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Article

Phytochemical Composition and Antimicrobial and Antioxidant Activity of Hedysarum semenowii (Fabaceae)

by
Anel Keleke
1,*,
Magdalena Maciejewska-Turska
2,
Martyna Kasela
3,
Tomasz Baj
2,
Liliya Ibragimova
1,
Zuriyadda Sakipova
1,
Olga Sermukhamedova
1 and
Agnieszka Ludwiczuk
2,*
1
Department of Engineering disciplines and Good Practices, Asfendiyarov Kazakh National Medical University, 94 Tole Bi Str., 050012 Almaty, Kazakhstan
2
Department of Pharmacognosy with the Medicinal Plant Garden, Medical University of Lublin, 1 Chodźki Str., 20-093 Lublin, Poland
3
Department of Pharmaceutical Microbiology, Medical University of Lublin, 1 Chodźki Str., 20-093 Lublin, Poland
*
Authors to whom correspondence should be addressed.
Molecules 2025, 30(23), 4503; https://doi.org/10.3390/molecules30234503
Submission received: 30 September 2025 / Revised: 12 November 2025 / Accepted: 19 November 2025 / Published: 21 November 2025
(This article belongs to the Special Issue Biological Evaluation of Plant Extracts)
Browse Figure
Versions Notes

Abstract

This paper provides a comprehensive phytochemical analysis of extracts obtained from the leaves and roots of Hedysarum semenowii using HPLC/PDA-ESI-QToF/MS-MS techniques. The study identified 53 compounds, with flavones and isoflavones as the primary polyphenols. Notably, flavones were predominant in the leaves, while isoflavones were found mainly in the roots, potentially serving as chemotaxonomic markers. Medicarpin and its glucoside were confirmed in the roots, while mangiferin and its derivatives were identified for the first time in both the roots and leaves. Isoflavones like formononetin, calycosin, and afrormosin, along with their glucosides, were exclusive to the roots. Flavonols such as quercetin and its glycosides were abundant in the aboveground parts. Our study also identified flavones like luteolin, flavanones (naringenin), and chalcones (liquiritigenin) in various parts. Additionally, the phenolic acids gallic and ferulic acids, as well as the organic acids malic and citric acid, were also detected. The extracts demonstrated differential antimicrobial and antifungal activities in a microbroth dilution assay, with the aerial part extracts showing superior efficacy, particularly against Staphylococcus epidermidis and Pseudomonas aeruginosa. Both aerial and underground parts exhibited comparable antifungal activity against Candida species. Antioxidant activity in the 1,1-diphenyl-2-picrylhydrazyl (DPPH•) radical scavenging test varied significantly, with ethanolic extracts from the aerial parts showing the highest potential (Antioxidant Activity Index (AAI) 2.07 ± 0.13). In contrast, root extracts had consistently low antioxidant activity. The results highlight the aerial parts of H. semenowii as a more promising source of biologically active compounds with antimicrobial and antioxidant properties compared to the roots.

Graphical Abstract

1. Introduction

Hedysarum L. is a genus within the third largest flowering plant family Fabaceae which is distributed across diverse habitats of the Northern Hemisphere, spanning Europe, Asia, North Africa, and North America [1,2].
The genus has significant medicinal importance, with species serving as sources of pharmaceuticals and diagnostic agents [3,4]. Moreover, these plants find utility in fodder cultivation [5,6] and ecological endeavors such as soil enrichment and sand fixation [7,8].
Among the notable species, Hedysarum polybotrys Hand.-Mazz., commonly known as “Hong Qi,” stands out for its utilization in traditional Chinese medicine, often as a substitute for the well-established medicinal plant Astragalus mongholicus Bunge (Fabaceae) [3,9]. Several pharmacological studies suggest that H. polybotrys exhibits pronounced effects compared to Astragalus mongholicus [10,11,12], combating aging and associated ailments [3,13], treating gastrointestinal and renal disorders [10,14], aiding postpartum recovery, and enhancing lactation [15,16]. Similarly, Hedysarum alpinum L. and Hedysarum flavescens Rgl. et Shmalh demonstrate pharmacological versatility, serving as antiviral agents and constituents of sedative, antipyretic, anti-inflammatory, and antimicrobial formulations [17,18,19]. Notably, Hedysarum neglectum Ledeb. is recognized for its role in atherosclerosis prevention, treatment of genitourinary system disorders, immune system stimulation, and adaptogenic properties [20,21,22]. Hedysarum sikkimense Benth. ex Baker is used in Mongolian traditional medicine to relieve edema of various etiologies [23].
The broad medicinal usage of Hedysarum species correlates with their rich chemical composition, encompassing polysaccharides, flavonoids, triterpenoids, coumarins, alkaloids, fatty acids, and other compounds [3,24]. In particular, the antioxidant, anti-aging, antitumor, and antidiabetic properties of H. polybotrys are attributed to its polysaccharide content, comprising heteropolysaccharides identified as Hedysarum polysaccharides 1-4 [14,24]. However, standardization proposals advocate for evaluating the phenolic compound content in H. polybortys roots, including calycosin, medicarpin, formononetin, 3-hydroxy-9,10-dimethoxypterocarpan, liquiritigenin, quercetin, formononetin-7-O-β-D-glucoside, calycosin-7-O-β-D-glucoside, and genistein, indicating that these compounds are responsible for the pharmacological activity of the plant [25]. H. alpinum and H. flavescens aerial parts are notable sources of 2-C-β-D-(glucopyranoside)-1,3,6,7-tetraoxyxanthone, a derivative of mangiferin [26], serving as the active constituent in the antiviral drug Alpizarin® [17,27]. Moreover, these species boast a rich phenolic compound profile, comprising xanthones, flavonols, and hydroxycinnamic and hydroxybenzoic acids: mangiferin, hyperoside, rutin, quercetin 3-L-α-rhamnofuranoside, avicularin, hyperoside, polystachoside, hedyserite-1, gallic acid, chlorogenic acid, and others [17,27]. Several Hedysarum species serve as alternative sources of mangiferin, including H. caucasicum M.Bieb., H. grandiflorum Pall., and H. daghestanicum Rupr. ex. Boiss [28].
The vast medicinal potential of Hedysarum species opens up opportunities to broaden their therapeutic uses. However, historically, taxonomic classification of Hedysarum posed challenges owing to uncertainties regarding the genus’s monophyly [1,29]. In view of the repeated taxonomic revisions within the genus [1,29], the study of scientifically recognized species closely related to those widely used in medicine is of particular importance. One such species, Hedysarum semenowii Regel & Herder, remains underexplored despite its wide distribution in the Central Tien Shan region across Kazakhstan, Kyrgyzstan, and China’s Xinjiang province [30,31]. H. semenowii, belonging to the Hedysarum section, shares taxonomic proximity with well-established medicinal species such as H. polybotrys, H. alpinum, H. flavescens, H. neglectum, H. theinum, and H. caucasicum [32,33]. According to the literature, the mentioned species contains compounds such as ononin, formononetin, mangiferin, calycosin, afromosin-7-O-β-D-glucopyranoside, calycosin-7-O-β-D-glucopyranoside, afrormosin, medicarpin, hedysarimpterocarpene B, betulinic acid, guanosine, and daucosterol [3,31,32]. Despite the existence of some data on the compounds identified in H. semenowii, comprehensive studies on the detailed phytochemical profile of extracts derived from the roots and aerial parts of this plant are still lacking. Moreover, there is insufficient information on the correlation of these findings with data on antimicrobial and antioxidant activities. Therefore, we investigated the chemical composition and antimicrobial and antioxidant activity of H. semenowii. These investigations could provide new data for chemotaxonomic differentiation within the genus Hedysarum and expand the understanding of the pharmacologically relevant metabolites, as well as revealing the species’ potential for standardization and medicinal use.

