**1. Introduction**

*Chelidonium majus* L. is a perennial herbal plant belonging to the family Papaveraceae that grows across Europe, western Asia, and North America [1–3]. It is a rich source of different biologically active substances (alkaloids, flavonoids, phenolic acids) and has been used in folk medicine for centuries. Over 27 alkaloids (e.g., chelerythrine, sanguinarine, chelidonine, protopine, allocryptopine, berberine, coptisine) were identified in its extracts and latex [4,5]. Plant laticifer cells are filled with latex (milky sap) that contains condensed defense substances [6,7]. The plant exudes latex immediately at the site of damage caused by an insect attack [7,8]. In folk herbal medicine, extracts and latex of *C. majus* are used to

**Citation:** Nawrot, R.; Warowicka, A.; Rudzki, P.J.; Musidlak, O.; Dolata, K.M.; Musijowski, J.; Stolarczyk, E.U.; Go´zdzicka-Józefiak, A. Combined Protein and Alkaloid Research of *Chelidonium majus* Latex Reveals CmMLP1 Accompanied by Alkaloids with Cytotoxic Potential to Human Cervical Carcinoma Cells. *Int. J. Mol. Sci.* **2021**, *22*, 11838. https://doi.org/ 10.3390/ijms222111838

Academic Editors: Andrzej Kutner, Geoffrey Brown and Enikö Kallay

Received: 2 October 2021 Accepted: 28 October 2021 Published: 31 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

treat warts and condylomas caused by human papillomavirus (HPV) [9]. The medicinal interest in *C. majus* is based mainly on small molecules such as alkaloids, flavonoids, and phenolic acids, which may also act synergistically [4,5,10]. These compounds exhibit antitumor, antiviral, and antibacterial activities [5,11]. The proteins of *C. majus* also show mitogenic, cytotoxic, antibacterial, and antiviral activities [7]. Nucleases present in milky sap were found to exert apoptotic effects on a human cervical cancer HeLa cell line [12,13]. The antiviral activity of milky sap was suggested to be associated with major latex proteins (MLPs) [7,14].

MLPs, pathogenesis-related 10 (PR-10) proteins, cytokinin-specific binding proteins (CSBPs), and norcoclaurine synthases belong to the Bet v 1 superfamily of proteins. Their characteristic hydrophobic cavity binds to secondary metabolites and hormones [15,16]. MLPs were discovered in *Papaver somniferum* as abundant, laticifer-specific peptides with unknown function [17]. MLPs constitute up to 50% of the soluble *P. somniferum* latex subproteome, which correlates with the relative abundance suggested by SDS-PAGE [18]. MLPs are also present in non-latex-bearing plants such as *Arabidopsis thaliana* and *Prunus persica* (peach) [15]. They influence fruit ripening in kiwi [16] and fruit and flower development in peach [19]. They protect cotton against *Verticillium dahliae* [20] and melon against cucumber mosaic virus [21]. The presence of MLP-like protein in the latex of *Chelidonium majus* L. was first reported using proteomic analysis against *C. majus* sequence database prepared after transcriptome sequencing and annotation [22]. The study identified previously uncharacterized nucleic acid binding protein and showed that it is highly overrepresented in the latex [22,23]. Statistical analysis confirmed that MLP is present in different stages of plant development until the fruit ripening period [14].

Our interest in *C. majus* originated from the traditional use of the fresh plant extracts and latex against warts and condylomas caused by the oncogenic human papillomavirus (HPV) infection. HPV infections can lead to cervical cancer in women, which can result from persistent infection with a group of "high-risk" HPVs [24]. However, previous studies on *C. majus* have shown that the proteins from this plant are also biologically active [3,13]. The antiviral activity of the *C. majus* milky sap is possibly linked with the presence of MLP [7,25]. Synergistic action of plant secondary metabolites with other components of the plant extracts is also postulated [10].

Hence, the goal of the study was to isolate the MLP from *C. majus* latex, identify accompanying low-molecular compounds and to analyze their joint cytotoxic activities against cervical cancer cell lines.

