Beyond the Scent: New Evidence about Micromorphological, Phytochemical and Biological Features of Plumeria rubra ‘Tonda Palermitana’ (Apocynaceae)

Plumeria rubra L. is an ornamental Caribbean plant widely known for its ethnobotanical uses and pharmacological activities. The ‘Tonda Palermitana’ cultivar, on which no data are to date available, is commonly cultivated in Sicily. The aim of our study was to characterize the micro-morphological features of leaves and flowers of this cultivar by light and Scanning Electron Microscopy and to investigate the phytochemical profile and the biological properties of their food-grade extracts (LE and FE, respectively) by LC-DAD-ESI-MS analysis and different in vitro assays. Numerous branched laticifers were observed, and their secretion contained alkaloids and lipophilic compounds as confirmed by histological analyses. Phytochemical analyses showed the presence of alkaloids (9%), terpenoids (13%) and fatty acids (6%), together with a very abundant presence of iridoids (28%) and polyphenols (39%). The most notable biological activity of both extracts appears to be the antioxidant one, showing half-inhibitory concentrations (IC50) about 5 times lower than those detected in anti-inflammatory assays (383.74 ± 5.65 and 232.05 ± 2.87 vs. 1981.23 ± 12.82 and 1215.13 ± 10.15, for FE and LE, respectively), with LE showing the best, and statistically significant (p < 0.001), biological activity. These results allow us to speculate promising nutraceutical and cosmeceutical applications for this old Sicilian cultivar.


Introduction
The genus Plumeria L. (Apocynaceae family) includes laticiferous shrubs and small trees widely cultivated as ornamental plants [1].In this genus, hundreds of scientific names used for species, varieties and cultivars are now regarded as synonyms.According to The Plant List [2], the accepted species within the Plumeria genus are 11, whereas according to Plants of the World [3], there are 18.Among these, the most cultivated species are P. obtusa L., P. pudica Jacq., and P. rubra L. [4].P. rubra, also known as 'Frangipani', is certainly the most used due to its beautiful and fragrant flowers of different colours and forms [1].The first description of this plant can be found in the Badianus Manuscripts of 1522, a book written by the Spanish priest Francisco de Mendoza on the medicinal uses of local plants by the Aztec population.However, the genus name Plumeria was given in honour of the seventeenth-century French botanist Charles Plumier [5][6][7].
Plants 2024, 13, 2479 2 of 21 P. rubra is native to Central America but it has been spread across tropical and subtropical areas of the world [1].In Hawaii, its flowers are traditionally used for the famous flower necklaces (Lei), which have become a symbol of brotherhood and hospitality [4].
In India, the plant is a symbol of immortality, and in the past, it was commonly planted near temples and graveyards [8].The species is also cultivated in Italy, along the coastal areas of Sicily, especially in Palermo, where it has been renamed 'Pomelia' [4].It is also called 'Frangipani', from the French word 'frangipanier', coagulated milk, in relation to its latex (or sap).It is not known exactly when the first plant of P. rubra arrived in Palermo.The first notices date from the second decade of the nineteenth century and concern the Boccadifalco Botanic Garden, which, together with the one in Palermo, seems to have constituted the nucleus of the propagation of this species in the city [4].Since its arrival in Palermo, the plant was immediately appreciated for the beauty and intense scent of its flowers, so much so that it quickly spread in private and public gardens or was grown in containers on balconies and terraces, becoming one of the symbols of the city's floral displays.However, in Palermo, P. rubra has not only ornamental use but also a profound cultural significance.It is an auspice of fertility formulated by analogy with the remarkable example of the vegetative force of the plant.Therefore, its white flowers are often used for the bouquets of Palermo brides and traditionally the bride who goes to live in a new house receives a plant from her mother as a wish for family continuity [4].
P. rubra is a deciduous tree of about 7-8 m, with a thin and grey-green bark and swollen branches which are leafy at the tips [1].All the portions of the plant are characterized by the presence of a white milky latex, which can be irritating to the skin [9].Leaves are lanceolate to oblong, having an acute base and acute to acuminate apex, while the large fragrant flowers can range from pink-red to white-yellow in colour [7].
The typical form of the plant cultivated in Palermo is characterized by a white corolla, yellow in the centre, intensely scented, with reflected round petals; it is generically named 'Tonda Palermitana', 'Classica Palermitana' or 'Acutifolia'.
According to the literature, about 110 chemical compounds have been found in P. rubra.These include iridoids, terpenoids, flavonoids, alkaloids, coumarins, etc., whose presence and quantity can vary in different portions of the plants as well as in different types of extracts.Therefore, several pharmacological activities of P. rubra have been reported, such as antibacterial and anti-inflammatory, anxiolytic, antidiabetic, hypolipidemic and many other properties.Moreover, no mortality or signs of toxicity have been recorded for methanolic or ethanolic extracts of the leaves, pods and flowers in in vivo tests for the doses used, suggesting a high margin of safety [7].
Leaves are used in folk medicine to treat inflammations, ulcers, rheumatism, bronchitis, cold and other diseases [10].Leaf infusions or extracts have been reported for use against asthma [11], while their decoction is employed in South America as purgative/anthelmintic or for the treatment of various skin problems [12].Flower decoction is instead used in Mexico for the treatment of diabetes mellitus [13,14].Used in particular doses, flowers can be useful for birth control [15].Flowers eaten with betel leaves (Piper betle L.) are used as a febrifuge [16] while, together with other herbs, are part of the recipe of the 'Five Flower Tea', a popular cooling beverage considered as a remedy for 'Hot Qi' [17].Latex is used as a good rubefacient in rheumatism [18] or topically applied on blisters and sores [19].In addition, the petals of many Plumeria species and varieties are used as edible flowers in different dishes, as well as for cosmetic purposes [20].
However, no studies on the biological properties of extracts from the old Sicilian cultivar of P. rubra are to date available.Therefore, the aim of our study was to investigate, by a multidisciplinary approach, the macro-and micro-morphology of both the leaves and flowers of P. rubra 'Tonda Palermitana' and to evaluate the phytochemical features and biological properties of their hydroalcoholic extracts.