2. Results

2.1. HPLC/PDA-ESI-QToF/MS-MS Analysis

Phytochemical investigations of the underground parts of Hedysarum spp. identified flavonoids as the main group of biologically active compounds. Among them, different subclasses have been reported, including flavones, isoflavones, flavanones, xanthones, and pterocarpans [3,34]. Knowledge of the qualitative composition and content of polyphenols present in different parts of H. semenowii is relatively poor; to our knowledge there are only two reports published so far [35,36]. Therefore, a detailed phytochemical profile of extracts, obtained from leaves and roots using different extraction solvents and extraction techniques, was conducted. As shown in Table 1, five extracts from the leaves of H. semenowii and three extracts from the roots were prepared according to the methods described in Section 4.2.
High-performance liquid chromatography (HPLC/PDA) combined with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QToF/MS-MS) enabled the identification of a total of 53 compounds in both the above- and underground parts of the plant. In terms of chemical diversity, flavones and isoflavones were the most representative group of polyphenols detected. Interestingly, their distribution differed significantly; flavones were typical of H. semenowii leaves, while isoflavones accumulated mainly in the roots, which could serve as potential chemotaxonomic markers to distinguish between these plant parts. The third group of bioactive constituents commonly found in the Hedysarum genus are pterocarpans [3], and the presence of medicarpin and its 3-O-glucoside was confirmed exclusively in the subterranean parts of the plant. Additionally, this study reports that the presence of xanthones, namely, mangiferin and its derivatives, were identified in both the roots and leaves of H. semenowii, and a detailed analysis of these compounds is presented below. Compounds from each extract were separated chromatographically and subjected to LC-MS analysis in both negative- and positive-ionization mode. Their identification was performed considering retention time and acquired chromatographic (specific UV absorption maxima) and spectrometric data (summarized in Table 2) compared with those reported in the literature and in publicly available MS databases (Human Metabolome Database—HMDB, https://hmdb.ca/) [37].
Table 2. Phytochemicals identified in extracts of Hedysarum semenowi using HPLC/PDA/ESI-QToF/MS-MS.
Table 2. Phytochemicals identified in extracts of Hedysarum semenowi using HPLC/PDA/ESI-QToF/MS-MS.
No.Compound Rt (min)Extract *Molecular FormulaExp. (m/z)Calcd.
(m/z)		Delta (ppm)Product Ions (m/z)References
1.Quinic acid1.98Hs_M99, Hs_M70, Hs_M50, Hs_Et50M, Hs_Et50UC7H12O6191.0557191.5612.14173.0416, 127.0364, 111.0409; 85.0273[37]
2.Sucrose1.97HsR_M99, HsR_M70, HsR_M50C15H18O9341.1076––179.0549; 161.0452; 149.0429; 119.0336[38]
3.Malic acid 2.14Hs_M99, Hs_M70, Hs_M50, HsR_M99, HsR_M70, HsR_M50, Hs_Et50MC4H6O5133.0138133.01423.33115.0029; 89.0231; 71.0136[37]
4.Citric acid2.62Hs_M99, Hs_M70, Hs_M50, HsR_M99, HsR_M70, HsR_M50, Hs_Et50MC6H8O7191.0175191.019711.59111.0085; 87.0090; 57.0348[37]
5.Isocitric acid4.05HsR_M99, HsR_M70, HsR_M50C6H8O7191.0196191.01970.66155.009; 111.0075; 87.0076[39]
6.Gallic acid4.62Hs_M99, Hs_M70, Hs_M50, Hs_Et50M, Hs_Et50UC7H6O5169.0128169.01428.51125.0166; 79.0155; 51.0226[37]
7.Hydroxybenzoic acid-O-glucoside
isomer 1 		5.32Hs_M99, Hs_M70, Hs_M50, HsR_M99, HsR_M70, HsR_M50, Hs_Et50M, Hs_Et50UC13H16O8299.0783299.0772−3.53137.0186; 93.0300[37]
8.Unknown7.03Hs_M99, Hs_M70, Hs_M50, HsR_M99, HsR_M70, HsR_M50, Hs_Et50M, Hs_Et50U-231.1315––213.1220; 195.1123; 185.1262
9.Dihydroxybenzoic acid hexoside 8.23Hs_M99, Hs_M70, Hs_M50, HsR_M99, HsR_M70, HsR_M50, Hs_Et50M, Hs_Et50UC13H16O5315.0710315.07223.66153.0271; 109.0274[37]
10.Hydroxybenzoic acid-O-glucoside
isomer 2		10.77Hs_M99, Hs_M70, Hs_M50, HsR_M99, HsR_M70, HsR_M50, Hs_Et50MC13H16O8299.0745299.07729.13137.0129; 93.0251[37]
11.Galloyl glucoside12.11Hs_Et50MC13H16O10331.0659331.06713.52313.0541; 169.0096; 168.0043; 149.9948; 125.0224[37]
12.Kaempferol-C-glucoside12.43HsR_M99, HsR_M70, HsR_M50C21H22O10433.1106433.1147.88343.0830; 313.0667; 285.0764; 283.0667; 151.0391fragmentation
13.Tetrahydroxyxanthone-di-O,C-hexoside (neomangiferin)12.89Hs_M99, Hs_M70, Hs_M50, Hs_Et50M, Hs_Et50UC25H28O16583.1284583.13053.52493.0971; 463.0866; 421.0732; 331.0447; 301.0347; 271.0306; 259.0215[40]
14.Mangiferin14.87Hs_M99, Hs_M70, Hs_M50, HsR_M99, HsR_M70, HsR_M50, Hs_Et50M, Hs_Et50UC19H18O11421.0780421.0776−0.86331.0394; 301.0287; 271.0273; 259.0161[40,41] r.s.
15.Isomangiferin16.08Hs_M99, Hs_M70, Hs_M50, Hs_Et50M, Hs_Et50UC19H18O11421.0744421.07767.66331.0471; 301.0366; 259.0259fragmentation; [41]
16.Quercetin-O-dihexoside18.89Hs_M99, Hs_M70, Hs_M50, Hs_Et50M, Hs_Et50UC27H30O17625.1393625.1412.75301.0338; 300.0279; 271.0217; 255.0315; 179.0028; 151.0007[42]
17.Quercetin derivative19.17Hs_M99, Hs_M70, Hs_M50, Hs_Et50M, Hs_Et50U–755.1970––301.0225; 300.0190; 271.0142; 255.0173; 178.9828; 150.9940fragmentation
18.Ferulic acid19.79HsR_M99, HsR_M70, HsR_M50C10H10O4193.0510193.0506−1.89179.0314; 178.0255; 149.0607; 134.0367fragmentation; [42]
19.Myricetin-O-glucoside isomer 120.23Hs_M99, Hs_M70, Hs_M50, Hs_Et50M, Hs_Et50UC21H20O13479.0836479.0831−1.01317.0279; 316.0221; 287.0190; 271.0244; 178.9987; 151.0049fragmentation
20.Calycosin-7-O-glucoside20.85HsR_M99, HsR_M70, HsR_M50C22H22O10491.1174 *491.11954.71283.0634; 268.0429; 211.0309; 184.0499fragmentation
