Model of Possible Interactions of Metabolites in Secretory Cells and Phytopreparations

Secretions in plant secretory cells are complex mixtures of different components. Here, they interact with each other, and the relationships result in various fluorescent characteristics. A similar picture may occur in phytopreparations after extraction of the compounds and formulation of the final drugs for a market. One approach to the analysis of their fluorescence may be to model the interactions using a mixture of individual compounds. In the following, we consider some examples of the interactions between water-soluble components or ethanol-soluble components used in phytopreparations. In the first case, there may be interactions between phenols such as anthocyanins and phenolic acids or between alkaloids and aromatic acids.

Water Solutions

Figure 5.4 demonstrates how combinations of phenolic acids like aromatic acids and coumarins, or phenolic compounds and alkaloids, influence fluorescence spectra.

As seen in Figure 5.4, water solutions of anthocyanins as flavonoids show the fluorescence maximum at 425-430 nm, but the spectrum changes after the addition of phenolic acids such as o-coumaric acid. The spectra of the fluorescence in this case contain two maxima: 430 nm (the earlier peak specific to anthocyanins was 420 nm) and 515 nm (individual coumaric acid has peak 510 nm). Unlike the experiments with coumaric acid, in a mixture of anthocyanin with rutin (dissolved in basic solution), there are no maxima. Individual flavonol glycoside rutin fluoresces in yellow-orange with maximum 595-600 nm and anthocyanin may quench the emission in the mixture.

Unlike their combinations with coumarins, anthocyanins interacting with the alkaloid sanguinarine show no change in the fluorescence maximum of the compound. Similar behavior was seen with the alkaloid berberine, which is similar in chemical nature to sanguinarine.

Another picture was observed in the interactions of phenolic acids (Figure 5.4). In a mixture of ferulic acid and coumaric acid (maxima of the emission are 460 nm and 510 nm, respectively, for individual compounds), one can see an increase in height of maximum 460 nm. It is interesting that contacts of basic (pH 8.0) solution of flavo- noid rutin with ferulic acids or the alkaloid berberine lead to a short-wave shift in the rutin maximum at 595 nm, while sanguinarine does not have this effect.

FIGURE 5.4 The fluorescence spectra for the interactions of water-soluble compounds (1 tng/mL). Ant: sum of anthocyanins from Dianthus caryophyllus, purified on Whatman 1 and dried in air; Berb: berberine; Coum: o-coumaric acid; Ferul: ferulic acid; Rut: rutin, pH 8.9: Sang: sanguinarine. Excitation: 360 nm for all substances, besides rutin and sanguinarine excited by light at 470 nm on subject glasses.

Ethanolic and Oil Solutions

Interactions between compounds within model mixtures in ethanol and oil such as azulene-chlorophyll, anthocyanin-chlorophyll, or flavonoid anthocyanin-azulene— are shown in Figure 5.5. Azulene, soluble in ethanol, fluoresces from 380 to 430-460 nm depending on the excitation wavelength. The commercial azulene reagent also shows an additional maximum 675 nm, like chlorophyll. Unlike azulene, carotene is soluble only in a hydrophobic native medium such as oil. Films of azulene and carotene, air-dried on glass slides, fluoresce with maximum 430 nm in blue and 530 nm in greenish-yellow, respectively. In a mixture of both compounds in films, a peak 515 nm is observed. Anthocyanins fluoresce with maxima in blue (415 nm) and in yellow-red (595-600 and 650-660 nm). In mixtures of ethanolic solutions of azulene and anthocyanin, the spectrum does not contain a maximum at 675-695 nm. Of particular interest are the interactions between chlorophyll and azulene, the mixture of which shows red emission. This leads to a shift in the blue peak of chlorophyll, which moves to a shorter region of the spectrum, while the red maximum is at the same site as for chlorophyll. In other words, the changes are linked to the blue emission.

