Investigation of the extract's composition of Viper's bugloss (Echium vulgare)

The characteristics of Viper's bugloss (Echium vulgare) plant, its pharmacological properties, and extracts’ composition are presented in this study. Results of the literature analysis, data on the biologically active compounds and areas of use of this medicinal plant are summarized. Viper's bugloss (E. vulgare) is a species of flowering plant in the borage family Boraginaceae. It is native to most of Europe as well as western and central Asia. Viper's bugloss (E. vulgare) is a plant that has been utilized as food (honey), medicine, a poison, an oil, and as a dye and tannin-producing ornamental plant. Viper's bugloss (E. vulgare) is especially rich in pyrrolizidine alkaloids, flavonoids, phenolcarboxylic acids, sterones and naphthoquinones. In traditional medicine, Viper's bugloss (E. vulgare) is utilized as exhilarant and a mood stimulant. That is why one of the possible uses of this plant is considered to be treatment of depressive states. Like most representatives of Boraginaceae family, it has been insufficiently studied. No previous work quantifying flavonoids content of aerial parts of Viper's bugloss (E. vulgare) growing in Ukraine has been presented. Continuing the studies of this species, the aqueous and ethanolic extracts from Viper's bugloss (E. vulgare) aerial parts were obtained and their phytochemical composition was investigated. For the first time, the qualitative analysis of biologically active compounds in Viper’s bugloss’s extract as well as the quantitative analysis of flavonoids by aluminum chloride spectrophotometric method are reported. The experimental results showed that the total concentration of flavonoids was 2.59% in the extract. The maximum yield of extractives was found to be 16%. The obtained research data will be used in future investigations.


Introduction
Depressive disorders, including major depression and dysthymia, are serious, disabling, and often difficult-to-treat illnesses. A promising direction in the treatment of depressive disorders is the study of existing and creation of new effective herbal remedies. A large number of herbal antidepressants on the Ukrainian market, such as "Life 900", "Sedariston", "Neuroplant", "Deprivit" and others, based on the extract of St. John's wort (Hypericum perforatum). dry up during the flowering stage. Both stems and leaves are covered with stout. The stem terminates in a panicle inflorescence, and each branch of the panicle forms a short helicoid cyme subtended by an upper foliage leaf. There can be as many as 50 cymes per stem, and each cyme bears up to 20 flowers on the top. The showy flowers range in size from 1 to 2 cm. The funnel-shaped, five-lobed flowers are typically bright blue, but may also be purple, pink or rarely, white ( Figure 1). The seeds of Viper's bugloss (E. vulgare) are called nutlets. They are brown or gray with a rounded pyramid shape and are quite small. [3]. Viper's bugloss (E. vulgare) grows throughout Ukraine. It grows best in sunny areas, such as meadows, overgrazed pastures, poorly drained slopes and roadsides. It usually will not do well in cultivated ground. [4].
E. vulgare belongs to dye and tannin producing, medicinal, ornamental, and bee plants since it is a source of nectar and pollen forage. The flowers of E. vulgare are frequently visited by bumblebees and honey bees. Nectar contains 30-40% sugars and is mostly sucrose-dominated. Honey productivity is 300-400 kg/ha. Honey from E. vulgare has a light color, a pleasant smell, and a delicate taste. It contains a number of components that act as conservatives, such as vitamin C, flavonoids, and other phenols, as well as enzymes like glucose oxidase, catalase, and peroxidase, so it can remain preserved in a completely edible form for a long time. Basal leaves and young shoots of E. vulgare can be eaten in salads or stirfried.
The decoction of the Blueweed is used in folk medicine as an expectorant and soothing remedy for common or whooping cough, and as anticonvulsant and sedative for epilepsy. It is also used externally against rheumatic pains in joints, tendon sprains etc. [5]. E. vulgare contains hepatotoxic pyrrolizidine alkaloids such as cynoglossin and consolidin, which can be toxic to horses and cattle when consumed in large amounts. Its roots yield a water-insoluble carmine-red dye alkannin for wool, and its flowers contain anthocyanin, which appears as a red pigment in acidic and blue in alkaline conditions. Viper's bugloss (E. vulgare) has been planted as an ornamental plant. It also has the fatty acid composition of the seed oil (28-32%) and is grown as an oilseed crop. The seed oil from E. vulgare contains significant amounts of omega-3 and omega-6 polyunsaturated fatty acids (PUFA) such as g-linolenic acid (GLA) and rare stearidonic acid (SDA) [3].
Iranian scientists at Mashhad University of Medical Sciences have found that the aqueous and ethanolic flavonoids-containing extracts of E. vulgare inhibit monoamino oxidase enzyme (MAO) that can leads to a significant antidepressant effect [9]. The potential antidepressant effects of extracts of E. vulgare were investigated on mice using a forced swimming test. This test was performed in two sessions. In the pretest session (24 hours before the main session), each mouse (BALB/c male, weight 25-30 g) was forced to swim in a cylindricalshaped container (diameter 10 cm, height 25 cm, water height 11 cm, temperature 25±1 °C). This preliminary test is stress-inducing and mice gradually lose their movement behavior. After 15 minutes, animals were removed and dried. Then, 23.5 hours later the relevant extract sample was injected intraperitoneally (IP) into mice. The main test was performed 30 minutes later. In this test, each mouse was left in the same container for 6 minutes and the following behaviors were recorded: 1. Immobility: floating in the water without swimming.
2. Swimming: active movement of extremities and circling in the container.
3. Climbing: active movement of forelimbs on the container wall.
The tests were performed on three groups of mice, eight mice in each. The first group received the drug imipramine, which is a selective inhibitor of monoamine reuptake (antidepressant). Group 2 received aqueous and alcoholic extracts of Viper's bugloss (E. vulgare) in various concentrations. The results showed that the extracts have a clear antidepressant activity that is comparable to imipramine. [9].
The pyrrolizidine alkaloids previously identified in floral honey attributed to E. vulgare were detected by Boppre et al. in Germany. Pyrrolizidine alkaloids were isolated from the aqueous acid extracts of pollen by use of strong cation-exchange, solid-phase extraction and identified by liquid chromatographic/mass spectrometric (LCMS) analysis. The pyrrolizidine alkaloids in the pollen are present mainly as the N-oxides. In addition to the previously described pyrrolizidine alkaloids and/or their N-oxides (echimidine, acetylechimidine, uplandicine, 9-O-angelylretronecine, echiuplatine, leptanthine, and echimiplatine), one unidentified (echivulgarine), but previously found in honey, and two previously undescribed (vulgarine and 7-O-acetylvulgarine) pyrrolizidine alkloids and/or their N-oxides were identified in the pollen [10]. Pyrrolizidine alkaloid-containing plants are widely distributed throughout the world. The structural types and concentrations of the alkaloids vary among plant species. In addition, within a species of plant, concentrations vary with environment and location. Many pyrrolizidine alkaloids are toxic and cause poisoning in livestock and humans [11][12][13]. That is why to detect and determine the content of alkaloids in specific plants it is necessary to develop a rapid, sensitive, and specific method, which will be one of our goals of future research.

