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Article

Exploring the Phytochemical Profile and Biological Insights of Epilobium angustifolium L. Herb

by
Reneta Gevrenova
1,*,
Gokhan Zengin
2,
Gulsah Ozturk
2 and
Dimitrina Zheleva-Dimitrova
1
1
Department of Pharmacognosy, Faculty of Pharmacy, Medical University, 1000 Sofia, Bulgaria
2
Physiology and Biochemistry Research Laboratory, Department of Biology, Science Faculty, Selcuk University, Konya 42130, Turkey
*
Author to whom correspondence should be addressed.
Plants 2025, 14(3), 415; https://doi.org/10.3390/plants14030415
Submission received: 18 December 2024 / Revised: 22 January 2025 / Accepted: 27 January 2025 / Published: 31 January 2025
(This article belongs to the Section Phytochemistry)
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Abstract

:
The aerial parts of Epilobium angustifolium L. (fireweed) (Onagraceae) are renowned for their use in the treatment of prostatic, kidney and urinary tract diseases, and skin infections. In this work, a comprehensive phytochemical profiling of the methanol-aqueous extract from E. anfustifolium aerial parts was performed by the means of liquid chromatography–Orbitrap high-resolution mass spectrometry. Annotation and dereplication of 121 secondary metabolites were achieved, including acylquinic acids, gallo- and ellagitannins, flavonoids, phenolic acids, and their glycosides. Forty-six compounds are reported for the first time in the species. Total phenolic and flavonoid content were 85.04 ± 0.18 mg GAE/g and 27.71 ± 0.74 mg QE/g, respectively. Antioxidant capacity assessment revealed that the extract actively scavenged DPPH and ABTS radicals (310.74 and 466.82 mg TE/g) along with a high reducing power in CUPRAC and FRAP assay (442.83 and 291.50 mg TE/g), respectively, and metal chelating (48.20 mg EDTA/g). The extract also had a distinct impact on α-glucosidase (3.48 mmol ACAE/g) and moderate activity towards α-amylase (0.44 mmol ACAE/g) and lipase (8.03 OE/g). It inhibited acetyl- and butyrylcholinesterase (2.05 and 1.67 mg GALE/g) and had a prominent anti-tyrosinase effect (61.91 mg KA/g). Our results contribute to establishing fireweed as a multifunctional agent for use in herbal preparations.

1. Introduction

Epilobium angustifolium L. (=Chamerion angustifolium Scop.) (Onagraceae) is renowned for its usage in nutraceuticals, diet supplements, and cosmetic products [1]. The species is distributed widely in the temperate zones of Europe, Asia, and North America [2]. E. angustifolium is commonly referred to as rosebay willowherb, fireweed, or great-willowherb [3]. It is a perennial plant with an erect stem up to 2 m high and which develops large racemes with a number of pink flowers [2].
Overall, more than 250 secondary metabolites, especially flavonoids, ellagitannins, and phenolic acids, have been reported in E. angustifolium extracts [1,2,4]. Fireweed also contains lignans, triterpenoids, steroids, fatty acids, essential oil, and alkaloids [1]. A high level of ellagitannins was determined, which represents about 15% of the dry mass of the herb [5]. Monomeric tellimagradin I and II along with macrocyclic dimer oenothein B and trimer oenothein A have been isolated from E. angustifolium aerial parts [5,6]. Oenothein B reached up to 50% of the total oligomeric ellagitannin level [6]. Its concentration varied from 2 to 4.5% in the raw plant material [7,8,9]. Flowers contained 10% more oenothein B as leaves, while the content of oenothein A was significantly lower [5]. In addition, gallotannins have been evidenced, including galloyl-glucose and galloyl-HHDP-glucose derivatives [10].
A variety of kaempferol-, quercetin-, and myricetin-glycosides has been reported, being present at 1–2% of the dry plant mass [1,3]. It is worth noting that the flavonol-rhamnosides have been found, especially in the flowers [5]. Recently, conjugates of flavonol glycosides with hydroxycinnamic acids have been reported (acylated flavonoids) [7]. Quercetin 3-O-glucuronide was the most abundant within the flavonol glycosides [11], reaching up to 4.75% [4,10]. Consequently, oenothein B and quercetin 3-O-glucuronide have been ascribed as marker compounds for raw material standardization [12,13]. Phenolic acids in the fireweed herb were delineated by gallic, ellagic, protocatechuic, caffeic acid, and acylquinic acid isomers [3,7,14]. HPLC-DAD-MS, HPLC-ESI-MS/MS, and UHPLC-DAD-ESI-QqQ-MS have been applied for identification/annotation and the quantitative determination of ellagitannins, flavonoids, and phenolic acids [5,9,10,14,15,16,17]. Interestingly, the inflorescence stem (from the apex) accumulated the highest content of total polyphenols (up to 250 mg/g dw) [5]. Oenothein B dominated these parts, being present at 148 mg/g dw. Quercetin 3-O-rhamnoside and kaempferol 3-O-rhamnoside reached 5.16 and 7.91 mg/g dw in flower buds, respectively, while they were not evidenced in the leaves [5].
E. angustifolium aerial parts have been used in ethnopharmacological approaches as an astringent and wound-healing agent, and for the treatment of skin infections, urinary disorders, and migraine headaches [1,2,13]. In Europe, the main use is to treat prostate inflammation, and kidney and urinary tract diseases [2]. The phytochemistry, pharmacology, and traditional usage of Epilobium L. species are the subject of literature reviews by Granica et al. [3] and Vitalone and Allkanjari [18] which strongly emphasize the anti-inflammatory potential of E. angustifolium extracts and the beneficial effects of plant formulations and oenothein B on benign prostate hyperplasia (BPH) and prostatitis, and the cytotoxic activity on human prostate cell lines. Adamczak et al. [2] summarized progress in phytochemical studies, and highlighted the variability of bioactive compounds in relation to plant parts, geographical origin, and phenological phases. In the review of Schepetkin et al. [13], particular attention was paid to the potential clinical use of the fireweed oenothein B, flavonoids, and other polyphenols. The effects of oenothein B on abnormal prostate cells, where it inhibits cell proliferation and prostate-specific antigen (PSA) secretion, sustain traditional claims relating to fireweed use for the mitigation of prostate issues.
Significant advances have been made in understanding the mechanisms of fireweed extract-mediated effects on BPH, which has been related to the modulation of prostate enzyme activity (5-α-reductase, aromatase, metalloproteinases), the regulation of androgen levels, the activation of the mitochondrial pathway of apoptosis, and pro-inflammatory cytokine production [13,16]. In addition to evoking an antioxidant response, oenothein B from E. angustifolium enhances the activity of various types of immune cells [13]. Herbal teas and dietary supplements from E. angustifolium are available in European Union countries [16]. In the monograph on E. angustifolium published by the European Medicinal Agency (EMA), these products are shown to be consistent with the requirements for “traditional use” (https://www.ema.europa.eu, accessed on 1 November 2024).
In monocentric, randomized, double-blind, placebo-controlled clinical trials, E. angustifolium food supplements, standardized to contain ≥15% oenothein B, improved the quality of life of subjects with BPH by reducing nocturia and aiding general renal function [16].
E. angustifolium aqueous extract did not lose its biological activity after in vitro digestion and did not affect intestinal bacteria growth [4]. Taking into consideration its cytotoxic activity towards colon cancer cell lines, the infusion has been evaluated as a supporting agent in colon cancer therapy. Dreger et al. [2] reviewed the antibacterial and antimycotic activity of fireweed extracts, emphasizing the potential use of the herbal drug as antimicrobial agent in wound healing, in cosmetic products, and in food preservatives.
It is worth noting that the aforementioned studies emphasize the main flavonoids and ellagitannins, while there is not a comprehensive metabolite profile of fireweed provided by means of liquid chromatography–Orbitrap high-resolution mass spectrometry (LC-HRMS).
The cited studies generate further interest in fireweed and have prompted us to undertake a comprehensive profiling of the secondary metabolites of methanol-aqueous extract from E. anfgustifoliun aerial parts by means of ultra-high-performance liquid chromatography–Orbitrap high-resolution mass spectrometry, integrated with an assessment of its antioxidant and enzyme inhibitory potential. Owing to the fact that crucial enzymes are considered to be targets in the pharmaceutical therapies, the enzyme inhibitory activity of the studied extract towards cholinesterases, tyrosinase, α-amylase, α-glucosidase, and lipase was determined. In addition, the evaluation of the antioxidant capacity via different mechanisms may have an important contribution in evoking an antioxidant response in the aforementioned conditions.

2. Results and Discussion

2.1. UHPLC-HRMS Profiling of Secondary Metabolites in E. angustifolium Extract

An in-depth UHPLC-HRMS analysis of E. angustifolium methanol-aqueous extract was performed, allowing for the identification/annotation of 121 secondary metabolites (Table 1). The complete workflow, combining UHPLC-HRMS data annotation and biological potential, is presented in Figure 1. Identification confidence levels for metabolite profiling were performed according to Sumner et al. [19] and were as follows: level 1–compounds identified by comparison to the reference standard; level 2–putatively annotated compounds. Twenty-six compounds were assigned to phenolic acids. Nineteen compounds were ascribed as acylquinic acids. Twenty-four tannins and fifty-two flavonoids were also identified in the studied extract. Among all these 121 metabolites, 11 phenolic acids and derivatives, 10 acylquinic acids, 3 tannins, and 22 flavonoids are reported for the first time in E. angustifolium aerial parts.