21.Quercetin-O-galactoside21.16Hs_M99, Hs_M70, Hs_M50, Hs_Et50M, Hs_Et50UC21H20O12463.0897463.0882−3.23301.0368; 300.0295; 271.0262; 255.0310; 178.9994; 151.0041[43,44]
22.Quercetin-3-glucoside-7-rhamnoside21.41Hs_M99, Hs_M70, Hs_M50, Hs_Et50M, Hs_Et50UC27H30O16609.1473609.1461−1.95301.0282; 300.0190; 284.0272; 271.0198; 255.0251; 178.9949; 150.9998fragmentation
23.Kaempferol-O-dirhamnoside-glucoside21.68Hs_M99, Hs_M70, Hs_M50, Hs_Et50M, Hs_Et50U-739.1830––593.1187; 284.0193; 285.0321; 255.0178; 227.0255; 150.9893[45]
24.Quercetin-3-O-glucoside22.46Hs_M99, Hs_M70, Hs_M50, Hs_Et50M, Hs_Et50UC21H20O12463.0897463.0882−3.23403.0546; 343.0350; 301.0266; 178.9930; 150.9969[43,44]
25.Quercetin-3-O-arabinoside-7-glucoside22.94Hs_Et50MC26H28O16595.1278595.13054.46463.0855; 301.0284; 300.0226; 271.0184; 179.0165[46]
26.Rutoside23.48Hs_M99, Hs_M70, Hs_M50, Hs_Et50M, Hs_Et50UC27H30O16609.1473609.1461−1.95301.0316; 300.0227; 271.0230; 255.0296; 178.9974; 151.0001[47,48]
27.Nicotiflorin24.18Hs_M99, Hs_M70, Hs_M50, Hs_Et50M, Hs_Et50UC27H30O15593.1500593.15122.01285.0345; 284.0272; 255.0228; 227.0324; 150.9953[45]
28.Hyperoside24.66Hs_M99, Hs_M70, Hs_M50, Hs_Et50M, Hs_Et50UC21H20O12463.0841463.08828.83301.0368; 300.0295; 271.0262; 255.0310; 178.9994; 151.0041[49]
29.Pentahydroxyflavone-O-pentoside (Quercetin-O-pentoside)28.27Hs_M99, Hs_M70, Hs_M50, Hs_Et50M, Hs_Et50UC20H18O11433.0862433.07764.23301.0271; 300.0232; 271.0219; 255.0246; 243.0297; 178.9921; 151.0034[46]
30.Hexahydroxyflavone-O-hexoside (Myricetin-O-hexoside isomer 2) 28.51Hs_M99, Hs_M70, Hs_M50, Hs_Et50M, Hs_Et50UC21H20O13479.0808479.08314.82317.0135; 316.0078; 178.9947; 151.0001; 137.0207fragmentation
31.Tetrahydroxyflavone-
3-O-hexoside (Kaempferol-O-
hexoside) 		29.17Hs_M99, Hs_M70, Hs_M50, Hs_Et50M, Hs_Et50UC21H20O11447.0933447.0933−0.03285.0364; 284.0328; 255.0289; 151.00[45,47]
32.Tetrahydroxyflavone-3-O-hexoside-pentoside (Kaempferol-3-O-
hexoside-pentoside) isomer 2		29.44Hs_Et50MC27H30O15593.1474593.15126.39285.0345; 284.0272; 255.0354; 151.0004[45,47]
33.Isorhamnetin-3-O-
hexoside		30.07Hs_Et50M, Hs_Et50UC22H22O12477.1033477.10381.15447.0863; 315.0404; 314.0410; 271.0492; 151.0006fragmentation
34.Quercetin-3-O-
malonylglucoside		31.75Hs_Et50M, Hs_Et50UC24H22O15549.0917549.0886−5.65505.0982; 463.0928; 301.0329; 300.0272; 271.0245; 255.0308; 179.0063; 151.0041[45]
35.Pseudobaptigenin-O-
glucoside		32.93HsR_M99, HsR_M70, HsR_M50C22H20O10489.1021 *489.10383.94281.0468; 253.0516; 225.0576; 135.0144[49]
36.Ononin34.40HsR_M99, HsR_M70, HsR_M50C22H22O9475.1259 *475.1246−3.06267.0630; 252.0438[48]
37.Afrormosin-O-glucoside (wistin)36.29HsR_M99, HsR_M70, HsR_M50C23H24O10505.1339 *505.13512.72297.0775; 282.0533; 267.0293; 254.0803; 239.0333; 195.0482[50]
38.Quercetin-O-acetyl-
glucoside		36.57Hs_Et50M, Hs_Et50UC23H22O13505.1002505.09882.84445.0757; 427.0680; 343.0412; 301.0327fragmentation; [49]
39.Liquiritigenin38.14HsR_M99, HsR_M70, HsR_M50C15H12O4255.0656255.0663−2.66135.0086; 119.0492[42]
40.Calycosin40.62Hs_M99, Hs_M70, Hs_M50, HsR_M99, HsR_M70, HsR_M50, Hs_Et50M, Hs_Et50UC16H12O5283.0606283.06122.1268.0387; 224.0413; 211.00402; 195.0403; 184.0519; 148.0168; 135.0100; 120.0214[49]
41.Medicarpin-3-O-
glucoside		40.86HsR_M99, HsR_M70, HsR_M50C22H24O9477.1411477.1402−1.81431.1238; 269.0807; 254.0481; 161.0228; 133.0298; 132.0207[51]
42.Quercetin42.78Hs_M99, Hs_M70, Hs_M50, Hs_Et50M, Hs_Et50UC15H10O7301.0362301.0354−2.73273.0402; 257.0402; 229.0485; 178.9985; 151.0026; 149.0323; 121.0290; 107.0137[52,53]
43.Luteolin43.11Hs_Et50M, Hs_Et50UC15H10O6285.0428285.0405−8.17151.0004; 133.0224[37,54]
44.Isoformononetin43.30HsR_M99, HsR_M70, HsR_M50C16H12O4267.0659267.06631.43252.0393; 223.0393; 208.0530; 195.0461; 135.0315; 132.0214[55]
45.Naringenin
45.16Hs_M99, Hs_M70, Hs_M50, HsR_M99, HsR_M70, HsR_M50, Hs_Et50M, Hs_Et50UC15H12O5271.0616271.0612−1.48227.0837; 177.0212; 151.0034; 119.0478; 107.0177; 93.0314[42]
46.Sissotrin-4′-O-glucoside42.84HsR_M99, HsR_M70, HsR_M50C22H22O10491.1209 *491.1195−3.14445.1154; 283.0598; 268.0292[49]
47.Pseudobaptigenin50.06Hs_M99, Hs_M70, Hs_M50, HsR_M99, HsR_M70, HsR_M50, Hs_Et50M, Hs_Et50UC16H10O5281.0462281.0455−2.32253.0499; 251.0360; 223.0396; 195.0449; 135.0093[49,56]
48.Formononetin50.95Hs_M99, Hs_M70, Hs_M50, HsR_M99, HsR_M70, HsR_M50, Hs_Et50M, Hs_Et50UC16H12O4267.0654267.06633.29252.0389; 223.0407; 208.0541; 195.0457; 135.0251; 132.0215[49]
49.Afrormosin 51.37HsR_M99, HsR_M70, HsR_M50C17H14O5297.0743297.07688.54282.0533; 267.0293; 254.0803; 239.0333; 195.0482fragmentation
50.Isoliquiritigenin 52.03Hs_M99, Hs_M70, Hs_M50, HsR_M99, HsR_M70, HsR_M50, Hs_Et50M, Hs_Et50UC15H12O4255.0656255.06632.66135.0081; 119.0466;
91.0184		[42]
51.Irilone52.62HsR_M99, HsR_M70, HsR_M50C16H10O6297.0385297.0405−6.58282.438; 269.0455; 267.0289; 241.0502; fragmentation; [56]
52.Medicarpin54.13HsR_M99, HsR_M70, HsR_M50C16H14O4269.0790269.081910.86254.0598; 161.0279; 145.0306; 133.0288; 132.0223[51]
53.Biochanin A59.06HsR_M99, HsR_M70, HsR_M50C16H12O5283.0637283.0612−8.81268.0381; 240.0419; 239.0377; 211.0410; 224.0489; 240.0419; 195.0441; 183.0440; 163.0036; 151.0054; 135.0097; 132.0208; 109.0303[49]
* Hs_M99—H. semenowii leaf extract in methanol 99.8%, Hs_M70—H. semenowii leaf extract in methanol 70%, Hs_M50—H. semenowii leaf extract in methanol 50%, Hs_Et50M—H. semenowii leaf extract in ethanol 50% (maceration), Hs_Et50U—H. semenowii leaf extract in ethanol 50%50% (ultrasonification), HsR_M99—H. semenowii root extract in methanol 99.8%, HsR_M70—H. semenowii root extract in methanol 70%, HsR_M50—H. semenowii root extract in methanol 50%, *—[M + HCOO]−, r.s.—identity of the compound additionally confirmed using a reference standard.

