Chemical Composition and Antifungal Activity of Chelidonium Majus Extracts – Antagonistic Action of Chelerythrine and Sanguinarine Against Botrytis Cinerea

Chemistry and Ecology

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Chemical composition and antifungal activity of Chelidonium majus extracts – antagonistic action of chelerythrine and sanguinarine against Botrytis cinerea

D. Wianowska, S. Garbaczewska, A. Cieniecka–Roslonkiewicz, R. Typek & A. L. Dawidowicz

To cite this article: D. Wianowska, S. Garbaczewska, A. Cieniecka–Roslonkiewicz, R. Typek & A. L. Dawidowicz (2018): Chemical composition and antifungal activity of Chelidonium majus extracts – antagonistic action of chelerythrine and sanguinarine against Botrytis cinerea, Chemistry and Ecology, DOI: 10.1080/02757540.2018.1462345 To link to this article: https://doi.org/10.1080/02757540.2018.1462345

Published online: 09 Apr 2018.

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RESEARCH ARTICLE Chemical composition and antifungal activity of Chelidonium majus extracts – antagonistic action of chelerythrine and sanguinarine against Botrytis cinerea D. Wianowskaa, S. Garbaczewskab, A. Cieniecka–Roslonkiewiczb, R. Typeka and A. L. Dawidowicza aDepartment of Chromatographic Methods, Faculty of Chemistry, Maria Curie-Sklodowska University, Lublin, Poland; bDepartment of Synthesis, Technology and Biotechnology of Biologically Active Products, Institute of Industrial Organic Chemistry, Warsaw, Poland

ABSTRACT ARTICLE HISTORY This study discusses the similarities and differences between the Received 4 November 2017 antifungal activity of Chelidonium majus L. extracts and Final Version Received 28 chelerythrine/sanguinarine, the main alkaloids in these extracts, March 2018 against the plant pathogenic fungus, Botrytis cinerea.Theobtained KEYWORDS data show that the Ch. majus L. extracts inhibit the growth of Natural fungicide; enhancing B. cinerea very effectively. The extracts activity is significantly antifungal activity; synergistic greater than that of chelerythrine/sanguinarine standard solutions action; celandine alkaloids alone and it does not always reflect the total content of alkaloids in the extracts. It has been proved that B. cinerea exhibits very diverse resistance to chelerythrine and sanguinarine. The IC50 values for chelerythrine and sanguinarine equal 5.41 mg/mL and 1.76 mg/mL, respectively. In the mixture the antifungal activity of chelerythrine and sanguinarine is antagonistic in the concentration dependent way. Based on the presented data it is not clear, however, which alkaloids present in the Ch, majus extracts at significantly lower concentration levels enhance the activity of chelerythrine and sanguinarine so that the extracts activity is considerably greater than that of both main alkaloids mixtures.

Introduction

Chelidonium majus L., commonly known as greater celandine, is a herbaceous perennial plant of the family Papaveraceae, that is native to Europe and Asia but has spread through- out the world. The main constituents of this plant are alkaloids such as benzophenanthridines (e.g. chelidonine, chelerythrine and sanguinarine) and protoberberines (e.g. coptisine and berberine) [1–4]. These compounds exhibit numerous biological properties, including choleretic, colagogue, spasmolytic, antitumour, antiinflammatory, antibacterial, antiviral and fungicidal effects, both in vitro and in vivo [5–9]. Of the above mentioned, fungicidal activity of benzophenanthridines deserves special attention since in the aspect of rapid development of pathogens resistance to the commonly used synthetic fungicides there is a great demand for alternative and effective natural antifungal agents.

CONTACT D. Wianowska [email protected] Department of Chromatographic Methods, Faculty of Chemistry, Maria Curie-Sklodowska University, Pl. Maria Curie-Sklodowska 3, Lublin 20-031, Poland © 2018 Informa UK Limited, trading as Taylor & Francis Group 2 D. WIANOWSKA ET AL.