2.1.1. Phenolic Acids, Their Glycosides, and Coumarins

The phenolic acids frequently exist in the form of glucosides (the bond with sugars occurs via one of the phenolic functions) [20,21]. The basic compounds of the hydroxybenzoic acid series were gallic (2) and protocatechuic acid (8) and their hexosides (3, 5, 6), along with hydroxybenzoic, gentisic, vanillic, syringic, and hydroxyphenyllactic acid—hexosides (4, 7, 9, 11, and 16). The hydroxycinnamic acid series was represented by coumaric and caffeic acid-hexoside (12, 13, 18, and 22) together with the respective free acids (17, 23, and 24). Based on the accurate masses and conformity of reference standard retention times and fragmentation patterns, 2, 8, 15, 17, 23, 24, and 26 were unambiguously identified in the studied extract (Table 1).
Table 1. Secondary metabolites in Epilobium angustifolium methanol-aqueous extracts.
Table 1. Secondary metabolites in Epilobium angustifolium methanol-aqueous extracts.
No.Identified/Tentatively Annotated CompoundMolecular FormulaExact Mass
[M-H]−		Fragmentation Pattern in (-) ESI-MS/MStR
(min)		Δ ppmReferences
Hydroxybenzoic, Hydroxycinnamic Acids, Phenylethanoid Glycosides, and Coumarins
1.galloyl O-hexose C13H16O10331.0671331.0676 (100), 271.0463 (10.2), 241.0349 (3.1), 211.0244 (10.4), 169.0133 (45.8), 151.0024 (13.4), 125.0230 (17.7), 107.0124 (4.7)0.941.511[16,17]
2.gallic acid aC7H6O5169.0142169.0132 (33.9), 125.0230 (100), 107.01231.146.133[10,16,17,22,23]
3.gallic acid O-hexoside 1C13H16O10331.0687331.0676 (100), 271.0465 (0.7), 241.0353 (1.9), 211.0246 (0.6), 169.0131 (16.7), 168.0054 (31.9), 125.0229 (36.7), 107.0123 (2.1)1.171.511
4.hydroxybenzoic acid-O-hexosideC13H16O8299.0778299.0765 (0.5), 137.0231 (100), 93.0330 (65.2), 1.26−2.543
5.gallic acid O-hexoside 2C13H16O10331.0687331.0674 (8.1), 169.0132 (16.7), 125.0236 (38.3)1.561.118
6.protocatechuic acid-O-hexosideC13H16O9315.0727315.0725 (100), 153.0181 (27.1), 152.0103 (56.8), 123.0072 (2.4), 109.0285 (9.9), 108.0202 (91.8), 81.0332 (0.6)1.681.221
7.vanillic acid-O-hexosideC14H18O9329.0875329.0903 (2.3),167.0339 (100), 152.0102 (24.0), 123.0437 (14.5), 108.0202 (39.9)1.777.581
8.protocatechuic acid aC7H6O4153.0181153.0182 (15.4), 109.0280 (100), 91.0174 (1.2), 81.0331 (1.2)2.02−1.392[14,23]
9.p-hydroxyphenylacetic acid O-hexosideC14H18O8313.0932313.0915 (1.6), 151.0388 (98.2), 123.0436(1.0), 107.0487 (100)2.130.988
10.hydroxybenzoyl hexoseC13H16O8299.0778299.0774 (26.4), 239.0559 (25.4), 209.0447 (7.4), 179.0339 (28.6), 137.0230 (100), 109.0280 (23.3), 93.0329 (12.4)2.160.600
11.syringic acid-O-hexosideC15H20O10359.0985359.0991 (7.6), 197.0448 (100), 182.0212 (22.1), 166.9976 (8.3), 153.0545 (16.6), 138.0309 (28.5), 123.0073 (32.9)2.262.311
12.caffeic acid-O-hexoside 1C15H18O9341.0871341.0870(4.2), 179.0340 (100), 135.0438 (55.5), 107.0485 (0.3)2.40−2.332[16]
13.caffeic acid-O-hexoside 2C15H18O9341.0871341.0865 (25.7), 281.0658 (2.3), 251.0563 (5.7), 221.0454 (1.9), 179.0341 (26.5), 161.0233 (100), 135.0439 (9.3), 133.0282 (26.4)2.61−3.827
14.caffeoyl-O-hexoseC15H18O9341.0871341.0859 (19.5), 281.0668 (93.4), 251.0561 (50.7), 221.0452 (47.1), 179.0341 (100), 161.0233 (6.2), 135.0438 (69.4), 133.0281 (20.4)2.82−5.469
15.4-hydroxybenzoic acid aC7H6O3137.0230137.0230 (100), 119.0123 (2.5), 108.0200 (9.8), 93.0329 (13.4), 65.0380 (1.1)2.84−10.052[17]
16.gentisic acid O-hexosideC13H16O9315.0727315.0725 (7.9), 153.0181 (100), 125.0230 (5.1), 109.0279 (2.6), 81.0329 (0.4)2.841.221
17.p-coumaric acid aC9H8O3163.0389163.0390 (6.2), 119.0488 (100), 93.0331 (3.6)3.01−6.792[23]
18.caffeic acid O-hexoside 3C15H18O9341.0871341.0877 (27.3), 281.0663 (0.9), 251.0562 (2.2), 221.0456 (0.9), 179.0340 (100), 135.0438 (71.6), 107.0488 (0.5)3.07−0.602
19.methylgallateC8H8O5183.0299183.0289 (100), 168.0053 (11.9), 140.0103 (11.4), 111.0074 (6.2), 83.0122 (0.7)3.150.711[14]
20.quinic acidC7H12O6191.0549191.0552 (100), 173.0447 (1. 8), 155.0336 (0.2), 127.0387 (4.1), 111.0435 (1.1), 93.0330 (6.6), 85.0278 (18.4)3.19−5.817[16]
21.umbeliferoneC9H6O3161.0244161.0233 (81.1), 133.0281 (100), 115.0173 (0.7), 105.0331 (1.5), 89.0381 (0.8), 77.0381 (0.8)3.19−7.250
22.coumaric acid-O-hexosideC15H18O8325.0930325.0922 (2.3), 163.0389 (100), 145.0284 (0.3), 119.0488 (90.0), 93.0331 (0.3)3.34−3.355
23.caffeic acid aC9H8O4179.0339179.0341 (17.8), 135.0438 (100), 107.0488 (1.2)3.54−6.044[23]
24.o-coumaric acid aC9H8O3163.0389163.0388 (8.3), 119.0487 (100), 93.0330 (1.3)4.55−7.467[23]
25.galloyl-(caffeoyl)-hexoseC22H22O13493.0988493.1006 (100), 341.0881 (5.9), 281.0667 (32.9), 251.0560 (24.8), 221.0449 (13.9), 161.0233 (26.0), 135.0438 (31.4), 169.0132 (14.7), 125.0230 (10.4), 107.0123 (1.7)4.783.643
26.salicylic acid aC7H6O3137.0230137.0230 (8.3), 108.0202 (7.5), 93.0330 (100), 65.0380 (0.7)6.29−10.125
Mono- and Diacylquinic Acids
27.3-galloylquinic acidC14H16O10343.0671343.0675 (38.9), 191.0554 (13.6), 173.0446 (22.2), 169.0132 (100), 125.0230 (32.6), 107.0124 (6.5), 93.0331 (7.8), 85.0279 (4.6)1.251.370
28.neochlorogenic (3-caffeoylquinic) acid aC16H18O9353.0867353.0883 (39.5), 191.0552 (100), 179.0340 (62.7), 173.0446 (2.7), 161.0230 (4.1), 135.0438 (50.8), 111.0434 (1.6), 93.0331 (3.9), 85.0279 (8.2)2.361.458[5,10,23]
29.3-p-coumaroylquinic acidC16H18O8337.0928337.0934 (7.6), 191.0553 (14.4), 163.0390 (100), 135.0437 (0.6), 119.0488 (31.3), 111.0432(1.5), 93.0329 (2.6), 85.0278 (3.0)3.011.096[10,16]
30.chlorogenic (5-caffeoylquinic) acid aC16H18O9353.0874353.0884 (4.2), 191.0553 (100), 173.0447 (1.1), 161.0235 (1.2), 111.0434 (1.5), 93.0331 (2.8), 85.0278 (2.0)3.190.665[10,16,17,23]
31.4-caffeoylquinic acidC16H18O9353.0878353.0882 (33.3), 191.0553 (43.1), 179.0340 (68.6), 173.0445 (100), 135.0438 (57.5), 111.0434 (3.6), 93.0331 (21.0), 85.0279 (7.8)3.37−0.100
32.3-feruloylquinic acidC17H20O9367.1034367.1039 (18.2), 193.0497 (100), 191.0553 (2.6), 173.0445 (4.0), 149.0594 (3.4), 134.0359 (54.0), 111.0435 (1.5), 93.0330 (2.1), 85.0276 (0.4)3.441.157[10,16,17]
33.1-galloyl-3-caffeoylquinic acidC23H22O13505.09505.0999 (64.0), 353.0883 (38.8), 191.0553 (100), 179.0341 (52.5), 173.0443 (3.9), 161.0233 (4.2), 135.0438 (52.8), 111.0436 (2.0), 93.0330 (3.8), 85.0280 (8.0)3.582.348
34.4-p-coumaroylquinic acidC16H18O8337.0928337.0937 (10.2), 191.0551 (10.8), 173.0444 (100), 163.0390 (19.3), 119.0488 (14.8), 111.0430 (2.3), 93.0330 (18.7), 85.0279 (3.0)3.79
35.5-caffeoylquinic acid isomerC16H18O9353.0874353.0884 (6.4), 191.0550 (100), 179.0341 (0.9), 173.0446 (0.9), 161.0232 (1.9), 111.0436 (1.1), 93.0329 (2.5), 85.0279 (8.4)3.881.684[16]
36.5-p-coumaroylquinic acidC16H18O8337.0928337.0934 (8.1), 191.0552 (100), 173.0444 (7.1), 163.0389 (6.4), 145.0283 (1.2), 119.0487 (5.3), 93.0330 (19.0), 85.0278 (4.9)3.951.629[10]
37.1-caffeoyl-3-galloylquinic acidC23H22O13505.0988505.0999 (100), 353.0883 (5.7), 343.0675 (15.2), 191.0553 (26.5), 179.0342 (18.2), 173.0445 (18.2), 169.0123 (73.3), 161.0230 (6.4), 135.0440 (14.8), 125.0230 (33.1), 107.0124 (4.6), 93.0331 (8.5)4.101.913
38.1-galloyl-5-caffeoylquinic acidC23H22O13505.0988505.0998 (35.5), 353.0880 (18.2), 191.0551 (100), 179.0341 (3.1), 173.0447 (1.3), 161.0233 (2.4), 135.0437 (3.0), 111.0434 (0.9), 93.0331 (2.4), 85.0279 (6.9)4.192.032
39.5-feruloylquinic acidC17H20O9367.1034367.1040 (20.6), 193.0500 (11.3), 191.0553 (100), 173.0445 (49.6), 149.0598 (0.5), 134.0360 (15.5), 111.0437 (4.1), 93.0330 (29.2), 85.0279 (4.5)4.391.402[10]
40.3-caffeoyl-5-galloylquinic acidC23H22O13505.0988505.0996 (54.9), 353.0881 (71.0), 191.0552 (100), 179.0340 (56.8), 173.0447 (4.2), 161.0233 (4.7), 135.0438 (54.2), 111.0434 (0.7), 93.0329 (4.1), 85.0279 (8.4)4.561.735
41.5-p-coumaroylquinic acid isomerC16H18O8337.0928337.0935 (6.8), 191.0551 (100), 173.0443 (1.8), 163.0388 (1.8), 145.0279 (0.7), 119.0489 (1.1), 111.0435 (0.9), 93.0329 (4.8), 85.0279 (6.8)4.611.926
42.caffeoyl-hydroxybenzoylquinic acidC23H24O12491.1196491.1203 (94.4), 447.1305 (16.0), 329.0882 (0.9), 179.0340 (8.3), 161.0232 (100), 137.0235135.0438 (12.0), 133.0281 (40.6), 109.0281 (2.3), 85.0277 (1.5)4.891.691
43.5-feruloylquinic acid isomerC17H20O9367.1034367.1038 (9.5), 193.0498 (1.6), 191.0552 (100), 179.0341 (1.6), 173.0444 (2.3), 161.0229 (0.6), 134.0360 (2.8), 111.0434 (1.3), 93.0330 (6.3), 85.0279 (8.0)4.910.939
44.3-galloyl-5-p-coumaroylquinic acidC23H22O12489.1038489.1046 (44.3), 337.0936 (16.9), 191.0553 (2.9), 179.0704 (2.9), 173.0443 (1.5), 163.0397 (4.3), 146.0239 (2.7), 119.0488 (1.2), 111.0439 (0.9), 93.0327 (4.9), 85.0279 (6.8)5.481.555
45.3-feruloyl-5-galloylquinic acidC26H32O11519.1872519.1877 (100), 193.0498 (37.0), 178.0262 (16.0), 161.0235 (5.1)6.700.954
Tannins
46.galloyl-HHDP-hexose 1C27H22O18633.0733633.0746 (100), 300.9993 (73.0), 275.0201 (31.2), 249.0403 (22.4), 257.0088 (4.3), 245.0095 (2.5), 229.0141 (7.9), 185.0236 (4.6), 169.0132 (7.0), 145.0282 (1.9), 125.0231 (9.2), 107.0123 (2.5)1.112.074[10]
47.galloyl-HHDP-hexose 2C27H22O18633.0733633.0749 (100), 300.9994 (67.9), 275.0201 (36.5), 249.0405 (27.2), 257.0092 (4.8), 245.0085 (1.9), 229.0139 (8.7), 185.0235 (2.6), 169.0133 (8.5), 145.0282 (0.9), 125.0229 (9.5), 107.0124 (1.4)1.402.264
