INTRODUCTION:

The demand for medicinal natural products is significantly increasing, hence generating further interest in this expanding research area which involves extensive utilization of medicinal plants of various formulations as herbal remedies. Over the past decades, there has been a renewed interest in determining antimicrobial and antioxidant properties of biologically active plant-derived chemicals. It is generally known that antioxidants play a critical role in the process of free-radical transformations in the body, which fully justifies the search for naturally occurring antioxidants and highlights the relevance of the research^1-8. The best antioxidant sources are plants which contain biologically active substances (BAS) with a broad spectrum of antimicrobial properties^9-15.

It should be noted that the investigation of Scorzonera purpurea subsp. rosea (genus Scorzonera), a representative of the family Asteraceae, is an unjustifiably neglected area in pharmacological and pharmacognostic fields. Undeniably, plants of this family are known for being rich in various flavonoids, hence displaying significant antioxidant and antimicrobial effects. Thus, the review of literary sources on morphological features, chemical composition, and application of aerial and underground parts of plants of representatives of the genus Scorzonera has been carried out^16-22.

S. purpurea subsp. rosea is a perennial herbaceous plant 15-50cm high; stems are erect, simple, always with one anthodium, with black and brown clusters of old leaf fibers; a dense basal rosette of linear-lanceolate or lanceolate leaves, glabrous; the sepals are elongated and stretched out, inner leaves are longer, lanceolate, obtuse; petal-like flower, five-lobed petals, pale pink, often white, longer than the sepal; blooms in July – August²³. The fruits, reaching 1cm in length, are yellowish, without shin leaves, grooved in length, with a short peduncle at the base, which is part of the fruit²⁴. It grows in the mountains of Central and Southern Europe (Alps, Carpathians, Northern and Central Apennines, Balkan seas) at altitudes of 1200-2000 m above sea level²³.

S. purpurea subsp. rosea is used in traditional medicine in Ukraine in the form of water extract of rhizomes for snake bite treatment²³but isn’t studied the chemical component and sensitivity of the microorganisms against ethanolic extract of this plant. Moreover, there are data on high antimicrobial activity to multi-resistant strains of ethanol extract microorganisms from the above ground part of Scorzonera sandrasica¹⁸. Several studies have also been published on the chemical composition of methanol-water extract of the aerial part of S. cinerea, S. incisa, S. latifolia, S. mollis ssp. szowitsii, S. parviflora, and S. tomentosa, where by HPLC method and using standard samples: chlorogenic acid, coffeic acid, ferulic acid, kumaric acid, ruthenium, hyperosid, luteolin-7-glucoside, hesperidine, rosmarinic acid, quercetin, luteolin, apigenin; chlorogenic acid, hyperoside and luteolin-7-glucoside have been identified ^25-29.

According to the previous phytochemical studies dihydroisocoumarins, benzyl phthalides, lignans, neolignans, sesquiterpenes and triterpenes, benzylphtalides, stilbenoids and phenylbenzofuran derivatives were found in Scorzonera sp^29,30.

Different species of Scorzonera have been used in European traditional medicine against pulmonary diseases, colds, for the treatment of wounds as well as for their stomachic, diuretic, antipyretic, anti-inflammatory and appetizing effects; in Mongolian traditional medicine for the treatment of diarrhea, lung edema, parasitic diseases, and fever caused by bacterial, and viral infections; in Libyan folk medicine for the treatment of hepatic pains; and in Chinese, as well as in Tibetan folk medicine against breast inflammation and abscess. Additionally, in Turkish folk medicine different species of this genus have been reported to be used in treatment of rheumatism, pain, wound healing, as well as arteriosclerosis, kidney diseases, hypertension, and diabetes, poisonous ulcers and malignant stomach neoplasia^31-33.

The high anti-inflammatory and wound-healing activities of S. latifolia and S. mollis sp. szowitsii compared to extracts of other representatives of this class have also been reported³⁴.

Extracts from S. mackmeliana alone or synergistically with antibiotics could be tested as novel effective antibacterial agents against resistant bacterial strains^27,
35.

