Microscopic and spectroscopic characterization of nutlets and mucilage of Ocimum basilicum and Thymus vulgaris

evaluation

arrangement of the cells. Marin (1996) noted that oval/round, clearly differentiated cells were only found in Thymus zygis L. nutlets. Duletić-Laušević and Marin (1999) pointed out that Th. vulgaris has a strong mucilage reaction. The nutlets of O. basilicum are oval and smooth (Diklić, 1974). The pattern of ornamentation of the nutlets of O. basilicum is irregular and does not show any particular microstructures as observed in the other Lamiaceae taxa (Marin, 1996).
Basil mericarps have been reported to be a rich source of protein, fibers, and fatty acids, especially linoleic and linolenic acid, minerals, and phenolic compounds, making it a novel food and functional ingredient with beneficial properties (Bravo et al., 2021). The chemical composition of thyme nutlets showed the presence of proteins, lipids, and crude fibers, while the extracts contained tannins, alkaloids, phenols, saponins, etc., and showed antimicrobial effects (Abbas et al., 2011).
The aim of this study was to investigate the micromorphology of the nutlets of Thymus vulgaris and Ocimum basilicum grown in Serbia. Since the positive nutritional and health effects of the nutlets of both species are well known in the literature, this study was also aimed to investigate the chemical composition for the first time the chemical composition of the nutlets and mucilage of the samples from Serbia using Raman spectroscopy.

MATERIAL AND METHODS
Plant material. The plant material of O. basilicum and Th. vulgaris was obtained from the collection of the Institute for Medicinal Plant Research "Dr. Josif Pančić". The plants were collected at the fruiting stage, air-dried, and stored in paper bags until further processing. Immediately before analysis, the nutlets are separated from the flower parts.
Stereomicroscopic analyses. The nutlets of the studied species were examined and photographed with a Nikon SMZ18 stereomicroscope (Tokyo, Japan) without prior preparation. In addition, the length and width of the nutlets (N= 10) were measured using DIGIMIZER 4.3.4 and expressed as mean ± standard deviation. The intensity of myxocarpy was monitored for 8 hours after wetting and the nutlets were photographed with LEICA DMLS. In the first hour, myxocarpy was recorded at 15-minute intervals, while further changes were recorded at 60-minute intervals. After recording the intensity of mucilage formation, the nutlets of both species were moistened and the fully formed mucilage was individually stained by adding a drop of 0.05% ruthenium red and 0.05% methylene blue.
Scanning electron microscopy (SEM) analysis. For the SEM analysis, the nutlets of O. basilicum and Th. vulgaris were coated with a thin gold layerfor 100 seconds at 30 mA (BALTEC SCD 005 Sputter Coater), and then observed and photographed (JEOL JSM-6390W).
Raman measurements. Raman microspectroscopy of O. basilicum and Th. vulgaris nutlets and mucilage was done. The nutlets were longitudinally cut at room temperature and their mucilage was recorded using XploRA Raman spectrometer from Horiba Jobin Yvon. The mucilage samples were prepared according to the procedure described by Salgado-Cruz et al. (2013). Raman spectra for nutlets and mucilage were recorded using the laser at a wavelength of 532 nm equipped with a 1200 lines mm -1 grating, applying exposure time 20 s and scanning the sample 10 times, using a 100% filter. The spectral resolution was about 3 cm −1 and calibration was checked by a 520.47 cm -1 line of silicon. The spectral range in the interval from 200 and 1800 cm −1 was analyzed for nutlets from both species and from 250 to 3250 cm -1 for mucilage samples. Ten spectra were collected and averaged per each sample and species (Figures 4 and 5). Characteristic bands of specific functional groups were described from literature records. Raman spectra acquisitions were managed by the LabSpec software (Horiba Jobin Yvon). Raman data were analyzed by Origine Pro 8.6 software (OriginLab, Northampton, MA, USA) and were smoothed using the Savitzky-Golay method, based on 5 points.