#### **2. Results**

To the best of our knowledge, no joint research has been performed before in separate fields of plant compounds research of different structures and sizes for latex-bearing plants. Such an approach requires multidisciplinary research with the use of biological, biotechnological, chemical, as well as analytical and theoretical techniques (Figure 1). Therefore, we employed a research scheme, which started with the use of two kinds of source materials—*C. majus* whole plant extract and latex samples. The second step enabled fractionation of the material with the use of affinity chromatography on heparin. All fractions were monitored with the use of proteomic and zymography techniques, as well as with the use of LC-ESI-MS/MS techniques for the identification of proteins and non-protein substances. This step allowed to indicate specific protein and alkaloids co-existing in the fractions, which was confirmed with the use of bioinformatic tools of molecular modeling and docking. Finally, the fractions were monitored in terms of their cytotoxic activities on cervical cancer cells (Figure 1).

#### *2.1. Isolation of MLP from C. majus Whole Plant Extracts and Latex*

To isolate and purify the MLP from whole plant extracts and latex samples, heparin column was selected due to its high affinity to DNA-binding proteins, coagulation factors, lipoproteins, and protein synthesis factors. In-gel DN-ase zymography was used to assess

1

the presence of MLP in separated fractions. We observed a strong nucleolytic activity for the selected fractions eluted (Figure 2). We collected protein bands showing nucleolytic activity and analyzed them by LC-ESI-MS/MS. We then applied Mascot analysis to MS results using the annotated *C. majus* CDS database [22]. We observed that MLP was the main constituent of the nucleolytic bands (MLP-like protein 28, Table 1, band 1A; Table S1). We visualized the protein content of the fractions by using SDS-PAGE and silver staining (Figure 2). The most representative bands were then collected and analyzed by LC-ESI-MS/MS. MLP was again found to be the main constituent of the fractions—both for the whole plant extracts (Table 1, band 1B; Table S2) and latex samples (Figure S1; Table S3). *Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 3 of 17

**Figure 1.** Workflow for the multidisciplinary study (toolbox) for two kinds of plants source materials—*C. majus* extracts and latex. The research scheme enabled fractionation with the use of affinity chromatography, monitoring of all fractions with the use of proteomic, zymography, as well as LC-ESI-MS/MS techniques for the identification of proteins and nonprotein substances. Molecular interactions were confirmed with the use of bioinformatic tools of molecular modeling and docking, and their biological activities were finally analyzed using cell lines. *2.1. Isolation of MLP from C. majus Whole Plant Extracts and Latex*  **Figure 1.** Workflow for the multidisciplinary study (toolbox) for two kinds of plants source materials—*C. majus* extracts and latex. The research scheme enabled fractionation with the use of affinity chromatography, monitoring of all fractions with the use of proteomic, zymography, as well as LC-ESI-MS/MS techniques for the identification of proteins and non-protein substances. Molecular interactions were confirmed with the use of bioinformatic tools of molecular modeling and docking, and their biological activities were finally analyzed using cell lines.

To isolate and purify the MLP from whole plant extracts and latex samples, heparin

**Figure 2.** Fractionation of *C. majus* whole plant extract on a heparin column. Top: Absorbance at 280 nm—blue line indicates protein content in fractions. Red lines and numbers represent fraction numbers. Middle: In-gel DNase assay zymography of protein fractions with high nucleolytic activity. Band 1A of ca. MW 33–35 kDa was cut, and MLP presence was confirmed by MS/MS. Bottom: Protein profiles of each fraction in SDS-PAGE stained with silver. Band 1B of ca. MW 36 kDa was cut from the gel, and MLP presence was confirmed by MS/MS.


**Table 1.** MS/MS results of protein bands cut from an in-gel DNase assay zymography gel (Figure 2, Band 1A) and a silver-stained SDS-PAGE gel (Figure 2, Band 1B) after electrophoretic separation of protein fractions isolated from whole plant extract. The main constituent of the fractions was MLP.