48.digalloyl-hexose 1C20H20O14483.0780483.0786 (100), 331.0677 (16.8), 313.0569 (10.1), 211.0240 (2.9), 169.0131 (81.5), 151.0021 (2.4), 125.0230 (56.8), 107.0124 (7.7)1.601.266[23]
49.digalloyl-hexose 2C20H20O14483.0780483.0786 (100), 331.0670 (9.0), 313.0570 (22.0), 271.0458 (7.4), 211.0243 (9.5), 169.0132 (49.9), 151.0026 (3.0), 125.0230 (30.8), 107.0125 (4.3)2.601.204
50.oenothein B 1C68H46O441568.1518
783.0686 [M-2H]2−,		783.0699 (100), 765.0590 (17.3), 633.0719 (1.0), 597.0530 (4.3), 427.0310 (3.2), 399.0376 (1.9), 300.9992 (27.7), 275.0199 (20.0), 273.0337 (0.4), 249.0405 (4.3), 245.0090 (4.6), 229.0139 (10.3), 217.0137 (3.7), 201.0186 (7.6), 169.0123 (9.5), 145.0282 (4.2), 117.0332 (1.6)2.741.743[5,10,16,17]
51.digalloyl-HHDP-hexose (tellimagrandin I) 1C34H26O22785.0843785.0864 (84.5), 300.9993 (100), 275.0200 (45.2), 257.0092 (6.6), 249.0409 (34.2), 245.0078 (4.1), 229.0141 (8.8), 201.0185 (5.5), 185.0232 (5.3), 173.0235 (3.6), 169.0133 (15.4), 145.0286 (3.4), 125.0231 (15.2), 107.0124 (3.2)3.012.630[10]
52.digalloyl-hexose 3C20H20O14483.0780483.0788 (100), 331.0680 (5.7), 313.0571 (17.7), 271.0462 (44.6), 211.0242 (13.1), 169.0132 (39.2), 151.0027 (1.9), 125.0231 (29.0), 107.0124 (4.3)3.091.659
53.galloyl-HHDP- hexose 3C27H22O18633.0733633.0747 (77.9), 463.0529 (3.4), 300.9992 (100), 275.0202 (11.1), 249.0406 (4.0), 257.0093 (4.7), 245.0095 (1.7), 229.0139 (7.6), 185.0232 (3.4), 169.0133 (2.3), 145.0285 (2.1), 125.0233 (2.9), 107.0121 (0.4)3.242.169
54.oenothein B 2C68H46O441568.1518
783.0686 [M-2H]2−,		783.0699 (100), 765.0596 (17.3), 633.0742 (1.6), 597.0522 (3.7), 427.0315 (4.5), 399.0354 (1.7), 300.9992 (27.7), 275.0198 (19.7), 273.0337 (0.4), 249.0402 (4.3), 245.0090 (4.6), 229.0139 (10.3), 217.0137 (3.7), 201.0186 (7.7), 185.0235 (2.4), 169.0133 (9.2), 125.0230 (8.2)3.331.590[10]
55.brevifolin carboxylic acidC13H8O8291.0149291.0151 (13.2), 247.0246 (100), 229.0170 (0.2), 219.0296 (3.1), 203.0341 (0.7), 191.0342 (9.3), 173.0234 (3.7), 145.0282 (3.4), 119.0487 (1.6)3.341.511
56.digalloyl-hexose 4C20H20O14483.0780483.0787 (100), 331.0675 (59.0), 313.0568 (10.8), 271.0462 (1.3), 211.0247 (1.6), 169.0133 (13.8), 125.0230 (45.6), 107.0125 (2.8)3.351.390
57.oenothein A1C102H70O662352.2277
1175.6083 [M-2H]2−,		1175.6090 (100) [M-2H]2−, 785.0854 (4.9), 765.0599 (9.2), 633.0746 (2.9), 597.0533 (3.1), 463.0533 (0.6), 427.0313 (4.0), 399.0364 (2.3), 300.9991 (35.7), 273.0043 (10.9), 257.0092 (4.6), 245.0090 (5.5), 229.0140 (9.9), 217.0138 (3.7), 201.0186 (7.3), 185.0235 (2.5), 173.0235 (4.4)3.442.250[5,15]
58.digalloyl-HHDP-hexose (tellimagrandin I) 2C34H26O22785.0843785.0865 (100), 300.9993 (96.9), 275.0199 (38.7), 257.0089 (5.8), 249.0403 (32.8), 245.0088 (3.2), 229.0135 (10.0), 201.0186 (5.7), 185.0236 (2.8), 173.0235 (3.6), 169.0135 (15.4), 145.0282 (3.4), 125.0230 (15.8), 107.0124 (3.2)3.661.776[10]
59.oenothein A2C102H70O662352.2277
1175.6083 [M-2H]2−,		1175.6084 (100), 785.0846 (3.9), 765.0589 (8.0), 633.0752 (3.3), 597.0527 (3.6), 463.0486 (0.7), 427.0307 (3.5), 399.0361 (2.0), 300.9990 (45.0), 273.0043 (9.6), 257.0092 (4.1), 245.0090 (5.6), 217.0135 (2.8), 201.0186 (7.4), 169.0133 (2.1)3.891.731
60.trigalloyl-hexose 1C27H24O18635.0890635.0904 (81.4), 465.0680 (100), 313.0569 (50.4), 253.0372 (0.8), 223.0259 (2.9), 193.0137 (5.5), 169.0132 (80.4), 151.0022 (3.7), 125.0230 (57.1), 107.0123 (12.0)3.922.209[10,23]
61.trigalloyl-hexose 2C27H24O18635.0890635.0905 (100), 483.0791 (11.1), 465.0680 (25.5), 313.0568 (27.7), 223.0236 (0.7), 193.0131 (4.9), 169.0131 (84.1), 151.0023 (1.8), 125.0229 (64.1), 107.0122 (7.1)4.012.304
62.trigalloyl-hexose 3C27H24O18635.0890635.0902 (100), 483.0786 (21.5), 465.0683 (11.3), 313.0568 (12.6), 253.0346 (0.4), 193.0134 (3.4), 169.0131 (49.2), 151.0024 (2.1), 125.0229 (49.7), 107.0123 (5.4)4.111.926
63.trigalloyl-hexose 4C27H24O18635.0890635.0905 (100), 483.0799 (4.8), 465.0681 (15.9), 313.0572 (26.8), 253.0363 (0.7), 193.0139 (1.9), 169.0132 (70.2), 151.0027 (1.8), 125.0230 (57.1), 107.0124 (6.7)4.312.304
64.tellimagradin II 1C41H30O26937.0953937.0970 (100), 785.0782 (0.3), 300.9991 (85.3), 275.0199 (18.1), 257.0094 (4.0), 249.0204 (12.32), 245.0090 (3.6), 229.0140 (7.1), 201.0188 (3.9), 169.0132 (13.8), 125.0230 (9.2), 107.0123 (1.9)4.571.796
65.ellagic acid O-pentosideC19H14O12433.0412433.0416 (100), 300.9990 (69.1), 271.9979 (2.5), 257.0092 (1.4), 245.0090 (0.6), 229.0139 (2.2), 201.0190 (1.5), 185.0237 (1.4), 145.0277 (0.4), 4.660.811
66.tellimagradin II 2C41H30O26937.0953937.0970 (100), 785.0897 (3.8), 300.9992 (85.0), 275.0200 (19.2), 257.0091 (4.3), 249.0403 (11.3), 245.0087 (3.6), 229.0141 (6.3), 217.0139 (1.6), 201.0186 (3.8), 169.0131 (14.7), 125.0230 (10.4), 107.0124 (1.4)4.741.726
67.tetragalloyl-hexose 1C34H28O22787.0999787.1011 (100). 617.0776 (6.3), 465.0681 (32.2), 313.0572 (13.5), 295.0458 (7.8), 193.0135 (4.0), 169.0131 (73.7), 151.0025 (1.8), 125.0230 (70.0), 107.0122 (8.9)4.941.518[23]
68.ellagic acidaC14H6O8300.9991300.9990 (100), 257.0086 (1.6), 229.0143 (3.2), 217.0139 (0.9), 201.0182 (3.2), 185.0235 (2.3), 173.0235 (2.9), 145.0281 (3.1), 129.0332 (0.9), 117.0331 (1.0)5.01−0.101[10]
69.tetragalloyl-hexose 2C34H28O22787.0999787.1019 (81.1). 617.0794 (57.9), 465.0681 (24.1), 313.0571 (10.1), 295.0459 (9.5), 193.0135 (4.2), 169.0123 (100), 151.0023 (3.0), 125.0230 (84.3), 107.0124 (10.4)5.052.521
Flavonoids
70.procyanidin dimerC30H26O12577.1351577.1367 (100), 425.0887 (80.2), 407.0778 (66.3), 289.0721 (68.6), 245.0816 (4.7), 203.0710 (6.1), 179.0341 (6.0), 137.0232 (17.0), 125.0231 (89.8)2.922.617
71.catechin/epicatechinC15H14O6289.0718289.0719 (100), 245.0816 (41.3), 203.0707 (15.9), 179.0341 (10.1), 137.0231 (11.4), 109.0280 (34.0)3.120.583
72.myricetin 3-O-galloylhexosideC28H24O17631.0941631.0953 (100), 479.0839 (27.6), 317.0300 (15.9), 316.0226 (51.7), 299.0195 (3.1), 287.0197 (12.4), 271.0247 (20.5), 259.0253 (4.4), 243.0295 (2.3), 178.9972 (4.0), 169.0130 (4.5), 151.0022 (5.6), 125.0229 (5.7), 107.0125 (3.7)4.191.914[10,15,23]
73.kaempferol 7-O-deoxyhexosylhexoside 1C27H30O15593.1512593.1525 (95.1), 447.0926 (8.6), 431.0983 (5.7), 285.0406 (30.5), 255.0298 (30.5), 227.0342 (4.0), 211.0396 (2.9), 151.0025 (1.3), 107.0120 (0.5)4.272.254
74.patuletin 3-O-dihexosideC28H32O18655.1516655.1529 (100), 331.0439 (14.0), 330.0386 (73.2), 315.0151 (44.7), 287.0201 (16.2), 259.0246 (2.1), 243.0289 (3.9), 231.0300 (4.9), 215.0349 (5.6), 187.0393 (2.8), 165.9897 (4.3)4.371.989
75.myricetin 3-O-hexoside1C21H20O13479.0831479.0830 (100), 316.0226 (93.8), 317.0292 (20.8), 287.0199 (17.6), 271.0249 (26.7), 259.0247 (6.4), 243.0292 (3.6), 227.0351 (1.0), 178.9974 (3.4), 151.0025 (4.8), 107.0124 (1.6)4.48−0.217[5,10,15,16,23]
76.myricetin O-hexuronideC21H18O14493.0624493.0634 (85.1), 317.0305 (100), 299.0199 (2.7), 271.0248 (3.9), 243.0297 (1.9), 227.0344 (1.3), 199.0391 (1.0), 178.9977 (15.6), 151.0025 (25.1), 137.0231 (16.8), 107.0124 (8.7)4.511.423[5,16]
77.myricetin 3-O-hexoside 2C21H20O13479.0831479.0836 (100), 317.0288 (17.9), 316.0226 (90.5), 287.0200 (17.1), 271.0248 (27.4), 259.0244 (7.0), 243.0291 (4.0), 227.0342 (0.9), 178.9975 (3.4), 151.0023 (3.9), 107.0121 (1.8)4.570.994[10]
78.6-hydroxykaempferol methyl ether O-dihexosideC28H32O17639.1567639.1581 (100), 459.0948 (0.7), 315.0499 (15.3), 314.0436 (64.2), 299.0199 (49.5), 271.0250 (25.7), 243.0298 (5.4), 227.0351 (3.4), 215.0343 (10.8), 199.0396 (2.4), 183.0445 (2.3), 165.9896 (6.6), 164.9817 (4.2), 136.9872 (0.7), 133.0278 (3.5), 109.9994 (3.0)4.772.233
79.quercetin 3-O-galloylhexoside 1C28H24O16615.0992615.1002 (100), 463.0890 (30.7), 301.0354 (30.4), 300.0277 (49.7), 271.0247 (31.7), 255.0299 (14.9), 243.0294 (5.2), 178.9977 (2.3), 169.0134 (9.6), 151.0026 (6.0), 125.0231 (11.8), 121.0283 (1.2), 107.0123 (4.4)4.821.727[5,10,16,23]
80.kaempferol O-dihexosideC27H30O16609.1461609.1473 (100), 429.0834 (2.2), 285.0395 (26.8), 284.0328 (82.9), 255.0298 (47.9), 227.0346 (32.2), 211.0397 (1.7), 178.9974 (1.6), 163.0030 (0.3), 151.0025 (2.8), 107.0121 (1.7)4.852.023
81.quercetin galloylhexoside 2C28H24O16615.0992615.1003 (100), 463.0889 (29.2), 301.0353 (30.4), 300.0274 (37.9), 255.0298 (14.1), 243.0298 (5.2), 178.9977 (2.8), 169.0130 (6.6), 151.0024 (7.2), 125.0231 (7.5), 121.0280 (1.3), 107.0123 (4.3)4.961.825[5,10]
82.myricetin 3-O-deoxyhexosideC21H20O12463.0882463.0888 (91.6), 317.0291 (26.3), 316.0227 (100), 287.0202 (17.7), 271.0248 (27.0), 259.0248 (5.4), 243.0293 (3.7), 227.0344 (0.6), 178.9974 (4.2)5.111.384[5,15]
83.6-hydroxykaempferol methyl ether O-deoxyhexosylhexosideC28H32O16623.1618623.1630 (100), 315.0493 (9.1), 314.0436 (67.1), 299.0200 (51.3), 271.0248 (26.7), 255.0299 (3.9), 243.0294 (5.2), 227.0346 (3.6), 215.0345 (11.8), 165.9897 (7.6), 164.9811 (4.9), 136.9867 (1.8), 133.0284 (4.8)5.141.929
84.Isoquercitrin aC21H20O12463.0885463.0888 (100), 301.0349 (29.8), 300.0277 (63.8), 271.0249 (36.6), 255.0297 (14.9), 243.0296 (9.4), 227.0344 (2.6), 215.0343 (0.5), 199.0392 (0.9), 178.9978 (2.6), 163.0025 (1.8), 151.0025 (5.7), 121.0278 (1.0), 107.0124 (2.2)5.191.319[15,16,23];
85.quercetin O-hexuronideC21H18O13477.0675477.0682 (62.4), 301.0356 (100), 283.0245 (1.8), 255.0296 (3.6), 245.0450 (2.6), 227.0354 (1.3), 211.0396 (1.8), 178.9978 (9.7), 163.0022 (2.6), 151.0024 (21.7), 121.0281 (6.3), 107.0123 (8.2)5.221.543[5,10,15,16]