Macro-, Micro-Morphological and Anatomical Studies
The main characteristic features of the plant in bloom are shown in Figure 1A-C.The leaves, up to 30 cm in length, are elliptic with an entire margin and a pointed tip.The leaf blade is bright green and leathery, with parallel secondary veins running from the midrib to the margin (Figure 1B).P. rubra flowers are arranged in terminal or lateral cymes.Each inflorescence consists of many white flowers with a yellow throat.The fragrant flowers show five petals fused at the base in a short funnel-shaped tube (Figure 1C).

Macro-, Micro-Morphological and Anatomical Studies
The main characteristic features of the plant in bloom are shown in Figure 1A-C.The leaves, up to 30 cm in length, are elliptic with an entire margin and a pointed tip.The leaf blade is bright green and leathery, with parallel secondary veins running from the midrib to the margin (Figure 1B).P. rubra flowers are arranged in terminal or lateral cymes.Each inflorescence consists of many white flowers with a yellow throat.The fragrant flowers show five petals fused at the base in a short funnel-shaped tube (Figure 1C).The leaves are amphistomatic and show sunken paracytic (or rubiaceous) stomata (Figure 2A white arrow and Figure 2B,C) and prominent solitary stomata slightly longer than the paracytic ones, present only on the ribs (Figure 2A, red arrow).On both leaf surfaces, the cuticle is densely striated, and waxes are arranged in small spheres scattered over the epidermis, stomata and trichomes (Figure 2C, white arrows).The leaves are glabrous except for the ribs on the lower surfaces, where uniseriate, pluricellular non-glandular trichomes (NGTs) with an acute apex are located (Figure 2D).The right frame of Figure 2D shows a magnified view of the NGTs where spherical waxes are randomly distributed.The leaves are amphistomatic and show sunken paracytic (or rubiaceous) stomata (Figure 2A white arrow and Figure 2B,C) and prominent solitary stomata slightly longer than the paracytic ones, present only on the ribs (Figure 2A, red arrow).On both leaf surfaces, the cuticle is densely striated, and waxes are arranged in small spheres scattered over the epidermis, stomata and trichomes (Figure 2C, white arrows).The leaves are glabrous except for the ribs on the lower surfaces, where uniseriate, pluricellular non-glandular trichomes (NGTs) with an acute apex are located (Figure 2D).The right frame of Figure 2D shows a magnified view of the NGTs where spherical waxes are randomly distributed.The whole outer petal epidermis presents prominent and conical papillae (Figure 3A).The floral funnel-shaped tube is densely pubescent, showing numerous NGTs on both the inner and outer side of its basal zone (Figure 3B).However, the abundance of these trichomes decreases towards the apex of the petal (Figure 3C).Different types of unicellular NGTs are observed: (1) long, with a smooth or slightly wavy surface and a pointed or rounded apex (Figure 3D); (2) long, with an irregular surface due to the presence of small protuberances (Figure 3E) or of protruding knobs (Figure 3F).The protuberances and the knobs are clearly visible in the frame of the respective figures (Figure 3E,F).The whole outer petal epidermis presents prominent and conical papillae (Figure 3A).The floral funnel-shaped tube is densely pubescent, showing numerous NGTs on both the inner and outer side of its basal zone (Figure 3B).However, the abundance of these trichomes decreases towards the apex of the petal (Figure 3C).Different types of unicellular NGTs are observed: (1) long, with a smooth or slightly wavy surface and a pointed or rounded apex (Figure 3D); (2) long, with an irregular surface due to the presence of small protuberances (Figure 3E) or of protruding knobs (Figure 3F).The protuberances and the knobs are clearly visible in the frame of the respective figures (Figure 3E,F).In the transversal section, the leaves show a dorso-ventral mesophyll (Figure 4A).A bi-collateral, arc-shaped vascular bundle accompanied by two smaller accessory vascular bundles at each side is visible in the midrib (Figure 4A, yellow arrows).Additionally, in all examined organs, numerous branched laticifers are present.They are observed in the leaves (Figure 4B,C, black arrows) and petals (Figure 4D) and in the transversal section of the petioles, where they are detected by both light microscopy (Figure 4E) and Scanning Electron Microscopy (Figure 4F, white arrows).In this latter case, they appear as small portions of laticiferous tubes.Both in the leaf and in the petiole, the laticifers are irregularly dispersed in the parenchyma, occurring close to the phloem on either side of the In the transversal section, the leaves show a dorso-ventral mesophyll (Figure 4A).A bi-collateral, arc-shaped vascular bundle accompanied by two smaller accessory vascular bundles at each side is visible in the midrib (Figure 4A, yellow arrows).Additionally, in all examined organs, numerous branched laticifers are present.They are observed in the leaves (Figure 4B,C, black arrows) and petals (Figure 4D) and in the transversal section of the petioles, where they are detected by both light microscopy (Figure 4E) and Scanning Electron Microscopy (Figure 4F, white arrows).In this latter case, they appear as small portions of laticiferous tubes.Both in the leaf and in the petiole, the laticifers are Plants 2024, 13, 2479 6 of 21 irregularly dispersed in the parenchyma, occurring close to the phloem on either side of the vascular cylinder (Figure 4C, black arrows).In the transversal section, they appear polygonal or circular (Figure 4C, black arrows) and enclosed in a rosette of parenchymatic cells (Figure 4E).The latex reacted positively with the Dragendorff reagent (Figure 4B,E), indicating the presence of alkaloids, and with Sudan III staining (Figure 4D), showing the presence of lipophilic compounds.
Plants 2024, 13, 2479 6 of 22 vascular cylinder (Figure 4C, black arrows).In the transversal section, they appear polygonal or circular (Figure 4C, black arrows) and enclosed in a rosette of parenchymatic cells (Figure 4E).The latex reacted positively with the Dragendorff reagent (Figure 4B,E), indicating the presence of alkaloids, and with Sudan III staining (Figure 4D), showing the presence of lipophilic compounds.

