Kateryna O Khokhlova

Background/Objectives: Compounded vaginal creams are widely used for conditions such as hormone replacement therapy, vaginal dryness, low libido, vaginal infections, etc. Recent research highlights the potential of using anhydrous bases to extend shelf life, particularly when combined with self-emulsifying and mucoadhesive properties that improve mucosal retention and enhance drug bioavailability. This study provides in vitro and ex vivo evaluation of an anhydrous vaginal base. Methods: Key quality indicators such as irritation potential, leakage potential, pH compatibility, mucoadhesion, and self-emulsification were assessed using the chorioallantoic membrane Hen’s Egg Test, MTT assay, texture analysis, and fluorescence microscopy. Results: The anhydrous vaginal base demonstrated high cell viability (>78%) and non-irritant potential (IS = 2.5) in in vitro assays. It maintained physiological vaginal pH (4.56 ± 0.05), showed strong mucoadhesive properties comparable to commercial products, and exhibited minimal leakage. Ex vivo studies confirmed its prolonged retention on vaginal tissues. The anhydrous vaginal base formed stable emulsions upon contact with vaginal fluid simulant, effectively distributing both lipophilic and hydrophilic compounds. Conclusions: Compared to water-containing bases, an anhydrous vaginal base shows advantages: longer retention time and lower leakage; adaptability to varying vaginal fluid levels; and efficient dispersion of both hydrophilic and lipophilic active pharmaceutical ingredients. These features support its potential use in compounded vaginal products, minimizing stability risks and enhancing patient compliance and therapeutic outcomes.

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

The high-performance thin-layer chromatography (HPTLC) is widely used for quality control of herbal raw materials (HRM). The purpose of this work were to define the problematic issues of quality control of herbal drugs in Ukraine and to develop a systematic scientific and methodological approach for the standardization of HRM and herbal drugs using the modern analytical technique of HPTLC: to optimize the existing and develop new methods of identification and determination of adulterations, and to develop alternative methods of quantitative determination, as well as studying of the stability. As a result of a critical evaluation of existing approaches for the standardization of HRM and herbal drugs of Ukraine’s flora, numerous problematic issues were identified, including the existence of a limited number of national monographs / national parts of monographs on the HRM of the Ukraine’s flora, which were not previously standardized by other pharmacopeias; non-consideration of Ukraine’s flora species (non-pharmacopoeial) in monographs on closely related species that introduced in the State Pharmacopoeia of Ukraine; lack of specificity of combinations of physical and chemical methods used for quality control; lack of specificity and reproducibility, the labor intensity of methods; necessity of usage of toxic solvents and precursors; use of different groups of markers and methods for the HRM and its finished product or HRM and its closely related species, etc. The proposed systematic scientific and methodological approach includes: a preliminary collection of information about the object of study, markers/groups of bioactive substances, approaches to its standardization; theoretical evaluation of existing techniques of quality control in monographs on HRM and experimental approbation using samples of domestic plants, determination of the need for optimization of existed methods by the parallel development of specific, reproducible and documented methods for identification, adulterations’ detection, quantification, and bio-detection (if necessary) by HPTLC method. The approach is illustrated with examples. The proposed approach can be used for comprehensive HPTLC analysis of domestic HRM and herbal drugs for research, prevention of adulteration, standardization of initial HRM and its finished product, pharmaceutical development, and stability studies.