Botrytis cinerea (teleomorph: Botryotinia fuckeliana) is an airborne plant pathogen with a necrotrophic lifestyle that affects many plant species worldwide. This fungus causes the grey mold in vegetables, ornamentals and fruits, that is responsible for great damage in crops in the field or after the harvest period [10]. In addition, B. cinerea is not indifferent to humans since it can cause a respiratory allergic reaction in predisposed individuals [11]. In view of this, many attempts were undertaken to develop fungicidal agents inhibiting effectively the growth of this fungus. There are many fungicides for B. cinerea control, most of them are the synthetic agents. Yet due to the genetic plasticity of this fungus at present many of these agents are ineffective [11]. Therefore new fungicides are constantly being looked for, particularly among therapeutic plants and compounds isolated from them as they are considered to be non-toxic to mammals and non-target organisms [12]. They also are regarded to be safer for the environment, which is important taking into account the negative environmental impact of the majority of synthetic fungicides. In the past decade the fungicidal activity of alkaloids from Ch. majus attracted attention of the researchers. It was proved that they are equally active against resistant clinical yeast isolates as well as plant pathogens [13–16]. The Pârvu research team proved in [15,16] that Ch. majus extracts cause ultrastructural changes in B. cinerea conidia and B. tulipae hyphae. In [16] the ultrastructural changes were combined with the chemical composition of Ch. majus extracts. However, until now the activity of alkaloids alone and their mixture was not compared to that of the Ch. majus extracts. Sanguinarine and chelerythrine are the main alkaloids of Ch. majus extracts. The high content of chelerythrine and sanguinarine is also characteristic of the pink plume poppy extract (Macleaya cordata R. Br.). The extract was registered for the use as a fungicide with the trade name Qwel (this extract has the CAS registry number [112 025-60-2]) [12]. This fact additionally develops the scien- tists’ interest in research on the correlation between the activity of sanguinarine and chelerythrine alone, their mixture and, the fungicidal properties of Ch. majus extracts. Hence the purpose of this study is to present similarities and differences in the antifungal properties of the Ch. majus extracts and the sanguinarine and chelerythrine solutions of the same concentrations as those in the plant extracts. In the experiments the effect of different extraction conditions i.e. maceration or extraction in the Soxhlet apparatus using chloroform, ethyl acetate, acetone or methanol as extractants on the antifungal activity of the Ch. majus extracts against B. cinerea was tested in vitro.

Materials and methods

Materials and reagents Chloroform, ethyl acetate, methanol and acetone (all of analytical grade), acetonitrile (HPLC), were supplied by the Polish Chemical Plant POCh S.A (Gliwice, Poland). Sanguinar- ine and chelerythrine standard (98%) and formic acid were purchased from Sigma-Aldrich (Poznan, Poland). Sabouraud Dextrose Agar was supplied by Argenta, CM0139 Oxoid, (Poznan, Poland). The plant pathogenic fungus (B. cinerea Persoon) was obtained from the Department of Phytopathology and Entomology, University of Warmia and Mazury in Olsztyn, Poland. Water was purified on a Milli-Q system (Millipore, Bedford, MA, USA). Ch. majus was collected in July 2015 near Warsaw (Poland). Overground parts of celandine were air-dried at room temperature for 2 weeks. A sufficiently large representative CHEMISTRY AND ECOLOGY 3 sample of the material was pre-cut and then ground with a Braun cutting mill to obtain particle size of 0.2–0.4 mm. Exactly weighted portions of the material (40 g) were sub- jected to extraction.

Extraction in the Soxhlet apparatus A sample of the celandine herb (40 g) was submitted to the exhaustive extraction process with 200 mL of chloroform or ethyl acetate or methanol or acetone for 6 h using the Soxhlet apparatus. After cooling, the extract was removed and the same plant material was soaked with a fresh portion of the extractant and again extracted in the Soxhlet apparatus. The obtained extracts were pooled together and evaporated to dryness using a rotary vacuum evaporator (Buchi R II rotavapor; 40°C). For statistical purposes three independent extractions were performed.

Maceration A 40 g portion of the walnut green husks was soaked with 400 mL of chloroform or ethyl acetate or methanol or ethyl acetate for 24 h at room temperature. After this time the extract was removed, filtered through Whatman no. 4 paper and evaporated to dryness using a rotary vacuum evaporator (Buchi R II rotavapor; 40°C). For statistical purposes three independent extractions were performed.

