Acylated Flavonoid Glycosides are the Main Pigments that Determine the Flower Colour of the Brazilian Native Tree Tibouchina pulchra (Cham.) Cogn.

Tibouchina pulchra (Cham.) Cogn. is a plant native to Brazil whose genus and family (Melastomataceae) are poorly studied with regards to its metabolite profile. Phenolic pigments of pink flowers were studied by ultra-performance liquid chromatography with a photodiode array detector and electrospray ionization quadrupole time-of-flight mass spectrometry. Therein, twenty-three flavonoids were identified with eight flavonols isolated by preparative high-performance liquid chromatography and analysed by one- and two-dimensional nuclear magnetic resonance. Kaempferol derivatives were the main flavonols, encompassing almost half of the detected compounds with different substitution patterns, such as glucoside, pentosides, galloyl-glucoside, p-coumaroyl-glucoside, and glucuronide. Concerning the anthocyanins, petunidin p-coumaroyl-hexoside acetylpentoside and malvidin p-coumaroyl-hexoside acetylpentoside were identified and agreed with previous reports on acylated anthocyanins from Melastomataceae. A new kaempferol glucoside was identified as kaempferol-(2′′-O-methyl)-4′-O-α-d-glucopyranoside. Moreover, twelve compounds were described for the first time in the genus with five being new to the family, contributing to the chemical characterisation of these taxa.


Introduction
Tibouchina Aubl., the most representative genus within Melastomataceae, has approximately 460 species [1][2][3]. Melastomataceae can be recognised among eudicots by the characteristic leaf acrodromous venation pattern [4]. The family is the fifth largest group among Angiosperms in Brazil [5], comprising 4500 species and approximately 170 genera. In spite of pantropical distribution, the greatest diversity of species is found in the Neotropics (ca. 3000 species), with 929 species native to Brazil [6]. Out of the 166 Tibouchina species reported in Brazil, 105 are endemic [6], occurring mainly in the Atlantic Rainforest and in the Cerrado (Brazilian savannahs); both biomes are recognised as biodiversity hotspots [7]. This native vegetation is constantly under illegal deforestation and agribusiness expansion, generating a need for programmes for biodiversity conservation and, consciously, resources exploitation.
Tibouchina species occur in open areas, such as forest edges and clearings, and are considered important for restoration/reforestation purposes [8]. Moreover, Tibouchina granulosa (Desr.) Cogn. and Tibouchina pulchra (Cham.) Cogn. have been characterised as possible biomonitors of air pollution, such as particulate matter and ozone [9][10][11][12][13][14][15][16]. Despite the ecological importance of this genus, the main use of Tibouchina is urban ornamentation and nowadays, several cultivars are available in the flower market. A large contributor to the beauty and fascinating feature of T. pulchra is the colour change of the flowers from white to intense pink during development [17].
Due to the importance of Tibouchina species for ornamentation and ecological purposes, the present work aimed to assess the qualitative profile of acidified alcoholic extract from T. pulchra flowers. An ultra-performance liquid chromatography with photodiode array detector and electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-PAD-ESI-QTOF-MS) method was established, and thirty-two compounds were detected with twenty-three identified, many of them reported for the first time in the species, genus, and family, as well as a new flavonol: kaempferol-(2 -O-methyl)-4 -O-α-D-glucopyranoside.  [30] T. grandiflora Cogn. anthocyanin/flavonol peonidin 3-sophoroside, peonidin 3-sambubioside, malvidin 3,5-diglucoside,

Chemical Screening of Tibouchina pulchra Flowers
To explore the pigment profiling of T. pulchra petals, the first step was to analyse acidic alcoholic extracts from white and pink flowers by UPLC-PAD-ESI-QTOF-MS. The exclusive difference between the two floral stages was the presence of anthocyanins in pink flower extracts ( Figure 1). In order to perform a complete characterisation of floral pigments, the pink floral stage was chosen for isolation and identification of constituents.

