Phytochemical Analysis, Mineral Composition and GC-MS Profiling of Three Iris Species from the Southeastern Part of Kazakhstan

TL;DR

This study analyzed the chemical composition and antioxidant properties of three Iris species from Kazakhstan, revealing differences in their phytochemical and mineral profiles.

Contribution

The study provides a detailed phytochemical and mineral comparison of three Iris species from southeastern Kazakhstan using GC-MS and HPLC-DAD profiling.

Findings

I. alberti had the highest sterol content, including γ-sitosterol and campesterol.

I. pallasii contained significant amounts of hexacosanol and γ-sitosterol.

I. sogdiana was notable for octacosanol and γ-sitosterol, with distinct phenolic profiles.

Abstract

This study examined the phytochemical profiles and bioactive potential of Iris alberti, I. pallasii, and I. sogdiana collected from southeastern Kazakhstan. GC-MS analysis identified fatty acids, sterols, long-chain alcohols, and hydrocarbons in each species. The key phytosterols β-sitosterol, stigmasterol, and campesterol were present in all three. I. alberti showed the highest sterol content of γ-sitosterol (28.49%) and campesterol (6.51%). I. pallasii contained significant amounts of hexacosanol (19.74%) and γ-sitosterol (13.24%), while I. sogdiana was notable for octacosanol (22.87%) and γ-sitosterol (12.18%). Fatty acid composition varied: I. sogdiana was rich in α-linolenic acid (4.77%) and palmitic acid (2.77%), I. pallasii had 3.96% and (3.48%). I. alberti contained dodecanoic acid (3.83%) and branched-chain fatty acids. Tannin content was highest in I. alberti (1.88%), while alkaloid levels were moderate across species (0.63–0.77%). Mineral analysis showed I. pallasii had the highest Ca (586.62 mg/100 g), Mg and K, whereas I. sogdiana had the highest potassium (538.36 mg/100 g). HPLC-DAD analysis revealed distinct phenolic profiles. Water–alcohol extracts from rhizomes of I. alberti had an IC50 of 16.90 ± 6.04 µg/mL in DPPH and were inactive in ABTS. I. pallasii exhibited IC50 values of 28.77 ± 2.93 µg/mL (DPPH) and 10.93 ± 7.29 µg/mL (ABTS), while I. sogdiana showed negligible activity in both assays. Ethyl acetate extracts displayed higher IC50 values, consistent with lower phenolic content.

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1. Introduction

The genus Iris Tourn. ex L. (Iridaceae Juss.) encompasses over 300 species, many of which remain poorly explored chemically, particularly in Central Asia [1,2,3,4]. These species are cosmopolitan and have significant horticultural, medicinal (homoeopathy), cosmetic (perfumery), food, and ecological importance, and served as templates for hydrophobic surfaces [5,6,7,8,9,10,11]. Floristic studies indicate that Kazakhstan has 19–22 Iris species distributed across all natural zones and ecosystems [2,3,4,12,13,14,15]. Three rare species—Iris alberti Regel, I. ludwigii Maxim., and I. tigridia Bunge are listed in the Red Book of Kazakhstan (2014) [16], and only eight have been used in folk and experimental medicine [17].

Among Kazakhstan’s Iris flora, Iris alberti Regel, I. pallasii Fisch. ex Trevir., and I. sogdiana Bunge occur in southeastern Kazakhstan and represent ecologically distinct taxa adapted to different environmental conditions [18]. Long-term phytocenotic introduction of wild-growing iris species in this region began in 2018 and is ongoing [19,20,21], although comprehensive comparative chemical analysis of these species is limited. According to the herbarium collections of the Institute of Botany and Phytointroduction (IBPh), I. sogdiana is found across various floristic regions, whereas I. pallasii and I. alberti exhibit a more restricted yet ecologically distinct distribution.

Taxonomic revisions within the genus Iris complicate comparisons of previously reported chemical data. Iris alberti is consistently assigned to the subgenus and section Iris [22,23,24,25,26]. Central Asian Iris, based on both morphological and molecular evidence, places I. sogdiana and I. pallasii in the subgenus Limniris [26]. Consequently, chemical and pharmacological data are dispersed across different species names, limiting direct comparability.

