Leafy onions are called by the common name leeks and are widely used in European and Asian cuisines as a green seasoning with a distinct garlic aroma and specific taste. Known representatives of this onion group include species of the sections Ophioscordon (Allium ursinum L.) and Anguinum (A. victorialis L., A. ochotense Prokh., A. microdictyon Prokh.). The chemistry of species growing in European Russia, A. ursinum [1] and A. victorialis [2], and the Far-Eastern species A. ochotense [3] has previously been studied although A. microdictyon found in Siberia still remains an unstudied species. According to a spectrophotometric analysis, leaves of A. microdictyon accumulate flavonoids (2.90 ± 0.05/14.53 ± 0.31 mg/g recalculated for fresh/air-dried raw material), which is also characteristic of other Allium species [3,4,5]. The results indicated that the component composition of flavonoids from A. microdictyon should be studied so that became the goal of the present work.

Fresh leaves of A. microdictyon afforded EtOAc and BuOH fractions that were separated by column chromatography (CC) over polyamide, Sephadex LH-20, and RP-SiO2 and by preparative HPLC to isolate two new flavonol glycosides 1 and 2 in addition to the known flavonoids astragalin (3) [4], isoquercitrin (4) [6], kaempferol-3-O-glucoside-7-O-glucuronide (5) [7], kaempferol-3-O-neohesperidoside (6) [4], kaempferol-3,7-di-O-glucoside (7) [4], quercetin-3-O-glucoside-7-O-glucuronide (8) [7], quercetin-3,7-di-O-glucoside (9) [8], isorhamnetin-3-O-(2″-O-rhamnosyl-6″-O-glucosyl)-glucoside (10) [5], kaempferol-3-O-neohesperidoside-7-O-glucoside (11) [4], quercetin-3-O-(2″-O-rhamnosyl-6″-O-glucosyl)-glucoside (12) [9], quercetin-3-O-neohesperidoside-7-O-glucoside (13) [10], and quercetin-3-O-gentiobioside-7-O-glucuronide (14) [7].

Compound 1 had molecular formula C33H38O22 according to HR-ESI-MS (m/z 787.4271 [M + H]+; calcd for C33H39O22, 787.5990) and 13C NMR spectroscopy. Total hydrolysis of 1 produced quercetin, D-glucose, L-rhamnose, and D-glucuronic acid. UV spectroscopy in the presence of ionizing additives was indicative of substituted C-3 and C-7 hydroxyls of the aglycon [7, 8].

Mass spectra (ESI-MS) in positive-ion mode showed the protonated ion (m/z 787) and ions due to sequential loss of rhamnose (m/z 641), glucose (m/z 479), and glucuronic acid (m/z 303). Enzymatic hydrolysis by β-glucosidase did not cause changes, which indicated that the glucose was not terminal. Formation of quercetin-3-O-glucoside-7-O-glucuronide (8) [7] was observed after incubation with α-rhamnosidase, suggesting the rhamnose may have been in a terminal position.

Oxidation by H2O2 in alkaline solution formed a disaccharide, the chromatographic mobility of which agreed with that of neohesperidose (2-O-rhamnosylglucose). Loss of glucuronic acid as a result of the reaction with β-glucuronidase formed quercetin-3-O-neohesperidoside (calendoflavobioside) [11], which was previously isolated from Calendula officinalis L. (Asteraceae) [12].

The PMR spectrum displayed a series of doublets characteristic of C-3 and C-7 substituted quercetin (δH 6.43–7.93) [8] and resonances for three anomeric protons at 5.61 (d, J = 7.1 Hz), 4.92 (d, J = 1.8 Hz), and 5.14 (d, J = 7.7 Hz) that were assigned to β-glucose, α-rhamnose, and β-glucuronic acid [7]. The resonance for the anomeric proton of the 3-O-glucose was shifted to weak field more strongly (δH 5.61) than the corresponding resonance in 8 (δH 5.31), which indicated that the neighboring C-2″ atom was substituted. This was confirmed by the location of the C-2″ resonance at weaker field (δC 76.2) than that in 8 (δC 74.2) in the 13C NMR spectrum. Correlations between rhamnose proton H-1″′ (δH 4.92) and C-2″ in the HMBC spectrum confirmed that the glucose C-2″ hydroxyl was substituted by a rhamnose residue (Fig. 1). Additional correlations in the HMBC spectrum between glucose H-1″ (δH 5.61) and aglycon C-3 (δC 133.1) and between glucuronic acid H-1″″ (δH 5.14) and aglycon C-7 (δC 162.7) were consistent with attachment of glucose and glucuronic acid at the quercetin C-3 and C-7 positions, respectively. Thus, the results showed that 1 was quercetin-3-O-(2″-O-α-L-rhamnopyranosyl)-β-Dglucopyranoside-7-O-β-D-glucuronopyranoside (quercetin-3-O-neohesperidoside-7-O-glucuronide).

