Single Cell Plant Model of Equisetum arvense for the Study Antihistamine Effects of Azulene and Sesquiterpene Lactones

Abstract

Single cell of vegetative microspore from spore-bearing plant field Equisetum arvense L. has been presented as a single-cell experimental model for the screening of native compounds acting as antihistamine agents. The effects of azulene, sesquiterpene lactones austricine, gaillardine, grosshemine, inulicine, and desacetylinulicine as well as sesquiterpene alcohol ledol, on the content of histamine in germinating horsetail microspores has been investigated by the fluorescent method. It has been shown using microspectrofluorimetry that these compounds are able to regulate the germination of microspores to varying degrees, as assessed by the autofluorescence of chlorophyll, in a medium without and in the presence of 0.5–1% sodium sulfate as a salt stress factor. A fluorescent histochemical reaction to histamine with ortho-phthalic aldehyde in cells and secretory mucilage revealed the ability of the compounds studied to reduce the level of this biogenic amine depending on their structure in the following order: grosshemine > azulene > austricine > ledol. Gaillardine, inulicine, and desacetylinulicine showed weak antihistamine activity

1. Introduction

In modern medicine, natural antihistamine preparations are still not used due to the lack of research at the cellular level. Such work is difficult to carry out on multicellular systems of mammalians; however, the presence of histamine in plant tissues [1,2,3,4,5] allows this to be done on plant cells. On unicellular models of plant microspores as biosensors (vegetative microspores of spore-bearing plants and pollen of seed plants), it is possible to test various biologically active compounds, including possible natural drugs [6,7,8,9,10]. For this purpose, it is convenient to use the fluorescence techniques such as luminescence microscopy with modifications (microspectrofluorimetry and confocal microscopy) [9].

Among the pharmacologically promising compounds that have been little studied in unicellular models are azulenes and sesquiterpene lactones, many of which are proazulenes [11,12]. These compounds are found in many plants. The most studied types of medicinal plant raw materials, where proazulenes are contained in significant quantities, are chamomile pharmacy flowers, yarrow common grass, and wormwood bitter herb [12]. On the basis of the materials, as well as some synthesized pharmaceutical substances (guaiazulene), Romazulan^® (manufacturer K.O. Biopharm S.A., Romania), Pepsan-R^® (Pharmatis, France), Rotokan-VILAR^® (JSC “Pharmcenter VILAR”, Russia), Wormwood bitter herb tincture (LLC “BAGRIF”, LLC “Tula Pharmaceutical Factory” Russia) (State Register of Medicines, https://grls.rosminzdrav.ru, accessed on 1 January 2021) have been developed and used.

Previously, a number of laboratories studied their anti-radioactive [13] and anti-inflammatory properties [14,15]. For azulene, which has anti-allergic properties, the effects of considering it as an antagonist of histamine [16], formed under stress in plant cells [17,18], are shown. Information on possible antihistamine reactions of proazulene sesquiterpene lactones is fragmentary published in the scientific literature [19,20,21,22,23].

Besides azulene [16], there is no information about the possible antihistamine reactions of proazulenes such as sesquiterpene lactones, although perspectives in using them as natural anti-allergic drugs are of interest in pharmacy and medicine. The monograph [9] dealt with phytopreparations offered the autofluorescence and fluorescent histochemical methods to study similar compounds. In this connection, the purpose of this study was to analyze the possible antihistamine properties of not only azulene, but also some known proazulenes-sesquiterpene lactones on the model of vegetative microspores of horsetail using fluorescent methods. Reactions to the substances used were autofluorescence of cell estimated as chlorophyll intensity or fluorescence in histochemical procedure for histamine.

2. Materials and Methods

2.1. Objects of Research

The objects of study were vegetative microspores of the horsetail Equisetum arvense, collected in the period from April to May 2021 in a meadow near the Oka River.