Assay of antifungal activity The activities of the extracts of celandine as well as the chelerythrine and sanguinarine solutions against B. cinerea were tested in vitro. Acetone solutions of dry extracts (with the concentration 20.0 mg/mL) and chelerythrine/sanguinarine standards (with the concentration from 0.010 to 6.0 mg/mL) were used in the study. A 1 mL aliquot of the solution was uniformly distributed on the surface of Sabouraud dextrose agar and acetone was allowed to evaporate aseptically. The mycelial discs with a diameter of 5 mm were cut from a homogeneous culture of the fungus and placed on the agar medium surface situ- ated on the sterile Petri plates with a diameter of 90 mm (Bionovo, Legnica Poland). The plates on which only acetone was deposited on the agar medium were used as a negative control. All plates were incubated at 25°C for 5 days. The activity was determined by measuring the inhibitory zone diameter and it was expressed in percent using the equation elaborated by Deans and Svoboda [17]: Inhibition(%) =[(C − T) × C−1]×100 where: C – the colony diameter of the mycelium on the control dish (mm); T – the colony diameter of the mycelium on the treatment dish (mm). The assay was performed in six replications.

Total phenolics content measurement The total phenolic content in the obtained extracts was determined by using the Folin-Cio- calteu’s phenol reagent according to the method of [18] with slight modifications. Briefly, 4 D. WIANOWSKA ET AL.

0.3 mL of the extract solution was mixed with 0.3 mL of Folin-Ciocalteu’s reagent. After 3 min, 3 mL of 7% sodium carbonate aqueous solution was added to the mixture. After incu- bation for 30 min at 40°C, the absorbance was measured at 760 nm with the Jenway 6300 spectrophotometer (Bibby Scientific). Quantification was done with respect to the standard curve of gallic acid (0.001–0.010 mg/mL). The results were expressed as gallic acid equivalents (GAEs), milligrammes per gram of dry extract weight. The samples were measured in three replicates.

LC–MS analysis The chromatographic measurements were performed on the LC-MS system consisting of UHPLC chromatograph (UltiMate 3000, Dionex, Sunnyvale, CA, USA), a linear trap quadrupole-Orbitrap mass spectrometer (LTQ-Orbitrap Velos from Thermo Fisher Scientific, San Jose, CA) and an ESI source. The column used was a 100 mm × 4.6 mm i.d.,3μm, Gemini C18 (Phenomenex, Torrance, CA, USA). Chromatographic separation was performed using gradient elution. The mobile phase A was 25 mM formic acid in water. The mobile phase B was 25 mM formic acid in acetonitrile. The gradient programme started at 5% B increasing to 65% for 60 min, next 65% B to 100% B for 5 min, followed by isocratic elution (100% B) for 5 min. The total run time was 70 min with the mobile phase flow rate 0.5 mL/min. The column effluent was ionised by electrospray (ESI). ESI was operated in positive polarity modes under the following conditions: spray voltage – 3.5 kV; sheath gas – 40 arbitrary units; auxiliary gas – 10 arbitrary units; sweep gas – 10 arbitrary units; capillary temperature – 320°C. Nitrogen (>99.98%) was employed as sheath, auxiliary and sweep gas. The scan cycle used a full-scan event at the resolution of 60,000. For better visualisation the chromatographic separation Single Ion Monitoring (SIM) function was used with m/z = 348 for chelerythrine and m/z = 332 for sanguinarine. The quantification of chelerythrine and sanguinarine was based on the calibration curves obtained for their authentic standards. The calibration curves were linear in the range of 10–1000 µg/mL for both compounds with the correlation coefficients >0.997. The limit of quantification was 0.05 µg/mL for both compounds. Due to the lack of standards of other alkaloids identified in the examined extracts, the amounts of these compounds were calculated by relating their chromatographic response to the calibration curve of sanguinarine. The identification of other alkaloids contained in the analyzed extracts was based on their chromatographic behaviour (retention times), literature data [3,4,8,19–23] and simu- lations carried out in the ChemSketch programme. The mentioned software enabled des- ignate the molecular mass of compound and the m/z value necessary in LC-MS analysis.

Statistical analysis All data are expressed as the mean of three independent measurements ± standard devi- ation. The diameters of the inhibition zones were measured using a ruler, with an accuracy of 0.5 mm. The analysis of variance (ANOVA) and F-test were used to assess the influence of experimental factors on the inhibition of the mycelial growth. The mean values were considered significantly different when a result of compared parameters differed at P = CHEMISTRY AND ECOLOGY 5

0.05 significance level. P-values were used to check the significance of each Fisher coefficient.

