Application of Approach for Development of HPTLC Identification and Quantification Methods for Determination of Phenolic Compounds and Terpenoids of Several Thymus L. Species

The aim of this work was to improve the approaches for standardization of Thymus L. species by development of HPTLC identification of phenolic compounds (flavonoids and phenylpropanoids) and terpenoids (essential oils and triterpenoids) and quantitative determination of rosmarinic acid and flavonoids for six Thymus L. species of Ukraine flora, as well as determination of chromatographic profiles of Thymus L. species’ extracts obtained using different extraction methods. Materials and methods. Objects were: samples of dry herb of Th.serpyllum, Th.vulgaris, Th.Marschallianus, Th.Pallasianus, Th.calcareus, and Th.moldavicus of Ukraine origin collected in 2019-2021 years or acquired commercially on Ukraine market. Instruments: CAMAG HPTLC Herbal System, visionCats 2.5. The analytical grade reagents were used. Reference substances were purchased from Extrasynthese, Sigma Aldrich. Chromatography was performed on HPTLC plates Si 60 F254, Merck according to the developed methods. Results. A new approach for quality control of different Thymus L. species of Ukraine flora included the development of HPTLC methods for identification of main groups of bioactive substances of these species, such as flavonoids and phenylpropanoids, essential oils, triterpenoids; development of quantification method of rosmarinic acid and assay of total flavonoids, expressed as luteolin-7-O-glucoside. The characteristic HPTLC fingerprints of six Thymus L. species in three mobile phases of different polarities that cover a wide range of bioactive substances were established. The content of rosmarinic acid in different Thymus L. species samples was in the range of 0,11-0,72 %: Th.moldavicus – 0,11 %; Th.Marschallianus – 0,19-0,27 %; Th.serpyllum – 0,38 %; Th.vulgaris – 0,51 %; Th.calcareus – 0,56 %; T.Pallasianus – 0,72 %. The total flavonoids content, expressed as luteolin-7-O-glucoside, was in the range of 0,8-2,72 %: Th.moldavicus – 0,8 %; Th.serpyllum – 0,87 %; Th.vulgaris – 1,06 %; Th.Pallasianus – 1,28 %; Th.Marschallianus – 1,89 %; Th.calcareus – 2,72 %. Conclusions. The proposed scientific approach for quality evaluation of Thymus L. species using HPTLC allows to determine comprehensive information of chemical composition and content of active substances of multiple samples in parallel, in a cost and time-efficient manner


Introduction
The Thymus L. genus belongs to the Lamiaceae family and includes several hundred species distributed in Eurasia, North Africa, Greenland [1][2][3][4]. Despite this, only a few Thymus L. species -T.vulgaris L. and T.zygis L. or its mixture, and T.serpyllum L. [5,6] have pharmacopoeial standardization. At the same time, usually, the collectors of herbal raw material do not distinguish the species of Thymus, so its herbal raw material could be represented by different related species, as well as their combination in the various ratios [3,4].
The modern high-performance thin-layer chromatography (HPTLC) method is included in the pharmacopoeial analysis [7]. It gives the possibility to compare results of different species or extraction in parallel at the same HPTLC plate or to compare tracks from different plates using "virtual plate". Moreover, the results could be stored in a traditional form of an image (chromatogram) or as peak profiles from image/densitogram. Thus, HPTLC is a useful tool for conducting not only the qualitative, but also specific and non-specific quantitative determination [8,9]. That enables to use of the HPTLC method for comprehensive evaluation of the quality of closely related plant species.
The aim of this work was the improvement of approaches for standardization of Thymus L. species by the development of HPTLC identification of phenolic compounds (flavonoids and phenylpropanoids) and terpenoids (essential oils and triterpenoids) and quantitative determination of rosmarinic acid and flavonoids of six Thymus L. species of Ukraine flora -Th. serpyllum