Experimental section
The source material for phytochemical studies was the stems of Viper's bugloss (E. vulgare), which was collected in the Mykolaiv region during the period of maximum accumulation BAR (July 2019). The procurement of raw materials was carried out according to regulatory and analytical documentation [2]. Air-dried raw materials were prepared in an Excelsior-shredding Machine. Studies of the extracts were performed according to standard methods using reagents that were supplied by Sigma-Aldrich Corp. and Merk. Analytical scales AS 220 from RADWAG, Poland was used to weigh samples.
The amount of extractives was measured by the gravimetric method [2]. The maximum yield of extractives was 16%.
The method of UV spectroscopy (Hitachi U-2810 spectrophotometer) was used to detect the quantitative content of flavonoids. The aluminum chloride method was used to determine the total flavonoid compounds [3]. The content of flavonoids in terms of avicularin and absolutely dry raw material in percent (X) was calculated by the formula: where D is the optical density of the test solution; 330specific absorption rate of avicularin complex with aluminum chloride at 410 nm; mweight of sample in grams; Wweight loss during drying of raw materials, %.
The powdered plant materials in the amount of 1 g was loaded into a 150 ml flask, 30 ml of 70% alcohol was added and refluxed in a water bath for 30 minutes. The flask was cooled to room temperature, and the mixture were passed through filter paper into a 100 ml volumetric flask. The extraction protocol was repeated 2 times as described above. The combined extracts were filtered, the solids were washed with 70% alcohol and the volume of the filtrate was adjusted to the mark (solution A).
Solution A (4 ml) was loaded into a 25 ml volumetric flask, 2 ml of a 2% solution of aluminum chloride in 95% ethanol was added and brought the volume with a 95% ethanol to the mark; after 20 min, the optical density of the solution was determined using spectrophotometer at a wavelength of 410 nm in a cuvette with a layer thickness of 10 mm. For comparison, the following solution was used: 4 ml of solution A was loaded into a 25 ml volumetric flask, a 1 drop of a dilute hydrochloric acid was added and the volume of the solution was adjusted to the mark with 95% ethanol. The flavonoid content was calculated based on dry raw material and found to be 2.59%.
With the help of qualitative reactions, the analysis for the presence of certain groups of BAS of medicinal plant raw materials was performed. The results of the identified substances are presented in Table 1.
Therefore, the obtained data demonstrated the presence of BAS in the object under study and provide insentives for further study.
It should be noted that the presence echimidine in the extract of the Viper's bugloss (E. vulgare) requires additional studies on the toxicity of extracts from this plant. Because this plant is widely used as a honey plant and contains alkaloids, there are several studies on their presence in honey.

Conclusions
The literature review has shown that Viper's bugloss (E. vulgare) as a study object has several advantages and disadvantages. The positive characteristics of the raw material include its availability and high prevalence. Viper's bugloss (E. vulgare) grows throughout Ukraine (rocky slopes, steppes, dry meadows, crops, fields). Significant antidepressant effect is equal in power to the official drugs and confirmed by foreign preclinical studies as well as public sector research of this raw material. The negative characteristics include the presence of pyrolyzidine alkaloids among the plant's secondary metabolites, which are dangerous to human health due to their hepatotoxic effect. On the other hand, this opens new horizons in the study of the psychotropic activity of the Viper's bugloss (E. vulgare) along with the development of methods to diagnose the toxicity of raw materials. Studies of Viper's bugloss (E. vulgare) indicate the relevance and prospects for further pharmacognostic and pharmacological studies of the plant.