Phenolic compounds were extracted from S. undulata roots. The methanolic and ethyl acetate fraction of S. undulata roots have been compared The methanolic fraction exhibited the highest total phenol content to compare with ethyl acetate fraction. However, the highest flavonoids concentration was observed in ethyl acetate fraction. The strongest radical scavenging activities was observed in methanolic fraction by means of the DPPH, FRAP and ABTS assays³⁶.

MATERIALS AND METHODS:

Collection and processing of plant raw materials:

Air parts of S. rosea, dried under normal conditions (in a dark place, temperature 20-25°C, relative humidity 30-60%) were used as raw materials. Classical maceration with the ratio of raw material extragent 1:10 was applied. This method of extraction allows you to obtain an organic BAS complex, since it does not demand any additional heat supply. Dry raw materials were crushed to a particle size of 1-3mm, thoroughly soaked in alcohol-water mixture of given concentration, steeped at room temperature for a week, filtered and left in a cold place at +2-4°С for coagulation of suspended particles. The extracts obtained before the tests were centrifuged with a rotational speed 3000rpm.

Determination of the content of extractive substances:

1mL of the extract under study was placed into a glass weighing bottle. Nitrogen stream was used to evaporate the solvent at a temperature of 40°C on a RapidVap ® Vertex Adapteror. The content of extractive substances (ES) in one milliliter of extract was re-weighed and determined.

Total Phenolic Content (TPC):

Studies of the content of phenolic compounds in the extracts studied were carried out by spectrophotometric method on the Hitachi U-2810 spectrophotometer according to the Folina-Chocalteu method¹³. Extracts were diluted to a concentration of 1mg/mL. In parallel, a standard solution of gallic acid was prepared. The total content of polyphenolic compounds was expressed in mg equivalent of Galic acid (GA) per 1mL (mgGA/mL) of the extract. The calculation was carried out according to the data of the gauge graph of the standard sample of gallic acid. All measurements were carried out in three replicates.

Total Flavonoid Content (TFC):

Determination of the content of flavonoids in the extracts studied was carried out by spectrophotometric method^13,15 using 2% aluminum solution (3rd) chloride on the Hitachi U-2810 spectrophotometer. Concentrated extracts diluted with 70% ethanol to a concentration of 1 mg/mL. The total content of flavonoids expressed in mg equivalent of quercetin (Q) per 1mL (mgQ/mL) of the extract, the calculation was carried out according to the calibration schedule of the standard quercetin sample. All measurements were carried out in three replicates.

HPLC analysis:

The composition of ethanol-water extract (ethanol concentrations of 70%) was identified by highly efficient liquid chromatography on Agilent 1200 device with a diode array detector. Chromatography was carried out on a column filled with octadecylsilyl sorbent С18 with a particle size of 5 microns. Moving phase: acetonitrile - phosphate-acid pH buffer 2.8 with a gradient of acetonitrile from 10% to 50% rev.

A buffer solution was added to a tested solution, 2.0mL of herbal extract, to increase a volume of solution to 5.0 mL, it was filtered through a filter with 40 microns pore size.

Solutions of standard samples of corresponding biologically active compounds were used for identification.

Free radical scavenging activity by DPPH assays:

The radical scavenging activity of plant extracts was studied by using a reaction of 2.2 diphenyl 1-picrylhydrazyl (DPPH 2.2-diphenyl–1- picrylhydrazyl)^13,38. During the reaction of plant antioxidants with a stable radical, a shift in the color of the working solution of DPPH from purple to yellow has been observed. We measured the absorption coefficient on the spectrophotometer at a wavelength of 517nM. The experiment was conducted in three repetitions. Antioxidant activity was calculated by the formula: AOA(%) = 100*(A₀-A)/A₀, where: A₀- optical density of DPPH solution in ethanol with a concentration of 2 mg/50mL; A - optical density of the solution of the test extract. To determine the effective concentration of inhibition IC₅₀we prepared a series of dilutions of alcohol-water extract with 70% ethyl alcohol. We calculated anti-radical activity according to the following formula: ARA=1/IC₅₀. Total antioxidant activity was expressed in mg of Trolox (T) (Trolox equivalent (TE)) and calculated by formula AKE=APA Sample/APA Trolox. To determine the IC₅₀ of trolox, a series of dilutions of its solution with a concentration of 1mg/mL were prepared.