2.1.1. Isoflavones

Isoflavonoids are specialized metabolites characterized by biological activity resembling that of the endogenous hormone 17-β-oestradiol. These compounds have been previously detected in underground parts of several Hedysarum spp. Our results are in accordance with data reported in the literature, as the presence of formononetin (48), calycosin (40), afrormosin (49) and their corresponding O-glucosides, ononin (36), calycosin-7-O-glucoside (20) and wistin (37), has been confirmed exclusively in H. semenowii roots [3,34]. Aglycones of isoflavones were characterized by the successive loss of small moieties from the deprotonated [M-H]− ions. The identification of retro-Diels-Alder (rDA) reaction products (rDA product ions) provided information on the number and type of substituents attached to the aromatic rings [57]. The precursor ions of isoflavone O-glucosides were observed as solvent adducts [M+HCOO]−. Cleavage of the O-C bond (−162 Da) in their structure resulted in the formation of an intense peak in the MS/MS spectra, corresponding to the free aglycone. The elution order of chromatographically separated isoflavones was similar to that previously described by Maciejewska-Turska and Zgórka (2022) [49]. Among the identified isoflavones, formononetin (m/z 267.0654) and ononin (adduct ion at m/z 475.1259) were the major compounds. Minor peaks for biochanin A (53), pseudobaptigenin (47), and their corresponding glucosides, sissotrin-4′-O-glucoside (46) and pseudobaptigenin-O-glucoside (35), were identified based on our experience and information obtained from online MS databases [37]. Two compounds (49 and 51) had a similar [M-H]− deprotonated molecular ion at m/z 297. Compound 49, eluting at 51.37 min, generated a precursor ion at m/z 297.0743, consistent with the molecular formula of C17H14O5. The MS/MS spectrum of this compound contained abundant product ions at m/z 282.0533 and 267.0293, which matched the fragmentation pattern typical of methoxylated isoflavones (−15 Da). Among known isoflavones bearing two methoxyl groups, previously reported in the genus Hedysarum, afrormosin was proposed as the most likely structure [3]. For compound 51, the product ion at m/z 297.0385 suggested a molecular formula of C16H10O6. An intense product ion at m/z 269.0455 corresponded to the loss of 30 Da, prevalent among methylenedioxy-substituted isoflavones, such as pseudobaptigenin or irilone [56].
Therefore, compound 49 was assigned as afrormosin, whilst compound 51, with a later elution, could be assigned as irilone. For compound 44, a similar fragmentation pattern to formononetin was observed, and consequently it was proposed to be isoformononentin.

2.1.2. Flavonols, Flavones, and Flavanones

The flavonols identified in the extracts obtained from leaves of H. semenowii were represented mainly by quercetin and less abundantly by kaempferol derivatives (Table 2). Small shifts in their absorption maxima, together with diagnostic fragmentation ions observed in their MS/MS spectra at m/z 301 and 285, suggested that the aglycone cores originated from quercetin or kaempferol, respectively [45]. Product ions (1.3A− and/or 1.3B−) arising from rDA cyclization were found to be the most useful for flavonoid identification. Quercetin (42) and eleven of its glycosides (16, 17, 21, 22, 24, 25, 29, 34, and 38) were confirmed to be present. The MS spectrum of compound 42 at m/z 301.0362 (C15H10O7) and a series of fragmentation ions at m/z 273.0402 [M-H-CO]−, 257.0402 [M-H-CO2]−, and 229.0485 [M-H-CO-CO2]−, together with rDA ions at m/z 178.9985 (1.2A−), 151.0026 (1.3A−), 121.0290 (1.2B−), and 107.0137 (0.4A−), supported the quercetin structure [52,53]. A key step in the identification of quercetin glycosides was the observation of neutral losses of 162 Da or 146 Da, indicating the presence of glucose or rhamnose substituents, respectively. For these compounds, the aglycone was recorded at m/z 301.03, while similar fragmentation ions in their MS/MS spectra to those described for quercetin confirmed the quercetin core [46,52]. As a result, quercetin-O-dihexoside (16), quercetin derivative (17), quercetin-3-glucoside-7-rhamnoside (22), quercetin-3-O-arabinoside-7-glucoside (25), rutoside (26), hyperoside (28), and quercetin-O-pentoside (29) were tentatively identified [45]. Since the use of spectrometric (MS) techniques alone does not provide information on the specific type of hexose substituent, the elution behavior and available literature data on the flavonoid profile of several Hedysarum species allowed the preliminary identification of compound 21 as quercetin-3-O-galactoside, and compound 24 was eluted later as quercetin-3-O-glucoside, previously noted in methanolic extracts of Hedysarum vicioides leaves [43,44,45]. In addition, one quercetin-3-O-malonylglucoside (34) and one quercetin-O-acetyl-glucoside (38) were also identified in the aboveground parts of the plant, based on the characteristic losses of malonyl (−86 Da and −44 Da) and acetyl (−42 Da) moieties [45]. Compounds 12, 23, 27, 31, and 32 were tentatively identified as kaempferol glycosides, namely, kaempferol-C-glucoside, kaempferol-O-dirhamnoside-glucoside, nicotiflorin, kaempferol-O-hexoside, and kaempferol-3-O-hexoside-pentoside (an isomer of nicotiflorin), respectively [45,47]. Diagnostic fragment ions observed in their MS/MS spectra at m/z 285.0667 and 151.0391 (1.3A−) indicated that these compounds originated from kaempferol rather than luteolin. In terms of compound 12, the generated precursor ion at m/z 433.1106 was in accordance with the empirical molecular formula of C21H22O10. The most intensive product ions at m/z 343.0830 [M-H-90]− and 313.0667 [M-H-120]− confirmed that the hexose unit was attached to the aglycone core via a C-C bond and underwent cross-ring cleavage, characteristic of C-glycosides [54]. Two isomers of myricetin-O-glucoside (19, 30) and one peak identified as isorhamnetin-3-O-hexoside (33) were also noted. Luteolin (43) was detected solely in ethanolic (50%, v/v) extracts of the aboveground parts of H. semenowii. In MS/MS spectra, a precursor ion observed at m/z 285.0428 and two distinctive rDA product ions at m/z 151.0004 and 133.0224, obtained through 10-eV collision-induced dissociation (CID), confirmed the structure of luteolin [54]. In addition, one flavanone, namely, naringenin (45), was identified by comparing our recorded elution behavior and UV-vis and MS spectra with information available in the literature [42].

2.1.3. Chalcones

According to the LC/MS results, it was proposed that two compounds (39 and 50) were chalcone-type natural products. Compound 39, detected solely in root extracts, yielded a deprotonated molecular ion at m/z 255.0656 (C15H12O4), which upon further fragmentation produced two major fragment ions at m/z 135.0086 and 119.0492. Compound 50 shared a similar fragmentation pathway to that of compound 39, consistent with publicly available MS data reported for liquiritigenin and isoliquiritigenin [42,58]. Based on their retention behavior, compound 39, eluting at 38.14 min, was assigned as liquiritigenin, while compound 50, identified in all analyzed samples, was assigned as isoliquiritigenin. These findings are in agreement with previously reported data for several Hedysarum spp. [3].

2.1.4. Pterocarpans

Compound 52, due to its apolar structure, eluted at 54.13 min and yielded a dominant [M-H]− precursor ion at m/z 269.0775, which was in accordance with the empirical molecular formula C16H14O4. An intense fragment ion at m/z 254.0598 suggested the loss of one CH3 group. Further fragmentation corresponded well with data reported for medicarpin, previously identified in Coffea leaf extracts by Wang et al. (2019) [51] and in several Hedysarum species [34]. These authors suggested that the product ions observed in MS/MS spectra may correspond to the cleavage of 6.8 (m/z 161.0279) 6.7 (m/z 145.0306), and 3.4.7 (m/z 133.0288) bonds in the A-ring and a 3.5 bond in the D-ring (m/z 132.0223) of medicarpin. Based on these data and the MS spectrum of compound 52, detected exclusively in roots, it was assigned as medicarpin. Similar fragmentation behavior was observed for compound 41. The mass difference between the adduct ion [M+HCOO]− at m/z 477.1367 (C22H24O9) and the predominant product ion at m/z 269.0807 resulted from the typical neutral loss of one hexose moiety. Moreover, the experimentally determined accurate mass of the fragment ion at m/z 269.0807 was in good agreement with the proposed molecular formula of C16H14O4, corresponding to the medicarpin core rather than a trihydroxyflavone structure. Therefore compound 41 was assigned as medicarpin-3-O-glucoside.