Метою роботи була розробка методики визначення кількісного вмісту суми флавоноїдів настойки нагідок методом високоефективної тонкошарової хроматографії (ВЕТШХ), що характеризується специфічністю, експресністю і економічністю. Матеріали і методи. Дослідження було проведене методом ВЕТШХ в автоматичній системі виробництва CAMAG (Швейцарія) на базі навчально-наукової тренінгової лабораторії хіміко-технологічних досліджень НФаУ (Харків, Україна). Результати обробляли за допомогою програмного забезпечення visionCats, CAMAG. Як стандартний зразок (СЗ) використовували рутин, як об’єкти дослідження використовували три зразки настойки нагідок вітчизняних виробників. Результати й обговорення. При розробці методики були обрані оптимальні умови визначення, що включали: визначення характерних профілів піків настойки нагідок в обраній рухомій фазі за різних довжин хвиль і вибір СЗ для перерахунку, оптимізацію етапів підготовки проби випробуваного і СЗ, етапів дериватизації і детекції; визначення способу розрахунку і розробку формули розрахунку. Так, визначення вмісту суми флавоноїдів проводилось після розділення речовин в обраній рухомій фазі і дериватизації реагентом алюмінію хлориду за довжини хвилі 408 нм. Для розрахунку був обраний метод калібрувального графіка за площею піків. Згідно із розробленою ВЕТШХ-методикою, вміст суми флавоноїдів у перерахунку на рутин у зразках настойки нагідок склав: для зразка 1 – 0,26±0,02%; для зразка 2 – 0,14±0,01%; для зразка 3 – 0,26±0,02%. Висновки. Розроблена методика може застосовуватися як специфічний, зручний і економічно вигідний експрес-метод визначення флавоноїдів настойки нагідок.

The tincture is a traditional liquid dosage form for the pharmaceutical market of Ukraine. Today, the range of the tinctures of Ukrainian manufacturers has 21 different items, while only six monographs on different tinctures are included in the State Pharmacopoeia of Ukraine. For standardization of tinctures, different combinations of specific and non-specific physical and chemical methods are used. The high-performance thin-layer chromatography (HPTLC) is a modern and specific chromatographic method, that can be used for the identification of polyphenolic substances responsible for a wide range of pharmacological activities of the tinctures. The purpose of the study was to identify and compare chromatographic fingerprints (images of chromatograms and peak profiles from images) of flavonoids and phenylpropanoids of 10 tinctures of Ukrainian production in the harmonized mobile phases, to select for each tincture the specific marker zones, to determine the suitability of the specified chromatographic conditions for standardization of the tincture and the possibility to perform in these conditions the quality control for several different tinctures in-parallel. The objects of the investigation were Hawthorn tincture, Peppermint tincture, Sage tincture, Motherwort tincture, Calendula tincture, Sophora tincture, Echinacea purpurea tincture, Valerian tincture, Peony tincture, Eucalyptus tincture. The research was carried out using the HPTLC method in the HPTLC automatic herbal system (CAMAG, Switzerland) on the base of two laboratories. For each investigated tincture, the determination and comparison of HPTLC chromatographic fingerprints (images and peak profiles of images) of flavonoids and phenylpropanoids obtained in different harmonized mobile phases were performed. In the harmonized chromatographic conditions the difference in separation of the bioactive substances was determined; the characteristic marker substances of the tinctures were identified; the suitability of the mobile phase for individual analysis of the tincture or in-parallel analysis of different tinctures on the same plate were determined; the possibility to replace one mobile phase by another, if needed, was determined. Proposed HPTLC methods were developed using modern procedure and approach. These allow to reduce of the scope and time of analysis, optimize the economical and labor costs. The findings are of special importance for the pharmaceutical manufacturers and quality control laboratories that perform analysis on a large scale.