Phytochemical Analyses on Food-Grade Extracts
After a careful macro and micro-morphological analysis, food grade extracts (FE and LE, respectively) were prepared using an ethanol/water mixture (80:20, v/v) obtaining a good and reproducible extraction yield (7.59% ± 0.27 and 8.69% ± 0.43 for FE and LE, respectively).
The phytochemical profile was first investigated by several colorimetric assays aimed at quantifying the main classes of secondary metabolites such as total phenols, total flavonoids, flavan-3-ols, as well as other flavonoids that have a free meta-oriented hydroxy group on the B ring (vanillin index), and proanthocyanidins.This also allowed us to establish the polymerization degree (vanillin index/proanthocyanidins), which if it is higher than 1 shows a greater abundance of monomer molecules.These results are shown in Table 1.LE was the richest extract not only in terms of total phenols but also in terms of flavonoids, with statistically significant differences (p < 0.05) with respect to FE.On the contrary, no statistically significant difference was detected in terms of proanthocyanidins content.Both extracts show a predominance of monomeric molecules according to the high polymerization index detected, with LE showing the best value according to the vanillin index results (Table 1).This preliminary screening was followed by an in-depth phytochemical characterization by LC-DAD-ESI-MS analysis (Table 2).Compounds were detected and tentatively identified by comparison of mass and UV-Vis spectra with literature data and online free consulting spectra databases, as well as with commercially available reference standards (see Table 2 and relative footnote for details).
These results confirm what was previously detected by Dragendorff reagent and Sudan III staining in micro-morphological analyses, which is the presence of alkaloids and lipophilic compounds (see Section 2.1) such as terpenoids, fatty acids and sterols (Table 2).This is also consistent with the results of the preliminary phytochemical screening, which also highlighted the abundant presence of flavonoids and phenolic acids (Table 1).
Expressing the phytochemical data of FE and LE in terms of percentage distribution of the various classes of detected compounds, it is immediately evident that the two extracts show substantial differences.Indeed, as depicted in Figure 5, apart from the iridoid class, which remains almost unchanged, in percentage terms, in the two extracts under examination (25% and 29% in FE and LE, respectively), FE showed the highest content of flavonoids (23% vs. 18% in LE), alkaloids (14% vs. 6% in LE) and fatty acids (9% vs. 3% in LE).Furthermore, FE contains the coumarin scopoletin, which it did not find in LE.On the contrary, LE showed the highest content of terpenoids (20% vs. 9% in FE), phenolic acids (18% vs. 14% in FE) and sterols (6% vs. 3% in FE).
The agglomerative hierarchical clustering analysis allowed us to delve deeper into the phytochemical differences found between the two extracts under examination (Figure 6).Since a qualitative analysis was carried out in the present study, a 0 score was assigned for the not found metabolites and a 1 score for those found in the extracts under examination.The analysis returns a heatmap that identifies the presence of the metabolite with the red colour and its absence with the blue colour.This allows for a quick and easy comparison of the LE and FE phytochemical profiles.The agglomerative hierarchical clustering analysis allowed us to delve deeper into the phytochemical differences found between the two extracts under examination (Figure 6).Since a qualitative analysis was carried out in the present study, a 0 score was assigned As shown in Figure 6, three clusters were found, of which two (the first and the last one from the top to the bottom) identify the secondary metabolites present in LE and absent in FE, and the third (in the middle) identifies the 15 metabolites common to LE and FE.Specifically, these are the iridoids plumieride p-Z-coumarate, plumieride-p-E-coumarate, plumieridin A and plumieridin B; the flavonoids myricetin 3-O-glucoside, isorhamnetin-3-O-rutinoside (narcissin) and plumerubroside; the phenolic acids 4-O-(3′-O-α-Dglucopyranosyl)-caffeoyl quinic acid and stearyl dixylosyl methoxygallic acid; the terpenoids rubrinol and arjunolic acid; the alkaloids plumericidine/plumieranine and plumerinine; the fatty acid methyl stearate and the sterol β-Sitosterol 3-O-glucoside.
The results, which were expressed as half-inhibitory concentration (IC 50 ) with the respective confidence limits at 95%, are shown in Table 3.According to the quantitative phytochemical results (Table 1), LE showed the lowest half-inhibitory concentration (IC 50 ) values and therefore the strongest antioxidant and anti-inflammatory activity in all the assays carried out, although statistically significant results (p < 0.05 vs. FE) were observed only in the DPPH, ICA and ADA tests (Table 3).
Calculating the average of the IC 50 values, the most notable biological property of both extracts appears to be the antioxidant one, showing IC 50 values about 5 times lower than those detected in anti-inflammatory assays (383.74 ± 5.65 and 232.05 ± 2.87 vs. 1981.23± 12.82 and 1215.13 ± 10.15, for FE and LE, respectively), with LE showing the best, and statistically significant (p < 0.001), biological activity.
Finally, by plotting the FE and LE concentrations with respect to the obtained inhibition percentages (yellow and green bars, respectively), interesting concentration-dependent antioxidant and anti-inflammatory behaviour was detected for both extracts, as shown in Figures 7 and 8, respectively.