Results and discussion

The inhibition of mycelial growth of B. cinerea by the celandine extracts obtained by isolation in the Soxhlet apparatus and maceration using different extractants (chloroform, ethyl acetate, methanol or acetone) is presented in Figure 1. The F-value which determines the effects of different extraction conditions on the inhibition of the fungus growth is listed in Table 1. The results of high F-value (Fexp > Fcrit) and low P-value indicate that the factor was statistically significant. As results from Figure 1, all investigated extracts demonstrate high inhibition activity of the B. cinerea growth. This finding is valid for both the extracts obtained by isolation in the Soxhlet apparatus (Figure 1(A)) and maceration (Figure 1(B)). Comparing the results shown in Figure 1(A) with those in Figure 1(B), at first glance, it seems that the application of different extraction methods does not change the antifungal activity of the extracts obtained using the same extractant type. It seems that the precision of the activity measurements, evidently higher for the extracts obtained by maceration, is the only differ- ence. Statistical analysis of the presented data reveals, however, that only for the ethyl acetate extracts the isolation method does not affect the activity (Fexp < Fcrit, see Table 1).

Figure 1. Inhibition activity of B. cinerea growth by the Ch. majus extracts obtained by isolation in the Soxhlet apparatus (A) and maceration (B) using different extractants (chloroform, ethyl acetate, methanol or acetone) and chelerythrine/sanguinarine standard solutions alone and their mixtures.

Table 1. F-values and P-values obtained during the variance analysis concerning the effects of different extraction conditions on the inhibition of B. cinerea:E1– effect of extraction method for a given extractant type (experimental results – see Figure 1(A and B)); E2 – effect of extractant type in the Soxhlet apparatus (see Figure 1(A)); E3 – effect of extractant type in maceration (see Figure 1(B)); E4 – effect of chloroform and ethyl acetate used in the Soxhlet apparatus (see Figure 1(A)). E1a b b a CHCl3 EtOAc MeOH ACE E2 E3 E4 F-value 10.64 1.60 37.65 5.31 31.45 67.74 0.33 P-value 8.5 × 10−3 2.3 × 10−1 1.1 × 10−4 4.4 × 10−2 9.1 × 10−8 1.2 × 10−10 5.8 × 10−2 aFcrit. = 4.96. bFcrit. = 3.10. 6 D. WIANOWSKA ET AL.

For the other extractants the effect of isolation method on the extract antifungal activity is varied and statistically significant (5.31 < Fexp < 37.65; Fcrit = 4.96). The effect is smaller for the acetone extracts (Fexp = 5.31) but larger for the methanol ones (Fexp = 37.65). Analyzing the effect of different extractant types, applied using the same isolation method, on the activity of the Ch. majus extracts, it is clear that the change of the extractant type leads to significant modification of the extracts antifungal activity. The correct- ness of this conclusion is confirmed by the F-values (31.45 < Fexp < 67.74; Fcrit = 3.10). Furthermore, the higher F-value obtained for maceration (Fexp = 67.74) indicates that the effect of extractant type on the extract activity is more pronounced for this technique. It is worth noticing that regardless of the extraction technique the chloroform and ethyl acetate extracts exhibit the highest antifungal activity. Based on the presented data it is evident that the extraction conditions determine plant extracts properties, including antifungal ones. This fact has been extensively discussed in the literature in the context of altering the extraction efficiency of compounds having rel- evant characteristics [24–32]. Thus, to indicate unequivocally the extraction conditions that isolate the antifungal compounds more effectively, it is necessary to analyze the composition of the obtained extracts. According to the literature data [1–4] alkaloids are the main compounds of Ch. majus with proven antifungal activity. Yet, in [16,33,34] it was demonstrated that phenolic compounds also inhibit the growth of different fungi, including B. cinerea. This group of compounds is very ubiquitous in the plant kingdom and they may be well represented in celandine. To verify this hypothesis the total phenolics content in the extracts was determined spectrophotometrically. These data together with the sum of alkaloids established in the extracts by means of the LC-MS method are presented in Table 2. The results of the detailed analysis of alkaloids characteristic of the obtained celandine extracts is shown in Table 3. As follows from the data presented in Table 2, the content of phenolics and alkaloids in the extracts is distinctly different and, as expected, dependent on the applied extraction method and extractant type. Phenolics were found to be present in the surprisingly small amounts ranging from 0.44 to 2.48 mg GAEs/g. Hence the contribution of this group of compounds to the antifungal activity of celandine extracts against B. cinerea in this study can be neglected. It should be remember, however, that the biological activity of plant extracts is highly effected by the isolation conditions of active constituents. In [15]

