Seed Germination Inhibitory Activity of Alkaloid Fractions from Narcissus pseudonarcissus cv. Carlton and Narcissus poeticus Leaves

Abstract

Narcissus species have been cultivated for centuries around the world and are mainly used as cut flowers. Although the bulbs of these species have been widely examined as sources of alkaloids and biological activity, the leaves have been understudied. In the present study alkaloid fractions of leaves from Narcissus pseudonarcissus cv. Carlton and N. poeticus L. were evaluated for inhibitory activity against seed germination of Lolium perenne L. and Trifolium pratense L. Separately, the metabolic profiles from seedlings of the target species were analyzed after treatment with a lycorine solution. The composition of methanolic extracts from seedlings and alkaloid fractions of Narcissus leaves were determined using GC/MS. The N. pseudonarcissus alkaloid fraction was more active than that of N. poeticus. Complete inhibitory activity of the alkaloid fraction was established at a concentration of 1 or 2 mg/mL, depending on the target species. Lycoramine and galanthine were identified as the main alkaloids of N. pseudonarcissus. 8-O-Demethylmaritidine, maritidine and homolycorine were found to be the predominant alkaloids of N. poeticus. Increased accumulation of some amino acids, saccharides and polyols, indicating protein synthesis inhibition, was the most common response of target species seedlings treated with 0.17 µM lycorine. The results showed the promising potential of alkaloid fractions from the leaves of Narcissus species as seed germination inhibitors. The study contributes to full utilization of the resources of these species and presents, to our knowledge, for the first time data on changes in the metabolic profiles of L. perenne and T. pratense seedlings after treatment with lycorine.

1. Introduction

Weed control in crops is an important task in agriculture. The use of synthetic herbicides has an adverse effect on the environment, human and animal health, which has motivated the search for new natural remedies. The application of natural products, in particular plant extracts, for weed control is a distinguishing feature of modern farming [1]. Numerous studies have reported that essential oils from aromatic plants are potent inhibitors of seed germination and root growth and have potential as weed control products. The germination inhibitory activity of the essential oils is attributed to their relatively high content of compounds such as carvacrol, thymol, citral isomers, α-β-pinene, eucalyptol, limonene and camphor [2,3,4,5]. Citral is patented as an herbicide and is the active ingredient of a number of lemongrass (Cymbopogon citratus Stapf.) oil-based natural herbicides. An increasing number of essential oils from different plants such as Cinnamomum zeylanicum, Cymbopogon flexuosus, Mentha arvensis, Mentha spicata, Juniperus mexicana, Piper nigrum, Cistus ladaniferus, Ferula galbaniflua and Citrus aurantium have been homologated for use in agriculture [5,6]. Alkaloids are secondary metabolites with divergent toxicity effects on humans and animals [7,8]. Although the plant growth-inhibitory properties of narciclasine and lycorine have been studied [9,10], the other alkaloids are poorly studied in this regard.

The genus Narcissus is primarily found in the Mediterranean area, and the center of origin is Europe (Spain, Portugal and the Iberian Peninsula), but the species are widely cultivated. It became one of the important commercial bulbous crops during 2005 [11]. Narcissus species (daffodils) are mostly known as ornamental plants, and they have been produced at a large scale for cut flowers. Wahyuni et al. [9] reported that methanol extracts of Narcissus pseudonarcissus cv. Carlton and N. poeticus L. bulbs possess a herbicidal effect on Senecio vulgaris L. and Lolium perenne L. After bioguided isolation, the authors found that narciclasine (IC₅₀ 0.10 μM) and lycorine (IC₅₀ 0.93 μM) inhibit radicle growth. Narciclasine was also found to inhibit cell division in Arabidopsis roots in a dose-dependent manner by reducing auxin transport, probably acting on auxin signaling [12]. Recently, Kempthorne et al. [10] found that haemanthamine, isolated from emerging N. pseudonarcissus L. buds, has more potent phytotoxic activity than the commercial herbicide glyphosate but is less toxic than narciclasine. Other Amaryllidaceae alkaloids 1-O-acetyllycorine, pancratistatin, 7-deoxynarciclasine and 7- deoxypancratistatin have been shown to have similar plant growth-inhibitory properties to those of narciclasine [12,13]. Besides bulbs, the leaves of the Narcissus species are an important source of alkaloids [14,15]. Furthermore, the use of leaves as a raw material to obtain an alkaloid fraction is a useful and important technique in light of the modern circular economy’s priority for utilizing all parts of plants.