Phytochemical studies of the genus Iris indicate that its representatives are rich sources of structurally diverse secondary metabolites, including isoflavonoids, xanthones, stilbenoids, quinones, phenolic acids, and triterpenoids, many of which exhibit pronounced biological activity [27,28]. However, research has predominantly focused on species such as I. lactea, I. ensata, I. pseudacorus, and I. tenuifolia, while data on I. alberti, I. pallasii, and I. sogdiana remain fragmentary or indirect [29,30,31,32,33,34].

Despite limited data, Iris alberti has a phytochemical profile primarily characterised by vitamin C and phenolic acids (sinapic and ferulic) in the leaves, and its roots and leaves are rich in tannins (2.99 and 3.52% [29]) and xanthones (mangiferin and isomangiferin) [28]. Traditionally, the roots are used for digestive issues, and the flowers serve as a source of natural green dye [17,29]. In contrast, I. pallasii (I. lactea s.l. or I. pallasii var. chinensis) exhibits a diverse secondary metabolite profile that includes benzoquinones, C-glycosylflavones, xanthones, stilbenoids, phenolic acids, and a significant proportion of unsaturated fatty acids in its seeds [30] (Appendix A, Table A1). This chemical diversity suggests adaptability for harsh environments. The presence of anticancer benzoquinones, such as irisquinone and pallasone, within the Iridaceae family likely functions as potent defence agents against biotic stressors. The pronounced antiviral activity of oligostilbenes, including vitisin B and C, further emphasises the pharmacological significance of this species [27,28,29]. Iris sogdiana accumulates carbohydrates and polysaccharides in roots (0.62–0.63%) and leaves (1.8–2.4%), with seeds containing glucomannans [30]. Iris sogdiana is considered a synonym of I. spuria [25], which is characterised by glycosylated iridals, and iso-iridal type of triterpenes, and isoflavonoids (peltogynoids and rotenoid). Likewise, I. pallasii is frequently treated as I. lactea Pall. or I. pallasii var. chinensis; thus, all phytochemical and pharmacological findings published under these names are integrated to provide a consistent comparative framework [27,32,33,34].

Considering the ecological heterogeneity of southeastern Kazakhstan and the sensitivity of plant secondary metabolism to environmental factors, a targeted comparative phytochemical study of these species is justified. In this context, the present comparison of I. alberti, I. pallasii and I. sogdiana provides important insights into their phytochemical profiles.

Therefore, the objective of this study was to undertake a comparative phytochemical and biological assessment of Iris alberti, I. pallasii, and I. sogdiana collected from southeastern Kazakhstan, thereby contributing novel chemical data for underexplored taxa. The species nomenclature is provided in accordance with the floristic summary [2,3,4].

2. Results

2.1. Collection Sites

The studied plants were collected during expedition trips in April–May 2023 in different phases of vegetation in the valleys of the desert rivers Ili (Iris pallasii, I. sogdiana) and Karatal (I. sogdiana) and in the downhill of the gorges Kasquelen, Shamalgan ridge of Zayliskiy Alatau (I. alberti) (Figure 1). All collected species specimens were submitted to the herbarium of AA belonging to the Institute of Botany and Phytointroduction, under numbers AA0042423, AA0042424, AA0042425. The species was identified using key signs [2,3,4,12,13,14] by Ramazanova M.S. and Prof. Gemejiyeva N.G.

2.2. Qualitative and Quantitative Analysis

Phytochemical screening provides a preliminary basis for identifying bioactive natural compounds and assessing their potential. The leaves and rhizomes of the three Iris species examined were screened for flavonoids, alkaloids, terpenoids, and tannins, and all these bioactive classes were confirmed. However, qualitative analysis was performed on rhizomes, as these parts are most significant for therapeutic value and are widely used in folk medicine. Additionally, moisture and ash content were calculated (Table 1).

Quantitative phytochemical analysis of three Iris samples revealed significant variation in the measured parameters. Flavonoid content was relatively uniform across samples, ranging from 0.29% to 0.33%. Tannin level illustrates considerable variability, with the highest concentration in I.alberti (1.88%) and the lowest in I.sogdiana (1.06%). Alkaloid content remained moderate and comparable across the three samples (0.63–0.77%).