Discussion
This study focused on P. rubra 'Tonda Palermitana', a plant that in Italy is exclusive to Sicily, where it is renowned as an ornamental species or for its symbolic/ritual use.However, in different tropical areas of the world where the plant is spontaneous or has become naturalized, many other uses are known, e.g., medicinal, cosmetic and even as food [7,20].In folk medicine, the leaves of P. rubra are used to treat inflammation [10] and

Discussion
This study focused on P. rubra 'Tonda Palermitana', a plant that in Italy is exclusive to Sicily, where it is renowned as an ornamental species or for its symbolic/ritual use.However, in different tropical areas of the world where the plant is spontaneous or has become naturalized, many other uses are known, e.g., medicinal, cosmetic and even as food [7,20].In folk medicine, the leaves of P. rubra are used to treat inflammation [10] and various skin problems [12].Latex is topically applied on blisters and sores [19].In addition, flowers of Plumeria spp.are also used for cosmetic purposes [20].
Among the different pharmacological activities reported in the literature, extracts of P. rubra are noted for their antibacterial and anti-inflammatory activities [7].
Considering this, our investigation was conducted on leaf and flower extracts of plants growing in Sicily to verify their biological properties and their possible uses in medicinal and cosmetic applications.To characterize the plant material, a pharmacognostic study was carried out.Indeed, although different anatomical and phytochemical studies have been performed on many Apocynaceae species [21][22][23][24], limited data are available today on P. rubra leaf and flower extracts.
Our observations confirmed the presence of paracytic stomata, as already recorded in Plumeria species by Metcalfe and Chalk [25] and Toma et al. [26].Solitary stomata, that have been previously observed in other genus belonging to the Apocynaceae family such as Vinca minor e V. major [27], were found.
According to Toma et al. [26], the leaves of Plumeria sp. are hypostomatic; on the contrary, in the present study, amphistomatic leaves similar to those found in another Apocynaceae, i.e., Mandevilla coccinea [21], were observed.Furthermore, in P. rubra 'Tonda Palermitana', both leaf surfaces showed striate cuticles, unlike P. rubra var.alba which lacks them [28].Pluricellular non-glandular trichomes with an acute apex were present only in the rib's closeness on the leaf lower surface.Instead, Araujo et al. [28], also studying the leaves of P. rubra var.alba, found trichomes near the veins, but they were unicellular and located on both epidermises.The presence of NGTs on the petal surface of P. rubra agrees with the data collected in other Apocynaceae by Basir et al. [29] in three Hoya species, and by Ramos et al. [30] in Adenium obesum.Trichomes covered with knobs or small protuberances along their entire length can be called 'bosselated' according to Payne [31].The prominent knobs observed in bosselated trichomes of the P. rubra flower appeared similar to those described in the multicellular stalks of the capitate glandular trichomes on the inner surface of Lavandula spp.petals [32,33].
The P. rubra leaves showed a dorso-ventral mesophyll and a bi-collateral vascular bundle, which are common features in members of the Apocynaceae family [25].In particular, a bi-collateral vascular bundle arranged in an open arc was present both in the leaf and petiole.In the leaf, two other smaller accessory vascular bundles were visible on both sides of the midrib.On the contrary, Carvalho et al. [22], in other Apocynaceae genera such as Araujia and Morrenia, and Toma et al. [26] in Plumeria sp., found them only in the petiole.
Leaves, petioles and petals were rich in branched laticifers with a polygonal or round shape in the transversal section, as already evidenced by Murugan and Inamdar [34] and Toma et al. [26] in the Plumeria genus.Moreover, laticifers of P. rubra 'Tonda Palermitana' were surrounded by a ring of parenchymatic cells, as described by Araujo et al. [28] in P. rubra var.alba.