2.2. Cultivation of Microspores

The cultivation of the microspores on object slides in Petri dishes, as well as statistical analysis, are described earlier [24,25]. Vegetative microspores of Equisetum arvense germinated on nutrient medium, which included K phosphate 6.63, CaCl₂ 6.51, NaCl 3.47, MgCl₂ 5 μg/L. All tested compounds were dissolved in the above-mentioned nutrient medium (for vegetative microspores). Petri dishes, 10 cm in diameter, were lined with filter paper (5 mL of water was added to the bottom of every dish) to provide moisture for germination of vegetative spores. The germination and autofluorescence of the spores as the test-reactions on the object glasses (slides) were carried out for 2–3 h or 2 days after moistening with the grown medium. All experiments were performed at room temperature 20–22 °C on object glasses, which are put on in the Petri plates. The germination of microspore occurred in a drop of the solution studied (0.05 mL = 1 drop) on the slides that has been estimated as red fluorescence of the cell chlorophyll.

2.3. Determination of Histamine

For the fluorescent histochemical determination of histamine within cells and outside, o-phthalic aldehyde from Sigma-Aldrich (St. Louis, MO, USA) was used according to the method described for animal cells [26,27] and applied for plant cell as well [4,7,8]. Microspores were put on object glasses (slides) and moistened by drops of 0.5–1% solutions of o-phthalic aldehyde to determine histamine. After 10–20 min of staining with the reagent, samples were dried at 50–80 °C for 5–10 min. Fluorescence reaction of forming products was studied under luminescence microscope Leica DM 6000 B (Wetzlar, Germany) or by camera Levenhuk (Tampa, FL, USA) at excitation by light at 360–380 nm. Histochemical reactions were repeated (up to 3–5 times) for control variants and variants exposure to tested azulene and sesquiterpene lactones.

2.4. Observation and Measurement of Cell Fluorescence

The samples were observed and photographed using the Leica DM 6000 B luminescence microscope, and their fluorescence spectra were recorded by the MSF-15 microspectrophotometer/microspectrofluorimeter (LOMO, Sankt-Petersburg, Russia), with the Levenhuk M300 Base camera (Tampa, FL, USA). The autofluorescence spectra of the intact cells were recorded with microspectrofluorimeter MSF-15 (LOMO, Sankt-Petersburg, Russia) at excitation at 430 nm, while fluorescence after histochemical reaction for histamine—at 360–380 nm.

The autofluorescence intensity and germination of vegetative microspores of E. arvense were tested at 680 nm (I₆₈₀). Living cells (both dry and during development within 2–24 h after wetting the dry microspores with a nutrient solution) were observed and photographed with a Leica DM 6000 B luminescent microscope (Wetzlar, Germany), laser-scanning confocal microscopy Leica TCS SP-5 (Wetzlar, Germany, USA, Austria), or camera Levenhuk (USA) on MSF-15 microspectrophotometer/fluorimeter (LOMO, Sankt-Petersburg, Russia). Cell started to germinate producing chlorophyll, which emitted in red, while the non-germinated cells emitted in blue. It should be marked that in histochemical reaction for histamine determination, the fluorescence intensity at 460 nm (I₄₆₀) was recorded in the blue spectral region [7,8,9,10].

2.5. Statistical Analysis

In each object glass lying in one Petri dish, 100 fluorescing microspores were analyzed and counted at 680 nm for the autofluorescence of chlorophyll or at 460 nm for the fluorescent product for histamine in histochemical reaction. The fluorescence intensity is measured in relative units. Counting was performed in four or five replicates (the number of Petri dishes per treatment). Results were expressed as mean and standard deviation (SD) and has been also shown graphically on figures (n = 4–5 object glasses with 100 microspores per one variant; p = 0.95).

2.6. Sources of Azulene and Sesquiterpene Lactones-Proazulenes

Azulene (Fluka, Germany), sesquiterpene lactones, and sesquiterpene alcohol ledol produced by VILAR (Moscow, Russia) for special order were used in the work. Chemical formulae of the compounds are shown in Figure 1. The lipophilic compounds preliminary were dissolved in 96% ethanol and then water was added up to necessary volume (concentration of ethanol in final probe was not higher then 0.001%).