86.hyperoside aC21H20O12463.0885463.0891 (100), 301.0351 (40.3), 300.0278 (71.9), 271.0249 (32.3), 255.0298 (13.9), 243.0296 (8.2), 227.0340 (2.7), 215.0343 (0.5), 178.9979 (3.3), 151.0025 (4.9), 121.0278 (1.5), 107.0121 (1.8)5.291.837[5,10,15,16]
87.kaempferol-galloylhexoside 1C28H24O15599.1042599.1055 (100), 447.0935 (2.9), 285.0401 (12.6), 284.0327 (12.0), 255.0296 (9.3), 241.0350 (6.6), 227.0350 (5.2), 211.0243 (1.9), 169.0133 (37.2), 151.0027 (4.2), 125.0230 (26.5), 107.0122 (5.6)5.292.148[10,16,23]
88.quercetin O-pentosideC20H18O11433.0776433.0781 (100), 301.0348 (23.1), 300.0277 (95.0), 271.0248 (49.6), 255.0295 (20.3), 243.0300 (10.6), 227.0353 (3.1), 178.9979 (2.1), 151.0023 (6.1), 107.0119 (2.6)5.620.982[5,10,15,16,23]
89.kaempferol 3-O-glucoside aC21H20O11447.0934447.0945 (100), 285.0396 (15.6), 284.0328 (53.2), 255.0297 (38.2), 227.0345 (41.5), 211.0402 (1.5), 151.0029 (1.9), 107.0120 (0.5)5.652.607[16]
90.kaempferol-galloylhexoside 2C28H24O15599.1042599.1055 (100), 447.0931 (5.2), 285.0403 (17.4), 284.0326 (15.7), 255.0301 (11.9), 241.0349 (4.3), 227.0348 (7.0), 211.0230 (0.7), 169.0132 (31.6), 151.0026 (3.5), 125.0230 (24.5), 107.0124 (6.9)5.672.148[16]
91.myricetin 3-O-caffeoylhexoside 1C22H22O12641.1160641.1160 (100), 479.0840 (37.8), 317.0294 (23.6), 316.0227 (78.4), 287.0198 (17.6), 271.0249 (30.0), 259.0243 (2.9), 243.0291 (2.9), 178.9974 (4.6), 151.0022 (4.6), 107.0120 (1.1)5.711.813[16]
92.kaempferol O-hexuronideC21H18O12461.0725461.0732 (38.4), 285.0405 (100), 257.0450 (4.5), 229.0501 (6.6), 211.0393 (1.3), 175.0236 (5.0), 151.0021 (1.2), 107.01221 (2.1)5.831.368[5,10,15,16]
93.myricetin 3-O-caffeoylhexoside2C22H22O12641.1160641.1160 (100), 479.0840 (29.0), 317.0297 (27.7), 316.0227 (70.7), 287.0200 (16.8), 271.0249 (27.3), 259.0245 (4.9), 243.0294 (3.3), 178.9978 (5.1), 151.0024 (6.7), 107.0122 (2.2)5.851.906
94.quercetin 3-O-deoxyhexosideC21H20O11447.0933447.0940 (100), 301.0352 (71.7), 300.0279 (78.9), 271.0252 (31.7), 255.0297 (17.6), 243.0299 (7.3), 227.0351 (2.3), 178.9972 (4.4), 151.0023 (10.3), 121.0284 (2.9), 107.0123 (4.0)5.901.645[5,10,15,23]
95.isorhamnetin 3-O-glucoside aC22H22O12477.1038477.1052 (100), 315.0497 (11.9), 314.0436 (54.4), 301.0359 (7.3), 271.0251 (20.5), 257.0454 (4.8), 243.0296 (22.2), 227.0351 (3.6), 199.0396 (3.1), 151.0024 (3.9), 107.0126 (0.7)6.022.831
96.naringenin O-hexosideC21H22O10433.1140433.1165 (4.5), 271.0613 (100), 227.0711 (0.2), 193.0138 (0.9), 151.0023 (21.0), 119.0487 (22.3), 107.0122 (2.6)6.065.703
97.isorhamnetin O-hexuronideC22H20O13491.0831491.0838 (49.3), 315.0514 (100), 300.0279 (25.8), 287.0570 (0.9), 271.0249 (26.8), 255.0298 (10.9), 243.0296 (7.7), 227.0340 (1.7), 215.0358 (0.4), 199.0385 (0.4), 175.0237 (5.6), 151.0025 (3.1), 107.0122 (1.3)6.081.397
98.kaempferol 3-O-pentosideC20H18O10417.0827417.0833 (100), 285.0393 (16.4), 284.0328 (63.3), 255.0298 (41.5), 227.0345 (38.1), 211.0391 (1.0), 151.0031 (0.8)6.080.530[15,16]
99.kaempferol O-deoxyhexosylhexoside 2C27H30O15593.1512593.1522 (77.4), 285.0406 (100), 229.0503 (11.8), 211.0393 (2.6), 163.0022 (1.9), 151.0022 (0.3), 135.0076 (1.3), 107.0124 (1.9)6.321.697
100.myricetin O-p-coumaroylhexosideC30H26O15625.1199625.1210 (85.5), 479.0835 (13.4), 317.0294 (18.9), 316.0227 (100), 287.0200 (15.4), 271.0250 (26.7), 259.0248 (6.3), 243.0301 (3.5), 178.9975 (4.3), 151.0026 (4.0), 107.0123 (1.8)6.591.819
101.kaempferol O-deoxyhexoside C21H20O10431.0984431.0988 (100), 285.0405 (78.8), 255.0299 (35.6), 227.0346 (30.6), 211.0390 (1.6), 163.0029 (0.9), 151.0012 (0.5), 135.0075 (0.5), 107.0124 (0.9)6.590.380[5,10,15,16,23]
102.myricetin 3-O-feruloylhexosideC31H28O16655.1305655.1322 (92.2), 479.0842 (8.7), 317.0290 (18.1), 316.0226 (100), 287.0200 (18.4), 271.0250 (26.1), 259.0249 (5.9), 243.0291 (4.7), 178.9978 (3.0), 151.0023 (4.5), 107.0122 (1.7)6.742.659
103.kaempferol 7-O-caffeoylhexosideC30H26O14609.1250609.1262 (100), 447.0934 (3.1), 285.0407 (75.3), 284.0331 (16.1), 255.0297 (14.6), 227.0345 (11.0), 211.0402 (1.0), 179.0340 (12.9), 161.0233 (37.6), 135.0438 (18.8), 133.0282 (13.6)6.811.940
104.quercetin O-coumaroylhexoside 1C30H26O14609.1250609.1260 (100), 463.0891 (22.7), 301.0353 (37.7), 300.0278 (59.7), 271.0250 (33.4), 255.0296 (15.6), 243.0297 (7.1), 227.0347 (3.0), 211.0397 (1.2), 178.9977 (2.6), 151.0025 (6.4), 121.0280 (1.3), 107.0122 (2.6)7.050.643[15,23]
105.quercetin O-coumaroylhexoside 2C30H26O14609.1250609.1260 (100), 463.0887 (30.0), 301.0352 (41.2), 300.0277 (78.9), 271.0250 (38.0), 255.0298 (16.6), 243.0295 (8.9), 227.0345 (2.5), 211.0398 (1.8), 178.9977 (2.7), 163.0031 (0.8), 151.0023 (6.6), 121.0281 (0.7), 107.0121 (2.1)7.171.743[16]
106.quercetin 3-O-feruloylhexoside 1C31H28O15639.1355639.1367 (100), 463.0887 (16.2), 301.0352 (37.7), 300.0277 (73.2), 271.0250 (36.0), 255.0297 (17.4), 243.0297 (11.1), 227.0351 (2.6), 178.9978 (3.4), 175.0393 (0.9), 151.0026 (8.0), 121.0277 (0.7), 107.0123 (2.8)7.201.841[16]
107.quercetin 3-O-feruloylhexoside 2C31H28O15639.1355639.1367 (100), 463.0887 (15.1), 301.0351 (31.9), 300.0278 (72.4), 271.0250 (37.3), 255.0300 (15.5), 243.0295 (9.3), 227.0344 (2.7), 199.0401 (0.6), 193.0502 (0.5), 178.9977 (2.7), 175.0398 (0.5), 161.0231 (1.3), 151.0025 (6.9), 121.0280 (1.4), 107.0121 (3.0)7.321.841
108.quercetin O-coumaroylhexoside 3C30H26O14609.1250609.1260 (100), 463.0888 (22.8), 301.0352 (38.1), 300.0278 (77.5), 271.0250 (40.5), 255.0298 (18.5), 243.0295 (10.3), 227.0344 (3.1), 211.0396 (1.2), 178.9980 (2.8), 163.0027 (1.6), 151.0025 (7.4), 121.0281 (1.3), 107.0125 (2.9)7.541.743
109.kaempferol O-coumaroylhexoside 1C30H26O13593.1301593.1313 (100), 447.0948 (4.0), 285.0403 (57.7), 284.0328 (68.0), 255.0298 (41.3), 227.0346 (28.0), 211.0396 (2.3), 163.0389 (1.1), 151.0024 (3.1), 145.0283 (11.5), 135.0075 (1.0), 119.0488 (2.7), 107.0125 (2.3)7.582.168[16]
110.quercetin aC15H10O7301.0354301.0357 (100), 273.0410 (3.2), 257.0463 (0.6), 229.0505 (1.2), 211.0401 (0.4), 178.9978 (22.3), 151.0025 (44.3), 121.0281 (14.5), 107.0124 (15.4)7.62 [17,23]
111.kaempferol O-coumaroylhexoside 2C30H26O13593.1301593.1313 (100), 447.0934 (2.0), 285.0403 (5.2), 284.0328 (52.4), 255.0298 (33.5), 227.0345 (24.5), 211.0395 (1.0), 163.0388 (2.5), 151.0024 (3.9), 145.0282 (8.0), 119.0488 (2.6), 107.0123 (2.0)7.682.084[16]
112.kaempferol O-feruloylhexoside 1C31H28O14623.1406623.1421 (100), 447.0935 (1.7), 323.0779 (2.7), 285.0401 (35.5), 284.0328 (63.0), 255.0298 (39.3), 227.0346 (28.4), 211.0397 (2.0), 193.0503 (1.6), 175.0394 (4.3), 161.0233 (8.1)151.0025 (2.2), 134.0358 (1.6), 107.0124 (1.6)7.712.345
113.kaempferol O-feruloylhexoside 2C31H28O14623.1406623.1419 (100), 447.0924 (1.5), 323.0770 (3.7), 285.0403 (61.1), 284.0327 (55.0), 255.0297 (41.4), 227.0344 (28.1), 211.0395 (3.1), 193.0500 (1.9), 175.0388 (4.5), 161.0231 (5.5), 151.0025 (3.6), 134.0359 (1.5), 107.0126 (2.7)7.82
114.6-hydroxykaempferol methyl etherC16H12O7315.0510315.0515 (80.4), 300.0279 (100), 272.0326 (8.1), 255.0302 (4.2), 243.0307 (1.2), 227.0345 (1.6), 165.9896 (8.0), 139.0025 (4.4), 136.9865 (4.4), 8.821.441
115.naringenin aC15H12O5271.0612271.0612 (12.6), 151.0020 (12.5), 119.0496 (10.2)8.580.468
116.kaempferol aC15H9O7285.0406285.0404 (100), 257.0457 (0.9), 239.0352 (1.2), 229.0499 (0.9), 211.0399 (1.0), 187.0390 (1.4), 151.0024 (1.5), 107.0125 (1.2)8.83−0.215[17]
117.quercetin O-cinnamoylhexoside 1C30H26O13593.1301593.1313 (100), 301.0346 (23.2), 300.0278 (80.2), 271.0248 (41.4), 255.0295 (9.6), 243.0295 (14.3), 227.0342 (4.0), 178.9976 (2.7), 151.0024 (5.0), 121.0279 (0.6), 107.0124 (2.3)8.852.067
118.quercetin O-cinnamoylhexoside 2C30H26O13593.1301593.1313 (100), 301.0349 (27.3), 300.0278 (73.8), 271.0251 (36.5), 255.0300 (10.0), 243.0297 (10.9), 227.0341 (3.1), 199.0392 (0.6), 178.9971 (2.8), 151.0024 (5.3), 135.0074 (1.0), 121.0283 (0.7), 107.0124 (2.4)9.122.084
119.isorhamnetin aC16H12O7315.0510315.0515 (100), 300.0279 (47.0), 271.0241 (3.6), 255.0312 (1.2), 227.0346 (1.1), 164.0103 (3.2), 151.0023 (8.3), 107.0122 (7.0)9.101.441
120.kaempferol O-cinnamoylhexosideC30H26O12577.1351577.1361 (100), 285.0396 (17.2), 284.0328 (71.6), 255.0298 (49.7), 227.0345 (8.1), 211.0393 (1.1), 151.0024 (1.2), 9.50−0.310
121.myricetin aC15H10O8In (+)ESI-MS/MS
319.0448		319.0440 (100), 273.0383 (2.6), 245.0440 (4.4), 217.0491 (5.0), 165.0179 (2.5), 153.0180 (13.2)9.49−0.804[15,17]
a—Identified by comparison with an authentic standard.
Sugar esters 1, 10, 14, and 25 yielded prominent fragment ions resulting from the hexose cross ring cleavages and losses of 60 Da (0,4Hex), 90 Da (0,3Hex), and 120 Da (0,2Hex), as has been previously reported [20] (Figure S1). Methylgallate with [M-H]− at m/z 183.029 was deduced from the typical loss of the methyl radical (•CH3) (−15 Da) at m/z 168.005 [24] (Table 1). The extracted ion chromatograms of hydroxybenzoic and hydroxycinnamic acids and derivatives showed that the E. angustifolium profile was dominated by gallic acid (2) (26.84%), methylgallate (19) (13.98%), coumaric acid-O-hexoside (22) (11.64%), and quinic acid (22) (11.52%) together with galloyl O-hexose (1) (10.27%). Overall, phenolic acids accounted for 6.42% of the assayed compounds (Figure 2D).