Foliar and petiolar laticifers showed a distribution similar to that previously reported also by Murugan and Inamdar [31] in P. alba.The latex, commonly present in the Apocynaceae members, is rich in several chemical compounds [21,35], as confirmed by the histochemical tests performed, which showed a positive reaction for both alkaloids and lipophilic compounds.
These results were confirmed, in the present study, by LC-DAD-ESI-MS analysis of flower and leaf food-grade extracts.According to previous data, several compounds belonging mainly to the classes of iridoids, terpenoids, flavonoids, phenolics, alkaloids, fatty acids and coumarins were detected [7].Although soil and climate factors, as well as harvesting and storage times can determine variability from the phytochemical point of view, P. rubra 'Tonda Palermitana' flower and leaf extracts contain several previously identified phytochemicals with well-established biological properties [7].
Several studies have shown that different extracts of Plumeria sp.exert anti-inflammatory, antioxidant and free radical scavenging properties.These are mostly antioxidant in vitro studies carried out on leaf and flower methanolic extracts and anti-inflammatory in vivo studies conducted on murine models by administering leaf, flower and pod methanolic or ethanolic extracts [46,54,55].In the first case, a direct comparison with the results obtained with food-grade extracts is possible, since many of the tests are the same as those used in the present study.For example, P. rubra methanolic leaf extract had already demonstrated interesting anti-peroxidase activity on a linoleic acid emulsion, which appears to be about one-third (IC 50 75 µg/mL) of that found in the present study (IC 50 22.05 µg/mL).According to our results, concentration-dependent behaviour was already highlighted in radical scavenging assays such as DPPH [54,55] with P. acuminata leaf extract.Furthermore, similar antioxidant behaviour to that observed in the present study was observed for P. rubra methanolic flower extract, which showed greater activity in the FRAP test, followed by DPPH and ICA [46].
The methanolic leaf extract of P. rubra at a dose of 500 mg/kg b.w.exhibited maximum anti-inflammatory effects, significantly reducing the formation of granuloma tissue induced by the cotton pellet method in Wistar albino rats and Swiss albino mice models [56].Furthermore, at doses of 100 and 200 mg/kg b.w., it exhibited significant dose-dependent activity, suppressing carrageenan-induced paw edema in rats [57].
Bihani's review [7] summarizes the traditional uses of P. rubra, many of which can be at least partly ascribed to the strong antioxidant and anti-inflammatory activity of its extracts, which were confirmed by the results of the present work carried out on flower and leaf hydroalcoholic extracts.However, beyond the interesting antioxidant and anti-inflammatory activity found in in vitro and in vivo models, it is important to comment that toxicity studies have demonstrated the safety of these extracts.An acute toxicity assay carried out administering a single dose at six different concentrations (500-2000 mg/kg b.w.) of P. rubra methanolic leaf extract to six groups of Wistar albino rats and Swiss albino mice showed no deaths and no stereotypical symptoms over 72 h, with a calculated median lethal dose (LD 50 ) greater than 2000 mg/kg b.w.[56].A similar trend was observed also with P. rubra methanolic red flower extract on male Sprague Dawley rats, which showed no toxic signs, behavioural changes or mortality up to dose of 4000 mg/kg b.w.[46].

Plant Material
About 500 g of both leaves and flowers of P. rubra 'Tonda Palermitana' were collected from an old tree located in Mondello (PA, Italy) (38 • 11 ′ 53 ′′ N, 13 • 19 ′ 44 ′′ E, 27 m a.s.l., exposure South, flowerbed) during July 2022.The voucher specimen (100083) was stored in the SAF herbarium at the Department of Agricultural and Forest Science (University of Palermo).Fresh plant material was immediately sent to the University of Genova and to the University of Messina to be processed and analyzed.