Our preliminary study showed that Narcissus leaf extracts showed phytotoxic activity against seed germination [16]. Consequently, in the present study, chemically characterized alkaloid fractions from the leaves of N. pseudonarcissus cv. Carlton and N. poeticus L. were evaluated as inhibitors of seed germination of Lolium perenne L. (monocot) and Trifolium pratense L. (dicot). Target species are not typical weeds as they are valuable forage crops; however, they are considered wees in lawns, gardens and orchards when they grow unwanted. Also L. perenne is characterized by high vitality and productivity, fast growth after sowing, tolerance to intensive grazing, stamping and frequent mowing [17], so substances or extracts that inhibit its growth will have high potential as herbicides. Additionally, to obtain data on the mechanism of action of alkaloids as seed germination inhibitors, the metabolic profiles from seedlings of the target species were analyzed after treatment with lycorine solution.

2. Materials and Methods

2.1. Plant Material and Reagents

The bulbs of the plants were kindly provided by Holland Biodiversity BV, Lisse, the Netherlands. Leaves of Narcissus pseudonarcissus cv. Carlton and N. poeticus were collected in the flowering stage from a cultivated area near Sofia, Bulgaria. The seeds of Lolium perenne L. and Trifolium pratense L. (used as target weed species) were purchased from Florian Company (Ruse, Bulgaria). Methanol, chloroform, pyridine (HPLC grade, St. Louis, MO, USA, Alfa Aesar), sulfuric acid and ammonia (analytical grade) were purchased from Valerus (Sofia, Bulgaria). A hydrocarbon mixture (C9-C36, Restek, Cat no. 31614, Bad Homburg, Germany), N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA, Sigma-Aldrich, product no. 15222, St. Louis, MO, USA) and arbutin (Sigma-Aldrich) were supplied by FOT (Sofia, Bulgaria).

2.2. Extraction of Alkaloid Fraction

Alkaloid fractions (AFs) were obtained as described by Berkov et al. [18]. Air-dried, powdered leaves were macerated with methanol (1:5 w/v ratio of plant material and solvent) in triplicate for 24 h at room temperature. After evaporation of the solvent, the combined methanol extracts were dissolved in 50 mL 2% aqueous solution of sulfuric acid. The neutral compounds were removed by triplicate extraction with 50 mL of chloroform. The alkaloids were fractionated after basification of the extract to pH 8–9 with 10% of ammonia and triplicate extraction with 50 mL chloroform.

2.3. Isolation of Alkaloid Standard Lycorine

Lycorine was isolated from Narcissus cv. Hawera AF, as described by Berkov et al. [19]. AF fractions were dissolved in 10 mL methanol for precipitation and crystallization of lycorine at 4 °C. Lycorine was removed from the solution by filtration. The filtrate was washed with small portions of cooled methanol, which were added to the alkaloid solution. Then, the alkaloid solution was left again at 4 °C. This procedure was repeated in triplicate before the complete evaporation of the solvent.

2.4. Methanolic Extract Preparation from Seedlings of the Target Species

Seedlings treated with lycorine at a concentration of 0.17 µM and their respective controls (three replicates) were collected at the end of experiments. A total of 300 mg fresh material from each sample was extracted with 1.5 mL methanol in screw-top Eppendorf tubes (2 mL of volume) at room temperature for 24 h. Before extraction, 50 μL of arbutin (1 mg/mL stock solution) was added as an internal standard. After filtration, 800 µL of the methanol extract was placed in vials and dried.

2.5. Gas Chromatography–Mass Spectrometry (GC/MS) Analysis

The GC-MS spectra were recorded on a Thermo Scientific Focus GC (Rodano, Milan-Italy) coupled with a Thermo Scientific DSQ (Austin, TX, USA) mass detector operating in EI mode at 70 eV. A DB-5MS column (30 m × 0.25 mm × 0.25 μm) was used. The temperature program was 100–180 °C at 15 °C × min⁻¹, 180–300 °C at 5 °C × min⁻¹ and 10 min hold at 300 °C. The injector temperature was 250 °C. The flow rate of the carrier gas (Helium) was 0.8 mL × min⁻¹. The split ratio was 1:10, and 1 mL of the solution was injected.

