SOLVENT FRACTIONATION AND CHROMATOGRAPHIC QUANTIFICATION OF FLAVONOIDS IN Nepeta olgae Regel EXTRACTS

УДК 543.544

Abstract

This study investigated the solvent-dependent fractionation and chromatographic quantification of flavonoids in extracts obtained from the aerial parts of Nepeta olgae Regel. The plant material was collected during the flowering stage, shade-dried, ground, and extracted with 70% aqueous ethanol. The concentrated extract was diluted with water and successively partitioned with chloroform, ethyl acetate, and n-butanol. Preliminary monitoring was performed by thin-layer chromatography, while quantitative profiling was carried out using reversed-phase high-performance liquid chromatography on an Agilent 1200 system equipped with a diode-array detector and an Eclipse XDB C18 column. Six marker compounds were identified and quantified: dihydroquercetin, luteolin, quercetin, rutin, cynaroside, and salidroside. The ethyl acetate fraction contained the highest concentrations of dihydroquercetin and quercetin, whereas the n-butanol fraction showed the highest total marker content and was particularly enriched in salidroside, rutin, luteolin, and cynaroside. The chloroform fraction contained only low concentrations of the analyzed phenolic compounds, while the aqueous residue retained moderate amounts of several markers. The results confirmed that solvent polarity plays a decisive role in the selective distribution of flavonoids. The obtained data provide a scientific basis for selecting ethyl acetate and n-butanol fractions as compositionally distinct intermediates for further development of standardized phytochemical products.

Аннотация

В работе исследовано распределение флавоноидов между фракциями, полученными из надземной части Nepeta olgae Regel растворителями различной полярности. Растительное сырьё собирали в фазу цветения, высушивали в тени, измельчали и экстрагировали 70%-ным водным раствором этанола. Концентрированный экстракт разбавляли водой и последовательно фракционировали хлороформом, этилацетатом и н-бутанолом. Предварительный контроль осуществляли методом тонкослойной хроматографии, а количественное профилирование проводили методом обращённо-фазовой высокоэффективной жидкостной хроматографии на системе Agilent 1200 с диодно-матричным детектором и колонкой Eclipse XDB C18. В полученных фракциях идентифицированы и количественно определены шесть маркерных соединений: дигидрокверцетин, лютеолин, кверцетин, рутин, цинарозид и салидрозид. Этилацетатная фракция характеризовалась наибольшим содержанием дигидрокверцетина и кверцетина, тогда как н-бутанольная фракция имела максимальную суммарную концентрацию маркерных веществ и была обогащена салидрозидом, рутином, лютеолином и цинарозидом. Хлороформная фракция содержала незначительные количества исследуемых фенольных соединений, а водный остаток сохранял умеренные концентрации отдельных компонентов. Полученные результаты подтверждают определяющую роль полярности растворителя в селективном распределении флавоноидов и обосновывают перспективность этилацетатной и н-бутанольной фракций для дальнейшей разработки стандартизованных фитохимических продуктов.

Keywords: Nepeta olgae Regel, extraction technology, flavonoids, HPLC, standardization, solvent fractionation.

Ключевые слова: Nepeta olgae Regel, технология экстракции, флавоноиды, ВЭЖХ, стандартизация, фракционирование.

Introduction

Medicinal and aromatic plants are renewable sources of phenolic acids, flavonoids, terpenoids, essential oils, and other secondary metabolites used in pharmaceutical, food, and phytochemical technologies. The efficiency of their recovery depends on the botanical origin of the raw material, phenological stage, drying conditions, solvent composition, extraction mode, and subsequent fractionation. Recent studies have shown that environmental conditions can substantially alter the qualitative and quantitative metabolite profiles of Nepeta species, which makes controlled raw-material collection and analytical profiling essential [1–4].

The genus Nepeta L. belongs to the Lamiaceae family and includes numerous aromatic species with documented antioxidant, antimicrobial, anti-inflammatory, and other biological properties. Modern chromatographic studies of Nepeta extracts have identified complex profiles of phenolic acids, flavonoid aglycones, glycosides, and iridoids, while solvent selection has been shown to influence both extraction yield and chemical selectivity [3–6].