Microscopical Analyses
The micro-morphological analysis of the leaf, petiole and flower was carried out on both fresh samples and samples fixed with the alcoholic-based fixative FineFIX (Milestone SRL, Sorisole, Bergamo, Italy) for 24 h at 4 • C. The leaf and petiole were hand-sectioned using a double-edged razor blade, while the petal surface was peeled off gently with fine tweezers.Sections and epidermal peels were then treated with Phloroglucinol-HCl (Merck, Darmstadt, Germany) to stain lignin, Sudan III (Merck, Darmstadt, Germany) or Dragendorff reagent (Merck, Darmstadt, Germany) to detect the presence of laticifers.All these samples were then mounted in water and observed by a Leica DM 2000 transmission light microscope (Leica Microsystems, Wetzlar, Germany) coupled with a ToupCam Digital Camera with a CMOS Sensor, 3.1 MP resolution (ToupTek Photonics, Hangzhou, China).In addition, some sections of leaf and petiole were cleared with an aqueous solution of chloral hydrate and mounted in a chloral hydrate-glycerol solution to prevent crystallization of the reagent during observations, according to Jackson and Snowdon [58].
Small pieces of fixed leaf, petiole and flower were also dehydrated through a graded ethanol series (70, 80, 90 and 100%) and critical point dried in CO 2 (CPD, K850 2M Strumenti s.r.l., Rome, Italy).Two-sided adhesive carbon tapes were fixed on aluminium stubs, and then a small amount of the dried specimens was placed on this conductive substrate.The samples were finally covered with a 10 nm layer of gold particles and examined under a Scanning Electron Microscope VEGA3-Tescan-type LMU (Tescan USA Inc., Cranberry Twp, PA, USA), operating at an accelerating voltage of 20 kV.

Extraction Procedure
Fresh plant material was powdered by a blade mill (IKA ® A11, IKA ® -Werke GmbH & Co. KG, Staufen, Germany) with liquid nitrogen to inhibit the enzymatic activity and preserve the native phytochemical profile.Food-grade flower and leaf extracts (FEs and LEs, respectively) were obtained according to Cornara et al. [59] by adding 100 mL of ethanol/water mixture (80:20, v/v) to ten grams of powdered plant material and sonicating in an ice bath for 10 min using a titanium probe set to 200 W and 30% amplitude (Vibra Cell™ Sonics Materials, Inc., Danbury, CT, USA).After this, samples were left to macerate under stirring in the dark at RT for 2 h.Supernatants were recovered by filtration on Whatman paper filter n. 1 within round bottom flasks and the extraction was repeated two more times.Collected supernatants were dried by a rotary evaporator (Büchi R-205, Cornaredo, Italy) in the dark at 37 • C and stored overnight in a vacuum glass desiccator with anhydrous sodium sulphate.After extraction yield calculation (77.59% ± 0.27 and 8.69% ± 0.43 for FE and LE, respectively), dry extracts were stored in burnished sealed vials with nitrogen headspace until analysis, when they were resuspended and properly diluted in the same hydroalcoholic mixture reported above.Total phenolics were quantified according to Ingegneri et al. [60].Briefly, 10 µL of FE and LE (0.625-5.0 mg/mL) were added to 90 µL of deionized water and mixed with 100 µL of Folin-Ciocalteu reagent.After 3 min, 100 µL 10% anhydrous sodium carbonate was added and samples were incubated in the dark at RT for 60 min, shaking every 10 min.Absorbance was read at 785 nm by using a Multiskan™ GO Microplate Spectrophotometer (Thermo Scientific, Waltham, MA, USA) against the hydroalcoholic mixture as a blank.Gallic acid was used as a reference compound (0.075-0.6 mg/mL; y = 0.0016x, R 2 = 0.999), and the results were expressed as g gallic acid equivalents (GAEs)/100 g dry extract (DE).

Total Flavonoid Compounds (TFCs)
Total flavonoids were quantified according to Lenucci et al. [61].Briefly, 50 µL of FE and LE (2.5-20 mg/mL) was added to 450 µL of deionized water and 30 µL of 5% NaNO 2 .After 5 min, 60 µL of 10% AlCl 3 was added and samples were incubated for 6 min at RT.Then, 200 microliters of 1 M NaOH and 210 µL of deionized water were added, and the samples were vortex mixed.The absorbance was recorded at 510 nm by a UV-Vis spectrophotometer UV-1601 (Shimadzu, Kyoto, Japan) against the hydroalcoholic mixture as a blank.Rutin was used as a reference standard (0.125-1.0 mg/mL; y = 0.0007x, R 2 = 0.999), and the results were expressed as g of rutin equivalents (RE)/100 g DE.

Vanillin Index
Flavan-3-ols as well as other flavonoids that have a free meta-oriented hydroxy group on the B ring were quantified by vanillin index assay [62].FE and LE were diluted in 0.5 M H 2 SO 4 (absorbance ranging from 0.2 to 0.4) and loaded onto a conditioned Sep-Pak C18 cartridge (Waters, Milan, Italy), which was then washed with 2.0 mL of 5.0 mM sulphuric acid.Samples were eluted with 5.0 mL of methanol and 1 mL of each eluate was added to 6.0 mL of 4% vanillin methanol solution and incubated at 20 • C for 10 min.Once the HCl (3 mL) was added, samples were incubated for 15 min at RT and the absorbance was recorded at 500 nm using the same instrument and blank reported in Section 4.4.2.Catechin was used as a reference standard (0.125-0.50 mg/mL; y = 0.0027x, R 2 = 0.998).The results were expressed as g of catechin equivalents (CE)/100 g DE.

