Azulenes in Plant Cell: Clover as Their Useful Resource

Special Article: Plant Cell

Ann Agric Crop Sci. 2024; 9(1): 1147.

Azulenes in Plant Cell: Clover as Their Useful Resource

Victoria V Roshchina*

Institute of Cell Biophysics, Federal Research Centre, Pushchino Scientific Centre for Biological Research, Russian Academy of Sciences, Institutskaya Str. 3, Pushchino, Moscow Region, 142290, Russia.

*Corresponding author: Victoria V Roshchina Institute of Cell Biophysics, Federal Research Centre, Pushchino Scientific Centre for Biological Research, Russian Academy of Sciences, Institutskaya Str. 3, Pushchino, Moscow Region, 142290, Russia. Email: roshchinavic@mail.ru

Received: December 05, 2023 Accepted: January 18, 2024 Published: January 24, 2024

Abstract

Location of azulenes in plant cell has been studied on three clover species Trifolium repens L., T. pratense L., and T. medium L. (fam. Fabaceae). By using of microspectrophotometer/ microspectrofluorimeter the absorbance spectra were recorded from the surface of intact leaves and intact isolated chloroplasts. Here the maxima in the range 580-620 nm, peculiar to blue pigments azulenes, were observed in all cases. The pigments’ picks 590-610 nm also have been found in the absorbance spectra of ethanolic extracts from the leaf surface and from the chloroplast suspension. The protector function of the azulenes for the plant cell has been discussed. Clovers studied are recommended for agriculture and pharmacy as useful resource of azulenes.

Keywords: The absorbance spectra; Cell surface; Chloroplasts; Microspectrophotometry

Introduction

The blue pigments azulenes are found in many types of plants, fungi, and invertebrate organisms [1,2]. Due to its physicochemical properties, azulene and its derivatives with many double bonds and their conjugated biologically active compounds have found application in technology, especially in optoelectronic devices [1-6]. Azulenes are also included in polymers, oligomers and conjugates with furanes and phenols in order to create new chromophore materials [2,3,5].

History of azulene-containing plants is beginning in ancient times for medical and cosmetic purposes. Natural azulenes and synthesized, such as azulenic retinoids, may have antitumor activity, immunomodulatory, and antifungal activity (guaiazulene derivatives isolated from the marine gorgonian) [4] or have an antioxidant effect [5] or affect dopamine receptors [6]. Moreover, in some cases they can affect other living organisms, including negative effects [7]. The technological direction is associated with the study of the physicochemical properties of azulenes and their application in the optical and chemical-analytical industries [2,3].

Unlike a burst in technological use of azulenes, there is small information about biological role of azulenes in plants themselves, especially of medicinal ones. For many years, chemists discovered azulenes by distilling essential oils from the medicinal herbs of wormwood bitter Artemisia absinthium L. or common yarrow Achillea millefolium L, which acquired a blue color [8]. But such pigments were also found in extracts of organic solvents in mosses of the hepatic Calypogeia azurea and others [9,10], pollen of different flowering species and pollen collected by bees [11, 12], cells of horsetail microspores Equisetum arvense L. [13], needles of the blue spruce Picea excelsa [14], isolated chloroplasts of pea Pisum sativum L. and clover Trifolium repens L. [14,]. The role of azulenes for the plants themselves has not yet been considered in the literature, although there is only one interpretation of their properties as growth regulators belonging to water soluble azulene [15] and protocols of the studies of the artificial pigment on the germination of pollen and vegetative microspores of horsetail [16,17]. In experiments with exogenous azulene on vegetative microspores of horsetail, the properties of this sesquiterpene lactone as antioxidant and as a histamine antagonist are shown [18]. Moreover, it may be the electron donor in model experiments on isolated chloroplasts [19-21].