2.1.5. Xanthones

Regarding xanthones, three specific high-intensity absorption maxima in the range of 250–370 nm were observed [59]. Compound identification was performed by comparing fragmentation ions formed after CID with the literature MS data. Compound 14 yielded a deprotonated ion [M-H]− at m/z 421.0828, which was consistent with the empirical molecular formula of C19H18O11. The most abundant ions at m/z 331.0471 and 301.0366 were formed due to the losses of 120 Da and 90 Da, respectively, from the precursor ion through cross-ring cleavage of the sugar moiety [48]. It was therefore postulated that the substitution of one hexose moiety was via a C-C bond. Further fragmentation resulted in fragment ions consistent with data published by Trevisan et al. [60] for a specific C-glycosylated xanthone, namely, mangiferin, registered under the name Alpizarin®® in the Russian Federation [41]. Comparison of spectroscopic and spectrometric data with the MG, reference standard, enabled the identification ofcompound 14 as mangiferin. To our knowledge, this is the first study to report the presence of mangiferin in both H. semenowii aerial and underground organs. Compounds 13 and 15, observed exclusively in the aerial parts of H. semenowii, shared similar fragmentation behavior to mangiferin. The lack of prominent product ion at m/z 271 in the MS/MS spectra of compound 15 suggested a structural isomer of mangiferin, tentatively identified as isomangiferin [61]. Regarding compound 13, the mass difference between the precursor ion [M-H]− at m/z 583.1284 (C25H28O16) and two major fragmentation ions at m/z 493.0971 [M-H-90]− and 463.0866 [M-H-120]− supported the presence of a sugar at the C-2 position, whilst a product ion at m/z 421.0732 [M-H-162]− could be attributed to the cleavage of an O-glucosidic bond. Less abundant product ions typical of mangiferin were also detected. According to the literature data, compound 13 could correspond to neomangiferin, a naturally occurring xanthone derivative with antidiabetic activity, bearing both O- and C-glucosidic residues [40]. However, data obtained solely from MS/MS spectra were insufficient to unambiguously determine the position of the glycosylation site, so compound 13 was tentatively assigned as a tetrahydroxyxanthone-di-O, C-hexoside [61].

2.1.6. Phenolic Acids and Other Compounds

Out of the six phenolic acids identified in H. semenowii, one was dihydroxybenzoic acid hexoside (9) and two were isomers of hydroxybenzoic acid-O-glucoside (7 and 10). Ferulic acid (18) was identified exclusively in the roots, while gallic acid (6) and its glucoside (11) were detected only in the aerial parts.
In addition, several organic acids, such as malic (3) and citric acid (4), were present in all samples. Quinic acid (1), with a precursor ion at m/z 191.0513, was noticed in the aboveground parts, whilst isocitric acid (5) with an [M-H]− at m/z 191.0196 was detected solely in the roots [39].

2.2. Quantification of Mangiferin and Isomangiferin via RP-LC/PDA

Preliminary RP-LC/PDA analysis revealed that mangiferin was the dominant compound in extracts obtained from the aerial parts of Hedysarum semenowii. For mangiferin determination, the aforementioned H. semenowii samples were injected in triplicate and the results are presented in Table 3 as the mean of three independent analyses, expressed as the quantity per 1 g of the extracts [mg/g] ± standard deviation with relative standard deviation. Quantitative analysis showed considerable variation in mangiferin content among the extracts, with almost three-fold higher concentration in the ethanolic (50%, v/v) extract compared to the others (Figure S1). Comparison of the mangiferin concentrations in samples Hs_M99, Hs_M70, and Hs_M50 indicated that the alcohol concentration used during the extraction process did not significantly affect the amount of mangiferin detected. The content of isomangiferin was calculated using the calibration curves of mangiferin, due to the similarity of their UV-vis spectra and chromatographic behavior. Isomangiferin levels were relatively low and, like with mangiferin, were highest in ethanolic extracts. The amount of neomangiferin was undetectable in all samples.

2.3. Antibacterial and Antifungal Activity

The present study evaluated the antimicrobial and antifungal potential of extracts of Hedysarum semenowii (Table 4 and Table 5). Aerial part extracts demonstrated higher antibacterial activity compared to root extracts, as evidenced by lower Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values against various microorganisms.
Against Gram-positive reference bacteria from the Staphylococcus genus, extracts from H. semenowii leaves exhibited notable activity, with MIC values ranging from 0.5 to 4 mg/mL. Particularly, a significant inhibitory effect was observed against S. epidermidis ATCC 12228. Additionally, leaf extracts displayed considerable activity against the Gram-negative P. aeruginosa ATCC 27853 (MIC = 1–2 mg/mL), notably higher than for E. coli ATCC 25922 (MIC = 8 mg/mL).
Among the tested samples, root extracts exhibited noteworthy activity against S. epidermidis ATCC 12228; interestingly, the activity increased proportionally with the concentration of methanol used as the extraction solvent. However, their efficacy against other bacterial strains was comparatively lower, requiring MIC ≥ 4 mg/mL for effective inhibition.
In terms of antifungal activity against Candida species, both underground and aboveground part extracts demonstrated comparable efficacy, with MIC values ranging from 4 to 16 mg/mL.
Calculated MBC/MIC and MFC/MIC ratios indicated that extracts demonstrated bactericidal and fungicidal properties.
These findings underscore the differential antimicrobial and antifungal properties exhibited by various parts of H. semenowii, with the leaves demonstrating superior efficacy against bacterial pathogens, while both underground and aboveground parts displayed comparable activity against fungal strains.
Statistical analysis showed that the studied H. semenowii extracts varied significantly in terms of their activity (MIC values) towards certain bacterial reference strains; however, these differences were not observed in the case of antifungal activity (Table 6). Despite significant differences, post hoc pairwise comparisons only allowed us to identify specific group differences in antimicrobial efficacy in two species. The analysis showed that these differences were visible for antistaphylococcal activity of Hs_M99 and HsR_M50 extracts against S. aureus ATCC 29213, as well as for activity against B. cereus ATCC 10876 between Hs_Et50M and HsR_M50.

2.4. Antioxidant Activity

The antioxidant activity of the extracts of H. semenowii was evaluated, and notable differences in antioxidant potential between the leaves and root extracts were observed.
Extracts from the aerial part (leaves) displayed varying degrees of antioxidant activity (Table 7). Notably, ethanolic extracts (Hs_Et50M and Hs_Et50U) showcased strong antioxidant potential, with Antioxidant Activity Index (AAI) values of 2.07 ± 0.13 and 1.49 ± 0.26, respectively. Extracts obtained with methanol exhibited different levels of activity, as Hs_M99 showed strong antioxidant activity with an AAI of 1.6 ± 0.2, while Hs_M70 demonstrated moderate activity with an AAI of 0.90 ± 0.09. Conversely, Hs_M50 displayed poor antioxidant activity, as indicated by an AAI of 0.37 ± 0.05.
In contrast, extracts derived from H. semenowii roots exhibited consistently low antioxidant activity across all samples during initial screening. Thus, these results were not included in this work. These findings underscore the differential antioxidant capabilities of H. semenowii extracts, with leaf extracts displaying a spectrum of antioxidant activity, particularly high in ethanolic extracts, while root extracts showed uniformly low antioxidant potential.