Proanthocyanidins
Proanthocyanidins were quantified by hot acid hydrolysis [62] diluting FE and LE in 0.05 M H 2 SO 4 (2 mL).The solutions were loaded onto conditioned Sep-Pak C18 cartridges (Waters, Milan, Italy).The proanthocyanidin-rich fractions obtained were eluted with methanol (3 mL) and collected in 100 mL round-bottom flasks shielded from light and containing 9.5 mL of absolute ethanol.After this, 12.5 mL of 300 mg/L FeSO 4 •7H 2 O hydrochloric acid solution was added and samples were left to reflux for 50 min.After cooling, the absorbance was recorded at 550 nm using the same instrument and blank reported in Section 4.4.2.The starting anthocyanins content of samples was estimated by recording the absorbance of samples prepared under the same conditions but cooled in ice instead of warmed.Cyanidin chloride (Cy) was used as reference standard (ε = 34,700) and results were expressed as g of Cy equivalents (CyE)/100 g DE.

In Vitro Antioxidant and Anti-Inflammatory Assays
The antioxidant and anti-inflammatory activity of FE and LE were evaluated by several in vitro spectrophotometric and spectrofluorimetric assays based on different mechanisms and reaction environments.The results were expressed as inhibition (%) of the oxidative/inflammatory activity by calculating the half-inhibitory concentration (IC 50 ) and the respective confident limits (C.L.s) at 95% by Litchfield and Wilcoxon's test (PHARM/PCS 4, MCS Consulting, Wynnewood, PA, USA).The following reported concentration ranges refer to final concentrations in the reaction mixture.

BCB
The BCB assay was carried out according to Cornara et al. [59] with some modifications.Briefly, 80 µL of FE and LE (6.25-50.0µg/mL) were added to β-carotene emulsion consisting of 250 µL β-carotene chloroform solution (1 mg/mL), 1.6 µL linoleic acid, 16 µL Tween-40 and 2 mL of air-insufflated deionized water.A β-carotene free emulsion was used as a negative control, whereas a β-carotene emulsion with hydroalcoholic mixture was used as a blank.Samples were incubated for 120 min at 50 • C in a shaking water bath, monitoring the absorbance decay every 20 min at 470 nm using the same instrument reported in Section 4.4.1.Butylhydroxytoluene (BHT) was used as reference standard (0.06-0.5 µg/mL; y = 0.8376x, R 2 = 0.999).

ICA
The iron-chelating activity was evaluated according to Cornara et al. [59] with some modifications.Briefly, 25 µL of 2.0 mM iron (II) chloride tetrahydrate was added to 50 µL of FE and LE (0.150-1.2 mg/mL and 0.075-0.6mg/mL, respectively) and incubated at RT for 5 min.Then, 50 µL of 5 mM ferrozine was added and the reaction mixture was diluted to 1.5 mL with deionized water, vortex mixed, and incubated for 10 min at RT.The absorbance was read at 562 nm using the same instrument and blank reported in Section 4.4.2.EDTA was used as a reference standard (1.5-12.0µg/mL; y = −0.0009x+ 0.4161, R 2 = 0.998).4.5.7.ADA FE and LE (0.500-4.0 mg/mL) were added to 0.4% fatty-acid-free BSA solution and PBS pH 5.3 (4:5:1 v/v/v, respectively) [63].Once the starting absorbance was recorded at 595 nm, samples were incubated for 30 min at 70 • C in a shaking water bath, recording the final absorbance at the same wavelength using the same instrument and blank reported in Section 4.4.1.Diclofenac sodium was used as a reference standard (3.0-24.0µg/mL; y = −0.012x+ 1.049, R 2 = 0.999).

Statistical Analysis
Data were expressed as IC 50 with respective 95% C.L. (see Section 4.5) or as mean ± S.D. of three independent experiments in triplicate (n = 3).The statistical significance was evaluated using one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls method.Values of p < 0.05 were considered statistically significant.Data analysis was performed with SigmaPlot 12.0 software (Systat Software Inc., San Jose, CA, USA).Chemometric analysis, such as dendrogram and hierarchical clustering analysis (HCA), was carried out by JMP7 (SAS Institute Inc., Cary, NC, USA).To compare the differences between FE and LE, the Euclidean distance was taken, and a hierarchical clustering analysis was carried out using Ward's variance-minimizing method.

Conclusions
This is the first study which investigated the old Sicilian cultivar P. rubra 'Tonda Pa-lermitana'.The multidisciplinary approach used allowed us to conduct a complete pharmacognostic evaluation of this plant species, from micro-morphological to phytochemical and finally biological characterization.Some peculiar micro-morphological features of this ancient cultivar that have not been found before in other species belonging to the Plumeria genus were documented, as well the presence of some phytochemicals that have been identified for the first time in this plant species.Finally, the preparation of flower and leaf food-grade extracts has allowed us to evaluate the antioxidant and anti-inflammatory activities.This proves, for the first time, that the most interesting part of the plant from the biological point of view is the leaves.The overall data collected allow us to speculate that, beyond the scent of its splendid flowers, whose essential oil is widely used today in the cosmetic and pharmaceutical industries, P. rubra 'Tonda Palermitana' flower and leaf food-grade extracts represent very interesting plant complexes that could certainly find industrial applications.The anti-inflammatory properties and especially the high antioxidant power found for this species are promising for the treatment of free radical damage, which has a central role in the onset and development of ageing and related diseases.Therefore, considering the great interest of the industry in using extracts rich in natural antioxidants, our data allow us to assume future applications of P. rubra hydroalcoholic extracts for the formulation of nutraceutical, cosmetic or dermatological products with anti-ageing effects.