2.5.1. Derivatization of the Methanolic Extracts

Methanolic extracts before GC/MS analysis were derivatized with 100 μL of N,O-bis-(trimethylsilyl) trifluoro-acetamide (BSTFA) in 100 μL of pyridine for 2 h at 60 °C. After cooling, 300 μL chloroform was added to the derivatized fractions and analyzed by GC/MS.

2.5.2. Identification of Metabolites

The compounds of the methanolic extracts were identified as TMS derivatives with the help of the NIST 05 database (NIST Mass Spectral Database, PC-Version 5.0, 2005) and the Golm Metabolome Database [20], as well as literature data based on matching mass spectra and Kovats retention indexes (RIs) as described in Berkov et al. [17]. The alkaloids in the alkaloid fractions were identified by using the in-home MS library of Amaryllidaceae alkaloids as described in Berkov et al. [18,19]. The mass spectra were deconvoluted by AMDIS 2.64 before comparison with the databases. The spectra of individual components were transferred to the NIST Mass Spectral Search Program MS Search 2.0, where they were matched against reference compounds from the NIST Mass Spectral Library 2005 and the Golm Metabolome Database. The groups of unidentified compounds were determined based on their specific mass spectral fragmentation and compared with the mass spectra of known metabolites. The amounts of individual alkaloids were expressed as a percentage of the total ion current (TIC) of all alkaloids in the alkaloid fraction. The amounts of metabolites in methanol extracts are expressed relative to the amount of internal standard arbutine.

2.6. Assessment of Inhibitory Activity Against Seed Germination

The germination bioassay was carried out in Petri dishes by applying aqueous solutions of studied fractions at concentrations 0.1, 0.5, 1 and 2 mg/mL on seeds of L. perrene and T. pratense. Separately, the alkaloid lycorine was studied for seed germination inhibition in the concentration range of 0.03–3.48 µM. Seeds of the target species were placed in Petri dishes on filter papers (three replicates of 100 seeds each) moistened with the tested solutions. The samples were incubated at room temperature for 7 days. The rate of germination inhibition and inhibition of root elongation [%] were calculated as described by Attak et al. [21]

Germination energy (GE) was evaluated. GE is defined as the fraction of seeds germinated during the period in which most of the seeds tested germinated uniformly [22]. The number of germinated seeds was determined every 24 h. GE was determined on the 3rd day for L. perrene seeds and on the 2nd day for T. pratense seeds after the start of the test. The fraction of germinated seeds at the end of the test period (total number of germinated seeds) was defined as the germination percentage. Both germination energy and germination percentage were calculated as a percentage of the total number of seeds tested according to Tang et al. [23]:

GE = S/St × 100,

where S is the number of seeds germinated in specified period, and St is the total number of seeds tested.

2.7. Data Analysis

Statistical analyses were performed using Microsoft Excel software. The results are presented as means with standard deviation (SD). The statistical significance of the differences between mean values for the treated and untreated variants was determined by the t-test, with p ≤ 0.05 accepted as significant.

3. Results

3.1. Phytochemical Analysis of Alkaloid Fractions

The AFs of N. pseudonarcissus and N. poeticus leaves were analyzed for their composition by GC/MS. The results are presented in

Table 1. Identified alkaloids in the alkaloid fraction of the studied Narcissus species [%].

Alkaloids *	RT	N. pseudonarcissus	N. poeticus
		cv. Carlton
Miscellaneous type
UA1 **	19.24		0.11
UA2	19.53		0.53
Trisphaeridine	23.81	0.16	0.32
		0.16	0.97
Galanthamine type
Galanthamine	25.41	7.58	8.03
Lycoramine	25.78	21.70	0.59
Lycoraminone	26.09	1.23
Norlycoramine	26.44	0.33
Narwedine	26.67	0.47	0.13
		31.31	8.75
Haemanthamine type
Maritidine	27.36	0.31	15.37
8-O-Demethylmaritidine	27.81		28.90
Haemanthamine	29.19	7.16	5.38
UA3	29.74		0.30
UA4	29.97		0.33
UA5	30.33		1.49
UA6	31.39		2.72
UA7	32.09		3.88
		7.47	58.37
Lycorine type
Kirkine	27.92	1.94	0.06
Pluvine	28.08	0.86
Assoanine	28.30	2.00	1.98
9-O-Demethylpluviine	28.59	0.44
11,12-Didehydroassoanine	29.76	1.48	2.33
Lycorine	30.01		0.89
Galanthine	30.18	39.66	3.06
8-O-Methylpseudolycorine	31.33	1.50
Tortuosine	31.85	1.35
11,12-Didehydrotortuosine	33.58	3.95
2-Methoxypratosine	35.59	0.17
		53.36	8.32
Pretazettine type
Tazettine	29.38	4.24	1.10
		4.24	1.10
Homolycorine type
Lycorenine	26.16		1.88
Homolycorine	29.54		18.41
8-O-Demethylhomolycorine	31.71	3.44	2.30
		3.44	22.59
Monthanine type
Pancracine	28.65		0.55
			0.55

