate: 0.8 ml/min, pressure: 350 bar, DAD conditions, 254, 270 nm.

MS conditions: scan mode at 50-1250 Da, cone voltage 15V, capillar voltage for positive 0.8 kV, ESI+ (positive mode).

Gallic acid (1). C₇H₆O₅, m.p. 260°C, UV spectrum (λmax, EtOH, nm) 272.5; 171.028 m/z ([M+H]+) [4].

Methyl gallate (2). C₈H₈O₅, m.p. 199-203℃, 207.0264 m/z ([M+Na]+) [4].

Umbelliferone (3). C₉H₆O₃, m.p. 233-234℃; UV spectrum (λmax, EtOH, nm) 300, 305 and 325; 163.039 m/z ([M+H]+) [4].

Scopoletine (4). C₁₀H₈O₄, m.p. 203-205℃, UV spectrum (λmax, EtOH, nm) 300, 315; 193.031 m/z ([M+Na]+) [4].

Ellagic acid (5). C₁₄H₆O₈, m.p. 448-450℃; 303.006 m/z ([M+K]+) [4].

Luteolin (6). C₁₅H₁₀O₆, m.p. 328-331℃, UV spectrum (λmax, EtOH, nm) 260, 274 and 356 nm; 287.081 m/z ([M+ CH3OHH]+) [5].

Kaempferol (3,5,7,4’-tetrahydroxyflavone) (7). C₁₅H₁₀O₆, m.p. 275-277℃, UV spectrum (λmax, EtOH, nm) 294 and 367 nm; 287.081 m/z ([M+ CH3OHH]+) 6].

Quercetin (3,5,7,3’,4’-pentahydroxyflavone) (8). C₁₅H₁₀O₇, m.p. 313-315℃, UV spectrum (λmax, EtOH, nm) 257, 268 and 371 nm; 303.042 m/z ([M+K]+) [6].

Isorhamnetin (3,5,7,4’-tetrahydroxy-3’-methoxyflavone) (9). C₁₆H₁₂O₇, m.p. 305-307℃, UV spectrum (λmax, EtOH, nm) 255, 266 and 372 nm; 317.065 m/z ([M+H]+) [5].

Mericetin (10). C₁₅H₁₀O₈, m.p. 350-357℃, UV spectrum (λmax, EtOH, nm) 378 and 356 nm; 319.0037 m/z ([M+Na]+) [4,5].

Kaempferol-7-rhamnoside (11). C₂₁H₂₀O₁₀, m.p. 231-234℃, UV spectrum (λmax, MeOH, nm) 260 and 365 nm; 433.112 m/z ([M+H]+) [5].

Kaempferol 3-O-glycoside (12). C₂₁H₂₀O₁₁, m.p. 223-229℃, UV spectrum (λmax, MeOH, nm) 266, 301 and 350 nm; 449.107 m/z ([M+H]+) [5].

Cinaroside (13). C₂₁H₂₀O₁₁, m.p. 240-242℃, UV spectrum (λmax, EtOH, nm) 256, 268 and 352 nm; 449.107 m/z ([M+H]+) [5].

Hyperoside (14). C₂₁H₂₀O₁₂, m.p. 235-236⁰С, UV spectrum (λmax, EtOH, nm) 257, 269 and 362 nm; 465.102 m/z ([M+H]+) [5].

Isoquercetin (quercetin-3-O-β-D-glucoside) (15). C₂₁H₂₀O₁₂, m.p. 238-239℃, UV spectrum (λmax, EtOH, nm) 255, 268 and 362 nm; 465.102 m/z ([M+H]+) [5].

Kaempferol-3-O-rutinoside (16). C₂₇H₃₀O₁₅, m.p. 215-217℃, UV spectrum (λmax, EtOH, nm) 265 and 352 nm; 595.158 m/z ([M+K]+) [5].

Isorhamnetin 3-rutinoside (17). C₂₈H₃₂O₁₆, m.p. 178-180℃, UV spectrum (λmax, EtOH, nm) 254, 265 and 356 nm; 625.169 m/z ([M+K]+) [5].

By processing the chromatogram (Figure 1), compounds were established (Score threshold 0.85, tolerance units 0.50 Da) in accordance with the masses, ions were found in the form of adducts H+, K+, Na+. (Pubchem and NIST2017 database).