2.1.2. Mono- and Diacylquinic Acids

Overall, 12 monoacylquinic and 7 diacylquinic acids were annotated/dereplicated on the base of preferential fragmentation reported elsewhere [24,25]. Thus, 27, 28, 29, and 32 were assigned to 3-galloyl-, 3-caffeoyl (neochlorogenic), 3-p-coumaroyl-, and 3-feruloylquinic acid, respectively, while 30, 35, 36, 39, 41, and 43 were ascribed as 5-substitidet quinic acid conjugates (Table 1, Figure 2A). Within the subclass of galloyl-caffeoylquinic acids at m/z 505.100 [M-H]−, four isomers, 33, 37, 38, and 40, were annotated. Compound 33 (consistent with molecular formula C23H21O13) gave a prominent ion at m/z 353.088 [M-H-galloyl]- and a base peak at m/z 191.055 [quinic acid-H]−, along with the abundant ions at m/z 179.034 [caffeic acid-H]− and 135.044 [caffeic acid-H-CO2]−, as was seen for 3-caffeoylquinic acid (Table 1). In line with the fragmentation pattern of the 1,3-diacylquinic acids subclass [24], 33 was assigned to 1-galloyl-3 caffeoylquinic acid. Compound 37 afforded characteristic ions at m/z 353.088 (5.7%) and 343.068 [M-H-caffeoyl]− (15.2%), indicating a loss of a caffeoyl residue before the galloyl one. This assignment was also suggested by the abundant ion at m/z 169.012, as was registered in 3-galloylquinic acid. Thus, 37 was ascribed as 1-caffeoyl-3-galloylquinic acid.
The base peak at m/z 191.055 suggested 1,5- (38) and 3,5-diacylquinic acid (40), supported by the relative abundances of the ions at m/z 179.034 and 135.044: 3.1% and 3.0% in 1-galloyl-5-caffeoylquinic acid, and 56.8% and 54.2% in 3-caffeoyl-5-galloylquinic acid, respectively (Table 1; Figure S2). In the same manner, 44 (3-galloyl-5-p-coumaroylquinic acid) produced a base peak at m/z 191.055, accompanied with low abundant peaks at m/z 163.040 (4.35%) and 119.049 (1.2%), as was seen in 5-p-coumaroylquinic acid (Table 1).
Among acylquinic acids, being present at 16.64% of the studied compounds (Figure 2D), neochlorogenic acid (28) (35.11%), together with its isomer chlorogenic acid (30) (16.91%), and 3-p-coumaroylquinic acid (18.23%) appeared to be dominant for the E. angustifolium extract. Overall, neochlorogenic acid content in leaves was three times higher compared to the flowers, being present up to 5.78 mg/g dw and 1.82 mg/g dw, respectively [5].