RESULTS AND DISCUSSION
Morphological and micromorphological characteristics of the nutlets and mucilage of the studied species. The nutlets of O. basilicum and Th. vulgaris were examined with the stereomicroscope and the scanning electron microscope. All characteristics of the nutlets examined, including length, width, shape, color, abscission scar and ornamentation, are listed in table 1.
The nutlets of O. basilicum were larger than those of Th. vulgaris (almost three times as long and twice as wide). The nutlets of Mentheae are bilaterally symmetric, based on the position of the abscission scar, and their size varies from 0.6-4.3 mm in length (Moon et al., 2009). The nutlet dimensions of about 30 species of the Nepetoideae subfamily ranged from 0.6 mm for certain Mentha species to 2.7 mm for Hyssopus officinalis (Duletić-Laušević and Marin, 1999). Fully mature nutlets in Mentheae are usually brown, except for the genus Salvia, which has grey and yellow nutlets in several species (Moon et al., 2009). According to the classification of length/width ratio proposed by Clopton (2004), the nutlets of O. basilicum were ovoid, and those of Th. vulgaris were broadly ovoid (Table 1, Figures 1 and 2).
Most often the abscission scar is located either at the center of the basal end or slightly shifted towards the ventral side; without an extended area in Salvinae or with the U-or V-shaped extended area at the ventral side (Moon et al., 2009). According to figures 1 and 2, the species studied also differ in terms of the shape and position of the abscission scar. The abscission scar on the nutlets of Th. vulgaris (tribe Mentheae) was round and located on the ventral side, just below the nutlet base (Figure 1d   The ornamentation of the nutlets of O. basilicum was characterized by the presence of secondary sculpturing elements, such as striae (Figures 2b and 2c). This ornamentation type was previously identified in some species of the genera Salvia, Clinopodium, Cyclotrichium, etc. Other species of the tribe Mentheae commonly possessed papillae or both striae and papillae as secondary surface elements (Moon et al., 2009). On the other hand, the nutlets of Th. vulgaris had a primary ornamentation pattern (Figures 2e and 2f) which is mostly recognized as a type E according to Moon et al. (2009). These authors defined that type E includes a rounded cell arrangement; each cell is rather flat and the cell boundary is slightly thick. Similarly, Marin (1996) indicated that certain Thymus species, such as Th. zygis had a round arrangement of exocarp cells. Both species lacked trichomes on the nutlets, which is consistent with the findings of Duletić-Laušević and Marin (1999) for the majority of the examined species of the Nepetoideae subfamily. These authors also pointed out that hairy or glandular nutlets do not show extensive mucilage production, so we expected the absence of trichomes in thyme and basil nutlets to correlate with an extensive mucilage reaction. (1999) found that the nutlets of most Nepetoideae species showed mucilage production upon wetting, although the reaction varies in intensity. The nutlets of both species showed a very fast reaction of myxocarpy, forming the maximum amount of mucilage 15 minutes after wetting. The nutlets of O. basilicum formed a mucilage layer four times thicker than Th. vulgaris (Table 2). According to Duletić-Laušević and Marin (1999), the mucilage layer >0.5 mm (O. basilicum) indicates a strong reaction, and 0.1-0.5 mm a moderately strong reaction (Th. vulgaris). Both species produced non-transparent, milky opaque mucilages with fibrils present (Table 2, Figure 3a and 3d). The mucilaginous cells are located in the exocarp of Lamiaceae species' fruit, and their cell walls contain spiral fibrils (Ryding, 1992;Duletić-Laušević and Marin, 1999;Ryding, 2010). The cellulose fibrils are attached to the surface of the nutlet, preventing the loss of the mucilage layer (Kreitschitz and Gorb, 2018).  The addition of ruthenium red (staining of pectins) and methylene blue (staining of cellulose) showed that the mucilage of Th. vulgaris consists of a pectin matrix and cellulose fibrils (Figures 3e and 3f). Ryding (2010) pointed out that mucilaginous cells present in the pericarp of tribe Mentheae species were colorless and contained globose bodies without starch as was confirmed in our study.