Antioxidant activity by FRAP method:

The method of determining antioxidant activity using FRAP reagent (ferric reducing antioxidant power) is characterized not only by the low-price and simplicity of preparation of reagents but also by the rapid course of analysis. It is based on the determination of recovered iron ions. During the interaction, the color of the substrate changes, the colorless solution becomes deep blue with a maximum absorption at a wavelength of 593 nm. Antioxidant activity is described by comparing the absorption changes of the analyzed sample and the sample solution. For the experimental stage the 1500μl of FRAP solution, 150μl of water and 50μl of the test extract were mixed. After mixing, the samples were aged for 4 minutes. For control, 50μl of 70% ethanol and 1650μl of FRAP solution were mixed. We measured the absorption coefficient on the spectrophotometer at a wavelength of 593nm. The experiment was conducted in three replicates. To compare the antioxidant activity of the extract under study, an aqueous solution of iron (ІІ) sulfate was used, which is known for its high antioxidant activity.

Study of antibacterial property:

Determination of antibacterial properties was carried out by the method of interaction of BAS extract with dye 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The method is based on the ability of the enzyme succinate dehydrogenase of the mitochondrial cell membrane to restore MTT yellow salt to purple formalized crystals, which accumulates as a result of this reaction in the cytoplasm of living cells. The amount of formazan formed is proportional to the available number of living cells. Determination of the relationship between living and dead cells is carried out using a spectrophotometer For the study of antibacterial activity, test strains of microorganisms E. coli dH5a, S. aureus ATCC 25923 and P. aeruginosa ATCC 9027 were used. All studies for the accuracy of the results were carried out in three repetitions. The suspension of bacteria was sowed in tubes with a peptone-glucose environment and cultivated overnight. After that, the culture of bacteria was incubated with the samples studied for 4 hours. After that, MTT dye was added in the amount of 10μl for each test tube, incubated for 1 hour at 37°C. After incubation, the samples were centrifuged at a speed of 3000rpm. for 10min. Then took away the supernatantand added 1mL of dimethyl sulfoxide to each test, incubated for 1 hour at 37°C. The optical density at a wavelength of 570nm and was determinet calculated the percentage of live bacteria³⁹.

Statistical analysis:

All experiments were conducted three times with three parallel repeats. The Analysis of Variance (ANOVA) was used as a statistical test for the comparison of the experimental groups. One-way ANOVA with Bonferroni post tests was applied in order to compare replicated means by rows using GraphPad Prism 6.0 software.

RESULTS:

Air parts of Scorzonera purpurea subsp. rosea was carried out during the flowering period in July in the Carpathian region: village Slavske, Lviv region and identified ³⁷. The raw materials were used to obtain water-ethanol extracts. Different concentrations of ethyl alcohol to obtain liquid dosage forms are widely used in the pharmaceutical industry. As an extragent, it can significantly influence bioactive compounds extraction, if compared to water. Its extraction ability depends on the concentration, i.e. with the concentration of alcohol getting higher the possibilities for hydrolytic processes in its environment are getting lower, which also entails enzymes inactivation. To obtain the extract with a maximum content of phenolic compounds, alcohol-water extracts of these herbs with an ethyl alcohol content of 40%, 70% and 96% alcohol were investigated ^15. The obtained liquid extracts from dark green to yellow-green colour, depending on the concentration of ethanol-water mixture, with a characteristic odour. The highest yield of extractive substances was obtained using ethanol-aqueous mixture with an ethanol content of 40%. Accordingly, the lowest yield - in the extract with a concentration of ethanol of 96% (Table 1).

It is known that for the extraction of the maximum amount of phenolic compounds often use 70% ethanol-aqueous mixtures ^{13, 15}. In the case of extracts, this pattern is also observed for E70 (Table 1). The least phenolic compounds are extracted with 96% ethanol E96. The calculation was performed according to the calibration curve of a standard sample of chlorogenic acid (equation of the curve y = 0.757x + 0.011, R² = 1), because it is the dominant compound among the phenols found in the test extract.

The content of flavonoids in the three extracts correlated with the content of phenolic compounds (Table 1). Accordingly, the extract of E70 had the highest content of flavonoids in terms of quercetin (equation of the calibration graph y = 0.757x + 0.011, R² = 0.998).

