OBTAİNİNG AND RESEARCHİNG FLAVONOİD-SAPONİN
COMPLEX FROM MEDİCAGO FALCATA L. RAW MATERİAL
*Tahir A. Suleymanov, **Aytakin S. Garibli
*Azerbaijan Medical University, Baku, Azerbaijan
**Azerbaijan State Agricultural University, Gandja, Azerbaijan
**garibli90@mail.ru
Abstract. In this study, we choose Medicago falcata L., also known as “alfalfa” or “Lucerne”, it belongs to the family Fabaceae. M. falcata L. is a perennial flowering plant. M. falcata L. produce different kinds of secondary metabolites such as saponins, flavonoids, coumarins, alkaloids, phenols which have antimicrobial and antibacterial properties which play medicinally vital role. The chemical constituents obtained from plant can cure high cholesterol, kidney problems, asthma, stomach, bladder problems and menopause issues. The aim of the study is to obtain the flavonoid-saponin complex of M. falcata L., which is common in the Azerbaijani flora, and to examine its antioxidant activity. The isolated flavonoid-saponin fraction when analyzed by A.B.T.S. and D.P.P.H. showed high activity.
The technical effect of the claimed invention consists in expanding the raw material base of medicinal plants, namely the base of crescent alfalfa (M. falcata L.) and the sum of the flavonoid-saponin complex of biologically active substances with antioxidant activity isolated from it.
Keywords: M. sativa L., biological active compounds, antioxidant, flavonoid, saponin
Introduction. Medicinal plants are producers and carriers of many biologically active substances. It is known that the flora of Azerbaijan is very rich of species composition. These species are not used enough to meet their health needs. The main reason for this is that phytochemical and pharmacological studies on plants have not been sufficiently studied. Especially after the independence of our republic, this problem has become even more important.
Antioxidants are substances that can neutralize the oxidative effects of free radicals and other substances. The role of antioxidants in our lives cannot be overestimated. They help limit the aggression of free radicals and repair the damage caused by them, resist free radicals, interrupting the destructive process of oxidation, strengthen the body’s own antioxidant system, and help restore metabolism [Mahdieh Raeeszadeh et al., 2022]. The plant world is diverse in the range of biologically active substances with an antioxidant effect, which are of considerable interest for the further search for new raw materials sources [Martyna Zagórska-Dziok et al., 2020].
A large number of natural antioxidants are known: ascorbic acid, tocopherol, beta-carotene, retinol, biogenic amino-serotonins, histamine, polyphenols and phospholipids, etc., contained in medicinal plants, on the basis of which various drugs are being developed.
In this study, we choose Medicago falcata L., also known as “alfalfa” or “Lucerne”, it belongs to the family Fabaceae [Flora of Azerbaijan, 1954]. M. falcata L. is a perennial flowering plant [Sadowska-Bartosz and Bartosz, 2014]. M. falcata L. produce different kinds of secondary metabolites such as saponins, flavonoids, coumarins, alkaloids, phenols which have antimicrobial and antibacterial properties which play medicinally vital role [Martyna Zagórska-Dziok et al., 2020, D. Tungmunnithum et al., 2018]. The chemical constituents obtained from plant can cure high cholesterol, kidney problems, asthma, stomach, bladder problems and menopause issues [Suleymanov and Garibli, 2019].
As a continuation of our research, the flavonoid-saponin complex of M. falcata L. was studied.
The aim of the study is to obtain the flavonoid-saponin complex of M. falcata L., which is common in the Azerbaijani flora, and to examine its antioxidant activity.
Materials and methods. In the present study the plants collection was performed from Shamakhi and identified. Plants were processed for different extractions and antioxydant activity in the laboratory of Aix Marseille University of France.
Bioactive substances of flavanoid-saponin complex is obtained as follows.
The dried and crushed aboveground part of the plant material M. falcata L. (0.6 kg) is extracted at room temperature 3 times with 6.0 l of 80% ethanol. The combined extract is evaporated under vacuum to a dry residue (52.9 g), then separated into fractions by sequentially extracting the resulting dry residue with petroleum ether to remove lipophilic substances and obtaining a dry residue; dichloromethane to remove chlorophyll; and ethyl acetate with the release of a complex of flavonoids and saponins, which is dried and washed with water from triglycosides and hydroxycinnamic acid. The resulting product, in the amount of 2.48 g, containing a complex of bioactive substances with antioxidant activity, is a yellowish powder, odorless and tasteless [Garibli et al., 2021]..