Duletić-Laušević and Marin
Myxocarpy was found in about 80% of the species of the tribe Ocimeae. The mucilage was often colorless, with spiral cellulosic fibrils (Ryding, 1992). The mucilage of O. basilicum contained pectins, fibrils, and distinguishable starch grains (Figures 3b and 3c). Our findings  Ryding (1992), who reported that spherical starch grains have been found in the mucilaginous cells of several probably unrelated genera of tribe Ocimeae, including Ocimum spp. Kreitschitz and Gorb (2018) reported that the mucilage of O. basilicum nutlets is covered by round starch grains spread as protrusions under the mucilaginous layer.
Raman signature of nutlets and mucilage. The nutlets from O. basilicum showed a high content of lipids (33%) and carbohydrates (43%) and a low protein content (10%) (Nazir et al., 2017). Previous results on the chromatographic profile of O. basilicum nutlet samples (Angers et al., 1996), indicated the predominance of unsaturated fatty acids and were verified with the major fatty acids being linolenic (up to 64.8%) and linoleic acid (up to 31.3%), with lesser amounts of oleic acid (up to 13.3%). On the higher content of linolenic fatty acids in seed samples indicated many reports ( These results showed that there are two distinct spectral patterns ascribed to unsaturated (α linolenic, linoleic, and oleic) and saturated (palmitic and stearic) fatty acids. The medium intensity bands were responsible for the saturated and unsaturated fatty acids observed at 1439 and 1299 cm -1 (Figure 4), assigned to the CH 2 scissoring deformation vibration and C-H bending, respectively (Baranski et al., 2006;Fei et al., 2017). In the Raman spectra of α-linolenic and linoleic acid, as predominant acids in both species, (Baeten et al., 2001;Lv et al., 2016) bonds with higher wavenumbers are marker features for the presence of double bonds (Martini et al., 2018). These acids differ mainly in the position of the double bonds, consequently, their Raman spectra are highly similar (De Gelder et al., 2007). The bands at 1652 and 1261 cm -1 (Figure 4), could be assigned to the cis stretching vibration of C=C and the bending of C-H, respectively, all involving the unsaturation moieties of unsaturated fatty acids cis isomers, which relative intensity is in accordance with the level of unsaturation, especially in a case of the band at 1652 cm -1 (Martini et al., 2018).
The degree of fatty acid unsaturation can be also estimated from the peak area of the bands at 1261 and 1299 cm -1 , which are due to in-phase C=C symmetric rocking and methylene twisting vibration, respectively (Baranski et al., 2006). The previous Raman studies of fatty acids found that the bands in the region from 800-900 and 1000-1150 cm -1 indicated on presence of C-C stretching bonds (Lv et al., 2016;Fei et al., 2017;Martini et al., 2018), such as the bands at 860 and 1025 cm −1 (Figure 4 and Supplementary Table). The band at 1742 cm -1 was assigned to the stretching of C=O from triacylglycerol structure that was present in all Raman spectra of different plant oil samples (Martini et al., 2018).
The highest intensity signals seen in the region from 1595-1630 cm -1 could be attributed to lignin from pericarp cell wall compounds. In this range, lignin gives a doublet band, with one maximum at 1598 cm -1 (aromatic vibrations) and a peak of lower intensity at 1626 cm -1 ( Baranski et al., 2006). Higher intensity of the bands at 1598 and 1626 cm -1 observed for Th. vulgaris could suggest that they contain higher lignin content than for O. basilicum. The lower and sharp intensity band at 999 cm -1 could attributed to the presence of protein fraction in nutlet samples, probably to phenylalanine (Li-Chan, 1996) while the intensity of the band in O. basilicum could be infuenced by the slight increase in proteins.
Raman spectroscopy of mucilage. The chemical composition of O. basilicum mucilage arises from a total carbohydrate (up to 80%), fats (4-11.55%), starch (1.5%), proteins (1.3-2%) and soluble sugars (0.6%) (Razavi et al., 2009;Hosseini-Parvar et al., 2010). Previously, several studies of the mucilage polysaccharides extracted from O. basilicum nutlets (Anjaneyalu and Dowda, 1979;Hosseini-Parvar et al., 2010) have reported the highest presence of glucomannan, (1,4)-linked xylan and a minor present of glucan. Azuma and Sakamoto (2003) also reported the presence of highly branched arabinogalactan in addition to glucomannan and (1,4)-linked xylan. According to staining reactions of the mucilage envelope in O. basilicum, it was shown that mucilage was comprised of starch grains, cellulose, and pectin (Kreitschitz and Gorb, 2018). There is no published results about chemical composition based on the carbohydrate  (Fernández-Alonso et al., 2003). Raman spectra of O. basilicum and Th. vulgaris nutlet mucilage are shown in figure 5. The bands in the region 1100-1460 cm −1 could be primarily due to the glucose as well as hemicellulosic polysaccharides, e.g. glucomannan and xylan (Himmelsbach and Akin, 1998;Salgado-Cruz et al., 2013). The bands at 1457 and 1333 cm −1 are due to coupled vibrations of hydrogen atoms corresponding to CH, CH 2 , and COH from the glucose as well as in the xyloglucan spectrum with characteristic bands at 1376 cm -1 (Himmelsbach and Akin, 1998;Malekfar et al., 2010;Synytsya et al., 2003). The spectral region between 1200 and 400 cm −1 is very often assigned in literature as the fingerprint of the glycosidic bond (Thygesen et al., 2003;Baranska et al., 2005). The bands at 478 and 1122 cm -1 were observed as a marker for starch and cellulose (Himmelsbach and Akin, 1998;Chylinska et al., 2014). The vibration originating from α-1,4 glycosidic linkages can be observed at 936 cm −1 (Almeida et al., 2010), while the characteristic band at about 859 cm -1 can be considered to be a marker band for α-glycosidic bonds in pectin (Synytsya et al., 2003;Chylinska at al., 2014). Vibrational C-H stretching was observed at 2908 cm -1 which is highly correlated with glucose as reported by Malekfar et al. (2010).
These results support the advantages of the Raman spectroscopy in methodology and provide, for the first time, characterization of dominant polysaccharides inside mucilage from O. basilicum and Th. vulgaris, without any specific and time-consuming procedures.

CONCLUSION
Thyme and basil are well-known medicinal plants from the Lamiaceae family of great economic importance. In contrast to the aerial parts, their nutlets were scarcely studied, so this work aimed to provide data on the micromorphology and chemical composition of the nutlets and their mucilages. Differences were found in the morphological characteristics as well as in the surface ornamentation of the nutlets of the species studied. Mucilage production was faster and more extensive in basil, but the properties of the mucilages were quite similar in both species. The mucilage was composed of a pectine matrix and cellulose fibrils, with additional starch grains visible in basil. As far as we know, there is no published report on the chemical composition of nutlets of both species and the mucilages contained in them, as determined by the Raman spectroscopy. Raman spectroscope, in combination with a microscope, is a suitable technique for the analysis of this type of samples, as it allows the visual identification of the tissues before spectroscopic analysis and then the spatial distribution of the plant metabolites. It was shown that the examined nutlets are good sources of phenols, unsaturated fatty acids, and polysaccharides and that the region below 1800 cm -1 in the Raman spectra is most important for their characterization.