The changes related to the behavior of azulene in mixtures are not understood. It is difficult to explain the received spectral data. Perhaps, the non-stable maxima in blue and the presence of a peak in 675 nm are related to the structure of the compounds,

FIGURE 5.5 The fluorescence spectra for the interactions of water-insoluble compounds (1 mg/mL) for mixtures in a glass cuvette or in a film (a, c-d) on slides (a and b). Ant: sum of anthocyanins from Begonia acetosa, purified on Whatman 1 and dried in air; Az et and Az oil: azulene as a film in ethanol and in a mixture with mentholic oil; Car: carotene in sunflower oil; Chi: chlorophyll. Excitation: 360 nm for all substances.

with an 8-carbon chain and many double bonds. These fluorescence peculiarities must be kept in mind when formulating compositions of natural drugs. Azulenes are not products of distillations of essential oils; they are present in the chloroplasts of spring leaves in legumes (Roshchina 1999) and are found in pollen cells (Roshchina et al. 1995). According to Kasha’s rule, fluorescence from organic compounds usually originates from the lowest vibrational level of the lowest excited singlet state (Si). Azulene, in exception to Kasha’s rule, shows fluorescence from the state S₂ and displays the fluorescence originating from the second excited singlet, which is much stronger than that from Si (Big Chemical Encyclopedia). This behavior may be explained by considering that the azulene molecule has a relatively large S,-Si gap, which is responsible for slowing down the normally rapid S₂ to Si internal conversion, such that the fluorescence of azulene is due to the S₂ -> S₀ transition. The fluorescence emission spectrum of azulene is an approximate mirror image of the S₀-S₂. Some fluorescence lifetimes are observed in picoseconds, although these are unusual cases. In organic molecules, the Sj—S₀ fluorescence has natural lifetimes of the order of nanoseconds, but the observed lifetimes can be much shorter if there is some competitive non-radiative deactivation (as seen for the case of cyanine dyes). A few organic molecules show fluorescence from an upper singlet state (e.g., azulene) and here, the emission lifetimes come within the picosecond time scale, because internal conversion to S and intersystem crossing compete with the radiative process. Fluorescence quenching may occur at the interaction of azulene with trans-stilbenes. Saltiel (1968) studied the effect of fluorescence quenching by azulene. Due to the structure of azulenes, which is rich in double bonds, they may interact with many compounds. For example, nanoparticles, like fullerenes, interact with azulene, and according to changes in the fluorescence registered by fluorescence spectroscopy, this has potential for pharmacy (Lorenzo et al. 2008).

Relations between the accumulation of azulenes and anthocyanins in vivo have been considered in only one publication (Bashirova et al. 2005). According to the data, there is a correlation between the increase of both compounds during the growth of medicinal plant species in the genus Artemisia belonging to various lines and sorts. This may be due to changes in metabolism, which depend on various factors. In Achillea asiatica, there is a correlation between anthocyanin and azulene synthesis. If the flowers were rich in anthocyanin, the accumulation of azulenes was 300% higher than control (Bashirova et al. 2003).

Similar changes may be seen in the fluorescence of secretory cells of the Achillea genus on excitation, for example, in A. millefolium (Roshchina et al. 2011b). Here were four examples of flower petals with different colors: white petal, white-rose petal enriched in various flavonoids, rose petal with weak anthocyanin pigmentation, and red-rose petal with bright anthocyanin pigmentation. The components were extracted by water, ethanol, and chloroform. Water extracts of all samples showed no emission, and neither did ethanol extracts of white petals. As petal color increased, their ethanol extracts demonstrated (1) weak emission in white-rose petal, (2) small clear maximum at 590-600 nm in weak rose petal, specific to some anthocyanins, and (3) high maximum at 590-600 nm in bright red-rose petal. When chloroform was used for extraction of more hydrophobic components, such as azulenes, in all samples, only one and the same maximum at 403-405 nm was seen. This maximum, perhaps, relates to proazulenes and azulenes that were both present in the species.

Correlation in Fluorescence between Cells and Extracts

Drugs in extracts and their localization in the raw phytomaterial may be compared for fluorescence between intact cells, cells after extraction by solvent, and the extract itself. Figures 5.6 and 5.7 and Table 5.1 represent examples of similar comparisons.