Figure 1. TIC chromatogram of the butanol fraction

These detected compounds are shown in Table 1.

Pharmacological studies were carried out on a methanol extract of the aerial part of Epilobium hirsutum L. (hereinafter referred to as “fireweed”), growing in the Republic of Uzbekistan.

The purpose of the study is to study the combined extract as a low-toxic analgesic and antipyretic with pharmacological action (analgesic, antipyretic and anti-inflammatory effects). This goal is achieved through the use of total fireweed extract as a non-narcotic analgesic, anti-inflammatory and antipyretic agent.

The study of anti-inflammatory activity was carried out in experiments on white mice. The groups for the experiments were selected according to the principle of analogues according to the criterion of body weight, 6 animals in each group.

Table 1.

Anti-edematous effect of total fireweed extract in comparison with ketoprofen

The generally accepted model of inflammation caused by 2% formaldehyde was used. Formalin edema of the paw of mice suggests the development of a local exudative inflammatory reaction, characterized by an increase in the volume of the limb exposed to phlogogen. In formaldehyde edema, the trigger point in the development of exudative inflammation is the protein destruction of membranes [7, 8].

Phlogogen formalin was administered subplantarly in a volume of 0.05 ml, the total fireweed extract was administered orally at a dose of 50-100-150-200 mg/kg. The non-steroidal anti-inflammatory drug ketoprofen was used as a comparison drug at a dose of 1.0-50.-10.0 mg/kg body weight.

3 hours after the introduction of phlogogen, the animals were removed from the experiment, and swollen and non-swollen hind feet were amputated at the level of the hip joints.

The anti-inflammatory activity of the studied compounds was calculated using the formula:

А=100% -

Here, A - anti-inflammatory activity, %; M_sf- the mass of the swollen foot in the experiment, mg; M_hf – weight of a healthy foot in the experiment, mg; M_sc – weight of the swollen foot in the control, mg; M_hc is the weight of the healthy foot in the control, mg [9].

The activity of the test substances was determined by their ability to reduce the development of edema compared to the control and expressed as a percentage of the control (Table 1).

Conclusion. As a result of experiments to study the anti-inflammatory effect on the generally accepted model of formalin inflammation, it was established that the claimed total extract of fireweed has a pronounced anti-inflammatory effect and is not inferior in activity to the reference drug Ketoprofen.

Reference:

1. М.Д. Машковский. «Лекарственные средства». Ташкент, Медицина, 1998 г., Т.1, с 159-179.
2. Регистр лекарственных средств России. 2002 г., выпуск-5, с. 399-400.
3. Регистр лекарственных средств России. 2015 г., №23, выпуск-23, с. 510.
4. Г.Р. Бушуева, А.В. Сыроешкин, Т.В. Максимова, А.В. Скальный. Кипрей узколистный-перспективный источник биологиччески активных соединений. Микроэлементы в медицине. 2016 г., 17 (2), с. 15-23.
5. М.П. Юлдашев. Флавоноиды растений семейств Lamiaceae, Fabaceae, Apiaceae и Scophulariaceae: Дис. докт. хим. наук (02.00.10-Биооррганическая химия, химия природных и физиологически активных веществ). 2000 г., с. 184.
6. А.М. Каримов, М.П. Юлдашев, Э.Х. Ботиров. Флавоноиды Scutellaria adenostegia Briq. Химия растительного сырья. 2015, № 1, с. 63-68.
7. Г.А. Востроилова. Экспериментальная и клиническая фармакология препаратов плаценты, получаемых методом криофракцинирования: Дис. докт. биол. наук (16,00,04-ветеринарная фармакология с токсикологией; 03.00.04-биохимия). Воронеж, 2007 г., с.319.
8. В.А. Бузлама, В.А. Николаевский, Ю.Н. Чернов, А.И. Сливкин: ВГУ.-Воронеж: Изд-во ВГУ, 2013 г.
9. Г.А. Востроилова, П.А. Паршин, Н.А. Хохлова, Н.А. Григорьева, А.В. Топольницкая, Н.М. Федорова, А.Ю. Калугина. «Изучения противовосполительного действия тканевых препаратов на белых мышах», Ветеринарный фармакологический вестник 2018 г., №3 (4), с. 40-45.