2.2.1. Extract Yield (%)

The extract yield was isolated from creeping rhizomes of three Iris species by solvent extraction. Water–ethanol mixture (50–70%) was applied as a solvent in the extraction process. In all cases, 50% ethanol solution yielded higher extractive values than a 70% ethanol solution, indicating better solubility of the major constituents in the polar solvent system. For I. pallasii the extractive value decreased from 18.9% with 50% water–ethanol to 15.5% (70% w–ethanol). A similar trend was observed for I. alberti (36.7% to 30.3%) and I. sogdiana (31.2% to 29.1%). Among the studied species, the abundance of ethanol-soluble constituents follows the order I. alberi > I. sogdiana > I. pallasii.

2.2.2. Mineral Composition (mg/100 g)

Quantitative analysis of minerals in rhizomes of the three Iris species revealed interspecific differences (Table 2). All samples contained higher levels of macronutrients (Ca, Mg, Na, K) compared to trace elements (Fe, Mn), indicating a consistent trend of decreasing content from major to minor elements across species. However, notable species-specific differences were observed.

Across species, a tendency toward accumulation of Ca, K, and Na was observed. Iris pallasii accumulates the highest levels of Ca (586.62 mg/100 g), Mg (167.99 mg/100 g), and Na (267.97 mg/100 g), considerably surpassing both I. alberti and I. sogdiana. In contrast, potassium content is highest in I. sogdiana (538.36 mg/100 g), followed by I. pallasii (385.63 mg/100 g) and I. alberti (200.76 mg/100 g). Among trace elements, iron concentration is highest in I. alberti at 22.11 mg/100 g. Manganese levels are relatively low and consistent across all species, with I. pallasii showing a slight increase at 1.16 mg/100 g.

2.3. GC-MS Analysis of the Native Nonpolar Extracts from the Leaves

GC–MS analysis of native CH_2_Cl_2_ leaf extracts from Iris alberti, I. pallasii, and I. sogdiana showed complex yet similar metabolite profiles dominated by lipophilic compounds. Compounds were identified using retention time, linear retention indices (LRIs), and mass spectral matching. Relative abundances were reported as percentage peak areas. Figure 2 compares compounds identified in native and TMS-derivatised extracts. Derivatisation significantly influenced the number of detected metabolites in a species-specific manner, reflecting differences in derivatisable functional groups. In I. alberti, TMS derivatisation increased detectable compounds from 54 to 77, indicating a higher proportion of hydroxylated or carboxyl-containing metabolites. In contrast, I. pallasii showed a decrease (74 to 48), while I. sogdiana had a slight increase (70 to 74), highlighting species-specific differences in metabolite composition.

Sterols, triterpenoids, fatty acids, and fatty acid esters were the dominant chemical classes in all three species. Quantities of identified compounds varied; many compounds had consistent retention times across species, demonstrating analytical reliability and a conserved core lipophilic metabolome in Iris. For instance, dodecanoic acid and squalene were present in all samples with nearly identical retention times, confirming accurate compound identification.

Identified compounds in CH_2_Cl_2_ leaf extracts from three Iris species were classified according to the chemical classes (Figure 3). Fatty acid esters and fatty acids were the most abundant chemical classes in all three species. Iris sogdiana had the highest number of fatty acid esters, while I. pallasii showed a similar but slightly lower diversity in this class. Sterols and terpenes/terpenoids were especially prominent in I. pallasii, which contained the highest number of these compounds among the species analysed. Aliphatic hydrocarbons were another major class, with I. sogdiana containing more identified compounds than I. alberti. In contrast, polyphenols and flavonoids, aromatic heterocycles (benzofuran, 2,3-dihydro-; 1,1,5-trimethyl-1,2-dihydronaphthalene; naphthalene, 1,2,3,4-tetrahydro-1,1,6-trimethyl-), alcohols, aldehydes, and ketones were present in limited quantity across all species, with only minor interspecies differences observed and grouped in the other category. In the other category, different classes of compounds were listed, including isoprenoid γ-lactone (4,8,12,16-Tetramethylheptadecan-4-olide), simple ethers, and unidentified compounds.

The following list comprises the 26 compounds that were found in all three samples (Table 3), and full peak assignments are provided in Supplementary Tables S1, S3 and S5.