Fig. 1
figure 1

Structure and HMBC correlations in 1 and 2.

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The molecular formula of 2 was determined as C33H38O21 (m/z 771.5742 [M + H]+), calcd for C33H39O21, 771.6000). Total hydrolysis formed kaempferol, D-glucose, L-rhamnose, and D-glucuronic acid. The PMR spectrum contained a series of doublets typical of kaempferol with substituted C-3 and C-7 (δH 6.46–8.11) [8]. UV spectroscopy with ionizing additives was also indicative of this. Enzymatic hydrolysis by β-glucosidase did not cause changes while α-rhamnosidase formed kaempferol-3-O-glucoside-7-O-glucuronide (5) [7]. Oxidative degradation by H2O2 produced neohesperidose. The reaction with β-glucuronidase gave kaempferol-3-O-neohesperidoside (6) [4]. Otherwise, mass spectrometric and NMR spectroscopic data were similar to those of 1, indicating that 2 was a kaempferol derivative with the same set of carbohydrate residues and had the structure kaempferol-3-O-(2″-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside-7-O-β-D-glucuronopyranoside (quercetin-3-O-neohesperidoside-7-O-glucuronide).

3-O-Rutinoside-7-O-glucuronides of quercetin and kaempferol isomeric to 1 and 2 were observed earlier in flowers of Tulipa gesneriana L. (Liliaceae) [7]. Flavonol 3,7-di-O-glycosides were found in other leek species in the section Anguinum, A. victorialis L. [13] and A. ochotense Prokh. [3], which was probably their chemotaxonomic signature.

Experimental

Leaves of A. microdictyon were collected before flowering in Pribaikalsky District, Republic of Buryatia (May 28, 2019; 52°10′35.2182″ N, 107°42′14.7780″ E, 612 m above sea level; humidity 80.02 ± 2.62%). The species was determined by Dr. N. K. Chirikova (North-Eastern Federal University, Yakutsk, Russia). A specimen of the raw material is preserved in the herbarium of the IGEB, SB, RAS (No. BU/AMA-0519/12-19). The total flavonoid content in leaves of A. microdictyon was determined by spectrophotometry in the presence of AlCl3 (recalculated as rutin) [14]. Column chromatography (CC) used polyamide, Sephadex LH-20, and reversed-phase silica gel (RP-SiO2, Sigma-Aldrich, St. Louis, MO, USA). Spectrophotometric studies used an SF-2000 spectrophotometer (OKB Spectr, St. Petersburg, Russia). Mass spectrometric studies used an LCMS-8050 TQ-mass spectrometer (Shimadzu, Columbia, MD, USA) [15]. NMR spectra were recorded on a VXR 500S NMR spectrometer (Varian, Palo Alto, CA, USA). Preparative HPLC was performed on a Summit liquid chromatograph (Dionex, Sunnyvale, CA, USA) using a LiChrospher RP-18 column (250 . 10 mm, ∅ 10 μm; Supelco, Bellefonte, PA, USA); mobile phase H2O (A) and MeCN (B); flow rate 0.8 mL/min; column temperature 26°C; and UV detection at λ 350 nm.

Extraction and Isolation of 1–14 from A. microdictyon. Fresh leaves (4.5 kg) were ground in a blender and extracted with EtOH (90%, 1:5) with heating (90°C) for 4 h. The EtOH extract was filtered and concentrated to dryness. The dry solid was suspended in H2O (200 mL) and extracted with hexane, EtOAc, and BuOH to produce hexane (2.02 g), EtOAc (0.64 g), and BuOH fractions (3.62 g). The EtOAc fraction (0.6 g) was separated over polyamide (800 g) with elution by H2O and EtOH (80%). The EtOH eluate was concentrated and chromatographed over Sephadex LH-20 (CC, 70 × 1 cm, EtOH–H2O eluent, 90:10→0:100) and RP-SiO2 (CC, 30 × 1 cm, H2O–MeCN eluent, 70:30→50:50) and by prep. HPLC [gradient mode (%B): 0–90 min, 40–70%] to isolate seven compounds including astragalin (kaempferol-3-O-glucoside, 3 mg, 3) [4], isoquercitrin (quercetin-3-O-glucoside, 6 mg, 4) [6], kaempferol-3-O-glucoside-7-O-glucuronide (21 mg, 5) [7], kaempferol-3-O-neohesperidoside (9 mg, 6) [4], kaempferol-3,7-di-O-glucoside (52 mg, 7) [4], quercetin-3-O-glucoside-7-O-glucuronide (20 mg, 8) [7], and quercetin-3,7-di-O-glucoside (15 mg, 9) [8].