Chemical Screening of Tibouchina pulchra Flowers
To explore the pigment profiling of T. pulchra petals, the first step was to analyse acidic alcoholic extracts from white and pink flowers by UPLC-PAD-ESI-QTOF-MS. The exclusive difference between the two floral stages was the presence of anthocyanins in pink flower extracts ( Figure 1). In order to perform a complete characterisation of floral pigments, the pink floral stage was chosen for isolation and identification of constituents.  In the chromatograms shown in Figure 1, two classes of phenolics were found: phenolic acids (including cinnamic derivatives, constituents 1 to 6) and flavonoids (flavonols and anthocyanins, constituents 7 to 30). Based on analysis of the MS data ( Figure S1), the presence of thirty-two compounds is suggested (Table 2), due to the co-elution of some compounds in the chromatographic analysis.
In the chromatograms shown in Figure 1, two classes of phenolics were found: phenolic acids (including cinnamic derivatives, constituents 1 to 6) and flavonoids (flavonols and anthocyanins, constituents 7 to 30). Based on analysis of the MS data ( Figure S1), the presence of thirty-two compounds is suggested (Table 2), due to the co-elution of some compounds in the chromatographic analysis.
The main flavonols identified in the petal extract were kaempferol, quercetin, and myricetin (Tables 2 and 3), which were previously described in leaves of T. pulchra but only with hexosyl and pentosyl substituents [10,36]. The most abundant flavonol skeleton was kaempferol (m/z 287.0548) with different substituents as glucuronyl methyl ester (constituent 23), galloylhexosides (constituents 13, 16 and 19), and p-coumaroylhexosides (21, 27, 28 and 30). Quercetin derivatives (m/z 303.0496, constituents 10, 11, 12 and 25) were the second most abundant flavonol identified, followed by myricetin derivatives (m/z 319.0445, constituents 8 and 9).      Compounds 8, 9, 11, 15 and 17 showed the loss of 162 amu during MS analysis, indicating the presence of a hexose, probably a galactosyl or a glucosyl group ( Figure S1). Pentoses, as arabinose, apiose or xylose, were also found as substituents in the case of compounds 18 and 20, which exhibited a mass loss of 132 amu. Compounds 12 and 23 showed hexauronic acids as substituents, identified by the mass loss of 176 and 190 amu, respectively. The difference is presumably the methyl group in compound 23 as consequence of solvent artefact during extraction procedures. Compound 23 was analysed by NMR and confirmed as glucuronic acid methyl ester substituent. Literature describes glucose as the most commonly identified sugar in flavonoids, while galactose, rhamnose, xylose, and arabinose are less frequent. Yet, mannose, fructose, glucuronic, and galacturonic acids are rare [41][42][43].
Mass loss of 314 amu indicates the presence of galloylhexoside. This group was identified in compound 10, a quercetin derivative, and in the isomers 13, 16 and 19, which are kaempferol derivatives. The fragment m/z 153.0181 was intense for these compounds, which can be ascribed to a galloyl substituent and to a typical ion signal from a fragment of A-ring+ [41], generated by retro Diels-Alder fragmentation of the C-ring (  [41,44,45]. Moreover, the additional mass loss of 162 amu confirmed the presence of hexoside (glucoside or galactoside).
Regarding the p-coumaroyl group, we identified eight compounds with this acylation pattern: 21, 22, 24, 25, 26, 27, 28, and 30. A mass loss of 308 amu is indicative of p-coumaroylhexose substitution, but it can also indicate a rutinosyl group (6-rhamnosylglucose) as substituent. The fragment of m/z 147.0439 found in compounds 25, 27, 28, and 30 confirmed the presence of either a p-coumaroyl or a rhamnosyl substituent. Furthermore, acylation with hydroxycinnamic acids, as p-coumaric acid, shifts the band I from ultraviolet/visible (UV/VIS) spectra of the flavonols to a lower wavelength, resulting in a peak or shoulder at 305-310 nm. In addition, acylation of the sugar moiety also increases retention time in a chromatographic analysis [46][47][48], as shown in Table 2.