The TLC essential oils profiles were being used conventionally for identification of herbal raw materials (HRM) of Lamiaceae family. Despite this, in recent years the European Pharmacopoeia to call in question the appropriateness of TLC on essential oils determination of Thyme and Wild Thyme, as well as Peppermint and Peppermint dry extract; these methods were replaced by HPTLC using flavonoid profile. This work aimed to establish the possibility of conducting HPTLC identification of methanolic extracts of 13 members of the Lamiaceae family in harmonized chromatographic conditions using chromatographic profiles on essential oils. The objects were methanolic extracts of HRM of Ukraine flora – Salvia officinalis, Salvia aethiopis, Thymus serpyllum, Thymus vulgaris, Origanum vulgare, Rosmarinus officinalis, Ocimum tenuiflorum (foreign origin), Mentha × piperita, Melissa officinalis, Orthosiphon stamineus, Agastache foeniculum, Leonurus cardiaca, Hyssopus officinalis, Marrubium vulgare, prepared from HRM that were purchased in Ukraine. The study wоas done by standardized HPTLC procedure using automatic HPTLC herbal system of CAMAG, Switzerland, at the base of CAMAG laboratory, Muttenz, Switzerland, and laboratory of ESTCCTI ESIPP, NUPh, Ukraine. Chromatography conditions: stationary phase – HPTLC plates Si 60 F254; mobile phase – toluene-ethylacetate (95:5); reference standards – borneol, bornyl acetate, cineole; derivatization – anisaldehyde reagent; detection: WRT, 254, 366 nm – before derivatization; WRT, 366 nm – after derivatization; sample preparation – methanolic extracts (100 mg/ml), extracted by shaking technique. In unified chromatographic conditions the HPTLC chromatographic fingerprints of essential oils of 13 Lamiaceae family species were established and evaluated. The most specific, intensive and reproducible HPTLC fingerprints were for Sage and Rosemary extracts. Moreover, fingerprints of Sage, Thyme, Rosemary, Origanum, Ocimum and Peppermint shown specific zones useful for differentiation of different genera of Lamiaceae family. For setting relative acceptance criteria for each of the species the validation for specific zones that were detected should be done.

The plants from the Lamiaceae family are traditional remedies of folk and scientific medicine. They are present at the domestic pharmaceutical market as herbal raw material (HRM); dietary supplements; aromatherapy; as well as being included in the composition of drugs for oral use etc. According to the modern direction of international harmonization of conditions for standardizations of herbal drugs, the topical issue is the development of unified methods of HPTLC identification for HRM, including the Lamiaceae family. The aim of the work was the selection of the unified HPTLC chromatographic conditions for identification of chromatographic profiles of flavonoids and hydroxycinnamic acids of HRM of some Lamiaceae species of Ukraine’s flora, presented at domestic pharmaceutical market, such as Sage, Wild Thyme, Thyme, Oregano, Rosemary, Java tea, Peppermint, Lavender, Anise hyssop, Melissa, Hyssop, White horehound, Motherwort; and comparison of their chromatographic fingerprints. As objects of investigation, the HRM of Lamiaceae family presented at the Ukrainian pharmaceutical market were used. The investigation was conducted by HPTLC procedure using an automatic herbal system of CAMAG, Switzerland at the base of CAMAG laboratory, Muttenz, Switzerland and laboratory of ESTCCTI ESIPP, NUPh, Kharkiv, Ukraine. The harmonized chromatographic conditions for determination of flavonoids and hydroxycinnamic acids for HRM of Lamiaceae species were chosen, these were: stationary phase – HPTLC Si 60 F254; mobile phase – ethyl acetate, formic acid anhydrous, water (8:1:1); sample preparation: solvent – methanol; method of extraction – shaking for 10 min; relative humidity: 33%, temperature 22 ± 5 °C; derivatization with Natural product and PEG reagents; detection – 254 nm, 366 nm, white light before derivatization: 366 nm and white light after derivatization. The reproducibility of results in these chromatographic conditions was ∆RF ≤ 0,05. It was established, that for all investigated species of the family the zone corresponding with the position and colour to rosmarinic acid was detected, thus it can be used as standard substance for «Identification», «Limit-test» and for chromatographic system suitability test. For investigated samples of domestic HRM, the specific zones that can be used for setting acceptance criteria of the «Identification» and «Foreign matters» tests were conducted. After validation investigation involving greater numbers of samples of each species from different growing regions of Ukraine, the obtained HPTLC chromatographic fingerprints could be recommended as reference images for quality control of home-grown HRM of Lamiaceae family.