Nepeta olgae Regel is distributed in Central Asia and occurs in the foothill areas of the Namangan Region. Previous investigations of this species established the presence of essential oils, amino acids, lipids, fatty acids, macro- and microelements, sterols, and flavonoids. Gas chromatography–mass spectrometry identified 24 volatile compounds in material collected from G‘ova and 58 compounds in material from Kosonsoy, indicating marked geographic variability [1, 7, 8].

The previously reported chromatographic dataset for N. olgae included six flavonoid and phenolic markers distributed among chloroform, ethyl acetate, n-butanol, and aqueous fractions. However, earlier presentation of the data as relative peak-area units limited interpretation. The present revision therefore reports the original quantitative values in mg/g, provides the complete chromatographic conditions, restricts the analytical claim to quantitative profiling rather than full regulatory method validation, and places the technological scheme within the Methods section.

The aim of this study was to evaluate the solvent-dependent distribution of selected flavonoids in N. olgae extracts and to identify compositionally distinct fractions suitable for further phytochemical development.

Materials and methods

Plant material and preliminary quality control. The aerial parts of N. olgae (stems, leaves, and flowers) were collected during flowering in May–June 2021–2022 from the G‘ova foothill area of Chust District and the Kosonsoy foothill area, Namangan Region, Uzbekistan. Foreign matter and damaged plant parts were removed. The material was shade-dried under natural ventilation, ground, and stored in closed containers protected from light.

Moisture, total ash, acid-insoluble ash, and extractive substances were determined by gravimetric procedures described in the dissertation study. These measurements were used as preliminary quality-control criteria before extraction [1].

Extraction and sequential liquid–liquid fractionation

The powdered plant material was extracted with 70% aqueous ethanol. The combined ethanolic extracts were concentrated under reduced pressure and diluted with water. The aqueous concentrate was extracted eight times with an equal volume of chloroform. The combined chloroform layers were separated, dried over sodium sulfate, and evaporated. The remaining aqueous phase was subsequently partitioned with ethyl acetate and then n-butanol. Organic solvents were removed under reduced pressure to obtain the chloroform, ethyl acetate, and n-butanol fractions; the residual aqueous phase was retained as the fourth fraction [1].

The technological sequence used in this study was: raw-material collection and preparation → shade drying → grinding → 70% ethanol extraction → filtration → vacuum concentration → dilution with water → chloroform partition → ethyl acetate partition → n-butanol partition → separate concentration of fractions → chromatographic analysis.

Figure 1. Technological scheme for extraction, sequential fractionation, and chromatographic analysis of N. olgae aerial material

Thin-layer and liquid chromatographic analysis. Fractions were preliminarily examined on silica-gel thin-layer chromatography plates and visualized under ultraviolet light using ammonia vapour, iodine, and vanillin–sulfuric acid reagents.

Quantitative profiling was performed using an Agilent 1200 liquid chromatograph equipped with an autosampler, diode-array detector, and Eclipse XDB C18 column (5 μm, 4.6 × 250 mm). The column temperature was 30 °C, the injection volume was 10 μL, and the flow rate was 1.0 mL/min. Detection was conducted at 254, 276, and 320 nm. A phosphate-buffer–acetonitrile mobile phase was applied according to the following gradient: 0–5 min, 95:5; 6–12 min, 70:30; 12–13 min, 50:50; and 13–15 min, 95:5. Reference solutions were injected before the prepared samples. Compounds were identified by comparison of retention times and ultraviolet spectra with reference standards, and concentrations were calculated from calibration relationships [1, 7, 8].

The reported values represent the quantitative concentrations contained in the original dissertation dataset and are expressed in mg/g. The available source records did not contain complete regulatory validation data for limits of detection and quantification, recovery, intermediate precision, or robustness. Therefore, the method is described here as a quantitative profiling procedure and not as a fully validated compendial assay. Full validation should be performed before routine quality-control or regulatory application in accordance with current analytical-validation guidance [9].

Figure 2. Extract solutions and the Agilent 1200 chromatographic system used in the study

Data treatment. Concentrations of individual markers were compared across the four fractions. The total quantified marker content of each fraction was calculated as the sum of the six measured compounds. Because replicate-level chromatographic data and standard deviations were not available in the dissertation record, no inferential statistical analysis was performed. The results are interpreted as comparative quantitative profiles.