Compounds in the alkaloid fractions comprising more than 0.10% of TIC. * Expressed as a % of TIC of all alkaloids. ** Unidentified alkaloid.

. In total 32 alkaloids were detected, and 7 of them, from the AF of N. poeticus, were not identified due to the lack of reference MS spectra in the literature and available databases. The alkaloid profiles of N. pseudonarcissus cv. Carlton and N. poeticus were dominated by galanthamine–lycorine- and haemanthamine–homolycorine-type compounds, respectively. The main compounds were lycoramine and galanthine in N. pseudonarcissus cv. Carlton and maritidine and 8-O-demethylmaritidine and homolycorine in N. poeticus. Structures of the main alkaloids in the studied fractions are presented in Figure 1.

3.2. Inhibition of Seed Germination

Alkaloid fractions from the leaves of the studied Narcissus species were evaluated as inhibitors of the germination of L. perenne and T. pratense seeds. The results are presented in

Table 2. Inhibition of seed germination of target species by alkaloid fractions of Narcissus species.

Narcissus Species	Target Species	Inhibition of Seed Germination [%] *
		Concentration [mg/mL]
		0.1	0.5	1	2
N. pseudonarcissus	L. perenne	18 ± 10 a	55 ± 15 b	98 ± 2 c	100 ± 0 c
	T. pratense	12 ± 9 a	23 ± 11 a	75 ± 14 b	92 ± 7 c
N. poeticus	L. perenne	14 ± 9 a	40 ± 11 b	77 ± 14 c	91 ± 6 d
	T. pratense	11 ± 5 a	32 ± 12 b	50 ± 9 c	89 ± 7 d

* values represent mean ± SD, n = 3. Values with the same letter within each row are not significantly different.

. The studied fractions inhibit the germination of L. perenne seeds more strongly than T. pratense seeds. Also, the fraction of N. pseudonarcissus showed stronger activity. At concentrations of 2 mg/mL, the alkaloid fraction of the species showed 100% and 92% inhibition of seed germination, whereas at 1 mg/mL, the inhibition of L. perenne and T. pratense seeds was 98% and 75%, respectively.

Solutions of pure substance lycorine were evaluated as an inhibitor on seed germination and root elongation in the concentration range 0.03–3.48 µM. The results are presented in Figure 2. Strong inhibition of seed germination and root growth was found by applying aqueous solutions at a concentration of 0.7 µM on L. perenne seeds and 1.04 µM for T. pratense. Root elongation was inhibited more than seed germination when applying the same concentration of lycorine solutions.

Germination energy of the seeds of target species treated with AF and lycorine solution was evaluated. The proportion of germinated seeds after a period in which most of the tested seeds germinated uniformly (3 days for L. perrene seeds and 2 days for T. pratense seeds) was defined as GE. The results are presented in Figure 3. The graphs show that increasing concentrations of both the AF and lycorine consistently decreased the germination energy of the treated seeds.

3.3. Metabolite Profiles of Target Species Treated with Lycorine

Treatment of T. pratense and L. perenne seeds with lycorine solution (0.05 mg/mL) induced a widespread metabolic response in both seedlings. Metabolites identified in the methanolic extracts of T. pratense and L. perenne seedlings using GC/MS are presented in

Table 3. Metabolites identified and quantified through GC/MS analysis in target species seedlings exposed to lycorine solution *.

