Alkaloids inyoung and mature
G. arborea petiole and stem samples were observed in cortical parenchyma cells surrounding the vascular tissue, epidermis and sieve-tube elements. Alkaloids were also observed in the epidermis and cortical collenchyma of the midrib and mesophyll cells of the lamina in both samples (Fig 1).
Epidermis and cortical collenchyma cells in the stem and the midrib, cortical parenchyma cells of the petiole of
G. arborea young and mature samples showed positive result for saponins. Localization of saponins were also observed in the mesophyll cells of the lamina in young samples (Fig 2).
Cortical parenchyma cells were positive for flavonoids in the stem, petiole and midrib of
G. arborea young and mature samples. Positive results were also observed in the petiole of young samples, some trichomes of the midrib and, epidermis and trichomes in the abaxial portion in the lamina of mature samples (Fig 3).
In the stem of
G. arborea, tannins were observed in the cortical collenchyma cells of the mature samples and some parenchyma cells between vascular tissues of young samples. Epidermis, cortical parenchyma cells and phloem parenchyma in the petiole, the epidermis, cortical collenchyma cells and some trichomes in the midrib and epidermis and mesophylls cells in the lamina of young and mature sample showed positive results (Fig 4).
Cardiac glycosides were observed cortical collenchyma and parenchyma cells and some sieve-tube elements in the stem of both samples and trichomes in the stem of mature samples (Fig 5).
The above findings coincide with the histochemical findings of other researchers. The distribution of alkaloids, as observed by
Hashimoto et al. (1992), are common in active tissues near the vegetative points, ovule, epidermis and the layer just inside of it, trichomes, peripheral layers of fruits and seeds, vascular bundles, cork cambium, cork tissues and laticifers. Some observations are similar with histochemical analysis of
Catharanthus roseus wherein the distribution of alkaloids is observed in phloem-associated parenchyma and epidermis
(Yamamoto et al., 2016). The epidermis and outer cortical layer, the parenchyma within and adjacent to the phloem and the periphery of the pith near xylem of
Atropa belladonna was also observed to contain abundant alkaloids
(James, 1950). Detection of alkaloids in young and mature samples may vary as plant age affects the production of these compounds and, more often than not, young samples have higher concentration than mature samples
(Koomson et al., 2018). Although with slow histochemical reaction due to low concentration, detection of alkaloids in the trichomes of mature samples is still possible
(Jing et al., 2014).
Localization of saponins was commonly observed in the epidermis and cortex of the stem and the midrib and mesophyll cells of the lamina in
G. arborea. This observation are similar to other histochemical studies of
Alphonsea zeylanica,
Ruta graveolens (Dhale, 2011),
Polygala sibirica (Teng et al., 2009a) and
Polygala tenuifolia (Teng
et al., 2009b). Detection of saponins in the leaves may indicate its concentration as
Chen et al., (2019) observed highest accumulation of saponins in the palisade layer of
Cyclocarya paliurusleaves.
Francis et al., (2002) indicated that there is a trend of higher concentration of saponins in young as compared to mature plants but these compounds occur in both samples and has no significant difference according to
Koomson et al., (2018) which supports the results of this study.
Tissue localization of flavonoids in
G. arborea were observed mostly in parenchyma cells. Parenchyma cells in the stem of
Morinda citrifolia contain flavonoids as observed by
Mownika et al., (2020). Flavonoids are also localized in the parenchyma cells and hypodermal layers of the stem and petiole of
Centratherum punctatum (Chitra
et al., 2014). Flavonoids can be detected in the leaves as they largely accumulate in the adaxial epidermal layer and sub-epidermal cells
(Agati et al., 2002) and palisade parenchyma
(Tattini et al., 2004). Localization of flavonoids can be observed in young and mature samples as these compounds occur in juvenile stage of plants and continuously synthesized as the plant mature. The concentration of flavonoids may vary across different developmental stages but can still be detected through histochemical analysis
(Chang et al., 2018; Luengas-Caicedo et al., 2007).
Localizations of tannins in the stem, petiole and midrib observed in this study is in agreement with the observations of
Dhale (2011),
Mahendra et al., (2017) and
Salatino et al., (1993). The detection of tannins in parenchyma cells around or near vascular tissues of the stem can be supported by the study of
Robil and Tolentino (2015) on
Medinilla magnificastem as tannins specifically accumulate in these tissues. On the other hand, tannins in the leaves of
Struthantus vulgaris (Salatino et al., 1993) and
Medinilla magnifica (Robi and Tolentino, 2015) are more abundant in the palisade parenchyma and in the more compact spongy parenchyma adjacent to the lower epidermis which is also similar to the result of this study. Some epidermal structures, like trichomes, are also known to contain tannins.
Sudipta et al., (2014) observed tannin localization in the leaf trichomes in some members of Combretaceae including
Terminalia chebula, Terminalia catappa, Quisqualis indica, Terminalia bellirica and
Combretum decandrum. Occurrence of tannins in some tissues of young and mature samples was also observed by Fleurat-Lessard
et al. (2016) in different cellular compartments of the pulvinus of young and mature
Mimosa pudica. Different types of condensed tannins also occur in the young and mature branchlets of
Casuarina equisetifolia as observed by
Zhang et al., (2009).
Cardiac glycosides were detected in both
G. arborea only in the cortical collenchyma and parenchyma cells of the stem and petiole and some glandular trichomes of young and mature samples. It implies that these substances occur in few tissues of these plants. Localizations observed in this study can be supported by the study of
Morsy (2017) as these compounds occur in the seeds, leaves, stem, roots and bark of plants in small amount. The study of
Hollman (1985) enumerated some plant families with representatives known to exhibit cardiac glycosides, however, Lamiaceae and Malvaceae are not included. Moreover, the observed localization of cardiac glycosides in both young and mature samples indicate that age is not a determining factor on the detection of this compound in both species. This is in congruent with the study of
Agrawal et al., (2012) wherein cardenolides of
Asclepias syriaca were detected by in both mature plant and seedlings but of different concentrations.
Based on the results of histochemical analysis, plants secondary products are localized in the different cells and tissues of different part of
G. arborea. This implies that these secondary products can be consumed by livestock, particularly goats when this plant species are utilized as forage. Both nutritional and toxic effects to goats can be attributed to the phytochemical constituents of this plant species. These effects are dependent on the amount of these phytochemicals consumed which is one of the main considerations in using
G. arborea as sole forage for goats.