It was known that hawthorn - Crataegus L. is a polymorphic genus. Two hawthorn species and their hybrids are included in the European Pharmacopoeia, twelve – in Ukrainian pharmacopoeia. Determination of chromatographic profiles of hawthorn fruits species native to Ukraine and other countries that are non-pharmacopoeial, but have sufficient plant raw material base, is essential for quality control of drugs. Aim. To analyze and compare the chromatographic profiles of fruits of 23 Crataegus L. species on phenolic compounds, evaluated by means of high-performance thin-layer chromatography procedure (HPTLC), and determine the specific features of chromatographic fingerprints. Materials and Methods. A total of 39 samples of fruits of 23 hawthorn species that are native to Europe, Asia and North America, such as Crataegus monogyna , C. laevigata/C. oxyacantha, C. leiomonogyna , C. curvisepala , C. pseudokyrtostyla , C. fallacina , C. subrotunda , C. ambigua , C. pentagyna , C. sanguinea , C. chlorosarca , C. almaatensis , C.pseudoheterophylla subsp. turkestanica , C. pinnatifida , C. pentagyna subsp. pseudomelanocarpa , C. punctata, C. pringlei, C. festiva , C. douglasii, C. holmesiana , C. submollis , C. flabellata , C. canadensis were investigated. The analysis has been done following the TLC method from European Pharmacopeia modified into HPTLC, using automated HPTLC herbal system (CAMAG, Switzerland). The results have shown that chromatographic profiles of phenolic constituents of nine Crataegus L. species of Europe, both pharmacopoeial and non-pharmacopoeial, were quite similar, despite the significant morphological distinctions. The chromatographical profiles of three species of Asia were similar to the pharmacopoeial species; three other species looked different and had specific marker zones. In addition, eight Crataegus L. species of North America had specific markers helping for discriminative analysis from pharmacopoeial species. Conclusion. The findings could help to identify the possible adulterations and prevent the falsification of finished products. The results will be taken into consideration during revision of the Ukrainian national pharmacopoeial monograph for hawthorn fruits.

Rhodiola rosea and Rhodiola quadrifida are widely distributed and sold in Eastern Europe. The purpose of this paper was to identify R.rosea, R.quadrifida and Rhodiola rosea liquid extract (RRLE) in the Ukrainian market and bring out adulteration cases using chromatographic characterisation by HPTLC. The multiple samples of R.rosea, R.quadrifida and RRLE were compared; the optimal chromatographic conditions for identification of R.rosea and RRLE based on the presence of rosavins and salidroside as well as for identification of R.quadrifida based on the presence of salidroside were proposed; the specific HPTLC fingerprints were obtained; the acceptance criteria for each product were set. The adulteration cases for R.rosea and RRLE samples were established. The dependence on handling R.rosea and presence of rosavins was determined. It was assumed that low-quality raw materials or inefficient technology process were used for RRLE. The consistency of HPTLC fingerprints for R.quadrifida samples was established.

The Hawthorn (Crataegus L.) is the most commonly used herb for treating cardiovascular diseases. The purpose of this paper was the analysis and comparison of HPTLC chromatographic profiles of flavonoids and triterpenes of leaves and flowers of 15 Crataegus L. species occurring throughout Eurasia and North America, and determination of specific distinguishing markers of its fingerprints. The optimal chromatographical conditions for evaluation of flavonoids and triterpenes of 15 Hawthorn species were proposed, the specific HPTLC fingerprints were obtained. The flavonoids fingerprints were qualitatively much the same for the pharmacopoeial species C.laevigata/oxyacantha and C.monogyna and other related non-pharmacopoeial European and Asian Hawthorn species including C.curvisepala, C.pseudokyrtostyla, C.fallacina, C.leiomonogyna, C.ambigua, C.sanguinea, C.almaatensis, C.turkestanica. The specific distinctive features for Hawthorn species native to North America, including C.punctata var aurea, C.pringlei, C.festiva, C.douglasii and C.holmesiana, were determined. The evaluation of the triterpenes profiles showed that all 15 analyzed Hawthorn species had quite similar fingerprints.