Callus cultures of Thymus vulgaris and Trifolium pratense as a source of geroprotectors

. Introduction. Geroprotectors are biologically active substances that inhibit the aging process. Many plant species are natural geroprotectors. For instance, Thymus vulgaris and Trifolium pratense are callus cultures with strong geroprotective properties. Study objects and methods. The present research featured T. vulgaris and T. pratense grown in vitro on various nutrient media. Their extracts were obtained by aqueous-alcoholic extraction using the following parameters: water-ethanol solvent S e = 30, 50, and 70 %; temperature Т e = 30, 50, and 70°C; time τ e = 2, 4, and 6 h. The quantitative and qualitative analysis was based on high-performance liquid mass spectrometry, gas mass spectrometry, and thin-layer chromatography. Results and discussion. The optimal extraction parameters for T. vulgaris were τ e = 4 h, Т e = 50°C , S e = 70 %, for T. pratense – τ e = 6 h, Т e = 70°C , S e = 70 %. The chromatography detected flavonoids, phenylpropanoids, simple phenols, higher fatty acids, mono- and sesquiterpenes, and aliphatic hydrocarbons. T. vulgaris appeared to have the highest content of thymol (23.580 ± 1.170 mg/mL); its thymol, apigenin, gallic, chlorogenic, and caffeic components demonstrated geroprotective properties. The extract of T. pratense possessed the highest rutin content (10.05 ± 0.35 mg/mL), and it owed its geroprotective characteristics to rutin, chlorogenic and p -coumaric acids. Conclusion. The callus cultures of T. vulgaris and T. pratense proved to be promising sources of geroprotective biologically active substances. https://doi.org/10.21603/2074-9414-2021-2-423-432 TLC T. pratense callus


Introduction
Environment directly affects human health [1]. Urban environment is a reliable source of various noxious factors [2,3]. One's health status depends on how well the body has adapted to the environment, while one's functional capabilities are based on the physiological profile, age, and character [4]. Any disease comes from this or that violation of adaptive mechanisms, i.e. when the body fails to resist unfavorable environmental conditions, e.g. air pollution, water contamination, vibration, radiation, noise, electromagnetic, etc. [5][6][7].
Biologically active compounds of plant origin can be divided into several chemical groups. Glycosides include cardiac glycosides, cyanogenic glycosides, glucosinolates, saponins, and anthraquinone glycosides. Phenolic compounds involve phenolic and hydroxycinnamic acids, stilbenes, flavonoids, and anthocyanins. Tannins are divided into condensed tannins, e.g. large polymers of flavonoids, and hydrolysable tannins, which consist of a monosaccharide nucleus with several catechin derivatives attached. The list of biologically active compounds also includes mono-, di-and sequiterpenoids, phenylpropanoids, lignans, resins, alkaloids, furocoumarins and naphthodianthrons, proteins, and peptides [15,16].
Extraction is the main technological process that produces biologically active compounds from plant raw materials [18]. Extraction process includes three main stages: (1) interaction of plant material with the extractant, (2) destruction of plant cell components, and (3) transfer of solutes to the extractant [19,20].
Extractants have to be able to penetrate cell walls and selectively dissolve biologically active compounds inside the cell. Therefore, a good extractant has to meet certain requirements, e.g. maximum solubility of active substances; selectivity; high penetrating power;
Plants are known to synthesize and accumulate secondary metabolites of various phytochemical groups [24,25]. Callus, suspension, and root cultures are induced for analytical and quantitative comparative analyses of the secondary synthesis of metabolites between plant material and callus, suspension, and root extracts [22,23].
Among all the medicinal wild plants of the Siberian Federal District, Thymus vulgaris and Trifolium pratense contain the most impressive amount of geroprotective biologically active substances, including such antioxidants as flavonoids, coumarins, etc. [26].
T. vulgaris has antiseptic, antimicrobial, and antioxidant properties [27]. Its extracts are used to treat dyspepsia and gastrointestinal disorders, cough, whooping cough, bronchitis, laryngitis, and tonsillitis, since it contains benzyl alcohol, rutin, apigenin, thymol, gallic acid, luteolin, etc. [28,29]. T. vulgaris owes its high antimicrobial and antifungal activity to such phenolic compounds as thymol and carvacrol [26]. The yield of essential oil ranges from 0.3 to 6.3% [30]. The content of thymol in the essential oil can reach 60%, which is significantly higher than the content of carvacrol (up to 6%) [31]. The antiseptic effect of thymol is 30 times higher than that of phenol, while its toxic effect is 4 times lower [32]. Phenolic compounds of T. vulgaris can form oxygen free radicals [33].
T. pratense is used as animal feed. This melliferous plant is very popular in agriculture [34]. As a result, its biologically active substances have become focus of constant scientific attention. T. pratense contains some flavonoids, isoflavonoids, and phenolic compounds, e.g. quercetin, rutin, genistein, formononetin, etc. [32]. This plant is used as an antioxidant, antimicrobial and diuretic medicine, as well as a remedy against coronary and nephric edema [34][35][36].
The research objective was to perform a quantitative and qualitative analysis of callus extracts of T. vulgaris and T. pratense to evaluate their geroprotective prospects.