Sterols and terpenoids were the most prominent compound classes identified across all analysed species. Among these, γ-sitosterol was the dominant constituent, accounting for 12.18% in I. sogdiana, 13.24% in I. pallasii, and 28.49% in I. alberi. Saturated and unsaturated fatty acids were found in the analysed species. Among the saturated fatty acids, n-hexadecanoic acid was particularly notable, comprising up to 2.29% and 1.68%, while tetradecanoic acid accounted for up to 1.24%. Dodecanoic acid was also detected in significant amounts in I. alberti at 3.83%. The presence of polyunsaturated fatty acids and their esters was exceptionally high. 9,12,15-octadecatrienoic acid reached concentrations of 4.77% in the I.sogdiana and 1.88% in the I.pallasii, whereas 9,12-octadecadienoic acid represented 2.77% and 0.92%. Their ethyl esters were also abundant; for example, the ethyl ester of 9,12,15-octadecatrienoic acid accounted for 2.32% in I.sogdiana and 0.85% and 1.04% across the other species. Other significant sterols included stigmasterol, which comprised 3.84% and 6.49% in I. sogdiana and I. pallasii, and campesterol, which contributed 1.48%, 3.28%, and 26.51% in I. alberti.

The study also considered hydrocarbons and aldehydes, such as pentacosane, heptacosane, nonacosane, octacosane, and triacontanal, as well as esters such as phytyl decanoate. A total of sterols, triterpenoids, tocopherols, and isoprenoid-type compounds were identified across all three analysed samples (Table 3 and Table 4). While the overall qualitative composition was broadly similar, the relative proportions of individual metabolites varied considerably. Sterols were the predominant class of compounds across all species. γ-Sitosterol and stigmasterol accounted for a substantial portion of the sterol profile, with the highest concentration in I. alberti (28.49% and 3.67%), nearly double that in I. sogdiana and I. pallasii. Campesterol increased from 1.48% in I.sogdiana to 3.28% in I. pallasii, peaking at 6.51% in I.alberti. Ergostan series sterols were observed in the studied species: γ-ergostenol (I. sogdiana, 0.63%), ergostanol (I. pallasii, 0.62%), and ergosta-4,6,22-trien-3α-ol (I. alberti, 0.69%) (Supplementary Tables S1–S6).

Tocopherol accounted for 3.58–5.75% of the total GC–MS profiles among the three Iris species. The highest tocopherol level was observed in I. sogdiana and was approximately 1.6 times greater than in I. pallasii, indicating moderate interspecific variation in tocopherol accumulation within the genus. Triterpenoids, including lupeol and lup-20(29)-en-3-one, have been identified as exclusive to I. sogdiana.

GC-MS Analysis of the TMS Derivatives of Nonpolar Leaf Extracts from Three Iris Species

The analysis showed that all three species possess complex but variable chemical profiles, comprising more than 70 identified compounds (Supplementary Tables S2, S4 and S6). These compounds predominantly belong to the fatty acids, sterols, long-chain alcohols, and hydrocarbons.

The studied species contain key phytosterols, including β-sitosterol, stigmasterol, and campesterol, as well as a wide range of common saturated fatty acids, such as palmitic, stearic, and myristic acids. Additionally, all samples contain long-chain alkanes (e.g., nonacosane and heptacosane) and alcohols such as 1-octacosanol. For example, the sterol composition differed significantly among the three species. I. alberti exhibited the highest overall sterol content, with β-sitosterol, which represents the main peak (16.85%) of the GC–MS profile. Furthermore, campesterol had the highest relative abundance (2.95%), and stigmasterol was present at 0.30%. In comparison, I. pallasii also contained substantial amounts of β-sitosterol (12.08%) and showed the highest relative concentration of octacosanol among the studied species (28.23%) (Figure 4). Meanwhile, I. sogdiana displayed the lowest relative abundance of β-sitosterol (3.39%). This species was characterised by the presence of stigmast-8(14)-en-3β-ol (0.30%) and lup-20(29)-en-3-one (0.25%).

A comparative analysis of fatty acids (FAs) and their derivatives revealed notable differences between species in both the amount and types of fatty acids present, although several key fatty acids were shared among all three species. Iris sogdiana showed the highest accumulation of unsaturated fatty acids, with α-linolenic acid as the main component of the fatty acid profile (17.45%), along with palmitic acid (9.37%) and a significant amount of fatty acid esters, suggesting active fatty acid metabolism. In contrast, I. pallasii had relatively low levels of α-linolenic acid (3.96%) and palmitic acid (3.48%), which aligns with the dominance of long-chain alcohols in this species. Iris alberti consistently contained dodecanoic acid (7.73%), and unique to this species were branched-chain (iso-) fatty acids, including iso-pentadecanoic and iso-palmitoleic acids, as well as oleic acid (Supplementary Table S2).