3. Discussion

Phytochemical analysis of Hedysarum semenowii extracts revealed a range of phytochemicals, consisting predominantly of flavones, isoflavones, xanthones, and pterocarpans. These results mainly support previously published studies on this plant and its closely related species, as well as reports indicating that extraction using moderately polar solvents (50–70% methanol or ethanol) provides optimal yields of biologically active constituents [3,32,48].
The H. semenowii leaf extracts were rich in flavonols and xanthones, such as quercetin, kaempferol, mangiferin, and their derivatives. Flavonols are a major subclass of polyphenols known for their antioxidant activity, primarily due to the presence of phenolic hydroxyl groups capable of donating hydrogen atoms to free radicals [62]. Quercetin, kaempferol, luteolin, and their glycosides identified in the analyzed extracts are known to have significant biological activities, particularly against diseases and conditions related to aging, inflammations, tumors, and oxidative stress [21,63,64,65]. Quercetin and its derivatives have displayed significant antioxidant activity compared to butylated hydroxytoluene and Allium cepa extract in DPPH• scavenging and ferric reducing ability in plasma (FRAP) tests [66]. Similarly, kaempferol and its derivatives, as well as plant extracts containing these compounds, have shown high antioxidant activity in DPPH•, superoxide radical, and nitric oxide (NO) inhibition assays, with results comparable to those of ascorbic acid [67,68]. The ability of these compounds to counteract oxidative stress plays a key role in their therapeutic potential against autoimmune, cardiovascular, and certain neurological diseases, as well as various cancer types [67].
Mangiferin and its derivatives, previously reported to be present only in the aerial parts of Hedyrasum L. species [31], were also detected in the roots of H. semenowii, although in significantly lower quantities compared to the leaves. The antiviral activity of mangiferin and isomangiferin is well-documented [17,27]. Some studies report their efficacy to be greater than that of acyclovir, idoxuridine, and cyclocytidine against herpes simplex virus, with isomangiferin demonstrating superior activity [69]. In addition to their antiviral properties, mangiferin and its derivatives have shown moderate to mild antimicrobial activity against strains such as Staphylococcus aureus, Escherichia coli, Candida albicans, and Aspergillus niger, as evidenced by disk diffusion assays [70]. Furthermore, these compounds are known for a broad spectrum of pharmacological effects, including antioxidant, antidiabetic, anti-inflammatory, and anticancer activities [71].
According to this study, the identified isoflavones, pterocarpans, and chalcones were found exclusively in the root extracts of H. semenowii. Isoflavones such as formononetin, calycosin, and afrormosin are known phytoestrogens, typically present in subterranean parts of plants belonging to the Fabaceae family, with Astragalus membranaceus being a primary source [72,73]. Their biological activity is associated with binding to estrogen receptors, which contribute to their potential effects against various cancer types, as well as their anti-inflammatory, neuroprotective, and hepatoprotective properties [72,73]. Medicarpin, a pterocarpan, has been reported to exhibit anti-osteoporotic activity by suppressing osteoclastogenesis in bone marrow cells [74]. It also shows antifungal activity against Trametes versicolor and antigonococcal activity against Neisseria gonorrhoeae [75,76] and neuroprotective effects [77]. Chalcones identified in the root extracts, such as liquiritigenin and isoliquiritigenin, are known to exhibit hepatoprotective and anti-inflammatory effects, the latter one of which is primarily attributed to their ability to reduce the production of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines [78,79].
Notably, this study reports for the first time the presence of ferulic, malic, quinic, citric, and isocitric acids in a species of Hedysarum L., specifically, H. semenowii [3]. These compounds, commonly found in various medicinal and edible plants, are known for their antioxidant, anti-inflammatory, and metabolism-regulating properties. For instance, ferulic acid has demonstrated strong radical-scavenging activity and is associated with cardiovascular and neuroprotective effects [80,81,82]. Organic acids such as malic and quinic acids have also been linked to antioxidant and antimicrobial activities [83,84]. Similarly, citric and isocitric acids are well-known for their antioxidant potential, with citric acid widely used in the pharmaceutical industry as a metal-chelating and pH-regulating agent [85,86]. The detection of these organic acids reveals a broader phytochemical profile in H. semenowii and plants of Hedysarum L., as well as highlighting the potential of this underexplored species as a valuable source of bioactive compounds.
However, despite literature reports documenting the presence of compounds such as hedysarimpterocarpene B, betulinic acid, guanosine, and daucosterol in H. semenowii [3], these compounds were not detected in the analyzed extracts. Their absence may be attributed to several factors, including differences in the collection region or vegetative stage, the extraction methodology used, the concentration of the compounds, or variations in sample preparation and taxonomic identification [1,29]. This study was conducted using a single population sample of H. semenowii, which represents a limitation, as phytochemical composition may vary among different ecological sources.
When analyzing the antimicrobial and antioxidant activities of H. semenowii extracts, it was found that the leaf extracts exhibited more pronounced effects compared to those obtained from the roots. These effects align with the known properties of the compounds identified in the extracts [21]. The root extracts of H. semenowii contained isoflavonoids, pterocarpans, and chalcones, which, according to published data, are not typically associated with strong antimicrobial or antioxidant properties. Instead, they are more recognized as phytoestrogens and anti-osteoporotic agents, with potential applications in the treatment of inflammatory conditions and chronic diseases such as cancer [3,34,72]. Although some studies have reported on medicarpin’s antimicrobial activity, its efficacy is considered relatively weak in comparison to flavonols [75].
The results of this study emphasize that the biologically active phenolic profile of H. semenowii is more concentrated in the aerial parts, which demonstrated superior performance in antioxidant and antimicrobial assays. Even though the biological activity assays used in this study were based on simplified conventional methods and do not reflect the complexity of in vivo activity mechanisms, they provide a useful preliminary screening of the comparative activity of extracts from different parts of the plant. This suggests that aboveground biomass of the plant, being more accessible for harvesting, may serve as a promising natural source of bioactive compounds with these properties. Conversely, the roots, as a source of phytoestrogens, may be better suited to potential therapeutic applications related to hormone modulation.
Given the phytochemical similarities between H. semenowii and other well-studied Hedysarum species such as H. alpinum, H. flavencens, and H. polybortys, these findings support the idea that this underexplored species holds significant pharmacological potential. Future research should prioritize the isolation of individual compounds for activity-guided studies, assessment of synergistic interactions, and in vivo evaluation of therapeutic safety and efficacy.

4. Materials and Methods

4.1. Plant Material

The leaves of Hedysarum semenowii Regel & Herder were collected during the flowering period in July 2023 from mountainous areas of the Kungey Alatau ridge, Kegen district, Almaty region, Republic of Kazakhstan (coordinates: N 43°00′18″, E 78°22′58.6″, elevation 1740 m), when aboveground parts had reached maximum biomass and metabolite content. Subsequently, root specimens of H. semenowii were collected in the same areas in September 2023, following the conclusion of the flowering period and accumulation of secondary metabolites later in vegetation. The collected samples underwent identification by specialists from the Republican State Enterprise with the Right of Economic Management (RSE REM) “Institute of Botany and Phytointroduction”, which operates under the Forestry and Wildlife Committee Ministry of Ecology, Geology and Natural Resources of the Republic of Kazakhstan. Identification was conducted using morphological characteristics and other relevant methods.

4.2. Extraction

Eight extracts of H. semenowii were prepared from both leaves and roots. Aerial parts were finely chopped and subjected to an extraction process using anhydrous methanol, 70% methanol, and 50% methanol (v/v) through ultrasonic maceration. The maceration was performed at room temperature with three 30 min cycles of ultrasonic treatments each time, using a fresh portion of the solvent at a ratio of 1:10 (weight/volume). All extracts were concentrated to dryness using a rotary evaporator at a temperature not exceeding 50 °C. Similarly, H. semenowii root extracts were prepared following the same procedure.
In addition, two types of aerial part extracts were prepared. The first extract was obtained through maceration using 50% ethanol at room temperature, while the second extract additionally underwent ultrasonic treatment in three short 5 min pulses. Both extracts were subsequently freeze-dried to dryness. The abovementioned solvents were chosen to represent different levels of polarity, enabling the extraction of a broad range of constituents, as well as the comparison of extraction efficiency and solvent selectivity.

4.3. LC and LC-MS Analysis

4.3.1. Chemical Reagents

HPLC-grade solvents (acetonitrile and formic acid) were supplied by Sigma Aldrich (Steinheim, Germany). Purified water (18.2 MΩ) was prepared by the Direct-Q system (Millipore, Molsheim, France). Solvents of spectroscopic purity, used for LC/PDA/ESI-QToF/MS-MS analysis (acetonitrile, formic acid, and water), were provided by J.T. Baker (Gross-Gerau, Germany). All other chemicals were of analytical reagent grade. The reference standard of mangiferin (MG) used for qualitative analysis had purity greater than 93% and was purchased from ChromaDex Inc. (Santa Ana, CA, USA). A stock solution of mangiferin was prepared by dissolving the standard in 50% methanol (HPLC-grade) to obtain a concentration of 0.2 mg/mL.

4.3.2. RP-LC/PDA Profiling

Extracts were analyzed both quantitatively and qualitatively using an Agilent 1100 (Agilent Technologies, Inc., Santa Clara, CA, USA) chromatograph equipped with an autosampler, a photodiode-array detector (DAD), and a thermostatic column. Separation of polyphenols was carried out on a Zorbax Eclipse XDB-C18 column (250 × 4.6 mm I.D., dp = 5 μm) at 25 °C. An optimized gradient of the mobile phase, consisting of acetonitrile (B) (v/v) and water (A), with the addition of 0.1% formic acid to both eluents, was used as follows: 0/10; 13/25; 15/95; isocratic run at 95% from 15 to 20 min and back to 10% in 25 min/%B in A. The flow rate was set to 1 mL/min, and a sample injection volume of 10 μL was used. The identification of compounds was based on their specific UV spectra recorded at 254 nm and retention time. The concentration of mangiferin (MG) in extracts from Hedysarium semenowii leaves was quantitatively determined using the external standard method. The content of its structural isomer, isomangiferin, was calculated as mangiferin, due to their nearly identical UV-vis spectra and chromatographic behavior. For this purpose, a set of methanolic solutions of MG was prepared in triplicate at different concentrations, ranging from 0.0125 to 0.2 mg/mL. A five-point calibration curve was constructed (peak areas versus increasing concentrations of reference solution). The calibration curves obtained were highly linear, with a correlation coefficient (R2) value of 0.9993 within the test ranges.
Regarding the method precision, the standard solution of MG was prepared in triplicate at a concentration of 0.0125 mg/mL. The prepared solutions were injected on the same day and on three consecutive days of the study for the determination of intra- and interday precision. The precision, expressed as the relative standard deviation (RSD, %) was below 2%, which confirmed the high stability of the method (Table 8). The lowest concentration of the MG standard was diluted with methanol and injected at a volume of 10 µL to determine the limit of detection (LOD) and limit of quantification (LOQ). Under the optimized chromatographic conditions, the LOD was defined as a signal-to-noise ratio (S/N) of 3:1 and the latter as an S/N of 10:1.