Study objects and methods
The research featured callus cultures obtained from seeds of Thymus vulgaris and Trifolium pratense grown in vitro. The seeds were washed in soapy water for 30 min and then washed in bidistilled water three times for 20 min. After that, they were treated with 70% ethanol for 1 min and washed three times in bidistilled water for 20 min. Finally, the seeds were washed with 5% sodium hypochlorite solution for 50 min and washed three times in bidistilled water for 20 min [37]. After sterilization, the seeds were planted on agar media. Table 1 shows the composition of the nutrient media.
The first seedlings of T. pratense appeared on week 1-2, and those of T. vulgaris -on week 4-5. The experiment featured sterile seedlings that were 2-5 weeks old. The explants were cut into pieces and planted in agar media. The first calli appeared during 14 days of cultivation. Further callus formation involved Murashige-Skoog (MS), Gamborg (B5), and Schenck Hildebrandt (SH) mineral bases with casein hydrolyzate (0.5 g/L), inositol (0.1 g/L), 3% sucrose or glucose, and 2% agar. The media varied in the composition of growth regulators, which included indoleacetic acid, 2,4-dichlorophenoxyacetic acid, kinetin, and 6-benzylaminopurine ( Table 2). The explants were incubated for 25 days.
The primary callus was separated from the remains of the explants and transferred to a fresh nutrient medium to grow for 4-5 weeks.
The callus cultures were extracted by the standard method of liquid aqueous-alcoholic extraction using ethanol (State Standard 5962-2013. Rectified ethyl The callus cultures of T. vulgaris and T. pratense were dried and ground in an LZM-1M rotary mill (Olis, Russia). The extraction samples weighed 3.000 ± 0.001 g.
The volume of ethanol was 260 mL. The extraction was carried out in a water bath (Elmasonic S60H, Germany) with an ascending refrigerator at a given temperature, time, and ethanol concentration. The obtained extracts T. vulgaris and T. pratense were stored at room temperature in the dark.
The antioxidant activity of the extracts was determined to define the total biologically active substances. It was expressed as the content of the sum of biologically active substances of a reducing nature in terms of quercetin   [43]. The experiment involved a liquid chromatograph (Shimadzu LC-20 Prominence, Japan) with a Shimadzu SPD-20-MA diode array detector and a RID-10A refractometric detector, a chromatographic column Kromasil 5 μm C18, 250×4.6 mm, a Guard Column Security Guard Gartridge (C18) Phenomenex (USA) with injection volume 20 μL. The column temperature was 30°C; the elution mode was isocratic; the mobile phase consisted of AcCN:isopropyl alcohol:H 2 O-H 3 PO 4 (20:5:75, pH 3.5).
Gas chromatography with mass spectrometry (GC-MS) and thin layer chromatography (TLC) were carried out at the same time as HPLC [44].
The analysis of biologically active substances involved Sorbfil PTS-AF-A TLC plates. The obtained extract was applied to the start line, dried, and placed in a chromatographic chamber filled with a mix of n-butanol, acetic acid, and water at a ratio of 60:15:25. After 10 min, a 25% solution of phosphoric-tungstic acid was added at 95°C. The densitometric analysis of the plate was performed using a Handycam HDR-CX405 densitometer with a Sony photofixation system (OOO IMID, Russia).
The T. vulgaris callus extracts underwent a GC-MS using a 30 m column with an inner diameter of 0.25 mm and helium as a carrier gas. The main parameters for GC-MS were as follows: carrier gas flow rate -1.4 mL/min; interface temperature -280°C; injector temperature -240°C; column temperature -100-270°C; volume of the injected sample -3 μL. The sample was introduced without dividing the carrier gas flow.