The BuOH fraction (3.5 g) was separated over polyamide, Sephadex LH-20, and RP-SiO2 as above and by prep. HPLC [gradient mode (%B): 0–20 min, 5–23%; 20–45 min, 23–34%; 45–90 min, 34–52%] to isolate 1 (195 mg), 2 (173 mg), isorhamnetin-3-O-(2″-O-rhamnosyl-6″-O-glycosyl)-glucoside (14 mg, 10) [5], kaempferol-3-O-neohesperidoside-7-O-glucoside (22 mg, 11) [4], quercetin-3-O-(2″-O-rhamnosyl-6″-O-glucosyl)-glucoside (11 mg, 12) [9], quercetin-3-O-neohesperidoside- 7-O-glucoside (56 mg, 13) [10], and quercetin-3-O-gentiobioside-7-O-glucuronide (12 mg, 14) [7].

Quercetin-3-O-neohesperidoside-7-O-glucuronide (1). C33H38O22. UV spectrum (70% MeOH, λmax, nm): 255, 267 sh, 352; +NaOMe 270, 395; + NaOAc 264, 329 sh, 397; +AlCl3 272, 297 sh, 437; +AlCl3/HCl 268, 295 sh, 360 sh, 403. HR-ESI-MS, m/z 787.4271 (calcd for C33H39O22, 787.5990 [M + H]+). ESI-MS, m/z (%): 787 [M + H]+ (2), 641 [(M + H) – C6H10O4]+ (40), 479 [(M + H) – C6H10O4 – C6H10O5]+ (100), 303 [(M + H) – C6H10O4 – C6H10O5 – C6H8O6]+ (4). Table 1 gives the PMR spectrum (500 MHz, MeOH-d4, 298 K, δ, ppm) and 13C NMR spectrum (125 MHz, MeOH-d4, 298 K, δ, ppm).

Table 1 PMR (500 MHz) and 13C NMR Spectra (125 MHz) of 1 and 2 (MeOH-d4, 298 K, δ, ppm, J/Hz) (*DMSO-d6)
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Kaempferol-3-O-neohesperidoside-7-O-glucuronide (2). C33H38O21. UV spectrum (70% MeOH, λmax, nm): 265, 347; +NaOMe 242, 270, 345 sh, 385; + NaOAc 265, 379; +AlCl3 272, 301 sh, 352, 401; +AlCl3/HCl 272, 300 sh, 348, 401. HR-ESI-MS, m/z: 771.5742 (calcd for C33H39O21, 771.6000 [M + H]+). ESI-MS, m/z (%): 771 [M + H]+ (20), 625 [(M + H) – C6H10O4]+ (22), 463 [(M + H) – C6H10O4 – C6H10O5]+ (100), 287 [(M + H) – C6H10O4 – C6H10O5 – C6H8O6]+ (5). Table 1 gives the PMR spectrum (500 MHz, MeOH-d4, 298 K, δ, ppm) and 13C NMR spectrum (125 MHz, MeOH-d4, 298 K, δ, ppm).

Total hydrolysis was performed in TFA (2 M) followed by separation of the reaction mixture over polyamide [16] and analysis of the hydrolysis products by GC-MS (aglycons) [17], derivatization with 3-methyl-1-phenyl-2-pyrazolin-5-one and reductive amination with L-tryptophan [18] (monosaccharides of the D/L-series) [19]. The hydrolysis products of 1 included quercetin [20], D-glucose, L-rhamnose, and D-glucuronic acid; of 2, kaempferol [20], D-glucose, L-rhamnose, and D-glucuronic acid.

Hydrolysis with β-glucosidase and α-rhamnosidase was conducted as described earlier [16]. Hydrolysis by β-glucosidase did not change 1 and 2. The products from hydrolysis of 1 by α-rhamnosidase consisted of quercetin-3-Oglucoside-7-O-glucuronide (8) [7]; of 2, kaempferol-3-O-glucoside-7-O-glucuronide (5) [7], which were identified using UV and NMR spectroscopic and mass spectrometric data.