2.4. HPLC Analysis of Extracts from Rhizomes

The phytochemical profiling of the summary 70% water–alcohol extracts from rhizomes belonging to the species of I. alberti, I. pallasii, and I. sogdiana, as well as ethyl acetate extracts from I. pallasii (EA-IP) and I.sogdiana (EA-IS), revealed significant qualitative and quantitative variations in their phenolic composition. Only 11 compounds were identified across the analysed samples (Table 5). While certain marker compounds were consistently present, the extracts exhibited distinct chemical signatures, especially in the distribution of simple phenolics, flavonoids, and phenolic esters. These compositional differences were also evident in the antioxidant activities assessed through DPPH and ABTS assays.

The HPLC-DAD analysis revealed that I. alberi exhibited a profile dominated by coumarin (35.92 mg/g) and trans-2-hydroxycinnamic acid (15.82 mg/g), which together represent the highest concentration of simple phenolics across all samples. Additionally, the presence of genistein (3.84 mg/g) introduces a flavonoid component to the overall profile, although at comparatively low levels.

In contrast, I. pallasii was characterised by flavan-3-ols, most notably catechin (4.72 mg/g) and epicatechin (1.98 mg/g), which were absent from other extracts. These compounds, commonly associated with potent radical scavenging activity, suggest a mechanistic basis for the observed antioxidant potency of this extract.

Iris sogdiana showed an extraordinarily high propyl gallate content (250.83 mg/g), a well-known synthetic phenolic antioxidant often encountered as a natural artefact or an extraction-derived constituent. Its overwhelming abundance suggests either deliberate enrichment during sample processing or an intrinsic conversion of native gallic acid derivatives during extraction. The presence of trace amounts of genistein further supports the notion that the extract retains limited native phenolic diversity.

Among the ethanol extracts, samples EA-IP and EA-IS showed broader but lower-intensity phenolic profiles. EA-IP contained protocatechuic and vanillic acids, as well as caffeic acid in trace amounts, while both incorporated genistein in concentrations of 0.58 mg/g and 7.76 mg/g, respectively. Sample EA-IS additionally contained low levels of vanillic acid, caffeic acid, and ferulic acid, indicating partial hydrolysis of phenolic glycosides or liberation of bound phenolics during ethanol extraction.

Overall, the phenolic landscape clearly demonstrates that the samples differ not only in specific phenols present but also in the structural classes they represent—simple phenolic acids in the extracts of I. alberti and EA-IP, flavan-3-ols in sample 2, phenolic esters in I. sogdiana, and mixed phenolics with flavonoids in EA-IS.

Correlation Between Phenolic Composition and Antioxidant Activity

The antioxidant assays revealed significant differences in activity across samples, with IC_50_ values closely correlating with the presence of specific phenolic groups (Table 5). In the DPPH assay, Iris alberti exhibited the lowest IC_50_ value among the native extracts (16.90 ± 6.04 µg/mL), whereas I. pallasii showed a slightly higher IC_50_ (28.77 ± 2.93 µg/mL), and I. sogdiana demonstrated negligible activity (IC_50_ > 400 µg/mL). In the ABTS assay, I. pallasii displayed the lowest IC_50_ (10.93 ± 7.29 µg/mL), I. sogdiana showed moderate activity (114.42 ± 3 µg/mL), and I. alberti was inactive under the tested conditions (NA).

Ethyl acetate (EA) extracts generally exhibited lower radical scavenging potential than the native extracts. EA–I. pallasii had IC_50_ values of 64.38 µg/mL (DPPH) and 150.21 µg/mL (ABTS), while EA–I. sogdiana ranged from 120 to >300 µg/mL in both assays. These results are consistent with the lower total phenolic content in the EA fractions.

Across all samples, IC_50_ values corresponded with the relative abundance of phenolic compounds. Extracts enriched in flavan-3-ols, coumarins, and cinnamic acid derivatives generally had lower IC_50_ values, reflecting higher radical scavenging potential, whereas extracts with lower phenolic content or dominated by other compounds showed higher IC_50_ values and reduced activity. Differences between DPPH and ABTS assays indicate that both hydrogen-atom transfer and electron-transfer mechanisms contribute to the observed antioxidant activity, depending on each extract’s phenolic composition.