2.1.3. Gallotannins and Ellagitannins

Four compounds (48, 49, 52, and 56) were assigned to digalloyl-hexose isomers. The MS/MS fragmentation pathway afforded indicative ions, resulting from the losses of galloyl residue at m/z 331.068 [M-H-C7H4O4]− and gallic acid (GA) at m/z 313.057 [M-H-C7H6O5]− along with abundant ions at m/z 169.013 [GA-H]− and 125.023 [GA-H-CO2]−. Peaks 57–60 were tentatively identified as trigalloyl-hexose isomers, owing to their [M-H]− at m/z 635.090 and typical MS/MS fragments at m/z 483.079 [M-H-galloyl]−, 313.057 [M-H-2galloyl-H2O]−, and 169.013 [M-H-2galloyl-Hex]− (Table 1). Regarding 67 and 69, four galloyl residues were deduced from the following transitions: 787.101→617.078 [M-H-GA]−→465.068 [M-H-2galloyl-H2O]−→313.057 [M-H-3galloyl-H2O]− (Table 1). Accordingly, the aforementioned compounds were annotated as tetragalloyl-hexose isomers [24]. Three isobars, 46, 47, and 53, shared the same [M-H]−at m/z 633.075; the abundant fragment ion at m/z 300.999 (ellagic acid, EA) indicated the loss of a galloyl-hexose moiety (332 Da, C13H16O10) (Table 1). This assumption was in line with the presence of a series of indicative ions for EA, including 275.020 257.009 [EA-H-CO2]−, 229.014 [EA-H-CO-CO2]−, 185.023 [EA-H-2CO-2CO2]−, and 145.029 [EA-H-4CO-CO2]−(Table 1) [21]. Consequently, the abovementioned compounds were ascribed as galloyl-HHDP-hexose isomers. In the same way, tellimagradin I isomers 51 and 58 at m/z 785.086(7) [M-H]− were discernable by the characteristic loss of 484.087 Da (C20H20O14) and the prominent ion at m/z 300.999 (Table 1). Compounds 64 and 66 at m/z 937.0970 differ from the aforementioned compounds with 152 Da (galloyl residue) and were consistent with tellimagradin II [26].
The MS/MS spectrum of compound 55 ([M-H]− at m/z 291.045, C13H7O8) was acquired. The fragmentation pattern was delineated by the base peak at m/z 247.025 [M-H-CO2]− and the following transitions at m/z 219.030: [M-H-CO-CO2]−→191.034 [M-H-2CO-CO2]−→173.023[M-H-2CO-CO2-H2O]−→145.028 [M-H-3CO-CO2-H2O]−, as has been seen in brevifolin carboxylic acid [24].
Compound 50 was ascribed as the macrocyclic ellagitannin dimer oenothein B, previously reported from Epilobium species [3]. The dimer consists of two 784 Da units (tellimagradin I) and has a molecular mass of 1568.1518 Da (C68H48O44). In the MS/MS spectrum, 50 afforded prominent ions [M-2H]2− at m/z 783.070 (consistent with C68H46O44) and at m/z 300.9992 [EA-H]−, resulting from the loss of C20H20O14 (484.087 Da = 152 + 152 + 162 + 18 Da). It should be noted that in the extracted ion chromatogram, Oenonthein B showed two isomers—the minor one could be seen eluting after the main isomer at tR 3.74 (both isomers had identical MS/MS patterns) (Figure S3). In this respect, Karonen et al. [27] reported that in HPLC-MS analysis, the purified oenothein B shows a mixture of two isomers in a 95:5 ratio.
Compounds 57 and 59 were assigned to oenothein A isomers. The ellagitannin trimer oenothein A has an additional monomer (784 Da) compared to oenothein B and a molecular mass of 2352.228 Da, consistent with a molecular formula C102H72O66. Both isomers were tentatively identified by the double-charged ions at m/z 1175.609 [M-2H]2−. They gave indicative fragment ions at m/z 785.085 [tellimagradin I-H]−, 633.075 [galloyl-HHDP-hexose]−, 427.031 [galloyl-HHDP-hexose-4H2O]−, and 399.036 [galloyl-HHDP-hexose-4H2O-CO]−, along with the abundant fragment ion at m/z 300.9992 ([EA-H]−). The trimeric Oenothein A showed a major isomer at tR 3.44 and a minor one, in contrast to the study of Karonen et al. [27], where two major isomers in a 50:50 ratio were recorded. Among gallo- and ellagitannins, reaching levels of up to 10.53%, oenothein B isomer 1 (50) (53.29%), followed by ellagic acid acid (68) (14.12%), galloyl-HHDP-hexose isomer 3 (53) (4.62%), and ellagic acid O-pentoside (65) (3.61%), were found to be dominant for E. angustifolium extract (Figure 2B).
It is well known that the polyphenol profiling of fireweed is dominated by oenothein B [5,10,16,17]. In the study of Baert et al. [5] on the intra-organ distribution of ellagitannins, both oenothein B and A have been found in significant levels, reaching 148.28 mg/g dw and 52.14 mg/g dw, respectively, in the inflorescence apex of the species. It is noteworthy that the oenothein B was twice as much as that determined in the leaves. Moreover, oenothein B was more abundant in the flowers (up to 102.27 mg/g dw) in comparison with the leaves.

2.1.4. Flavonoids

Procyanidin dimer (70) at m/z 577.137 and catechin/epicatechin (71) at m/z 289.072 were deduced from the indicative fragment ions at m/z 245.082, 203.071 and 137.023 (Table 1) [26].
A variety of acylhexosyl flavonols were tentatively identified, including kaempferol, quercetin, and myricetin glycosides (Figure 2C). The approach for flavonol annotation has been delineated elsewhere [20]. Compounds 72, 79, 81, 87, and 90 shared similar fragmentation patterns, affording indicative fragment ions at m/z 447.093 (87 and 90), 463.089 (79 and 81), and 479.084 (72), resulting from the loss of a galloyl moiety (152 Da, C7H4O4) from kaempferol, quercetin, and myricetin glycosides, respectively (Table 1). Moreover, the galloyl residue was evidenced by the fragment ions at m/z 169.013, 151.002, and 125.023. The glycosylation position at C-3 was discernable by the low abundant Y0− aglycone ions compared with the radical aglycone ions [Y0-H]−●, as typical MS/MS fragmentation behavior of flavonoid 3-O-glycosides [24]. Consequently, 72 was ascribed as myricetin 3-O-galloylhexoside, while 79 and 81 were assigned to quercetin 3-O-galloylhexosides, and 87 and 90 to kaempferol 3-O-galloylhexosides (Figure S4). In the same manner, 91/93 and 103 were annotated as caffeoyl esters of myricetin- and kaempferol-hexoside, respectively. The caffeoyl residue was deduced from the losses of 162 Da (C9H6O3) and 324 Da (C15H16O8), supported by the prominent fragments at m/z 179.034, 161.023, and 135.044 (Table 1). Coumaroyl esters of myricetin- (100), quercetin- (104, 105, and 108), and kaempferol-hexoside (109 and 111) were discernable by the transitions—[M-H]−→(Y0-H)−●—resulting from the losses of 309 Da (C15H17O7). Accordingly, feruloylhexosides 102, 106, 107, 112, and 113 were witnessed by the indicative losses of 176 Da (C10H8O3) and 339 Da (C16H19O8) from the corresponding precursor ions. Two quercetin-cinnamoylhexosides (117 and 118) and kaempferol-cinnamoylhexoside (120) were evidenced on the basis of the loss of 293 Da ([M-H]−→(Y0-H)−●).
Compounds 76, 85, 92, and 97 were closely related with the same fragmentation pathway, giving ([M-H-HexA]−, suggesting hexuronides of myricetin, quercetin, kaempferol, and isorhamnetin, respectively. In the same manner, deoxyhexosylhexoside of kaempferol (73, 99) and 6-hydroxykaempferol methyl ether (83) were tentatively identified (Table 1).
In (-) ESI-MS/MS compounds, 74 and 78 afforded prominent fragment ions at m/z 331.044 and 315.050, respectively, indicating the presence of a dihexosyl moiety. The deprotonated aglycone molecules underwent the loss of a methyl radical at m/z 315.015 and 299.020, respectively. Low abundant RDA ions at m/z 165.990 (1,3A−-●CH3) (70 and 74), 164.982 (1,3A−-CH4), and 136.987 (1,3A−-CH4-CO) (78) a suggested 6-methoxylated A-ring of the flavonoid aglycone. Accordingly, the aforementioned compounds were annotated as patuletin 3-O-dihexoside (74) and 6-hydroxykaempferol methyl ether O-dihexoside (78).
Based on the comparison with the fragmentation patterns and retention times of reference standards, 84, 86, 89, 95, 110, 116, 119, and 121 were unambiguously identified as isoquercitrin, hyperoside, kaempferol 3-O-glucoside, isorhamnetin 3-O-glucoside, quercetin, kaempferol, isorhamnetin, and myricetin. Overall, kaempferol O-deoxyhexoside (101) (23.19%), quercetin 3-O-deoxyhexoside (94) (14.73%), and quercetin O-hexuronide (85) (13.84%) were found to be the predominant flavonoids, followed by kaempferol O-hexuronide (92) (7.10%), 6-hydroxykaempferol methyl ether O-dihexoside (78) (3.99%), and isoquercitrin (84) (3.68%) (Figure 2C).
According to the comparison of the relative abundance and the percentage ratio of all annotated compounds, flavonoids were found to be the main classes’ secondary metabolites found in E. angustifolium aerial parts (66.53% ± 6.23%) (Figure 2D).
These results were consistent with the earlier reports on the E. angustifolium distribution of the main polyphenols, where the highest value of the sum of hyperoside, quercetin 3-O-glucuronide, and quercitrin was determined with an intra-population mean content of 9.4 mg/g dw [3]. Quercetin 3-O-glucuronide has been previously reported as the major compound in E. angustifolium [5,11]. Baert et al. [5] reported up to 6.19 mg/g dw in the leaves, while significantly lower values were found in the flowers (up to 2.83 mg/g dw). Both, quercetin- and kaempferol-3-O-glucuronide were identified as chemotaxonomic markers of the species; the latter was present in similar amounts in leaves and flowers [5,11]. It is noteworthy that in our study, the flavonoid profile was dominated by kaempferol 3-O-deoxyhexoside. The highest levels of this compound have been recorded in fireweed flowers (up to 6.76 mg/g dw), while it was completely absent from the leaves [5]. In Baert’s study, hyperoside showed similar levels in the majority of the studied populations, being present from 0.15 to 0.38 mg/g dw (up to 1.48 mg/g dw in the leaves). Myricetin 3-O-glucoside and myricetin 3-O-glucuronide exhibited the greatest inter-population variability, ranging from below the limit of quantitation to the average content of 2.31 mg/g dw. In the same study, quercetin 3-O-(6-galloyl)-galactoside reached up to 0.56 mg/g dw in the flowers, while it was found in a very low concentration in the leaves. Furthermore, kaempferol-, quercetin-, and myricetin 3-O-glucuronide were unique to the flowers, accounting for 70–85% of flower flavonoid content.