3. Results and Discussion

Vegetative microspores of field horsetail Equisetum arvense L. as unique unicellular model for the study in cell biology demonstrated visible autofluorescence with maximum 680 nm, peculiar to chlorophyll that served as indicator of their development [7,24]. Figure 2 shows the common images of the development of single microspore cell seen under luminescence and laser-scanning confocal microscope from dried sample after moistening by medium—from 2–3 to 24 h period in dependence on the time of collection. Cell of microspores is covered with thick envelope and has elaters served for the anchoring to soil. Both structures fluoresce in blue or blue-green as seen with luminescence microscope (a, f) at excitation 360–380 nm. Confocal microscopy with the laser excitation 405 nm showed the emission in stack of three channels and separately in every channel—blue, green, and red if the spore moistened. After 15–30 min after the moistening with water the spore started to fluoresce in red that seen through the transparent envelope and then missed elaters and later this envelope (f–h). This period of the development during first 24 h is used to study the influence of various factors including sesquiterpenes as in our work.

3.1. Autofluorescence of Germinating Cells with Sesquiterpenes

Figure 3 shows the changes in the germination of unicellular horsetail spores in the first two hours or two days after wetting with a nutrient medium. In variants with the presence of 2 mg/mL of studied sesquiterpene lactones in two hours of germination on the medium containing 0.5 or 1% sodium sulfate salt, a decrease in the formation of chlorophyll (test for the germination of spores) was noted compared to growth on a medium without sodium salt (taken as 100%).

Only in the version with a 1% salt solution, azulene had no deviations from control, while desacetylinulicine had the highest value of chlorophyll emission compared to other samples. After two days, a decrease in the germination rate was noted, compared to the option of 2 h of germination.

3.2. Formation and Estimation of Fluorescent Histamine Product

After one–two days of germination in the spores, a noticeable amount of histamine is formed, which was determined by a fluorescent histochemical reaction with ortho-phthalic aldehyde when excited by light at 360–380 nm (Figure 3). The blue emission reaction to histamine appeared both in the cell itself and to a greater extent in the mucilage secreted during salt stress (Figure 4). The reaction for histamine was well seen in nucleus and chloroplasts. The fluorescence was in region 460–470 nm, characteristic for emitted product of histamine reaction. It is different from the self-emission of azulene and proazulene sesquiterpene lactones at 400–430 nm [28].

Among the studied lactones (Figure 5) were compounds that dramatically reduce the formation of histamine in the cell compared to control (by 40–60%). These are azulene, austricine, grosshemine, and ledol, as well as to a lesser extent inulicine and desacetylinulucine. Similar inhibition is noted for gaillardine at 1% sodium sulfate in the medium. In the secretion that came out of the cells, the concentration of histamine was higher than the control in the variants with gaillardine, inulicine, and desacetylinulucine and almost equal control in the variant with azulene.

These results confirm previously published data from austricine and grosshemine studies in models releasing β-hexosaminidase from rat leukemia cells RBL-2H3 [20,21].

Figure 6 shows how cell fluorescence looks in the presence of sesquiterpene lactones. An extensive field of mucilage around the microspore is noted in the presence of sodium sulfate in the medium. The addition of the most active lactones such as austricine or azulene, significantly reduced the fluorescent halo around the cell and the intensity of fluorescence related to histamine presented in the medium with sodium sulfate. The addition of the most active lactones, austricine, or azulene, significantly reduces the fluorescent halo around the cell and the intensity of this fluorescence related to histamine.

Figure 7 shows the concentration dependence of their action on the content of histamine both in the cell and in the secretion with a maximum (1%) amount of sodium sulfate as a stress factor. The effects are more stable in the grosshemine variant both inside and outside the cell. Grosshemine reduces the amount of biogenic amine both in cell and in secretion. In the azulene, austricine, and ledol variants, the reaction with the presence of sodium sulfate reduced histamine mainly in the mucilage of secretion at some concentrations of these compounds.