Table 2. Total amounts of alkaloids and phenolic compounds in the Ch. majus dry extracts obtained by maceration (MAC) and Soxhlet extraction (SOX) using different extractants: chloroform (CHCl3), ethyl acetate (EtOAc), methanol (MeOH) and acetone (ACE). Extractant type Extraction method Total alkaloids content [mg/g] Total phenolics content [mg GAEs/g]

CHCl3 MAC 23.19 ± 0.68 0.44 ± 0.04 SOX 74.80 ± 0.79 1.83 ± 0.01 EtOAc MAC 10.99 ± 0.28 2.07 ± 0.03 SOX 23.60 ± 0.56 2.42 ± 0.01 MeOH MAC 17.72 ± 0.15 1.68 ± 0.04 SOX 32.25 ± 0.38 1.67 ± 0.09 ACE MAC 10.82 ± 0.14 2.48 ± 0.23 SOX 6.43 ± 0.21 1.79 ± 0.14 The data are expressed as (mean values ± SD) (n = 3). Table 3. Composition of alkaloids identified in the Ch. majus extracts obtained by maceration (MAC) and the Soxhlet extraction (SOX) using different extractants: chloroform (CHCl3), ethyl acetate (EtOAc), methanol (MeOH) and acetone (ACE). Alkaloids content in [mg/g] of dry extract obtained using

CHCl3 EtOAc MeOH ACE No. Alkaloids name m/z MAC SOX MAC SOX MAC SOX MAC SOX 1 Noroxyhydrastinine 192 0.028 ± 0.005 0.062 ± 0.005 nd nd nd nd nd nd 2 Norsanguinarine 318 0.018 ± 0.002 0.046 ± 0.003 nd nd nd nd nd nd 3 Coptisine 321 0.28 ± 0.02 1.32 ± 0.03 0.096 ± 0.012 0.047 ± 0.006 0.94 ± 0.04 1.15 ± 0.03 1.00 ± 0.03 0.67 ± 0.03 4 Dihydrocoptisine 322 n.d. n.d. n.d. n.d. 0.48 ± 0.03 0.027 ± 0.004 0.42 ± 0.03 0.067 ± 0.005 5 Stylopine 324 0.53 ± 0.03 1.31 ± 0.02 0.36 ± 0.03 0.24 ± 0.01 0.88 ± 0.04 0.88 ± 0.03 1.39 ± 0.02 0.83 ± 0.03 6 Sanguinarine 332 9.04 ± 0.10 27.34 ± 0.58 4.43 ± 0.21 4.13 ± 0.26 5.15 ± 0.19 13.13 ± 0.16 0.28 ± 0.02 0.43 ± 0.03 7 Norchelerythrine 334 0.063 ± 0.005 0.21 ± 0.01 0.32 ± 0.02 0.30 ± 0.02 0.28 ± 0.01 0.36 ± 0.03 0.44 ± 0.03 0.25 ± 0.01 8 8-oxycoptisine 336 0.13 ± 0.01 0.51 ± 0.03 0.055 ± 0.004 0.026 ± 0.004 0.27 ± 0.02 0.12 ± 0.01 0.36 ± 0.03 0.13 ± 0.01 9 Berberine 337 0.025 ± 0.003 0.024 ± 0.003 0.031 ± 0.002 0.025 ± 0.004 0.024 ± 0.003 tr tr tr 10 Dihydroberberine 338 0.15 ± 0.01 0.58 ± 0.03 0.11 ± 0.01 0.077 ± 0.005 0.43 ± 0.03 0.32 ± 0.03 0.44 ± 0.03 0.31 ± 0.03 11 Norchelidonine 340 0.036 ± 0.003 0.051 ± 0.003 0.094 ± 0.005 0.12 ± 0.01 0.045 ± 0.005 0.039 ± 0.004 0.026 ± 0.004 tr 12 Canadine 340 0.033 ± 0.002 0.063 ± 0.004 0.026 ± 0.004 0.041 ± 0.003 0.034 ± 0.003 0.047 ± 0.005 0.023 ± 0.003 0.027 ± 0.004 13 Corydine 342 n.d. n.d. n.d. n.d. 0.088 ± 0.006 0.42 ± 0.03 0.066 ± 0.005 0.026 ± 0.004 14 Chelerythrine 348 11.71 ± 0.13 40.33 ± 0.61 4.43 ± 0.24 17.33 ± 0.36 6.95 ± 0.15 13.73 ± 0.27 0.40 ± 0.03 0.48 ± 0.03 15 Oxysanguinarine 348 0.27 ± 0.01 0.12 ± 0.01 n.d. n.d. tr 0.13 ± 0.01 tr 0.024 ± 0.003 16 Dihydrochelerythrine 350 n.d. n.d. n.d. n.d. 0.039 ± 0.005 tr 0.028 ± 0.005 n.d. 17 8-hydroxydihydro-sanguinarine 350 0.032 ± 0.002 0.051 ± 0.003 0.064 ± 0.003 0.068 ± 0.007 n.d. n.d. n.d. n.d. 18 Didehydrochelidonine 351 0.045 ± 0.004 0.17 ± 0.01 0.24 ± 0.01 0.50 ± 0.03 0.17 ± 0.01 0.42 ± 0.03 n.d. 0.14 ± 0.01