4.3.3. RP-LC/DAD/ESI–QToF/MS/MS Analysis

Extracts were subjected to LC-MS analysis performed on an Agilent 1260 Infinity chromatograph connected to a 6530B Accurate Mass QTOF/MS mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). As a soft ionization technique, the ESI ion source operated in both positive- and negative-ionization modes. The chromatographic separation was attained on a Zorbax SB-C18 narrow-bone column (2.1 × 150 mm, dp = 3.5 μm). The chromatographic and MS parameters were applied as previously described by Maciejewska-Turska, M., & Zgórka, G. [49]. Data acquisition was performed using MassHunter Qualitative Navigator B.08.00 software. Compounds of interest were tentatively identified based on retention time, UV spectra, and self-analysis of their potential fragmentation patterns, supported by the comparison of obtained MS/MS spectra with records found in the literature or publicly available databases like Metlin (https://metlin.scripps.edu) [87] and HMDB (https://hmdb.ca) [37]. The identification of mangiferin isomers was performed based on the fragmentation pattern of the mangiferin reference standard.

4.4. Antimicrobial Evaluation

The antimicrobial activity of the H. semenowii extracts was tested using the microbroth dilution method according to the European Committee on Antimicrobial Susceptibility Testing [88], against a panel of reference microorganisms. The method involves determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) of the extracts and categorizing their action as bactericidal/fungicidal (MBC/MIC ratio or MFC/MIC ratio ≤ 4) or bacteriostatic/fungistatic (MBC/MIC ratio or MFC/MIC ratio > 4). The panel of microorganisms consisted of the reference strains from the American Type Culture Collection (ATCC): Staphylococcus aureus ATCC 29213 (MSSA; methicillin-susceptible S. aureus), S. epidermidis ATCC 12228, Enterococcus faecalis ATCC 29212, Bacillus cereus ATCC 10876, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 and fungi (yeasts) Candida albicans ATCC 10231 and C. glabrata ATCC 90030. Reference strains were selected to encompass clinically relevant bacteria and yeasts that reflect the broad diversity of pathogens encountered in healthcare settings. Gram-positive representatives included S. aureus, chosen for its virulence factor production and frequent involvement in skin infections, S. epidermidis, notable for biofilm-associated pathogenicity, E. faecalis, selected owing to intrinsic antibiotic resistance, and B. cereus, recognized for its endospore formation and role in foodborne disease. Gram-negative bacteria were represented by E. coli (Enterobacteriaceae), chosen for its clinical significance, and Pseudomonas spp., chosen for its high intrinsic resistance to antimicrobials. C. albicans and C. glabrata were included for their prevalence in human candidiasis and distinct antifungal resistance profiles. This strain selection ensured comprehensive and clinically relevant assessment of antimicrobial activity. The detailed methodology of the assay was outlined in detail in prior studies [89]. All experiments were carried out in triplicate, and the values are presented as the mode.
Statistical analysis was conducted to determine whether certain extract types exhibit different activity towards tested reference microorganisms. The analysis was conducted using a non-parametric Kruskal–Wallis test (significance set as p < 0.05) followed by a post hoc Dunn’s multiple comparisons test (GraphPad Prism version 6.0 for Windows; GraphPad Software, Boston, MA, USA).

4.5. DPPH• Radical Scavenging Assay

The antioxidant activity of H. semenowii extracts was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH•) radical scavenging method according to the methodology described in prior studies [89]. An initial test solution was prepared by dissolving 20 μL of extract in 180 μL of methanol, followed by a series of dilutions in the same solvent at concentrations ranging from 0.16 to 10 mg/mL. Each dilution was then combined with a methanol solution of DPPH• in a 96-well plate, which was incubated in the dark at room temperature for 30 min. For each extract, the concentration that scavenged 50% of the initial DPPH• radicals (EC50) and antioxidant activity index (AAI) were calculated based on absorbance measurements at 515 nm using a Biotek Epoch Microplate Spectrophotometer (Winooski, VT, USA; Software Version 3.08.01). AAI values were then compared to the criteria for assessment of antioxidant activity proposed by Sherer and Godoy [90] according to which AAI < 0.5—weak antioxidant activity, 0.5 < AAI < 1.0—moderate activity, 1.0 < AAI < 2.0—strong activity, AAI > 2.0—very strong activity. The experiments were conducted three times, and the results were reported as mean values with standard deviation (±SD).

5. Conclusions

The chemical composition of Hedysarum semenowii extracts was investigated by thorough comprehensive phytochemical analysis using HPLC/PDA and ESI-QToF/MS-MS. A total of 53 compounds were identified in extracts from both the aerial and underground parts of the plant, with flavonoids representing the most abundant class of bioactive substances. Their distribution was organ-specific—flavones predominated in the aboveground parts, while isoflavones were mainly concentrated in the roots. Flavones and isoflavones were the most representative polyphenols, highlighting their potential as chemotaxonomic indicators. Furthermore, xanthones (e.g., mangiferin and its derivatives) were found in extracts from the entire H. semenowii plant, including both roots and aerial parts, for the first time.
Preliminary evaluation of the antioxidant and antibacterial activities of H. semenowii extracts revealed that the aerial parts exhibited higher activity compared to the roots, which correlates with differences in their phytochemical composition. However, the presence of isoflavonoids and pterocarpans in H. semenowii roots suggests potential for further studies on biological activities related to their phytoestrogenic properties.
The results of this study provide comprehensive information on the phytochemical composition of H. semenowii, which may help distinguish this species from others and demonstrate its potential as a source of diverse pharmacologically active substances, depending on the plant part. Moreover, they emphasize the significance of further biological analyses and future potential pharmaceutical and medical applications.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/molecules30234503/s1, Figure S1: RP-LC/PDA chromatogram of major compounds detected at λ = 254 nm in Hedysarum semenowii extracts.