In Figure 5.6, fluorescent images of a leaf cell surface are seen before and after drug extraction for 1 h at excitation by light at 360-380 and 400-430 nm. The reader will observe how water-soluble and ethanol-soluble compounds have disappeared, liberating parts of the surface. In all cases, glandular structures, primarily seen in the control as dark spots, become more visible as secretory glands with a center and surrounding cells as a sheath. After water extraction, the whole surface weakly fluoresces, while treatment with ethanol opens internal cells containing chlorophyll

FIGURE 5.6 Fluorescent images of the surface of Eucalyptus cinerea before (a) and after extraction with water (b) and ethanol (c). Left: excitation by light at 360-380 nm, right: excitation by light at 400-430 nm.

FIGURE 5.7 The fluorescence spectra of leaves and extracts from leaves of Eucalyptus cinerea and Ginkgo biloba. Water and ethanol extracts from the first species were at 2:100 and 10:100 w/v for 1 h, respectively, while the same extracts from the second species were at 3:100 and 5:100 w/v, respectively. For leaf: (1) control, (2) after extraction with water, (3) after extraction with ethanol, (4) after extraction with chloroform. For extracts: (5) water extract, (6) ethanolic extract.

(chlorenchyma, parenchyma), which emit in red. In officinal medicine, an ethanol tincture from various species of Eucalyptus is used. As seen in Figure 5.7, the control variant has emission maxima at 450,470, and 685 nm. Water quenched the leaf emission such that only the smallest peaks at 450 and 470 nm were seen, while after the ethanol extraction, additional maxima at 530 and 680 nm were observed (flavins and chlorophyll, respectively). The spectrum of the water extract included a shoulder at 415 nm and the main maximum at 445 nm. In contrast, the ethanol extract emitted with peaks at 440, 580, and 680 nm. After treatment with the hydrophobic solvent chloroform, only maxima at 445 and 470 nm were seen in the leaf, as well as a chlorophyll peak at 680 nm (Table 5.1). After water extraction, leaf from Ginkgo biloba had no emission, but after the ethanol extraction, leaf fluoresced with maximum 423 nm, shoulder 470 nm, and maximum 685 nm. In the water extract, the emission demonstrated peak 445-450 nm, while ethanol extract - and small peak of chlorophyll 685 nm. The duration of extraction may result in a different picture of fluorescence, depending on the thickness of the leaf and surface components, which are extracted first.

Excitation by blue light at 360 and 380 nm induced mainly blue fluorescence of intact leaf tissue (Figure 5.7) with maxima at 430 nm (specific to terpenes such as pro- azulenes and azulenes) and 450-460 nm (due to some phenols and NADH/NADPH).

TABLE 5.1

Fluorescence Images and Fluorescence of Secretory Cells before and after Extraction with Various Solvents

In leaves of Actinidia chinensis and Nerium oleander under a luminescence microscope (Table 5.1), secretory hairs fluoresce due to surface components. After extraction with water, the hair emission decreased visually, and the fluorescence maxima shifted to shorter wavelengths. If to see the maxima of the extracts a reader marks that the 1-hour extraction completely evolve components fluoresced at 460 nm. It may be supposed that these are phenolic compounds, because they emit more intensely then NADH/NADPH usually do. When ethanol is used as a solvent, this maximum is also present, but new maxima at 500 and 680 nm appear due to flavins and chlorophyll, respectively. In the case of leaf of Eucalyptus cinerea, water extracts showed a maximum at 440-460 nm, possibly due to flavonoids. Nevertheless, ethanol extracts showed new maxima at 500 and 680 nm, like the two other-mentioned species. In the extract with chloroform, hydrophobic components extracted by chloroform had maxima in blue, and the glandular fluorescence disappeared completely. Only weak emission was seen with maxima at 410 and 680 nm. Terpenes, predominant in the leaf of the species, fluoresced with maxima < 460 nm, were extracted, and one can see their emission only in the chloroform extract.