19 Oxychelidonine 352 tr 0.038 ± 0.004 n.d. n.d. n.d. n.d. n.d. n.d. ECOLOGY AND CHEMISTRY 20 Chelidonine 354 0.47 ± 0.03 1.32 ± 0.03 0.097 ± 0.006 0.085 ± 0.006 0.29 ± 0.0.21 0.25 ± 0.01 0.39 ± 0.03 0.22 ± 0.01 21 Protopine 354 0.023 ± 0.003 0.099 ± 0.008 0.47 ± 0.03 0.34 ± 0.03 0.84 ± 0.03 0.74 ± 0.03 1.18 ± 0.03 0.65 ± 0.03 22 Chelerubine 363 0.033 ± 0.003 0.084 ± 0.006 0.055 ± 0.006 0.023 ± 0.003 0.044 ± 0.003 0.037 ± 0.004 n.d. n.d. 23 Oxynitidine 364 n.d. n.d. n.d. n.d. n.d. n.d. 0.075 ± 0.008 tr 24 6-methoxydihydrosanguinarine 364 n.d. n.d. n.d. n.d. n.d. n.d. 0.083 ± 0.006 tr 25 8-hydroxydihydrochelerythrine 366 n.d. tr n.d. n.d. 0.02 tr 0.025 ± 0.003 tr 26 Chelamine 368 n.d. n.d. n.d. n.d. tr n.d. 0.038 ± 0.004 tr 27 α-homochelidonine 370 0.055 ± 0.006 0.26 ± 0.01 n.d. n.d. 0.037 ± 0.004 0.024 ± 0.002 0.037 ± 0.004 tr

(Continued) 7 8 .WAOSAE AL. ET WIANOWSKA D.