The yield on raw materials is 1.8%. It dissolves well in methanol and ethanol. Qualitative and quantitative composition of the resulting complex, studied in the following way.
The ethyl acetate extract (1.9 g) was fractionated on a Combi Flash Rf flash chromatograph. C18 RediSep Rf 130 g (CV 133ml – 85ml/min.) was chosen as the column. Fractionation was carried out at a pressure of 225psi (15.5 bar) and a wavelength of 254mm. Flow rate 40 ml/min. with MeOH and H2O gradient. Fractions X, A, B, C, and D were obtained.
Results of the study. The obtained fractions were tested in the system ethyl acetate-formic acid – glacial acetic acid – water (100:11:11:26) by thin-layer chromatography and by spraying cumic aldehyde and the content of saponin-flavonoid compounds in them was determined.
fractions X 0.0050 g
fraction A 0.0078 g
fraction B 0.3473 g
fraction C 0.4153 g
fraction D 0.009 g
The resulting fractions were dissolved in methanol and analyzed by thin layer chromatography. Ethyl acetate-formic acid-glacial acetic acid-water (100:11:11:26) was used as a system, and cumaldehyde was used as a developer. Based on the obtained analysis, fractions B and C were taken for further study. Fraction B (0.34 g) was again processed by flash chromatography. As a result of flash chromatography of fraction B obtained from the ethyl acetate fraction, 10 new fractions (B-1 – B-10) were obtained. A C18 Redi Sep Rf 30 g column (CV 26.4 ml – 35 ml/min) was chosen for fractionation in flash chromatography. The assay is run at 350 psi (24 bar), wavelength 254 nm, flow rate 15 ml/min, gradient MeOH and H2O.
fractions B-1 0.0008 g
fractions B-2 0.0001 g
fractions B-3 0.0002 g
fractions B-4 0.0012 g
fractions B-5 0.0071 g
fractions B-6 0.022 g
fractions B-7 0.2026 g
fractions B-8 0.0315 g
fractions B-9 0.009 g
fractions B-10 0.0363 g
The resulting fractions are processed by thin layer chromatography. Ethyl acetate-formic acid-glacial acetic acid-water (100:11:11:26) was used as a system, and cumaldehyde was used as a developer. According to the obtained results Fraction B-10, Fraction B-8, Fraction B-7 and Fraction B-6 were taken for further research.
From fraction V-10 (20 mg) obtained by chromatography in a column with Sephadex (eluent: pure methanol), 3 fractions (V-10-1 – V-10-3) were obtained. Of which fractions B-10-2 and B-10-3 are identified as Kaempferol and Quercetin.
From fraction B-8 (20 mg) obtained by chromatography in a column with Sephadex (eluent: pure methanol), 4 fractions (B-8-1 – B-8-4) were obtained. Of which fraction B-8-3 was identified as Kaempferol-7-O-α-L-rhamnopyranoside-3-O-β-D-glucopyranoside.
As a result of chromatography in a column with polyamide fraction B-7, 8 new fractions were obtained (B-7-1 – B-7-8). 6 fractions (B-7-4-1 – B-7-4-6) were obtained from fraction B-7-4 by chromatography in a polyamide column (eluent: pure methanol and water). Of which fraction B-7-4-3 is identified as Naringenin.
From fraction B-6 (22 mg) as a result of chromatography in a column with Sephadex (eluent: pure methanol), 4 fractions were obtained (B-6-1 – B-6-4). Of which fraction B-6-2 is identified as Kaempferythrin.
As a result of flash chromatography of fraction C (85.2 mg) obtained from the ethyl acetate fraction, 5 new fractions (C-1 – C-5) were obtained.
As a result of flash chromatography of the C-2 fraction, 6 new fractions (C-2-1 – C-2-6) were obtained.