Taken together, these results demonstrate that antioxidant profiles are species-dependent and influenced by both the type and abundance of phenolic constituents. Native extracts consistently exhibited higher radical scavenging potential than the EA fractions, highlighting the importance of the complete phenolic matrix. All IC_50_ values were higher than those of the reference antioxidant BHA (5.09 ± 0.22 µg/mL for DPPH, 2.21 ± 0.09 µg/mL for ABTS), indicating lower activity than the standard.

3. Discussion

The complex taxonomy of Iris Tourn. ex L. remains a critical consideration when interpreting phytochemical data [28,34]. Notably, Iris sogdiana Bunge is frequently treated as a synonym of I. halophila var. sogdiana or I. spuria L. [25,35], while I. pallasii Fisch. ex Trevir. is often regarded as a synonym of I. lactea Pall. [25,28]. Despite these taxonomic ambiguities, and while retaining the species names as recognised in the flora of Kazakhstan [2,3,4,12,15], these species exhibit pronounced adaptations to local eco-geographical conditions, as evidenced by their successful acclimatisation at the MBG and the Ili Botanical Garden (IBG) [19,36].

A herbarium review of Iris collections from southeastern Kazakhstan at the Institute of Botany and Phytointroduction (herbarium AA) confirmed at least 65 species across five floristic regions: Balkhash-Alakol, Dzungarian Alatau, Zailiysky and Kungey Alatau, Ketmen and Terskey Alatau, and Chu-Ili mountains. I. sogdiana was found in eight floristic regions. I. pallasii was recorded in Dzungar Alatau, Zailiysky Alatau, Toraygyr mountains, and Balkhash-Alakol. I. alberti was identified in Zailiysky Alatau, the Kazakh part of the Western Tien-Shan, and other Tien-Shan regions in Kyrgyzstan (Appendix B, Table A2). These studies independently verify species identity and geographic distribution, supporting the relevance of the phytochemical and antioxidant analyses presented here.

This study is the first report on the quantitative (flavonoids, tannins, and alkaloids) and phytochemical profiles of CH_2_Cl_2_ extracts and their TMS derivatives from leaves, along with HPLC-DAD analysis of phenolic compounds and the antioxidant potential of summary extracts from rhizomes for the Kazakhstani three Iris species.

Quantitative analysis of rhizome extracts revealed relatively low total flavonoid content (0.29–0.33%) and tannin content (1.06–1.88%) across all three species, whereas their qualitative profiles exhibited clear species-specific differences.

This research provides the first detailed phytochemical profile of I. alberti rhizomes and leaves. HPLC-DAD analysis revealed that simple phenols, particularly coumarin (35.92 mg/g) and trans-2-hydroxycinnamic acid (15.82 mg/g), are the predominant compounds. According to [27,29], the leaves of I. alberti contain xanthones (mangiferin and isomangiferin) and phenolic acids (sinapic and ferulic), along with elevated tannin [29]. While GC-MS analysis showed that the leaves contain saturated fatty acids (dodecanoic acid, n-hexadecanoic acid) and the unsaturated fatty acid 9,12,15-octadecatrienoic acid, a profile characteristic of the lipophilic fractions of numerous Iris species, such as I. lactea and I. sibirica [28,37]. Squalene, terpenes and phytosterols, including campesterol (6.51%) and γ-sitosterol (28.49%), were also detected in I. alberti. Analysis of sterols in other species, including I. lactea, I. germanica, and I. pallida, revealed the presence of stigmasterol-3-O-β-D-glucopyranoside and β-sitosterol [30]. The occurrence of β-amyrin in I. alberti aligns with previous reports of this pentacyclic triterpenoid in the rhizomes of I. germanica [38]. Additionally, α-iron (0.11%) was detected in the lipophilic fraction of I. alberti; irons, including α-, β-, and γ-isomers, are characteristic of irises in the Iris section (bearded irises such as I. germanica and I. florentina) and contribute to their distinctive violet aroma [27,39].