Compounds	RT	Trifolium pratense		Lolium perenne
		Control	Treated	Control	Treated
Amino acids
L-Alanine	5.95	27.73 ± 3.7	51.11 ± 11.7	2.30 ± 0.3	7.21 ± 1.8
Glycine	6.85	7.69 ± 0.2	12.01 ± 3.6
L-Valine	8.23	39.83 ± 1.7	67.32 ± 15.1	2.05 ± 0.3	4.89 ± 1.4
L-Leucine	9.60	5.93 ± 0.6	21.31 ± 6.1	1.67 ± 0.1	5.91 ± 0.8
L-Isoleucine	10.04	30.82 ± 2.6	60.56 ± 16.3
L-Serine	11.83	72.48 ± 2.6	170.11 ± 24	2.89 ± 0.5	7.51 ± 2.8
L-Threonine	12.12	32.58 ± 3.0	38.72 ± 8.7	1.90 ± 0.2	6.23 ± 2.2
L-Aspartic acid	16.52	12.61 ± 7.2	16.98 ± 3.1	1.68 ± 0.1	1.96 ± 0.5
L-Glutamic acid	18.95	9.42 ± 2.8	27.97 ± 9.2	3.69 ± 0.4	8.45 ± 2.1
Pyroglutamic acid	19.33	3.82 ± 0.4	4.59 ± 2.0	3.08 ± 0.7	9.97 ± 3.9
L-Phenylalanine	20.23	82.76 ± 9.2	66.88 ± 14.8
L-Tyrosine	26.00	20.22 ± 1.1	20.49 ± 4.1
L-Tryptophan	32.50	5.60 ± 0.6	133.04 ± 47
Organic acids
Phosphoric acid	11.01	47.41 ± 4.9	97.22 ± 13.2	4.82 ± 0.4	22.90 ± 4.8
Glyceric acid	11.31	26.06 ± 5.0	7.82 ± 3.8
Succinic acid	12.74	3.28 ± 0.02	2.33 ± 0.8	2.50 ± 0.6	6.19 ± 2.5
Malic acid	15.83	11.19 ± 0.3	13.49 ± 7.8	14.33 ± 3.2	11.05 ± 3
Sugars and sugar alcohols
Glycerol	8.93	4.05 ± 0.2	4.16 ± 1.2	2.69 ± 0.8	14.43 ± 5.3
Fructose 1	19.84	0.43 ± 0.1	6.43 ± 2.4	80.67 ± 21	66.44 ± 18
Fructose 2	20.13	0.77 ± 0.3	31.06 ± 9.2	33.78 ± 11	160.03 ± 60
Fructose 3	20.40	90.51 ± 19	124.66 ± 34.6	90.44 ± 30	338.47 ± 90
Quinic acid	21.68			27.12 ± 7.5	52.55 ± 8.7
Monosaccharide	22.27	Trace	6.43 ± 2.4
Galactose	22.60	23.35 ± 1.7	31.06 ± 9.1	132.59 ± 40	442.06 ± 90
β-D-Glucopyranose	24.18	9.17 ± 3.9	27.31 ± 21.8	254.1 ± 70	769.60 ± 105
Disaccharide	31.77	1.43 ± 0.4	26.52 ± 12	17.46 ± 3.7	455 ± 177
Sucrose	32.19	6.77 ± 3.4	343.78 ± 107	77.80 ± 16	1127 ± 139
Disaccharide	32.39	2.10 ± 0.4	20.64 ± 9	9.24 ± 4.2	232.1 ± 65
Disaccharide	32.67			31.72 ± 9	794.5 ± 140
Disaccharide	32.93	2.15 ± 0.9	99.11 ± 9.8	35.04 ± 8.2	809.1 ± 109
myo-Inositol	24.98	0.4 ± 0.2	2.37 ± 1.2	1.38 ± 0.1	4.65 ± 2
Fatty acids
Hexadecanoic acid	27.71	9.63 ± 0.5	11.58 ± 9.0	3.01 ± 0.7	6.14 ± 1.8
9,12-Octadecadienoic acid	30.07	7.20 ± 0.3	13.16 ± 4.3	1.38 ± 0.4	1.72 ± 0.5
9-Octadecenoic acid	30.40	5.69 ± 0.2	2.82 ± 2.8	3.47 ± 0.7	4.04 ± 0.9
9,12,15-Octadecatrienoic acid	30.93	3.78 ± 0.2	4.66 ± 1.2	7.14 ± 0.9	8.43 ± 1.2
Phenolic acids
Caffeic acid	29.70	2.67 ± 1.6	4.44 ± 4.6	3.28 ± 1.9	1.83 ± 0.9
Ferulic acid	30.31	0.16 ± 0.1	0.22 ± 0.1	0.08 ± 0.04	0.34 ± 0.3
Chlorogenic acid	37.19			9.69 ± 3.2	8.71 ± 5.2
Sterols
Campestrol	38.90	0.50 ± 0.1	0.56 ± 0.3	0.89 ± 0.2	0.91 ± 0.2
Stigmasterol	40.16	1.80 ± 0.1	1.37 ± 0.4	0.37 ± 0.1	0.47 ± 0.1
β-Sitosterol	40.88	5.70 ± 0.7	4.37 ± 1.3	4.90 ± 1.1	6.76 ± 1.7

* Values represent mean ± SD, n = 3; amounts are relative based on internal standard (mg/g dry sample).