It is evident that the most active compounds, azulene, austricine, grosshemine, and ledol have a similar structure (Figure 1). This fact should be marked as follows. For most sesquiterpene lactones the structure–activity relationship (QSAR) concludes common effects with regard to the gamma–lactone cycle [12], except ledol. The pharmacological understanding of antihistamine activity is primarily related to the effect on specific receptors, but in a broad sense can be mediated by other mechanisms too.

3.3. Screening of Antihistamine Features of Azulene and Proazulenes as Possible Cellular Protectors

Determining most antihistamine compounds after short fluorescent screening permitted choosing them fast for medicinal experience without expensive biochemical procedures and vivisection on mammalians. Moreover, plant single model cell shows analogous processes on cell level in all other animal single cells that are difficult to cultivate in artificial culture.

Considering the role of histamine in plant cell, the main known feature [1,2,17] is regulating growth reactions of the microspores and seed germination. Azulene and proazulene sesquiterpene lactones in some plants may participate in preventing the accumulation of the biogenic amine at higher concentration during salt or ozone stress [7,17]. However, stress conditions which lead to illness in mammalians are often linked with the histamine formation too that induced allergic reactions. Therefore, azulene and proazulenes may be potential antihistamine drugs which prevent the reactions.

When discussing the data obtained, the attention should be also paid to the role of the structural features of azulene and the studied proazulene sesquiterpene lactones, which may be responsible for antihistamine properties. It is a fact that compound must bind to histaminic receptors, preventing an allergic reaction of mammalians. According to some researchers, the antihistamine properties of azulene are associated with the presence of a conjugate system of five double bonds in its structure [12], and proazulene sesquiterpene lactones are associated with the α-methylene-γ-lactone group, which has pronounced alkylating properties [29]. Anti-allergic effects of sesquiterpene lactones from Saussurea costus (Falc.) Lipsch are determined using in vivo and in vitro experiments [29]. Our study showed that sesquiterpene alcohol ledol has antihistamine-like activity, in a structure where there is no α-methylene-γ-lactone group. B.K. Lee with co-workers [29] also noted that although the three sesquiterpene lactones studied (alactolactone, kostunolide, and dehydrocusus lactone) have α-methylene-γ-lactone, they have shown different anti-allergic effects in mast cell degranulation in vitro and allergic asthma in vivo. H. Pyun and colleagues point to the ability of sesquiterpene lactones to inhibit the activity of the transcription factor NF-kB and enzymes [30] involved in the arachidonic acid pathway. Dehydrocostus lactone, a sesquiterpene from Saussurea lappa Clarke, suppresses allergic airway inflammation by binding to dimerized translationally controlled tumor proteins [29]. However, the exact mechanisms of antihistaminic and anti-allergic activity of sesquiterpene lactones have not yet been established, suggesting the need for further research in this area.

Based on information on the formation of histamine in adverse conditions and in plants [1,2,3], it is possible to assess the existence of such receptors at the plant cell level. Testing of antihistamine properties in unicellular plant models has some advantages over multicellular mammals, where the effects vary depending on the different tissues of the whole organism. In addition to antihistaminic characteristics, azulene compounds in essential oils also have a powerful antioxidant effect [31]. Some plant terpenoids are also considered as probable GABA receptor agonists [32]. Of course, the next stage in the analysis of the antihistamine properties of azulene and proazulenes should be their testing on patients in a medical hospital. However, preliminary experiments with plant single cells as a model system sensitive to the compounds and fast fluorescence changes in similar models give useful information on the screening of most perspective natural drugs. Minimal amount of materials and reagents, fluorescence technique, and absence of vivisection permit achieving the goal in a short period.

4. Conclusions

The approach to use the combination of model single plant cells having sensitivity to medicinal natural compounds with their fluorescence reactions demonstrates perspectives for application in pharmacy and medicine in the preliminary screening of similar compounds. The background consists in the measurement of the fluorescence intensity of analyzed living cells germinating under various conditions with and without added natural drugs. In this case, fluorescence technique, absence of animal vivisection, and minimal amount of materials and reagents made similar studies accessible for every research laboratory for the above-mentioned screening.