While the literature on Iris spuria (a syn. I. sogdiana) has focused on isoflavonoids (tectorigenin, irisolone) and rotenoids (12α-hydroxirotenoid) [27,34], our analysis revealed an exceptionally high content of propyl gallate (250.83 mg/g) in the summary extract of I. sogdiana. Additionally, in the EA extract, six phenolic compounds were identified and quantified: vanillic acid (0.92 mg/g), caffeic acid (0.64 mg/g), ferulic acid (0.61mg/g), and genistein (7.76 mg/g). Furthermore, I. sogdiana exhibited the highest potassium content (538.36 mg/100 g) among the studied species. GC-MS analysis of the non-polar extracts confirmed that sterols and triterpenoids are the predominant classes of compounds across all three species, with γ-sitosterol (53.24%) and stigmasterol (16.77%).

In the literature, Iris lactea s.l. is the most frequently described species, and, if considered a synonym of I. pallasii, it is among the most extensively studied taxa within the genus (Appendix A). Nevertheless, the studied phenolic profile reveals the presence of catechin (4.72 mg/g) and epicatechin (1.98 mg/g). The EA extracts contained protocatechuic, vanillic, and caffeic acids, which have also been identified in other species of the genus [17,28]. Compared to the sterol and terpenes profiles of the rhizome, which contains only stigmasterol derivatives according to [28], we semi-quantified the presence of campesterol (1.17–3.30%), γ-, β-sitostrol (1.29 and 16.85%), and fucosterol (0.66%).

This study presents the first comprehensive phytochemical and quantitative analysis of Kazakhstani Iris species, examining flavonoids, tannins, alkaloids, phenolic compounds, and antioxidant activity in rhizomes and leaves. Comparative analysis of I. albertii, I. pallasii, and I. sogdiana identifies both shared structural compounds characteristic of the genus and unique metabolites reflecting species-specific ecological adaptations. Sterols, triterpenoids, fatty acids, and phenolic compounds emerged as the dominant constituents across all studied species.

4. Materials and Methods

Quantitative analyses were performed in accordance with the protocols described in the State Pharmacopoeia of the Republic of Kazakhstan [40] and procedures described in [41].

4.1. Sample Preparation

The dried, ground leaves of the studied samples (0.2 g) were extracted with 2 mL of dichloromethane. Extraction was carried out for 2 h at room temperature, consisting of two ultrasonic treatments (5 min each) at 25 °C. The extract was decanted and evaporated to dryness without heating; the dry residue was redissolved in dichloromethane and analysed by GC–MS. The extractive yield was 10.33%.

4.2. Summary Extract Preparation

The summary extracts were prepared using 50% water–ethanol, and each sample underwent three extractions to ensure maximal recovery of phenolic constituents. The combined extracts were concentrated to a minimal volume and then sequentially partitioned with chloroform and ethyl acetate to obtain the corresponding solvent fractions.

Silylation: To the dry extract obtained using the previously described method, add 50 μL of N, O-bis(trimethylsilyl)acetamide (BSA). Incubate the mixture at 50 °C with constant stirring using a magnetic stirrer until the residue has fully dissolved and the silylation reaction is complete. Once the reaction is finished, dilute the resulting solution of silylated derivatives with dichloromethane and analyse it using GC–MS.

4.3. GC-MS Analysis

GC–MS analysis was performed using a Maestro gas chromatograph (“Interlab”, Moscow, Russia) coupled with a quadrupole mass spectrometer detector (Agilent 5975) and a capillary quartz column (HP-25, 30 m, “Interlab”, Moscow, Russia). The carrier gas was helium (1 mL/min), and the oven temperature was programmed from 50 °C (3 min) to 280 °C over 10 min, followed by 20 min isothermal at 280 °C (total run time 45 min). The injector temperature was 270 °C, injection volume 1 µL, with electron-impact ionisation at 70 eV. Components were identified using the NIST-2011 and Wiley-2008 libraries (match factor ≥ 85%) and confirmed by calculating linear retention indices (LRIs) from a homologous series of n-alkanes (C8–C37) under the same chromatographic conditions. The LRI for each compound was determined according to the Van den Dool and Kratz equation:

$$[eqn]$$

Here, t_x_ is the retention time of the compound, t_n_ and t_(n+1)_ are the retention times of the n-alkanes eluting immediately before and after the compound, and n is the carbon number of the preceding n-alkane. LRIs, together with mass spectral data, were used to confirm compound identities by comparing them with literature values and reference databases. Relative abundances were expressed as the percentage of peak area in the total ion chromatogram (TIC), providing a semi-quantitative comparison of constituents within and between samples.