Four fractions (C-2-3-1 – C-2-3-4) were obtained from the C-2-3 fraction by chromatography in a Sephadex column (eluent: pure methanol). Of which the C-2-3-3 fraction is identified as Apigenin.
6 fractions (C-2-5-1 – C-2-5-6) were obtained from the C-2-5 fraction by chromatography in a Sephadexm column (eluent: pure methanol). Of which the C-2-5-4 fraction is identified as Luteolin.
As a result of flash chromatography of the C-3 fraction, 7 new fractions (C-3-1 – C-3-7) were obtained.
From the C-3-5 fraction, chromatography in a Sephadex column (eluent: pure methanol) yielded 5 fractions (C-3-5-1 – C-3-5-5). Of which the C-3-5-3 and C-3-5-5 fractions are identified, respectively, Ursolic and Oleanolic acid.
Discussion of results. The development of new anti-bacterial agents by plant extracts is very much of importance nowadays. Phytotherapy is increasing by time to cure many diseases and serve humanity. The plant screened for phytochemical constituents appeared to have the ability to act as a source of useful drugs and improve the health status of consumers as a result of presence of various compounds that are essential and beneficial for good health. So it could be concluded that the M. sativa extract produce significant and remarkable phytochemicals which can be used as anti-microbial, anti-cancer and anti-fungal agents. The leaves, and stems are of great importance in the field of pharmacy. This identification of various phytochemicals leads to the production of more vaccines and supplements for many diseases and can promote better health and survival. However further work must still be conducted.
In recent years, great progress has been made in the field of chemistry and pharmacology of natural flavonoids and triterpene saponins, which are widely distributed in the plant world. Of particular interest are well-known valuable agricultural crops, namely representatives of the legume family, characterized by a wide range of biologically active substances (BAS), in particular, a representative of the alfalfa genus (Medicago L.) – alfalfa (M. sativa L.), which has long been used in folk medicine. However, another representative of this genus, sickle alfalfa (M. falcata L.), is little studied and is of considerable interest as a raw material source of biologically active substances with antioxidant activity [Xue-Gui L. et al., 2018].
The search and analysis of sources in this area showed that crescent alfalfa (M. falcata L.), as a medicinal raw material, is little studied, one source was found in which the herb M. falcata L is part of the medicinal collection.
The essence of the work consists in a complex of flavonoid-saponin biologically active substances with antioxidant activity isolated from sickle alfalfa (M. falcata L.), which includes: quercetin, kaempferol, kaempferythrin, kaempferol-7-O-α-L-rhamnopyranoside-3-O-β-D-glucopyranoside, apigenin, luteolin, naringenin, oleanolic and ursolic acids.
The components included in the sum of the complex are known in medicine as antioxidants and medicines:
Quercetin is a flavonoid, a non-carbohydrate biologically active component and belongs to the vitamin preparations of the P group. Quercetin has anti-inflammatory and antioxidant effects, reduces the synthesis of leukotrienes, serotonin and other inflammatory mediators.
Kaempferol – a flavonoid has anti-inflammatory, antimicrobial, cardioprotective, analgesic properties, reduces the synthesis of fatty acids in malignant cells, and this reduces the development of certain types of cancer, reduces “oxidative stress” in the body, enhancing protective functions and accelerating metabolism, prevents lipofuscin from accumulating in the body – aging pigment.
Kaempferitrin and kaempferol-7-O-α-L-rhamnopyranoside-3-O-β-D-glucopyranoside are kaempferol glycosides. They have a wide range of pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial, antitumor, cardioprotective, neuroprotective, antidiabetic, antiosteoporotic, estrogenic/antiestrogen, anxiolytic, analgesic, and antiallergic activities.
Apigenin is a flavonoid widely distributed in the plant world. It acts as a free radical scavenger and antioxidant to reduce oxidative stress.
Luteolin is a flavonoid with an anti-inflammatory effect.
Naringenin is a flavonoid with powerful antioxidant, anti-allergic and anti-inflammatory properties.
Oleanolic acid, a monounsaturated fatty acid, exhibits weak anti-HIV and weak anti-HCV activity in vitro and may slow the progression of adrenoleukodystrophy (ALD).