2.2. Total Phenolic and Flavonoid Content

Phenolic compounds are considered effective therapeutic aids for preparing health-promoting applications. In this sense, determining the total phenolic content provides a first insight into the potential of the plants [28]. In the present study, the total phenolic and flavonoid content of the methanol aqueous extract of E. angustifolium was determined using spectrophotometric assays (Table 2). Our results revealed a higher concentration of polyphenols (85.04 ± 0.18 mg GAE/g) and lower level of flavonoids (27.71 ± 0.74 mg QE/g) in comparison with the previous records [29]. Moreover, Monschein et al. [30] have found that the total content of phenolic compounds in E. angustifolium showed no correlation with the altitudinal level. The extracts contained phenolics between 97.6 and 135.2 mg/g (800 m: 113.7 ± 15.71 mg/g; 1000 m: 123.0 ± 11.23 mg/g; 1500 m: 114.8 ± 1.60 mg/g). [30]. In addition, Szwajgier et al. [17] reported that the concentration of total polyphenols and flavonoids in the freeze-dried infusion was 407.02 ± 7.10 mg GAE/g dry material and 53.04 ± 1.24 mg QE/g, respectively. However, oenothein B, the compound unique to fireweed, was not present, due to the different sample preparation and extraction solvent [17]. In another study by Ak et al. [31], the total phenolic content and flavonoid content in E. hirsutum extracts was determined as 43.52–254.55 mg GAE/g and 10.87–87.66 mg rutin equivalent (RE)/g. In recent times, spectrophotometric methods have faced criticism due to their lack of specificity, as reagents can interact with both phenolic and non-phenolic compounds, potentially yielding inaccurate results [32]. To ensure accuracy, spectrophotometric findings should be confirmed through chromatographic methods like LC-MS/MS or NMR.

2.3. Antioxidant Capacity

Antioxidants are effective defenses against attacks by free radicals, preventing the progression of serious chronic and degenerative diseases such as cancer, diabetes, and cardiovascular diseases. In the current study, the antioxidant properties of the methanol-aqueous extract of E. angustifolium were investigated using different methods. The results are presented in Table 2.
DPPH and ABTS are the most commonly used free radicals to evaluate scavenging ability. In particular, through the transfer of hydrogen from antioxidants, the radicals can be intercepted, and the changes can be determined using spectrophotometric measurements. CUPRAC and FRAP assays are used to evaluate the electron donation ability of antioxidants, referred to as their reducing ability. A high reducing ability reflects a high antioxidant effect. In addition, the phosphomolybdenum test involves the conversion of Mo(VI) to Mo(V). The ability to chelate metals is associated with controlling the production of hydroxyl radicals in the Fenton reaction. As can be seen in Table 2, the tested extract showed significant radical scavenging and reduction effects as well as metal chelation. The radical scavenging activity of E. angustifolium has been reported previously [11,14,33]. Hevesy et al. [11] evaluated ABTS radical scavenging activity of five Epilobium species, with EC50 ranging between 1.71 and 3 μg/mL—the highest activity levels possessed by E. parviflorum. Deng et al. [14] reported that the E. angustifolium ethanol extract possessed antioxidant activities in DPPH and ABTS assays with EC50 at 25.53 ± 0.40 and 45.71 ± 1.34 μg/mL, respectively, and inhibited lipid peroxidation (%inhibition 26.74 ± 2.03). The highest radical scavenging ability in DPPH and ABTS assays was found for the ethylacetate extract in comparison with the petroleum ether extract. Owing to the high content of oenothein B in many Oenothera, Epilobium, and Eucalyptus species, its biological activity has been widely studied [22]. Numerous investigations demonstrated that oenothein-rich Epilobium sp. significantly reduced the production of reactive oxygen species (ROSs), which may hold significance for reducing the risk of diseases associated with active oxygen damage [12,22,34]. These effects were ascribed to the radical scavenging capacity of the ellagitannins, which are able to terminate free radical chain reactions of lipids, proteins, and DNA by self-oxidation [22].
In vivo studies showed that oenothein B exerts neuroprotective effects on the central nervous system, mitigates neuroinflammation in the brain, and enhances neuronal signaling pathways [34]. Indeed, oenothein had the highest radical scavenging activity among the other polyphenols (quercetin 3-O-glucuronide and myricetin 3-O-rhamnoside) in fireweed methanol extracts [12,33]. In fireweed herb, a correlation between radical scavenging activity and total phenolic content, notably oenothein B, has been evidenced [33]. It is noteworthy that the metabolites of various ellagitannins may have more pronounced antioxidant activity compared to the corresponding compounds [13].

2.4. Enzyme Inhibitory Activity

Enzymes are important targets in pharmaceutical therapies, and many treatment approaches focus on modulating enzymes. By inhibiting critical enzymes, symptoms of diseases such as diabetes, obesity, and Alzheimer’s can be treated. For example, suppressing amylase and glucosidase activities helps control blood sugar levels in diabetics following a high-carbohydrate diet [35]. Although synthetic enzyme inhibitors, including acarbose, galanthamine, and kojic acid, have been synthesized, their adverse side effects remain a challenge. Therefore, it is important to identify safer and more effective natural inhibitors [36]. The enzyme inhibitory activity of the studied extracts was determined against acetyl- and butyrylcholinesterase, tyrosinase, α-amylase, and α-glucosidaseand lipase (Table 2). The studied extract showed moderate acetylcholinesterase (2.05 mg GALAE/g) and butyrylcholinesterase (1.67 mg GALAE/g) inhibitory activity.
In addition, the extract displayed very high potential against the enzyme tyrosinase (61.94 ± 0.05 mg KAE/g) (Table 2). This enzyme plays a key role in the biosynthesis of melanin and is responsible for skin pigmentation. Increased melanin formation leads to skin diseases such as hyperpigmentation and skin spots, amongst others. Tyrosinase inhibitors are becoming increasingly important as hypopigmenting active ingredients in cosmetics and pharmaceuticals [37].
Regarding enzymes involved in carbohydrate and lipid metabolism, E. angustifolium revealed low α-amylase (0.44 mmol ACAE/g) and high α-glucosidase (3.48 mmol ACAE/g) and lipase inhibitory (8.03 mg OE/g) effects. The inhibition of these enzymes is known to be an important therapeutic strategy to control blood glucose levels in diabetic patients who have had a carbohydrate-rich diet and metabolic disorders. In the literature, some researchers have reported enzyme inhibitory effects of the members of the genus Epilobium [12,38,39,40]. For example, Ak et al. [31] reported that the AChE and BChE inhibitory activity was 2.69–4.48 mg GALAE/g and 1.11–4.72 mg GALAE/g for the extracts of the aerial parts of E. hirsutum, respectively. In addition, the amylase and glucosidase inhibitory effects were found to be 0.17–1.02 mmol ACAE/g and 1.57–1.62 mmol ACAE/g, respectively, in their study. In terms of insights into the structure–ability relationship based on Table 1, some identified compounds in the tested extracts may contribute to the observed enzyme inhibitory effects. For example, quercetin, gallic acid, caffeic acid, kaempferol, and myricetin have been already recognized as enzyme inhibitors in previous reports [41,42,43,44,45,46,47,48,49]. In this sense, the tested E. angustifolium extract could be considered as a multifunctional bioactive agent from antioxidants to enzyme inhibitors, and, thus, the presented study could be valuable to provide an effective raw material in the pharmaceutical, nutraceutical, and cosmeceutical industries.

3. Materials and Methods

3.1. Plant Material

Fireweed is distributed in the Vitosha Mt. floristic region in Bulgaria, where the locality was chosen with the following criteria: (1) a representative subalpine region; (2) the species is common and extensive in the herbaceous community; (3) a lack of anthropogenic activity; (4) the diversity of higher plants in the Vitosha Mt. represents 41% of the total species for Bulgaria [50]. E. angustifolium aerial parts (Epilobii Herba) were collected at the locality “Platoto”, Vitosha Mt. (1906 m. a.s.l.), Bulgaria, during the full flowering stage in July 2023. The species taxonomic identity was confirmed by one of us (R. Gevrenova) according to www.worldforaonline.org. A voucher specimen was deposited at Herbarium Facultatis Pharmaceuticae Sophiensis, Medical University-Sofia, Bulgaria (Voucher specimen No. 11823). The plant material was dried at room temperature.

3.2. Sample Extraction

E. angustifolium aerial parts were powdered by a grinder (Rohnson, R-942, 220–240 V, 50/60 Hz, 200 W, Prague, Czech Republic). Powdered plant material (50 g) was extracted with 80% MeOH (1:20 w/v) by sonication (100 kHz, ultra-sound bath Biobase UC-20C) for 15 min (×2) at room temperature. The methanol was evaporated in vacuo (40 °C) and water residues were lyophilized (lyophilizer Biobase BK-FD10P; −65 °C) to yield 7.3 g of crude extract. Then, the lyophilized extracts were dissolved in 80% methanol (0.1 mg/mL), filtered through a 0.45 μm syringe filter (Polypure II, Alltech, Lokeren, Belgium), and an aliquot (2 mL) of each solution was subjected to UHPLC–HRMS analyses. The same extracts were used for in vitro antioxidant and enzymatic capacity tests.

3.3. Chemicals

Acetonitrile (hypergrade for LC–MS), formic acid (for LC–MS), and methanol (analytical grade) were provided from Chromasolv (Sofia, Bulgaria). The reference standards used for compound identification were obtained from Extrasynthese (Genay, France) (for gallic, protocatechuic, 4-hydroxybenzoic, 3-hydroxybenzoic, o- and p-coumaric, caffeic, and salicylic acids, myricetin, hyperoside, isoquercitrin, kaempferol 3-O-glucoside, isorhamnetin 3-O-glucoside, quercetin, isorhamnetin, and kaempferol) and Phytolab (Vesten-bergsgreuth, Bavaria, Germany) (ellagic acids and naringenin). A working solution containing 0.1 mg/mL of the assayed compounds was prepared from a stock solution in methanol containing 0.5 mg/mL.
The chemicals for antioxidant and enzyme inhibition assays were purchased from Sigma-Aldrich (Darmstadt, Germany). They were as follows: ABTS, DPPH, gallic acid, rutin, electric eel acetylcholinesterase (AChE) (type-VI-S, EC 3.1.1.7), horse serum butyrylcholinesterase (BChE) (EC 3.1.1.8), galantamine, acetylthiocholine iodide (ATChI), butyrylthiocholine chloride (BTChI) 5,5-dithio-bis(2-nitrobenzoic) acid (DTNB), tyrosinase (EC1.14.18.1, mushroom), glucosidase (EC. 3.2.1.20, from Saccharomyces cerevisiae), amylase (EC. 3.2.1.1, from porcine pancreas), sodium molybdate, sodium carbonate, Folin–Ciocalteu reagent, hydrochloric acid, sodium hydroxide, trolox, ethylenediaminetetraacetate (EDTA), neocuproine, cupric chloride, ammonium acetate, ferric chloride, 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), ammonium molybdate, ferrozine, ferrous sulfate hexahydrate, kojic acid, and acarbose. All chemicals were of analytical grade.