Author Contributions

Conceptualization, A.L. and A.K.; collection and identification of plant material, A.K., L.I., Z.S. and O.S.; extraction, A.K., L.I., Z.S. and A.L.; chromatographic analysis, M.M.-T., A.L. and A.K.; antimicrobial evaluation, M.K. and A.K.; DPPH radical scavenging assay, A.K. and T.B.; data curation, M.M.-T., M.K. and A.L.; writing—original draft preparation, A.K., M.M.-T. and M.K.; writing—review and editing, A.L. and A.K. visualization, A.K., M.K. and M.M.-T.; supervision, A.L. and Z.S.; project administration, A.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Statutory Funds DS25 of the Medical University of Lublin.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Table 1. Extracts prepared for phytochemical analysis of H. semenowii.
Table 1. Extracts prepared for phytochemical analysis of H. semenowii.
Part of the PlantExtraction SolventDesignated Abbreviation
LeavesMethanol 99.8%Hs_M99
Methanol 70%Hs_M70
Methanol 50%Hs_M50
Ethanol 50% (maceration)Hs_Et50M
Ethanol 50% (ultrasonification)Hs_Et50U
RootsMethanol 99.8%HsR_M99
Methanol 70%HsR_M70
Methanol 50%HsR_M50
Table 3. The content of mangiferin and isomangiferin in the extracts from H. semenowii leaves.
Table 3. The content of mangiferin and isomangiferin in the extracts from H. semenowii leaves.
CompoundExtract Number *
[mg/g Extract] and (SD; RSD)
Hs_M99Hs_M70Hs_M50Hs_Et50MHs_Et50U
mangiferin1.52
(0.018; 1.235)		1.89
0.062; 3.313)		2.00
(0.005; 0.259)		4.12
(0.017; 0.425)		5.46
(0.014; 0.256)
isomangiferin0.19
(0.004; 2.663)		0.10
(0.003; 3.331)		0.18
(0.002; 11.298)		0.59
(0.024; 4.178)		0.61
(0.004; 0.789)
* Hs_M99—H. semenowii leaf extract in methanol 99.8%, Hs_M70—H. semenowii leaf extract in methanol 70%, Hs_M50—H. semenowii leaf extract in methanol 50%, Hs_Et50M—H. semenowii leaf extract in ethanol 50% (maceration), Hs_Et50U—H. semenowii leaf extract in ethanol 50%50% (ultrasonification), HsR_M99—H. semenowii root extract in methanol 99.8%, HsR_M70—H. semenowii root extract in methanol 70%, HsR_M50—H. semenowii root extract in methanol 50%.
Table 4. Antibacterial and antifungal activity of the extracts from H. semenowii (mg/mL) leaves.
Table 4. Antibacterial and antifungal activity of the extracts from H. semenowii (mg/mL) leaves.
Reference MicroorganismHs_M99Hs_M70Hs_M50Hs_Et50MHs_Et50U
MICMBCMBC/MICMICMBCMBC/MICMICMBCMBC/MICMICMBCMBC/MICMICMBCMBC/MIC
G(+)Staphylococcus epidermidis ATCC 122280.5121221220.5120.512
Staphylococcus aureus ATCC 292130.512441242122122
Enterococcus faecalis ATCC 292122841616116161242284
Bacillus cereus ATCC 10876221441441122242
G(−)Escherichia coli ATCC 25922881881881441881
Pseudomonas aeruginosa ATCC 27853242242221121122
MICMFCMFC/MICMICMFCMFC/MICMICMFCMFC/MICMICMFCMFC/MICMICMFCMFC/MIC
YCandida albicans ATCC 10231482881482881482
Candida glabrata ATCC 90030816281624828162881
G(+)—Gram-positive bacteria; G(−)—Gram-negative bacteria; Y—yeast (fungi); MIC—minimum inhibitory concentration, MBC—minimum bactericidal concentration, MFC—minimum fungicidal concentration. Hs_M99—H. semenowii leaf extract in methanol 99.8%, Hs_M70—H. semenowii leaf extract in methanol 70%, Hs_M50—H. semenowii leaf extract in methanol 50%, Hs_Et50M—H. semenowii leaf extract in ethanol 50% (maceration), Hs_Et50U—H. semenowii leaf extract in ethanol 50%50% (ultrasonification), HsR_M99—H. semenowii root extract in methanol 99.8%, HsR_M70—H. semenowii root extract in methanol 70%, HsR_M50—H. semenowii root extract in methanol 50%.
Table 5. Antibacterial and antifungal activity of the extracts from H. semenowii (mg/mL) roots.
Table 5. Antibacterial and antifungal activity of the extracts from H. semenowii (mg/mL) roots.
Reference MicroorganismHsR_M99HsR_M70HsR_M50
MICMBCMBC/MICMICMBCMBC/MICMICMBCMBC/MIC
G(+)Staphylococcus epidermidis ATCC 12228144282482
Staphylococcus aureus ATCC 2921348288116161
Enterococcus faecalis ATCC 2921216>16nd8>16nd16>16nd
Bacillus cereus ATCC 10876441482881
G(−)Escherichia coli ATCC 25922161611616116161
Pseudomonas aeruginosa ATCC 27853881881881
MICMFCMFC/MICMICMFCMFC/MICMICMFCMFC/MIC
YCandida albicans ATCC 10231482482482
Candida glabrata ATCC 90030816281628162
G(+)—Gram-positive bacteria; G(−)—Gram-negative bacteria; Y—yeast (fungi); MIC—minimum inhibitory concentration, MBC—minimum bactericidal concentration, MFC—minimum fungicidal concentration. Hs_M99—H. semenowii leaf extract in methanol 99.8%, Hs_M70—H. semenowii leaf extract in methanol 70%, Hs_M50—H. semenowii leaf extract in methanol 50%, Hs_Et50M—H. semenowii leaf extract in ethanol 50% (maceration), Hs_Et50U—H. semenowii leaf extract in ethanol 50%50% (ultrasonification), HsR_M99—H. semenowii root extract in methanol 99.8%, HsR_M70—H. semenowii root extract in methanol 70%, HsR_M50—H. semenowii root extract in methanol 50%, nd—not determined.
Table 6. Results of Kruskal–Wallis test and post hoc Dunn’s multiple-comparisons test, showing significance of differences in antibacterial activity of Hedysarum semenowii extracts.
Table 6. Results of Kruskal–Wallis test and post hoc Dunn’s multiple-comparisons test, showing significance of differences in antibacterial activity of Hedysarum semenowii extracts.
Reference StrainsKruskal–Wallis TestDunn’s Multiple-Comparisons Test
H (7)p
S. epidermidis ATCC 1222818.010.012nd
S. aureus ATCC 2921321.630.003Hs_M99 vs. HsR_M50
E. faecalis ATCC 2921220.080.005nd
B. cereus ATCC 1087918.270.011Hs_Et50M vs. HsR_M50
E. coli ATCC 2592220.180.005nd
P. aeruginosa ATCC 2785319.680.006nd
C. albicans ATCC 1023112.990.072-
C. glabrata ATCC 9003010.430.165-
H (7)—test value, p—significance level, nd—not determined.
Table 7. Antioxidant activity of the extracts from H. semenowii leaves.
Table 7. Antioxidant activity of the extracts from H. semenowii leaves.
Hs_M99Hs_M70Hs_M50Hs_Et50MHs_Et50U
EC50, µg/mL48.3 ± 6.2784.6 ± 8.4206.6 ± 30.436.7 ± 2.3550.9 ± 8.81
AAI1.6 ± 0.20.90 ± 0.090.37 ± 0.052.07 ± 0.131.49 ± 0.26
Hs_M99—H. semenowii leaf extract in methanol 99.8%, Hs_M70—H. semenowii leaf extract in methanol 70%, Hs_M50—H. semenowii leaf extract in methanol 50%, Hs_Et50M—H. semenowii leaf extract in ethanol 50% (maceration), Hs_Et50U—H. semenowii leaf extract in ethanol 50%50% (ultrasonification), HsR_M99—H. semenowii root extract in methanol 99.8%, HsR_M70—H. semenowii root extract in methanol 70%, HsR_M50—H. semenowii root extract in methanol 50%.
Table 8. RP-LC/PDA method validation parameters.
Table 8. RP-LC/PDA method validation parameters.
Reference SubstanceRegression EquationR2Linearity Range (mg/mL)LOD (μg/mL)LOQ (μg/mL)Intraday Precision (n = 3) (%)Interday Precision (n = 3) (%)
mangiferiny = 4 × 107x + 128,2650.99930.0125–0.21.0423.1251.320.89
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Keleke, A.; Maciejewska-Turska, M.; Kasela, M.; Baj, T.; Ibragimova, L.; Sakipova, Z.; Sermukhamedova, O.; Ludwiczuk, A. Phytochemical Composition and Antimicrobial and Antioxidant Activity of Hedysarum semenowii (Fabaceae). Molecules 2025, 30, 4503. https://doi.org/10.3390/molecules30234503

AMA Style

Keleke A, Maciejewska-Turska M, Kasela M, Baj T, Ibragimova L, Sakipova Z, Sermukhamedova O, Ludwiczuk A. Phytochemical Composition and Antimicrobial and Antioxidant Activity of Hedysarum semenowii (Fabaceae). Molecules. 2025; 30(23):4503. https://doi.org/10.3390/molecules30234503

Chicago/Turabian Style

Keleke, Anel, Magdalena Maciejewska-Turska, Martyna Kasela, Tomasz Baj, Liliya Ibragimova, Zuriyadda Sakipova, Olga Sermukhamedova, and Agnieszka Ludwiczuk. 2025. "Phytochemical Composition and Antimicrobial and Antioxidant Activity of Hedysarum semenowii (Fabaceae)" Molecules 30, no. 23: 4503. https://doi.org/10.3390/molecules30234503

APA Style

Keleke, A., Maciejewska-Turska, M., Kasela, M., Baj, T., Ibragimova, L., Sakipova, Z., Sermukhamedova, O., & Ludwiczuk, A. (2025). Phytochemical Composition and Antimicrobial and Antioxidant Activity of Hedysarum semenowii (Fabaceae). Molecules, 30(23), 4503. https://doi.org/10.3390/molecules30234503

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