Table 3. Continued. Alkaloids content in [mg/g] of dry extract obtained using

CHCl3 EtOAc MeOH ACE No. Alkaloids name m/z MAC SOX MAC SOX MAC SOX MAC SOX 28 10-hydroxychelidonine 370 0.045 ± 0.004 0.29 ± 0.02 0.035 ± 0.004 0.057 ± 0.006 0.15 ± 0.01 0.12 ± 0.01 0.15 ± 0.01 0.074 ± 0.006 29 α-allocryptopine 370 0.14 ± 0.48 ± 0.03 0.077 ± 0.005 0.054 ± 0.005 0.088 ± 0.007 0.096 ± 0.007 0.20 ± 0.01 0.088 ± 0.006 30 6-methoxydihydrochelerythrine 380 n.d. n.d. n.d. n.d. 0.094 ± 0.006 n.d. 1.76 ± 0.04 1.00 ± 0.03 31 Dihydrochelelutine 380 n.d. 0.034 ± 0.004 n.d. n.d. 0.036 ± 0.004 n.d. 0.91 ± 0.03 0.38 ± 0.03 32 Angoline 380 n.d. n.d. n.d. n.d. 0.024 ± 0.003 0.048 ± 0.006 tr tr 33 6-acetonyl-5,6-dihydrosanguinarine 390 tr n.d. tr 0.063 ± 0.005 0.16 ± 0.01 0.089 ± 0.007 0.40 ± 0.03 0.26 ± 0.01 34 Macarpine 393 0.036 ± 0.004 0.11 ± 0.01 0.12 ± 0.01 0.036 ± 0.004 0.062 ± 0.004 0.047 ± 0.005 0.086 ± 0.007 0.057 ± 0.005 35 Dihydrochelerubin 396 n.d. n.d. n.d. n.d. n.d. n.d. 0.11 ± 0.01 0.045 ± 0.004 36 8-acetonyldihydrochelerythrine 406 n.d. n.d. n.d. n.d. n.d. n.d. 0.022 ± 0.003 tr 37 Methyl 2′-(7.8-dihydro-sanguinarine-8- 406 n.d. n.d. n.d. 0.040 ± 0.002 0.11 ± 0.01 0.026 ± 0.003 0.45 ± 0.03 0.25 ± 0.01 yl)acetate 38 Chelidimerine 721 n.d. n.d. n.d. n.d. n.d. n.d. 0.026 ± 0.004 0.021 ± 003 n.d.: not detected; tr.: traces. CHEMISTRY AND ECOLOGY 9 describing the activity of alcoholic extracts from Ch. majus, among the antifungal compounds the more polar phenolic compounds, mainly phenolic acids, were distinguished. Remembering the phenolics content in the obtained extracts, in contrast, the concentration level of alkaloids was much higher, up to 75 mg/g. Thus the alkaloids proved to be the main group of compounds responsible for the antifungal activity of the examined extracts. Taking into account that in the case of the majority of the applied extractants, the Soxhlet extraction revealed the alkaloids amount 2-3-fold higher than that obtained by maceration, the former technique should be recommended for the extraction of antifungal compounds from Ch. majus. Regarding the alkaloids composition, the data presented in Table 3 show how complex alkaloids mixture are the Ch. majus extracts and prove that chelerythrine and sanguinarine are the main alkaloids. Furthermore, chelerythrine and sanguinarine contents are various and strongly dependent on the applied extractant type and extraction method. Higher chelerythrine and sanguinarine contents, in general, are obtained by extraction in the Soxhlet apparatus. The last observation confirms the validity of the statement that the Soxhlet extraction should be recommended for the isolation of antifungal alkaloids from celandine. Taking into account the effect of extractant type on the efficacy of this extraction technique, it must be stated that the largest amounts of chelerythrine and sanguinarine are present in the chloroform extracts (40.33 mg/g and 27.34 mg/g, respectively), whereas the smallest in the acetone extracts (in the range of 0.28–0.48 mg per 1 g of dry extract). It is worth noticing that the chelerythrine and sanguinarine contents in the chloroform and ethyl acetate extracts are distinctly different, however, the differ- ence in the antifungal activity exhibited by both extracts is statistically insignificant

(Fexp < Fcrit, see Table 1) (compare 40.33 mg/g for chelerythrine and 27.34 mg/g for sanguinarine with 17.33 mg/g for chelerythrine and 4.13 mg/g for sanguinarine in the chloroform and ethyl acetate extract). This result may indicate that chelerythrine and sanguinarine are extremely effective antifungal agents even at low concentration levels or other alkaloids (or other compounds) exhibit/modify the antifungal activity of Ch. majus extracts. Yet from the above presented data it is difficult to state explicitly that the changes in the inhibition activity of B. cinerea growth demonstrated by the examined extracts reflect those in the sanguinarine and chelerythrine amounts. To facilitate the comparison of the antifungal activity of extracts with that of chelerythrine/sanguinarine alone and the mixture of these alkaloids another series of experiments was performed in which the antifungal activity of chelerythrine and sanguinarine standard solutions was determined. It should be emphasised that the concentrations of chelerythrine and sanguinarine standards in the applied solutions were the same as those of these compounds in the corresponding Ch. majus extracts (see Table 3). The ability of sanguinarine and chelerythrine standard solutions differing in concentrations for B. cinerea growth inhibition is presented in Figure 1 (bars with the horizontal lines and dots for chelerythrine and sanguinarine standard solutions, respectively, and white bars for their mixtures). Comparing the results obtained for the extracts as well as chelerythrine and sanguinarine solutions, it can be concluded that the ability of standard solutions for B. cinerea growth inhibition is clearly lower than that exhibited by the celandine extracts. Even in the case of the largest amount of chelerythrine and sanguinarine in solution, their antifungal activity is much lower than that demonstrated by the chloroform extract obtained in the Soxhlet apparatus. The fact that the antifungal activity of extracts is 10 D. WIANOWSKA ET AL. higher than that exhibited by the standard solutions is known from the literature (Wia- nowska et al. [33]). Yet the results presented in Figure 1, showing that the mixtures of chelerythrine and sanguinarine standard solutions applied in the same amounts as before exhibit lower activity than chelerythrine/sanguinarine alone, prove the antagonistic action of both alkaloids. As follows from Figure 1 this action depends on the ratio of individual alkaloids in the mixtures. According to the authors’ knowledge this experiment evi- dences the antagonistic action of chelerythrine and sanguinarine for the first time. Finally, it is worth mentioning that the less concentrated sanguinarine solution exhibits the higher activity than the more concentrated chelerythrine solution (compare Table 3 and Figure 1). This observation may suggest a very diverse resistance of B. cinerea to chelerythrine and sanguinarine. To check that the effect of the increase of chelerythrine and sanguinarine standards concentration in the solutions on the inhibition of B. cinerea growth was examined. The obtained data are collected in Table 4. They prove that sanguinarine is characterised by strong antifungal activity and effectively inhibits the growth of B. cinerea at much lower concentration level in comparison with chelerythrine. This can be clearly seen for instance considering the concentrations providing the same growth inhibition (the concentration resulting in 50% inhibition, IC50). The IC50 values for chelerythrine and sanguinarine equal 5.41 mg/mL and 1.76 mg/mL, respectively.