. The metabolites affected and the magnitude of the change varied between the two plant species. A significant increase in some amino acids (alanine, isoleucine, serine and tryptophan), fructose and sucrose was found in methanol extracts of lycorine-treated seedlings.

4. Discussion

The phytochemical analysis showed that AF of both studied Narcissus species differed in their alkaloid profiles. The alkaloid patterns of leaf AF from N. pseudonarcissus cv. Carlton and N. poeticus resemble those reported earlier by Torras-Claveria et al. [14] and Cahlíková et al. [24]. The dominance of galanthamine- and lycorine-type alkaloids in N. pseudonarcissus cv. Carlton and haemanthamine- and homolycorine-type alkaloids in N. poeticus provides an opportunity to screen the herbicide activity of different structural types of Amaryllidaceae alkaloids. Seven compounds in the AF of N. poeticus, comprising 9.36% of TIC, were left unidentified. Five of them (UA3–UA7), were characteristic of the haemanthamine-type alkaloids’ intensive molecular ([M]⁺) and [M-H]⁺ ions with low intensity, in contrast to the compounds of lycorine, galanthamine and homolycorine types [25]. The structural type of UA3–UA7 was assigned to the haemanthamine type due to the similarity of their fragmentation patterns to those of maritidine and 8-O-demethylmaritidine, which are the main compounds in the AF of the N. poeticus. Their structures are currently determined by other mass spectral techniques. The structural types of UA1 and UA 2 were not possible to be determine with the available literature data.

In the present study, the phytotoxic activity of the studied AFs was assessed by their ability to inhibit seed germination of L. perenne and T. pratense. N. pseudonarcissus cv. Carlton AF showed stronger inhibitory activity against seed germination than N. poeticus. Potent inhibitory activity of the alkaloid fractions from both species was established at a concentration of 2 mg/mL. Such a result has been found for extracts from other species applied in much higher concentrations [26]. Some examples are as follows: Inhibition of 97% and 91of roots and hypocotyl growth, respectively, after application of C. cardunculus crude extract has been observed at a concentration of 10 g L⁻¹ (10 mg/mL) [27]. Nerium oleander flower extract suppresses growth of Lolium multiflorum Lam. (Italian ryegrass) at 40 g L⁻¹ (40 mg/mL) of extract concentration [28]. At a concentration of 40 g/L, the germination of Plantago minor Gilib. showed high inhibition influenced by the extracts of Tamarix mannifera Ehrenb. ex Bunge (100%), Alhagi maurorum Medik. (65%), Echinops spinosissimus Freyn. (85%), Haloxylon salicornicum (Moq.) Bunge ex Boiss. (67%), Lactuca virosa L. (95%), Neurada procumbens L. (71%) and Ochradenus bacctus Delile (73%) [29].

The high activity of AF indicated that bioguided isolation of alkaloids from the studied species may reveal new potent phytotoxic agents. Earlier studies have shown that narciclasine and haemanthamine, characteristic of N. pseudonarcissus cv. Carlton, possess strong phytotoxic effects comparable to commercial synthetic herbicides [9,10]. Narciclasine, a hydrophilic compound with four hydroxyl groups, was not detected in the alkaloid fraction due to the liquid–liquid fractionation of the alkaloids with apolar chloroform. Haemanthamine’s contribution in the alkaloid fraction from N. pseudonarcissus cv. Carlton (7.16%) is similar to that of N. poeticus (5.38%), indicating that the activity is due to other alkaloid(s). The major alkaloid galanthine (39.66%) may have inhibitory activity like the structurally similar lycorine 89. In addition to haemanthamine and lycorine (0.89%) at low quantities, the AFs from N. poeticus contain other haemanthamine-type compounds as major compounds (maritidine and 8-O-demethylmaritidine), which also may have phytotoxic activity.