Quercetin-3-O-glucoside-7-O-glucuronide (8). C27H28O18. UV spectrum (70% MeOH, λmax, nm): 256, 268 sh, 351; +NaOMe 271, 396; + NaOAc 263, 330 sh, 399; +AlCl3 271, 298 sh, 435; +AlCl3/HCl 270, 296 sh, 361 sh, 401. HR-ESI-MS, m/z: 641.0231 (calcd for C27H29O18, 641.4674 [M + H]+). ESI-MS, m/z (%): 641 [M + H]+ (12), 479 [(M + H) – C6H10O5]+ (100), 303 [(M + H) – C6H10O5 – C6H8O6]+ (18). 1H NMR spectrum (500 MHz, MeOH-d4, 298 K, δ, ppm, J/Hz)Footnote 1: 6.45 (1H, d, J = 2.1, H-6), 6.78 (1H, d, J = 2.1, H-8), 7.91 (1H, d, J = 2.1, H-2′), 6.89 (1H, d, J = 9.0, H-5′), 7.50 (1H, dd, J = 2.1, 9.0, H-6′), 12.53 (1H, br.s, 5-OH)**, 9.55 (1H, br.s, 3′-OH)**, 9.70 (1H, br.s, 4′-OH)**; 3-O-β-D-glucopyranose – 5.31 (1H, d, J = 7.2, H-1′′), 3.70 (1H, m, H-2′′), 3.55 (1H, m, H-3′′), 3.48 (1H, m, H-4′′), 3.61 (1H, m, H-5′′), 3.90 (1H, dd, J = 2.1, 12.0, HA-6′′), 3.79 (1H, m, HB-6′′); 7-O-β-D-glucuronopyranose – 5.10 (1H, d, J = 7.5, H-1′′′), 3.52 (1H, m, H-2′′′), 3.38 (1H, m, H-3′′′), 3.45 (1H, m, H-4′′′), 4.08 (1H, d, J = 9.0, H-5′′′). 13C NMR spectrum (125 MHz, MeOH-d4, 298 K, δ, ppm): 157.6 (C-2), 133.3 (C-3), 177.5 (C-4), 160.1 (C-5), 99.3 (C-6), 162.9 (C-7), 94.6 (C-8), 156.2 (C-9), 105.7 (C-10), 120.4 (C-1′), 115.2 (C-2′), 144.0 (C-3′), 148.7 (C-4′), 116.1 (C-5′), 121.4 (C-6′); 3-O-β-D-glucopyranose – 102.6 (C-1′′), 74.2 (C-2′′), 78.1 (C-3′′), 70.4 (C-4′′), 77.7 (C-5′′), 60.5 (C-6′′); 7-O-β-D-glucuronopyranose – 99.7 (C-1′′′), 73.2 (C-2′′′), 75.6 (C-3′′′), 71.4 (C-4′′′), 75.2 (C-5′′′), 171.1 (C-6′′′).

Kaempferol-3-O-glucoside-7-O-glucuronide (5). C27H28O17. UV spectrum (70% MeOH, λmax, nm): 266, 345; +NaOMe 241, 272, 341 sh, 381; + NaOAc 266, 375; +AlCl3 272, 300 sh, 351, 400; +AlCl3/HCl 272, 301 sh, 347, 400. HR-ESI-MS, m/z: 625.0967 (calcd for C27H29O17, 625.4684 [M + H]+). ESI-MS, m/z (%): 625 [M + H]+ (33), 463 [(M + H) – C6H10O5]+ (100), 287 [(M + H) – C6H10O5– C6H8O6]+ (12).

Oxidation by H2O2. A weighed portion (5 mg) of a compound was suspended in a mixture (10 mL) of H2O2 (40%)–NH3 (25%)–H2O (75:14:11), incubated at 25°C for 48 h, treated with catalase (100 U, 1.11.1.6, ≥10,000 U/mg, No. C40; Sigma-Aldrich), and passed after 2 h through Amberlyst® 15 (H+-form, Sigma-Aldrich) that was eluted with H2O (100 mL). The aqueous eluate was concentrated and analyzed by HPLC on a Milichrom A-02 chromatograph (EcoNova, Novosibirsk, Russia) equipped with a Separon SGX NH2 column (2 . 75 mm, ∅ 5 μm; Tessek Ltd., Prague, Czech Rep.) in isocratic mode (MeCN–H2O, 75:25, flow rate 100 μL/min) at column temperature 30°C with UV detection (λ 190 nm).

The degradation products of 1 and 2 included neohesperidose (2-O-rhamnosylglucose, tR 25.43 min), the retention time of which differed from that of isomeric rutinose (6-O-rhamnosylglucose, tR 27.14 min).

Hydrolysis by β-Glucuronidase. A weighed portion of compound (10 mg) was dissolved in DMSO (50 μL), treated with phosphate buffer (990 μg, pH 5.0) and β-glucuronidase from Helix pomatia (100 U, HP-2, 3.2.1.31, ≥100,000 U/mL; No. G7017, Sigma-Aldrich), and incubated at 37°C for 3 h. Then, the mixture was treated with Me2CO (4 mL) and centrifuged. The supernatant was concentrated to dryness under vacuum. The dry solid was separated over polyamide (50 g) with elution by H2O (100 mL) and EtOH (70%, 200 mL). The EtOH eluate after hydrolysis of 1 held quercetin-3-O-neohesperidoside (calendoflavobioside, 5 mg) [11]; of 2, kaempferol-3-O-neohesperidoside (6 mg, 6) [4].