4.4. HPLC–DAD Analysis

Phenolic compounds in the extract were analysed together with 43 standard phenolics [42] using a Shimadzu HPLC–DAD system (Shimadzu, Kyoto Japan) consisting of an LC-20AT pump and an SPD-M20A diode array detector, controlled by LC-Solution software 5.117 software (CBM-20A). Separation was achieved on an Inertsil ODS-3 column (GL Sciences Inc., Shinjuku-ku, Tokyo, 163-1130 Japan) (4 μm, 4.0 × 150 mm) with a guard column at 35 °C. The mobile phase comprised 0.1% acetic acid in water (A) and 0.1% acetic acid in methanol (B) and was applied in gradient mode at a flow rate of 1.0 mL/min. Samples and standards (8 mg/mL) were filtered through a 0.45 μm MN filter, and 20 μL was injected. Detection was performed at 254 nm. Phenolic compounds were identified by comparing retention times, and results were expressed as mg/g dry extract.

4.5. Antioxidant Activities

The antioxidant activity of the extracts was evaluated spectrophotometrically using DPPH free radical scavenging and ABTS cation radical scavenging assays with a 96-well microplate reader (SpectraMax 340PC384, Molecular Devices, Silicon Valley, CA, USA). SoftMax Pro v5.2 software (Molecular Devices, Silicon Valley, CA, USA) was used for data analysis and bioactivity calculations. Stock solutions of the samples were prepared at a concentration of 4000 μg/mL. Radical scavenging activity was calculated using the following formula:

$$[eqn]$$

where Abs control represents the initial absorbance of DPPH or ABTS, and Abs sample represents the absorbance of the remaining radicals in the presence of the extract or positive control. Antioxidant activity was expressed as IC_50_ values (µg/mL), defined as the concentration of the extract required to inhibit 50% of DPPH or ABTS radicals. BHA was used as a positive control.

4.6. DPPH Radical Scavenging Assay

The free radical scavenging activity of the extracts was evaluated using the DPPH assay [43]. A 0.1 mM DPPH solution was prepared by dissolving 39.4 mg of DPPH in methanol. Aliquots of 40 μL of the extracts at various concentrations were mixed with 160 μL of DPPH solution. After incubation in the dark at room temperature for one hour, absorbance was measured at 517 nm. Butylated hydroxyanisole (BHA) was used as the reference antioxidant. All measurements were performed in triplicate.

4.7. ABTS Cation Radical Scavenging Assay

ABTS cation radical scavenging activity was determined according to [44] with slight modifications [45]. The ABTS cation radical was generated by reacting 7.0 mM ABTS with 2.45 mM potassium persulfate and incubating the mixture in the dark at room temperature for 12 h. Prior to analysis, the solution was diluted with distilled water to an absorbance of 0.70 ± 0.01 at 734 nm. In each well, 160 μL of ABTS solution was mixed with 40 μL of the extract at different concentrations. Solvent for the extract served as the negative control, and BHA as the standard antioxidant. Absorbance was recorded at 734 nm after 10 min of incubation at room temperature. All analyses were conducted in triplicate.

4.8. Statistical Analyses

HPLC and activity assay data represent the mean of three independent replicates and are expressed as mean ± SEM. Statistical analysis was performed using ANOVA, and significant differences between means were determined by Student’s t-test (p < 0.05).

5. Conclusions

The taxonomy of Iris species remains complex, with I. sogdiana and I. pallasii often considered synonyms of other taxa. Nevertheless, all studied species demonstrate clear local adaptations to Kazakhstani conditions. This study provides the first comprehensive phytochemical and quantitative analysis of these species, encompassing flavonoids, tannins, alkaloids, phenolic compounds, and antioxidant activity in rhizomes and leaves.

The results reveal species-specific phytochemical profiles:* I*. alberti is rich in xanthones, phenolic acids, fatty acids, squalene, terpenes, and phytosterols, including β-amyrin and α-iron, contributing to its aroma; I. sogdiana exhibits exceptionally high propyl gallate, multiple phenolic acids, and elevated potassium, with sterols and triterpenoids predominant in non-polar extracts; I. pallasii contains diverse phenolics and rhizome sterols, including campesterol, γ- and β-sitosterol, and fucosterol.

Overall, sterols, triterpenoids, fatty acids, and phenolic compounds are the dominant constituents across all Kazakhstani Iris species studied, reflecting both common genus-level features and species-specific adaptations to their ecological environments.