Research planning (methodology)
The design of the experiment for quality control of Thymus L. species of Ukraine flora using HPTLC included several steps: the selection and collection of objects for investigations (pharmacopoeial and non-pharmacopoeial species); determination of groups of bioactive substances/markers; determination and evaluation of existed approaches for standardization of objects if any; optimization of existed standardization approaches using comprehensive HPTLC.
For standardization of Thymus L. species different approaches exist. The SPhU and European Pharmacopoeia monographs for Thymus, describing 2 species -T. vulgaris and T. zygis or its mixture, include TLC/HPTLC method for flavonoids and phenylpropanoids (markers used are rutin and rosmarinic acid) identification and essential oils content determination (steam distillation (SD) and gas chromatography (GC), while monograph for T.serpyllum quality assessment requires: the identification of essential oils by TLC (markers are thymol and carvacrol), its content by SD method, determination of extractable matters. Thus, the approaches for TLC/HPTLC identification used [5,6] require the determination of different groups of bioactive substances for related Thymus species, neither consider related non-pharmacopoeial Thymus species of Ukraine flora, nor allow to distinguish them between each other or to show their similarity. Besides, the control of temperature and relative humidity that are necessary for reproducible HPTLC analysis, SST, intensity markers are not required; evaluation of results is performed according to the reference table, which is often difficult for interpretation. The approach for quantification of Thymus and T.serpyllum [5,6] includes determination of the content of essential oils by SD (2.8.12), which is cumbersome and unspecific assay method; besides, a monograph on Thymus requires determination of essential oils (sum of thymol and carvacrol) by GC method, that despite the specificity requires time-consuming sample preparation; a monograph on T.serpyllum requires determination of extractable matters, extracted with 30 % alcohol (non-specific assay). Another pharmacopoeial approach for standardization of T.serpyllum [10] includes TLC identification of flavonoids (specific marker luteolin-7-O-glycoside is used) and spectrophotometry assay of TFC, after complexation reaction with aluminium chloride, expressed as luteolin-7-O-glycoside; determination of extractable matters, extracted with 30 % alcohol and water (non-specific assay). Besides, for T.serpyllum different spectrophotometry assay methods of TFC, expressed as rutin [11], and total content of hydroxycinnamic acids, expressed as rosmarinic acid [3] were published.
Comparison of HPTLC fingerprints of methanolic and 70 % and 40 % alcoholic extracts showed their similarity, despite differences in chlorophylls appearance close to the front position ( Fig. 1, b). This is important for the transfer of the method for the finished herbal drugs of Thymus L. species. According to the results of identification obtained in different detection modes (Fig. 2), five Thymus L. species had a pair of orange (red arrows) and yellowgreen (green arrows) zones due to flavonoids in the middle part of the chromatogram, while the analyzed sample of Th.vulgaris had a slight difference in flavonoids fingerprints and showed a pair of orange zones in the middle part of the chromatogram (red and yellow arrows) and characteristic diffuse yellow zone in the upper third (orange arrow); all Thymus species had rosmarinic acid in the upper part of chromatogram (white arrows). At 366 nm at the middle part of the chromatogram, Th.Pallasianus and Th.calcareus showed more light-blue zones due to phenylpropanoids. The identification of essential oils was performed in mobile phase: dichloromethane, where orange zone due to thymol was in the middle part of chromatogram with R F ≈0,44 (Fig. 3, a). Cineol and thymol could be proposed for SST; thymol can be used as a reference solution and intensity marker (Fig. 3). Comparison of chromatographic fingerprints of samples with different extraction techniques and solvents (dichlormethane (DCM), methanolic, 70 % alcoholic and 40 % alcoholic extracts obtained by automated shaking and essential oils obtained by SD) showed differences in their content and composition, with more intense essential oils' zones in samples of essential oil obtained by SD (Fig. 3). Essential oils of Thymus L. species obtained by SD showed the presence of zone due to thymol (green arrows) in Th.vulgaris, Th.serpyllum, Th.Marschallianus and Th.moldavicus; the Th.Pallasianus and Th.calcareus samples did not show the zone due to thymol. Thymol was found to be the main essential oil in methanolic and alcoholic extracts of T.vulgaris, T.serpyllum, T.Marschallianus, while T.Pallasianus and Th.calcareus, which are xerophyte plants, did not show the zone due to thymol either or showed its low content (Th.moldavicus). Th.calcareus essential oil and extracts showed a violet zone of different intensity that was positioned slightly below the zone due to thymol (red arrows). The violet zone of different intensity (R F ≈0,22, violet arrows) was present in essential oils of all Thymus L. species besides Th.serpyllum; and was of light intensity in their methanolic extracts. The violet zone with R F ≈0,22 might be probably due to caryophyllene oxide, but this assumption requires confirmation with reference substance and its UV/MS comparison. The brownish zone with R F ≈0,16 (probably, due to borneol) was present in all Thymus L. species' samples obtained by SD and was not detected in their methanolic/alcoholic extracts.
The developed HPTLC identification of triterpenoids was performed in the mobile phase: tolueneethylacetate-formic acid (7:3:0,1). In this mobile phase, it was possible to detect both triterpenoid oleanolic acid and essential oil thymol. For SST ursolic acid and essential oil borneol can be used. As a reference solution and intensity markers oleanolic acid and thymol can be proposed.
As we can see in Fig. 4, oleanolic acid was present in methanolic and 70 % alcoholic extracts of all Thymus L. species (violet arrows), and absent in 40 % alcoholic extracts.   For quantitative determination of Thymus, the HPTLC methods for specific determination of phenylpropanoidrosmarinic acid, as well as TFC, expressed as luteolin-7-O-glycoside, were proposed.
Rosmarinic acid was present in all investigated Thymus L. species and according to the literature is responsible for antioxidant, anti-inflammatory, antiviral, anti-allergy activities [15], thus, it can be considered as an active marker. With the same mobile phase used for ID 1, the quantification of rosmarinic acid was performed at 330 nm by peak profile from scanning densitometry (PPSD). The detection mode at 330 nm was chosen as it corresponded to the maximum of rosmarinic acid. The specificity of the quantification method was proved by the comparison of R F of zones due to rosmarinic acid reference standard and Thymus L. species samples, as well as by the comparison of their UV spectrum (Fig. 5). The calculations were done using CAMAG visionCATs software. The validation parameters of the method such as linearity (calibration function: y=0,0077x+0,0015, correlation coefficient (R)=0,9998, LOD=0,0218 (µg/band), LOQ=0,0662 (µg/band), precision (n=6, RSD=0,1 %), repeatability (n=3, RSD=0,9 %), accuracy (95,9 %) were determined. According to the results the content of rosmarinic acid in different Thymus L. species samples, calculated on the dry raw material, was in the range of 0,11-0,72 %: Th.moldavicus -0,11 %; Th.Marschallianus -0,19-0,27 %; Th.serpyllum -0,38 %; Th.vulgaris -0,51 %; Th.calcareus -0,56 %; T.Pallasianus -0,72 %. Total flavonoids are responsible for antioxidant, anti-inflammatory, antimicrobial, hepatoprotective, antiviral activities of the plant [11]. The assay method for TFC, expressed as luteolin-7-O-glycoside, calculated on a dry basis, was developed. The separation of bioactive substances was performed in the mobile phase: ethyl acetate, formic acid anhydrous, water (8:1:1), then the plate was dipped in aluminum chloride reagent, and detected after 2 hours at 400 nm by scanning densitometry, subtracting peaks at 400 nm before derivatization (Fig.  6). The areas of all peaks of the flavonoids profile of the sample were measured and added. The expression was done using the standard substance luteolin-7-O-glycoside that was applied onto the same plate as sample solutions. The comparison of TFC of methanolic extracts of different Thymus L. species was done by the method (Fig. 6,  7). According to the results, the TFC was in the range of 0, 8 The comparison of the quantity of rosmarinic acid and total flavonoids of different Thymus L. species is shown in Fig. 7.