The antiradical activity extracts was determined by DPPH method (Table 1). For the E70 extract, the values of the effective concentration of IC₅₀ inhibition were determined by diluting the original extract to plot the percentage inhibition of the stable free radical DPPH from the concentration of biologically active substances in the extract (Table 1).

The concentration of active substances of E70 extract was determined in terms of iron (II) sulfate (equation of the calibration graph y = 0.741x + 0.106, R²= 0.995) by FRAP assay.

A large number of BAS was found in the extract, among which chlorogenic, caffeic, ferulic, rosmarinic acids, luteolin, quercetin, apigenin, apigenin-7-glucoside, rutin were identified (Figure 1). Chlorogenic acid, luteolin and apigenin - found in the extract in the largest quantities.

Chlorogenic acid is an important biologically active substance with antimicrobial, antiviral and high antioxidant activity. It has been suggested that flavonoids and chlorogenic acid are partly responsible for anti-inflammatory and antinociceptive activity of Scorzonera species^16,32. Quercetin, luteolin, apigenin - flavonoids that have high antioxidant potential.

In the composition of 70% ethanol extract of the herb were identified: organic acids (chlorogenic, coffee, ferulic), flavonoid glucosides (luteolin-7-glucoside, apigenin-7-glucoside, kaempferol-7-glucoside), flavonoids in - ruvonoidin, quercetin, apigenin (Figure 1). In the quantitative ratio of most chlorogenic and ferulic acids, and kaempferol-7-glucoside.

Taking into account the results of the study of the chemical composition of extracts with different ethyl alcohol content, the E70 extract was selected to study the antibacterial property (Figure 3).

From the above diagrams it is seen that the studied extract shows antibacterial activity against all analyzed test-strains of microorganisms. Antibacterial properties of the extract are not significant, but signals that in the list of extracted substances, there are antibacterial agents, which at the stage of the first screening allows to consider this source of biologically active substances as promising. Positive control in these experiments was ethanol 70%. Our next step will be to separate distinct flavonoids and test those biological activities.

Unfortunately, in the freely available literary sources there is only some bit information about the phytochemical composition, as well as the antioxidant and antibacterial activity of Scorzonera species. In this regard, it is impossible to assess the results we have obtained.

CONCLUSION:

The obtained results confirmed the potential of liquid extracts of herba Scorzonera purpurea subsp. rosea as a source of phenolic compounds among which chlorogenic, caffeic, ferulic, rosmarinic acids, luteolin, quercetin, apigenin, apigenin-7-glucoside, and rutin. Chlorogenic acid, luteolin and apigenin were the dominant substances in terms of quantity. The highest yield of extractive substances 24.7 mg/mL was obtained using ethanol-aqueous mixture with an ethanol content of 40%. Extract E70% was showed the maximum content of phenolic compounds 2.11 mgGA/mL including flavonoids 0.28 mgQ/mL and anti-radical activity of 86%. which was determined by the DPPH method, its effective concentration of inhibition IC₅₀ - 0.67 mg/mL as well as the strong ferric reducing potential (FRAP method) - 23.58 μmol/mL. Studies of the antibacterial effect of the extract E70 with respect to the test-strains of bacteria Staphylococcus aureus ATCC 25923, Escherichia coli dH5a, Pseudomonas aeruginosa ATCC 9027 showed the effectiveness of the extract compared to control.

Further research is needed to study in more detail the phytochemical composition and biological activity of Scorzonera purpurea subsp. rosea.

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Received on 21.11.2022 Modified on 19.02.2023

Research J. Pharm. and Tech 2023; 16(8):3945-3950.

DOI: 10.52711/0974-360X.2023.00649

Object	ЕS, mg/mL	TPC, mgGA/mL	TFC, mgQ/mL	ARA, %	ІС₅₀, mg/mL	FRAP, μmol/mL
E96	17.8±0.32	1.432±0.15	0.188±0.001	25±0.45	-	-
E70	19.4±0.38	2.111±0.17	0.276±0.005	86±0.98	0.67±0.008	23.58±0.38
E40	24.7±0.45	2.02±0.11	0.265±0.005	69±0.63	-	-