3.4. UHPLC-HRMS

The UHPLC-HRMS analyses were performed as previously described [51] on a Q Exactive Plus mass spectrometer (ThermoFisher Scientific, Inc., Waltham, MA, USA) equipped with a heated electrospray ionization (HESI-II) probe (ThermoScientific). The equipment was operated in negative and positive ion modes within the m/z range of 150 to 1500. The chromatographic separation was achieved on a Kromasil Eter-nityXT C18 (1.8 µm, 2.1 × 100 mm) reversed-phase column, at 40 °C. The UHPLC analyses were run with a mobile phase consisting of 0.1% formic acid (A) and 0.1% formic acid in acetonitrile (B). The run time was 33 min and the flow rate was 0.3 mL/min. The used gradient elution program was as follows: 0–1 min, 0–5% B; 1–20 min, 5–30% B; 20–25 min, 30–50% B; 25–30 min, 50–70% B; 30–33 min, 70–95%; 33–34 min 95–5% B. The equilibration time was 4 min. The injection volume was 1 µL, and the flow rate was 300 µL/min. Data were processed by Xcalibur 4.2 (ThermoScientific, Waltham, MA, USA) instrument control/data handling software. MZmine 2 software was applied to the UHPLC–HRMS raw files of the studied E. angustifolium extract for the semi-quantitative analysis. Results are expressed as the % peak area of the compound to the total peak areas of the corresponding group secondary metabolites and all metabolites.

3.5. Assay for Total Phenolic and Flavonoid Contents

According to the methods specified by Zengin and Aktumsek [52], total phenolics and flavonoids were quantified. The extract was prepared at a concentration of 2 mg/mL. Gallic acid (GA) and rutin (R) were used as positive equivalents in the assays, and the results were reported as gallic acid equivalents (GAEs) and rutin equivalents. The values of the calibration curves are as follows: for the total phenolic content, absorbance = 0.268 [μg gallic acid] (R2: 0.9988, concentration range: 0–3 μg gallic acid); for total flavonoid content, absorbance = 0.1274 [μg rutin] + 0.0506 (R2: 0.9968, concentration range: 0–20 μg rutin).

3.6. Assays for In Vitro Antioxidant Capacity

According to the methods provided by Zengin et al. [53], antioxidant tests were executed. The extract was prepared at concentrations of 0.1–2 mg/mL. The DPPH, ABTS radical scavenging, CUPRAC, and FRAP results were expressed as milligrams of Trolox equivalents (TE) per gram of extract. The antioxidant potential determined by the phosphomolybdenum (PBD) assay was presented in millimoles of Trolox equivalents (TE) per gram of extract. Metal chelating activity (MCA) was calculated as milligrams of disodium edetate equivalents (EDTAE) per gram of extract. The experimental details are presented in the Supplemental Materials.

3.7. Inhibitory Effects Against Some Key Enzymes

Enzyme inhibition experiments on the samples were carried out following established protocols [53]. The extract was prepared at a concentration of 0.1–2 mg/mL. Amylase and glucosidase inhibition were expressed as acarbose equivalents (ACAE) per gram of extract, while acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition were evaluated as milligrams of galanthamine equivalents (GALAE) per gram of extract. Tyrosinase inhibition was evaluated in milligrams of kojic acid equivalents (KAE) per gram of extract. Lipase inhibition was measured as the equivalent of orlistat (OE) per gram of extract. The experimental details are presented in the Supplemental Materials.

3.8. Statistical Analysis

The experiments for the evaluation of total phenolic and flavonoid contents and antioxidant and enzyme inhibitory capacity were performed in triplicate and the results were presented as the mean and standard deviation. GraphPad 9.1 was used to evaluate the obtained results.

4. Conclusions

Herein, an in-depth phytochemical profiling of the methanol-aqueous extract from E. anfustifolium aerial parts with Bulgarian provenance was performed by the means of liquid chromatography–Orbitrap high-resolution mass spectrometry. More than 120 secondary metabolites—notably, phenolic acids and their glycosides, acylquinic acids, gallotannins and ellagitannins, and flavonoids—were dereplicated/annotated. Forty-six compounds (11 phenolic acids, 10 acylquinic acids, 3 tannins, and 22 flavonoids) are reported for the first time in the species. Furthermore, links between the compounds having similar HRMS/MS fragmentation patterns were established, which greatly help the metabolite annotation of galloyl-caffeoylquinic acids, mono-, di-, and trigalloyl-hexose, galloyl, caffeoyl, feruloyl, and p-coumaroyl conjugates of flavonol hexosides. The percentage ratio of the main compound classes revealed the highest relative content of flavonoids (66.5 ± 6.23%), followed by mono- and diacylquinic acids (16.6 ± 1.2%) and tannins (10.53 ± 0.89%). Gallic and ellagic acids, methylgallate, neochlorogenic and chlorogenic acids, oenothein B, galloyl-HHDP-hexose, as well as deoxyhexosides of kaempferol and quercetin, along with a variety of acylated flavonol hexosoides, appeared to be characteristic for E. anfustifolium aerial parts. The strong antioxidant potential (DPPH•, ABTS•+, FRAP, and CUPRAC) could be assigned to dimeric and trimeric ellagitannins together with simple phenolics (gallic acid and methylgallate), and numerous caffeoyl, coumaroyl, and feruloyl conjugates. Indeed, the polyphenols are the main group in fireweed with an impact on the anti-cholinesterase and anti-tyrosinase activity. In addition to evoking an antioxidant response, E. angustifolim extract exhibited in vitro inhibitory activity towards key enzymes of melanin biosynthesis and carbohydrate and lipid metabolism, which generates further interest in the herbal drug Epilobii Herba as a potential therapeutic candidate for associated disorders.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14030415/s1, Figure S1: (-) ESI-MS/MS spectrum of galloyl-(caffeoyl)-hexose (25); Figure S2: ESI-MS/MS spectrum of 1-galloyl-5-caffeoylquinic acid (38); Figure S3: (-) ESI-MS/MS spectrum of oenothein B (50); Figure S4: (-) ESI-MS/MS spectrum of quercetin 3-O-galloylhexoside (79); Determination of Antioxidant and Enzyme Inhibitory Effects.

Author Contributions

Conceptualization, R.G. and D.Z.-D.; methodology, D.Z.-D., G.O., and G.Z.; software, D.Z.-D. and G.O.; validation, R.G., D.Z.-D., and G.Z.; formal analysis, D.Z.-D. and G.O.; investigation, D.Z.-D. and G.Z.; resources, R.G.; data curation, R.G.; writing—original draft preparation, R.G.; writing—review and editing, D.Z.-D. and G.Z.; visualization, D.Z.-D.; supervision, R.G.; project administration, D.Z.-D.; funding acquisition, D.Z.-D. All authors have read and agreed to the published version of the manuscript.

Funding

This research is financed by the European Union NextGenerationEU program, through the National Recovery and Resilience Plan of the Republic of Bulgaria, project BG-RRP-2.004-0004-C01, “Strategic research and innovation program for development of Medical University—Sofia”.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The workflow for the Epilobium angustifolium study.
Figure 1. The workflow for the Epilobium angustifolium study.
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Figure 2. Extracted ion chromatograms of acylquinic acids (A), tannins (B), and flavonoids (C) in the Epilobium angustifolium extract; the percentage ratio of the main classes’ secondary metabolites (D). For peak numbering, see Table 1.
Figure 2. Extracted ion chromatograms of acylquinic acids (A), tannins (B), and flavonoids (C) in the Epilobium angustifolium extract; the percentage ratio of the main classes’ secondary metabolites (D). For peak numbering, see Table 1.
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Table 2. Total bioactive compounds, antioxidant properties, and enzyme inhibitory properties of tested extract.
Table 2. Total bioactive compounds, antioxidant properties, and enzyme inhibitory properties of tested extract.
Total bioactive compounds
Total phenolic content (mg GAE/g)85.04 ± 0.18
Total flavonoid content (mg RE/g)27.71 ± 0.74
Antioxidant properties
DPPH scavenging ability (mg TE/g)310.74 ± 11.09
ABTS scavenging ability (mg TE/g)466.82 ± 23.60
CUPRAC (mg TE/g)442.83 ± 12.27
FRAP (mg TE/g)291.50 ± 4.32
Metal chelating (mg EDTAE/g)48.20 ± 0.44
Phosphomolybdenum (mmol TE/g)2.10 ± 0.09
Enzyme inhibitory properties
AChE inhibition (mg GALAE/g)2.05 ± 0.04
BChE inhibition (mg GALAE/g)1.67 ± 0.07
Tyrosinase inhibition (mg KAE/g)61.94 ± 0.05
Amylase inhibition (mmol ACAE/g)0.44 ± 0.01
Glucosidase inhibition (mmol ACAE/g)3.48 ± 0.08
Lipase inhibition (mg OE/g)8.03 ± 0.11
Values are reported as mean ± SD of three parallel measurements. GAE: gallic acid equivalent; RE: rutin equivalent; TE: trolox equivalent; EDTAE: EDTA equivalent; GALAE: galanthamine equivalent; KAE: kojic acid equivalent; ACAE: acarbose equivalent; OE: orlistat equivalent.
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Gevrenova, R.; Zengin, G.; Ozturk, G.; Zheleva-Dimitrova, D. Exploring the Phytochemical Profile and Biological Insights of Epilobium angustifolium L. Herb. Plants 2025, 14, 415. https://doi.org/10.3390/plants14030415

AMA Style

Gevrenova R, Zengin G, Ozturk G, Zheleva-Dimitrova D. Exploring the Phytochemical Profile and Biological Insights of Epilobium angustifolium L. Herb. Plants. 2025; 14(3):415. https://doi.org/10.3390/plants14030415

Chicago/Turabian Style

Gevrenova, Reneta, Gokhan Zengin, Gulsah Ozturk, and Dimitrina Zheleva-Dimitrova. 2025. "Exploring the Phytochemical Profile and Biological Insights of Epilobium angustifolium L. Herb" Plants 14, no. 3: 415. https://doi.org/10.3390/plants14030415

APA Style

Gevrenova, R., Zengin, G., Ozturk, G., & Zheleva-Dimitrova, D. (2025). Exploring the Phytochemical Profile and Biological Insights of Epilobium angustifolium L. Herb. Plants, 14(3), 415. https://doi.org/10.3390/plants14030415

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