In 2019-2023 there were works that search azulenes in plants, which have been connected with tolerance to tropospheric ozone and ultra-violet irradiation [22]. It was shown that short time-extracts by organic solvents from leaves having blue color in some woody plants contained azulenes [22]. The fact gives us the idea that their tolerant ability may be connected with the defensive role of azulenes. Short time of the azulene appearance in 10 min-extracts showed a possibility of the compounds’on the leaf surface as a barrier or filter for active ultra-violet radiation and ozone as the origins of dangerous oxidants [20-23]. The surface in transmitted light was analyzed by sensitive Invitro Evos M5000 microscope showed the presence of blue wax plates on the leaf surface of Eucalyptus cinerea, in both covered secretory structure (glands) and parts lack of the glands [20,22]. Also it has been carry out spectral analysis with use microspectrophotometer/microspectrofluorimeter for the receiving of spectra of autofluorescence and absorbance in order to test the composition of compounds outside the cell wall [21,23,26].

Although today this pigment is known and applied for medicine, cosmetics and technics, the attention to the functions and location of the natural metabolite for the plant itself was far from the study many years. It may be useful for practical use of our knowledge for agriculture of medicinal and nutritional plant species. In this connection we had a goal to analyse the location of azulenes in plant cell using three species of clover. For this task, it should need to observe the leaf surface and penetrate into the cells of model objects for the isolated organelles such as chloroplasts.

Materials and Methods

Objects

Leaves and isolated chloroplasts from three clover species were chosen: white clover Trifolium repens L., red clover Trifolium pratense L., and zigzag clover Trifolium medium L. collected on natural reservation “Lugovoi” near Oka river.

Isolation of Chloroplasts

Isolation of intact chloroplasts from leaves was carried out according to Robinson with co-workers [27] in the phosphate buffer medium containing 0.3 M mannitol,0.08 M KCl, 0.066 M KH2PO4, 0.066 M Na2HPO4 pH 7.25 .

Spectral Measurements

Absorption (absorbance) of the intact leaves was measured directly on slides using the microspectrophotometer/microspectrofluorimeter MSF-15 (LOMO, St. Petersburg, Russia) [20,21]. The position of the maxima in the absorption spectra of intact cell surfaces was determined according to the Zolotarev method by the option of the reflection spectra differentiation [28]. The absorption and fluorescence spectra of extracts with 100% acetone or 95% ethanol from cells (1:10 w/v for 10 min to 1 hour or more) in 1-0.5 cm cuvettes or on paper chromatograms were recorded using the Unicam Helios-epsilon spectrophotometer (USA), spectrophotometer Specord M-40 (Germany) and Perkin Elmer 350 MPF-44 B spectrofluorometer (Great Britain) [20-23].

Extractions of Azulenes

Azulenes in sample extracts with ethanol or acetone for 10-30 minutes of exposure were determined spectrophotometrically at 580 nm, as described earlier [22]. The average error of the experiment of three to four repetitions was calculated for each variant and control, respectively.

Detection of Azulenes

Azulenes in sample extracts with 95% ethanol and acetone for 10-30 minutes of exposure were determined spectrophotometrically at 580 nm, as described earlier [20-22]. To detect azulenes, the extracts were chromatographed on Whatman Paper No. 1 without or after the impregnation with Vaseline oil or on thin-layer plates of Silufol silica gel, as previously described for pollen of a number of seed plants and horsetail microspores [11-13]. Their absorbance spectra were then recorded to compare them with data obtained on intact cells. The concentration of azulenes (A) was estimated in solutions of ethanol or acetone extracts, according to the formula: A=D580 /e×l, where D580 is the optical density at 580 nm, e is the coefficient of molar extinction of 328 M–1 cm–1, l is the thickness of the liquid layer in the cuvette, cm. The average error of the experiment of three to four repetitions was calculated for each variant and control, respectively.

Results and Discussion

Since from 50-60 years of 20 century scientific interest to azulenes based on the pharmacological knowledge and experiments with the blue pigments as by -products of distillated plant oil [3]. Here we applied spectral methods to testing of presence of azulenes using microspectrophotometer/microspectrofluorimeter MSF-15 [20-22 below we represented new data received not only leaf surface, but also intact chloroplasts of three clover species.

Spectral Analysis of Plant Cells Surface and Isolated Chloroplasts

Spectral studies of leaf surfaces and isolated chloroplasts were carried out by microspectrophotometry, which makes it possible to record the absorbance spectra in individual cells. On Figure 1-3 visible leaf absorbance spectra of three clover species and isolated from intact chloroplasts with marked areas of absorption of azulenes and chlorophyll with significant maxima, indicated by circles and polygons, respectively.