Results and discussion
Nutrient medium 2 proved optimal for the callusogenesis of Thymus vulgaris, which included the following growth hormones: kinetin -2 mg, 6-benzylaminopurine -0.5 mg, peoxyethanolic acid -3 mg. When the callus culture of Trifolium pratense was cultivated on nutrient medium 2, which contained 1 mg of kinetin and 2,4-dichlorophenoxyacetic acid, the callus growth was slow. Nutrient medium 3 proved optimal for T. pratense callus culture: it contained the following growth hormones: kinetin -2 mg, 6-benzylaminopurine -0.1 mg, indoleacetic acid -2 mg, and 2.4-dichlorophenoxyacetic acid -2 mg. Tables 3 and 4 show the total content of biologically active substances in terms of quercetin in 1 mL of the extract under different extraction conditions.
After establishing the optimal extraction parameters, the next step was to analyze the qualitative and quantitative composition of biologically active substances in aqueousalcoholic extracts.   Figure 1 shows the results of HPLC analysis for T. vulgaris callus extract, while Table 5 demonstrates the results of the qualitative and quantitative analysis of biologically active substances.
The HPLC analysis (Fig. 1, Table 5) of T. vulgaris callus extracts revealed that the samples contained flavonoids, phenylpropanoids, and simple phenols. Figure 2 illustrates the GC-MS analysis of T. vulgaris callus extract and displays other individual biologically active substances.
The extracts of T. pratense callus culture underwent HPLC and TLC chromatography. Figure 3 demonstrates the HPLC chromatogram.
The content of biologically active substances in T. pratense samples changed depending on the extraction method, which was the main peculiarity of this extract. After ultrasonic extraction, the amount of flavanoids was 2.13%, isoflavonoids -4.42%; after heat maceration, the yield of flavonoids was 1.64%, isoflavonoids -3.24% [40,42]. Figure 4 shows the densitogram of the TLC analysis of the Trifolium pratense callus extract.  The TLC analysis showed that the T. pratense callus extracts contained such biologically active substances as quercetin-3-O-rutinoside (rutin), chlorogenic acid, and isoquercitrin.

Conclusion
The present research featured the quantitative and qualitative content of biologically active substances in aqueous-alcoholic extracts of callus cultures of Thymus vulgaris and Trifolium pratense. The optimal extraction parameters for T. vulgaris callus culture were as follows: τ e -4 h, Т e -50°C, S e -70%; for T. pratense: τ e -6 h, Т e -70°C, S e -70%.
As for the T. pratense callus extracts, the HPLC analysis revealed rutin, chlorogenic acid, p-coumaroyl-3-quinic acid, p-coumaric acid, isoquercetrin, biochanin A, ononin, daidzein, genistein, and melilotic acid. The selected extraction parameters produced a high yield of rutin (10.05 ± 0.35 mg/mL). According to the TLC chromatography, the T. pratense callus extracts contained rutin, chlorogenic acid, and isoquercitrin. Therefore, the callus cultures of T. vulgaris and T. pratense proved to be sources of geroprotective biologically active substances.

Contribution
The authors are equally responsible for the information published in this article and any possible cases of plagiarism.

Conflict of interest
The authors declare no conflict of interests regarding the publication of this article.