Expectedly, like the alkaloid fractions, the effects of lycorine on the seed germination and root elongation were species-specific. It showed stronger effects on L. perrene than on T. pratense. Lycorine is the most frequent and characteristic of the Amaryllidaceae alkaloid, and it has been proven to show multiple biological and pharmacological activities, including cell growth and protein synthesis inhibition [8,30]. Also, lycorine has been identified among the bioactive compounds with herbicidal potential [9,13], but its effect on the metabolic profiles of treated seedlings, which may indicate the mechanisms of action, has not been studied yet. Because galanthine (39.66%), the major alkaloid identified in AF of N. pseudonarcissus cv. Carlton, is structurally similar to lycorine, we conducted an experiment to study the effect of lycorine on the metabolic profiles of seedlings. The selected concentration of treatment (0.05 mg/mL) was such that a reduction in the number of germinated seeds was observed, but there was still a sufficient number of germinated seeds to monitor their metabolic composition. It has been reported that lycorine and its derivative1-O-acetyllycorine are highly phytotoxic at a concentration of 0.1 mg/mL [12].

Analysis of the metabolic composition of lycorine-treated seedlings showed that the amounts of some amino acids such as alanine, valine, serine and glutamic acid were about two times higher in both species as compared to the controls. Threonine was increased in lycorine-treated L. perenne seedlings but not in T. pratense ones. The amount of tryptophane was over 20 times higher in treated T. pratense seedlings as compared to the control. The increased amino acid content in the treated plants indicates that lycorine inhibits protein synthesis and usage of amino acids like some sulfonylurea herbicides [31]. Similarly, the amounts of monosaccharides (fructose), disaccharides (e.g., sucrose) and polyols (myo-inositol) were increased in the treated plants, indicating that lycorine disrupts carbohydrate metabolism and usage like some synthetic herbicides [32,33]. Considering the fatty acids, the amounts of 9,12-octadecadienoic acid and hexadecanoic acid were increased in lycorine-treaded T. pratense and L. perenne plants, respectively. The amounts of free phenolic acids and sterols were not affected by the lycorine treatment. Massive accumulation of saccharides and amino acids, which are osmolytes, has been reported for Aegilops geniculata Roth after treatment with coumarins. The authors relate this result to analogous changes observed during water stress, suggesting that coumarins act on the plant by causing an imbalance in its water content and the increased accumulation of osmolytes is crucial for abiotic stress tolerance [34]. Data on the mechanisms of action of various natural compounds or mixtures of them such as the effects of essential oils on seed germination inhibition are scarce. A few studies identify possible modes of action of essential oils such as disruption of mitochondrial respiration and oxidative pentose phosphate pathways, reducing the rate of cell division and cell elongation or altering several amino acidic pathways and the TCA cycle of treated plants [3].

Lycorine is known as potent protein inhibitor in eucaryotic cells [8,30]. The results from metabolic profiling of lycorine-treated seedlings indicate that the herbicidal activity of lycorine is most probably due to inhibition of the protein synthesis, which leads to disruption of amino acids and carbohydrate usage affecting the seed germination and growth.

Most crops use one part of the plant—flowers, fruits, leaves or underground parts—while in the case of daffodils, the flowers and bulbs are used. [35]. The current study also opens up the possibility of using the leaves, after harvesting the flowers and bulbs, to produce bioactive extract and compounds, thus allowing to utilize biomass that has not been used to date. More efficient use of raw materials and waste reduction are among the goals of the circular economy concept adopted in 2022 by the European Parliament.

5. Conclusions

The results showed that the alkaloid fractions from the leaves of the studied Narcissus species exhibit a strong inhibitory effect on seed germination, which is due mainly to the alkaloids contained in them. Higher inhibitory activity against seed germination was found for the fraction of N. pseudonarcissus cv. Carlton compared to that of N. poeticus. The difference in the activity perhaps is determined by a higher content of galanthine, which is structurally similar to lycorine in the first species. To the best of our knowledge, data about the effect of lycorine on the metabolic composition of ryegrass and clover seedlings are presented for the first time. Increased accumulation of some amino acids and saccharides, indicating inhibition of protein synthesis, was found to be the most common reaction of the treated seedlings. The results of the present study indicate that the alkaloid fractions from the leaves of the studied daffodil species have the potential to be further investigated for future application as bioherbicides. Field studies are needed, as well as an assessment of the impact of the alkaloid fractions on non-target organisms. Using the leaves to obtain extracts for application as a bioherbicide would allow for the full use of Narcissus species resources, which is in line with the principle of the circular economy.