Discussion
The proposed scientific approach for quality evaluation of Thymus L. species using HPTLC allows to determining the comprehensive information of chemical composition and content of active substances of multiple samples of various species, obtained by a different technology, in parallel, in a time and cost-efficient manner. The findings obtained using developed HPTLC methods, prove the presence of main groups of substances: flavonoids, phenylpropanoids, essential oils, and triterpenoids in all Thymus L. species [3]; show the influence of solvent and extraction technique for its composition; provide similarity and differences in HPTLC fingerprints of different Thymus L. species, that growth on different soils ( Fig. 1-7). For example, the comparison of essential oils fingerprints of Thymus L. species showed the presence of thymol as the main component in Th. serpyllum, Th. vulgaris and Th. Marshallianus and different essential oils composition with no or low content thymol found in three investigated Thymus L. species that grow in dry and poor soils Th.Pallasianus (sands), Th.calcareus (chalk mountains), and Th.moldavicus (shelly soil).
The results prove the replacing of TLC on essential oils by HPTLC using flavonoids profile by European pharmacopoeia for distinguishing of both pharmacopoeial species -Th.serpyllumand Th.vulgaris that have similar essential oils composition (thymol chemotype) (Fig. 3) [16]; confirm the possibility of standardization of Thymus L. species by flavonoids and hydroxycinnamic acids (rosmarinic acid) content (Fig. 1, 2) [3]; and show the prospect of inclusion of Th. Marshallianus, which is the most widespread Ukraine species, into pharmacopoeial analysis, based on similarity of HPTLC fingerprints of main bioactive substances with Th.serpyllum. Study limitations. The limitations of the approach are a necessity of usage of sophisticated equipment, cost of consumables.
Prospects for further research. The collection and evaluation of new Thymus L. species samples from different years and origins using proposed comprehensive HPTLC, and verification of results in different labs are of major importance.

Conclusions
The scientific approach for quality evaluation of six Thymus L. species using comprehensive HPTLC, with HPTLC methods that were respectfully developed, allowing determining: flavonoids, phenylpropanoids, essential oils and triterpenoids, and content of active substances: rosmarinic acid and flavonoids, of multiple samples in parallel, in a cost and time-efficient manner was proposed.
The similarity and differences in HPTLC fingerprints of different Thymus L. species that grow on different soils were shown. The findings are important for the selection of the place of growth of Thymus L. species during of implementation of GACP in Ukraine.
The prospect of inclusion of Th. Marshallianus, which is a mostly widespread Thymus L. species in Ukraine, into pharmacopoeial analysis, based on similarity of HPTLC fingerprints and content of its main bioactive groups of substances with Th. serpyllum was highlighted.