Ursolic acid – the compound belongs to pentacyclic triterpenes, which are characterized by high biological activity. It has found wide application in cosmetology, nutrition and bodybuilding.
As a result of chromatographic and spectroscopic studies, it was found that the flavonoid-saponin sum consists of the following substances: quercetin, kaempferol, kaempferythrin, kaempferol-7-O-α-L-rhamnopyranoside-3-O-β-D-glucopyranoside, apigenin, luteolin, naringenin, oleanolic and ursolic acids.
The antioxidant activity of the flavonoid-saponin fraction obtained from the species M. falcata L. was studied by three methods: 2, 2-diphenyll-1-picryl-hydrazyl hydrate (D.P.P.H.), acid (A.B.T.S.) and the iron reduction antioxidant power (F.R.A.P.) method. Gallic acid, ascorbic acid, quercetin, caffeic acid, trolox, and kaempferol were used as standard samples. Standards and sum of saponin flavonoids for D.P.P.H., A.B.T.S. and F.R.A.P. used in 6 different concentrations.
For D.P.P.H. analysis used solutions of substances in the following concentrations: gallic acid, caffeic acid, kaempferol and quercetin, respectively 0.5 µg/ml, 1 µg/ml, 1.25 µg/ml, 2 µg/ml, 2.5 µg/ml, 5 µg/ml, ascorbic acid 3 µg/ml, 5 µg/ml, 6 µg/ml, 7.5 µg/ml, 10 µg/ml, 15 µg/ml, Trolox 2.5 µg/ml, 3 µg/ml, 5 µg/ml, 7.5 µg/ml , 10 µg/ml, 15 µg/ml, flavonoid-saponin fraction 5 µg/ml, 10 µg/ml, 20 µg/ml, 25 µg/ml, 50 µg/ml, 100 µg/ml.
For A.B.T.S. analysis used solutions of substances in the following concentrations: caffeic acid 1 µg/ml, 2 µg/ml, 2.5 µg/ml, 5 µg/ml, 10 µg/ml, 12.5 µg/ml, gallic acid 0.25 µg/ml, 0.5 µg/ml ml, 1 µg/ml, 2 µg/ml, 2.5 µg/ml, 5 µg/ml, quercetin 0.5 µg/ml, 1 µg/ml, 2 µg/ml, 2.5 µg/ml, 5 µg/ml, 10 µg /ml, ascorbic acid, trolox and kaempferol 2.5 µg/ml, 5 µg/ml, 6.25 µg/ml, 10 µg/ml, 12.5 µg/ml, 25 µg/ml, flavonoid-saponin fraction 2.5 µg/ml, 5 µg /ml, 10 µg/ml, 20 µg/ml, 25 µg/ml, 50 µg/ml.
For F.R.A.P. analysis used solutions of substances in the following concentrations: gallic acid and quercetin 0.25 µg/ml, 0.5 µg/ml, 1 µg/ml, 1.25 µg/ml, 2 µg/ml, 2.5 µg/ml, caffeic acid and ascorbic acid 0.5 µg/ml ml, 1 µg/ml, 1.25 µg/ml, 2 µg/ml, 2.5 µg/ml, 5 µg/ml, Trolox 1 µg/ml, 2 µg/ml, 2.5 µg/ml, 5 µg/ml, 8 µg /ml, 10 µg/ml, kaempferol 2.5 µg/ml, 4 µg/ml, 5 µg/ml, 8 µg/ml, 10 µg/ml, 20 µg/ml, flavonoid-saponin fraction 2.5 µg/ml, 4 µg /ml, 5 µg/ml, 8 µg/ml, 10 µg/ml, 20 µg/ml.
The isolated flavonoid-saponin fraction when analyzed by A.B.T.S. and D.P.P.H. showed high activity.
The technical effect of the claimed invention consists in expanding the raw material base of medicinal plants, namely the base of crescent alfalfa (M. falcata L.) and the sum of the flavonoid-saponin complex of biologically active substances with antioxidant activity isolated from it.
Gratitude. Also, we would like to sincerely appreciate the professor Madam Evelyne Ollivier of Pharmacognosy and Ethnobotany faculty at the Aix-Marseille University.
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