Conclusions

The results presented in this paper showed that the Ch. majus L. extracts inhibit the growth of B. cinerea very effectively. The inhibiting activity of the extracts is highly affected by the type of the applied extractant and extraction method. The highest inhibiting activity is exhibited by the extracts obtained in the Soxhlet apparatus using chloroform. The obtained data confirmed that chelerythrine and sanguinarine are the main constituents of the Ch. majus extracts with proven antifungal activity. This fact is reported in the literature. However, owing to the comparison of the antifungal properties of the extracts with the activity of chelerythrine/sanguinarine alone and a mixture of these alkaloids applied at the same concentration as those in the corresponding extracts, it has been demonstrated for the first time that the extracts activity is significantly greater and it does not always reflect the quantity of alkaloids in them. It has been proved that in the mixture the antifungal activity of chelerythrine and sanguinarine is antagonistic in the concentration dependent way. Based on the presented data it is not clear, however, whether the observed greater antifungal activity of the extracts is a result of enhancing the

Table 4. Inhibiting properties of B. cinerea growth (in %) by the solutions of different concentrations of pure chelerythrine and pure sanguinarine. Inhibition (in %) of the B. cinerea growth by standard solution of Concentration [mg/mL] chelerythrine sanguinarine 1.0 27.15 ± 1.54 46.58 ± 3.43 2.0 32.01 ± 3.43 51.43 ± 0.00 3.0 38.89 ± 2.39 56.09 ± 4.88 4.0 42.94 ± 4.54 58.37 ± 7.44 5.0 47.19 ± 4.33 67.91 ± 5.77 6.0 53.08 ± 3.86 71.20 ± 6.38 The data are expressed as the mean value ± SD, (n = 3). CHEMISTRY AND ECOLOGY 11 antifungal properties by the other alkaloids, though they are present at very low concentration levels, or by other compounds. In order to resolve the problem of synergistic action of the Ch. majus extracts components explicitly and/or to indicate the extracts components enhancing their antifungal properties, another series of experiments is required. However, this task is very complicated taking into account how very complex mixtures are the extracts and that the synergistic action can depend on the ratio of individual compounds in the extracts.

Disclosure statement

No potential conflict of interest was reported by the authors.

Notes on contributors

Dorota Wianowska is a researcher and teacher at the Maria Curie-Skłodowska University in Lublin (Poland), with expertise in analytical chemistry, environmental chemistry, and medical chemistry. Sylwia Garbaczewska is a researcher at the Institute of Industrial Organic Chemistry in Warsaw (Poland). Her field of research is the natural protection of plants against fungi. Anna Cieniecka-Roslonkiweicz is a researcher at the Institute of Industrial Organic Chemistry in Warsaw (Poland). Rafal Typek is a researcher at the Maria Curie-Skłodowska University in Lublin (Poland). Andrzej L. Dawidowicz is a professor of chemistry at the Maria Curie-Skłodowska University in Lublin (Poland).

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