Results

Quantitative flavonoid distribution. Six marker compounds were quantified in the four solvent fractions. The ethyl acetate and n-butanol fractions showed markedly higher concentrations than the chloroform fraction and aqueous residue (Table 1).

Table 1. Quantitative distribution of flavonoid and phenolic markers in N. olgae fractions (mg/g); ND, not detected

Fraction-specific marker profiles. The ethyl acetate fraction contained the highest concentration of dihydroquercetin (3.930 mg/g) and quercetin (1.130 mg/g). Dihydroquercetin in this fraction was approximately fourfold higher than in the n-butanol fraction and more than sixfold higher than in the chloroform fraction and aqueous residue. These results indicate preferential transfer of the medium-polar aglycone-rich component into ethyl acetate.

The n-butanol fraction contained the highest total marker concentration (7.73 mg/g). Salidroside was detected only in this fraction (3.210 mg/g), and the highest rutin concentration was also observed in n-butanol (1.580 mg/g). Luteolin and cynaroside were concentrated mainly in the n-butanol fraction, with smaller amounts remaining in the aqueous phase.

The chloroform fraction had the lowest total concentration of the quantified markers (0.73 mg/g), while the aqueous residue contained 1.91 mg/g. The combined marker concentration of the ethyl acetate fraction was 6.47 mg/g. Thus, sequential fractionation produced chemically differentiated fractions rather than a uniform redistribution of all analytes.

Table 2. Total quantified marker content and dominant compounds in each fraction

Discussion

The observed distribution is consistent with the physicochemical differences between flavonoid aglycones and glycosides. Ethyl acetate preferentially extracts compounds of intermediate polarity, whereas n-butanol is more suitable for polar glycosides. Sequential use of solvents is therefore a rational approach for generating fractions with distinct phytochemical profiles [6, 10].

The high dihydroquercetin and quercetin concentrations support use of the ethyl acetate fraction as a targeted intermediate for further antioxidant-product research. In contrast, the n-butanol fraction is distinguished by salidroside and rutin and may be more appropriate when polar phenolic glycosides are the desired markers. These conclusions concern chemical composition only; biological efficacy and safety must be confirmed independently for each prospective product.

The findings also complement broader phytochemical evidence for the genus Nepeta. Recent metabolic profiling of N. nuda identified numerous phenolic acids, flavonoids, and iridoids and demonstrated that environmental and cultivation conditions alter metabolite abundance. This supports the inclusion of collection site, phenological stage, and raw-material quality among future standardization criteria for N. olgae.

The present work resolves the principal ambiguity of the earlier manuscript by reporting the chromatographic values as mg/g rather than relative peak-area units and by providing the full instrumental conditions. Nevertheless, the absence of replicate-level data and complete analytical-validation characteristics remains a limitation of the original dataset. Future studies should include independently prepared biological and analytical replicates, mean ± standard deviation, confidence intervals, recovery experiments, linearity ranges, detection and quantification limits, and robustness testing in accordance with ICH Q2(R2).

Another limitation is that the dry mass yield of each solvent fraction was not recorded in the available dissertation dataset. Consequently, the current results describe concentration within the fractions but do not permit calculation of total analyte recovery from the starting plant material. Future process-development experiments should report both fraction yield (%) and marker recovery (mg per 100 g dry raw material), because these parameters are required for a complete technological and economic assessment.

Conclusion

Sequential partitioning of a 70% ethanolic extract of N. olgae produced compositionally distinct chloroform, ethyl acetate, n-butanol, and aqueous fractions. Quantitative chromatography identified six marker compounds. The ethyl acetate fraction was enriched in dihydroquercetin and quercetin, whereas the n-butanol fraction contained the highest combined marker concentration and was characterized by salidroside, rutin, luteolin, and cynaroside. The results demonstrate that solvent polarity is the principal factor governing selective flavonoid distribution. The revised dataset provides a practical basis for selecting fractions for further phytochemical development, while full analytical validation, replicate statistics, fraction-yield determination, and marker-recovery studies remain necessary before routine quality-control or scale-up application.

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