Figure 2 .
Figure 2. Leaf anatomy under Scanning Electron Microscopy: (A) adaxial surface, evidencing the paracytic stomata (white arrow) and a solitary stomata on the rib (red arrow); (B) stomata on the abaxial surface; (C) magnification of the adaxial surface showing striated cuticle and waxes forming small spheres (white arrows) scattered on both the epidermal cells and stomata; (D) NGTs on the ribs with a detailed view of the waxes present on their surfaces (in the right frame).

Figure 2 .
Figure 2. Leaf anatomy under Scanning Electron Microscopy: (A) adaxial surface, evidencing the paracytic stomata (white arrow) and a solitary stomata on the rib (red arrow); (B) stomata on the abaxial surface; (C) magnification of the adaxial surface showing striated cuticle and waxes forming small spheres (white arrows) scattered on both the epidermal cells and stomata; (D) NGTs on the ribs with a detailed view of the waxes present on their surfaces (in the right frame).

Figure 3 .
Figure 3. Petal anatomy under Scanning Electron Microscopy: (A) conical papillate cells on the outer epidermis; (B) basal portion of the floral funnel-shaped tube with numerous NGTs on both surfaces; (C) apical portion of the petal showing few NGTs on its inner surface; (D) NGTs on the outer surface with a smooth or slightly wavy surface and a pointed or rounded apex; (E) NGTs with small protuberances on their surface; (F) NGTs with protruding knobs.(E,F): in the left frames are visible details of the two NGTs surface.

Figure 3 .
Figure 3. Petal anatomy under Scanning Electron Microscopy: (A) conical papillate cells on the outer epidermis; (B) basal portion of the floral funnel-shaped tube with numerous NGTs on both surfaces; (C) apical portion of the petal showing few NGTs on its inner surface; (D) NGTs on the outer surface with a smooth or slightly wavy surface and a pointed or rounded apex; (E) NGTs with small protuberances on their surface; (F) NGTs with protruding knobs.(E,F): in the left frames are visible details of the two NGTs surface.

Figure 5 .
Figure 5. Distribution percentage of phytochemical classes identified in P. rubra 'Tonda Palermitana' flower and leaf extracts (FE and LE, respectively).

Figure 5 .
Figure 5. Distribution percentage of phytochemical classes identified in P. rubra 'Tonda Palermitana' flower and leaf extracts (FE and LE, respectively).As shown in Figure 6, three clusters were found, of which two (the first and the last one from the top to the bottom) identify the secondary metabolites present in LE and absent in FE, and the third (in the middle) identifies the 15 metabolites common to LE and FE.Specifically, these are the iridoids plumieride p-Z-coumarate, plumieride-p-E-coumarate, plumieridin A and plumieridin B; the flavonoids myricetin 3-O-glucoside, isorhamnetin-3-O-rutinoside (narcissin) and plumerubroside; the phenolic acids 4-O-(3 ′ -O-α-D-glucopyranosyl)-caffeoyl quinic acid and stearyl dixylosyl methoxygallic acid; the

Figure 6 .Figure 6 .
Figure 6.Agglomerative hierarchical clustering analysis of the phytochemical data of P. rubra 'Tonda Palermitana' flower and leaf extracts (FE and LE, respectively) obtained by LC-DAD-ESI-MS analysis.The heatmap shows the expression pattern of the identified metabolites, indicating in

Figure 8 .
Figure 8. Anti-inflammatory concentration-dependent behaviour of P. rubra 'Tonda Palermitana' flower (yellow bars) and leaf (green bars) extracts (FE and LE, respectively) evaluated by PIA (panel (A)) and ADA (panel (B)) assays.The results, expressed as inhibition percentage (%), show the mean ± standard deviation of three independent experiments in triplicate (n = 3).* p < 0.05 vs. FE by one-way ANOVA followed by Student-Newman-Keuls Method.

Table 2 .
Phytochemical profile of P. rubra 'Tonda Palermitana' flower and leaf extracts (FE and LE, respectively) tentatively identified by LC-DAD-ESI-MS using both the positive and negative ionization modes.

Table 3 .
Antioxidant and anti-inflammatory activity of P. rubra 'Tonda Palermitana' flower and leaf extracts (FE and LE, respectively) in comparison with the reference standards.The results, which represent the mean of three independent experiments in triplicate (n = 3), are expressed as the concentration inhibiting 50% of the oxidant/inflammatory activity (IC 50 ) with 95% confidence limits (between brackets).
• C drying gas temperature, 9 L/min drying gas flow and 40 V skimmer voltage.Acquisition was carried out in fullscan mode (90-2000 m/z).Data were acquired by Agilent ChemStation software version B.01.03 and Agilent trap control software version 6.2.Identification was carried out by comparing the retention times, UV-Vis and MS spectra of each analyte with those of commercially available standards (see Table 2), literature data and UV-Vis and open-source mass spectra databases.