Structural Elucidation of Acylated Flavonoids by NMR and Identification of a New Flavonol Glucoside
Flavonoid acylation can influence the biological activity of compounds by altering their solubility, stability, reactivity, and interaction with cellular targets [49], and with regards to the colour of flowers, esterification typically enhances the intensity [50]. Thus, we further isolated the acylated flavonoids by preparative high-performance liquid chromatography (HPLC) to investigate their structure through NMR spectroscopy. Successful isolation was achieved for kaempferol The mass spectrum of 17 was indicative of a mixture of two compounds, which differed from each other in 14 amu, suggesting the presence of an additional methyl group in one of the structures (Figure 2). In the NMR spectrum of 17, seven signals typical of aromatic hydrogens were observed: three doublets at δ 6.22 (1H, d, J = 2.0 Hz, H6), δ 6.21 (1H, d, J = 2.0 Hz, H6), and δ 6.46 (2H, s, H8), corresponding to a meta-coupling of these protons which were attributed to the flavonoid A-ring; + *R1, R2, R3 and R4 indicate substituents. The chemical composition of polar extracts of T. pulchra revealed thirty-two compounds, with twenty-three identified by UV/VIS and MS. Nuclear magnetic resonance spectroscopy was used as additional technique to support the structural elucidation of eight compounds.

Structural Elucidation of Acylated Flavonoids by NMR and Identification of a New Flavonol Glucoside
Flavonoid acylation can influence the biological activity of compounds by altering their solubility, stability, reactivity, and interaction with cellular targets [49], and with regards to the colour of flowers, esterification typically enhances the intensity [50]. Thus, we further isolated the acylated flavonoids by preparative high-performance liquid chromatography (HPLC) to investigate their structure through NMR spectroscopy. Successful isolation was achieved for    (δ 136.10) positions of the flavonoid, which is consistent with the presence of a free hydroxyl group at C3 [51]. Astragalin is a common flavonol present in red wine and in many plants [52]. Flavonols are usually substituted at positions 3 and 7 [53]. The 4′ moiety is unusual but kaempferol 4′-O-β-D-glucopyranoside has already been described [54]. However, to the best of our knowledge, the 2′′ methylated, α-linked sugar in the 4′ position of the kaempferol-(2′′-O-methyl)-4′-O-α-D-glucopyranoside has not previously been reported in the literature.  The mass spectrum of 17 was indicative of a mixture of two compounds, which differed from each other in 14 amu, suggesting the presence of an additional methyl group in one of the structures (Figure 2). In the NMR spectrum of 17, seven signals typical of aromatic hydrogens were observed: three doublets at δ 6.22 (1H, d, J = 2.0 Hz, H6), δ 6.21 (1H, d, J = 2.0 Hz, H6), and δ 6.46 (2H, s, H8), corresponding to a meta-coupling of these protons which were attributed to the flavonoid A-ring; and another three doublets with ortho-coupling constants at δ 6.89 (2H, d, J = 8.4 Hz, H3 and 5 ), δ 6.93 (2H, d, J = 8.5 Hz, H3 and 5 ) and δ 8.04 (4H, d, J = 8.4 Hz, H2 and 6 ), suggesting two para-substituted B-rings of flavonoids. The anomeric protons appeared at δ 5.46 (1H, d, J = 7.6 Hz, H1 ) and δ 4.51 (1H, d, J = 3.6 Hz, H1 ). The smaller coupling constant of the latter suggests an α-linked carbohydrate. Signals between δ 3.09 and δ 3.58 were attributed to the hydrogens of the sugar moiety. The presence of a methyl group was confirmed by signals at δ 3.26 (s, 3H) and δ 54.74 (OMe). Furthermore, the heteronuclear multiple bond correlation (HMBC) spectrum showed two relevant correlations: the first between the anomeric protons at δ 5.46 with C3 (δ 133.63) from the flavonoid C-ring, confirming the position of the sugar moiety in kaempferol 3-O-β-D-glucopyranoside (astragalin), and the second one, the anomeric proton (δ 4.51) with the methyl group at δ 54.74, suggesting the presence of 2-methoxyglycosyl moiety (Figure 2). Although the correlation between the anomeric proton and carbon C4 of the flavonoid was not observed, the position of the glycoside was supported by the 13 C NMR chemical shifts at C2 (δ 147.27) and C3 (δ 136.10) positions of the flavonoid, which is consistent with the presence of a free hydroxyl group at C3 [51].

Flavonoids in Tibouchina and Melastomataceae
In this work, seventeen compounds were described for the first time in T. pulchra, with twelve described for the first time in Tibouchina. Flavonols, especially myricetin derivatives, are characteristic within Mytales [4]. In Tibouchina, the most common flavonols are quercetin and isorhamnetin and in T. pulchra, kaempferol was the main flavonol. Moreover, kaempferol derivatives have only been described before in T. ciliaris and T. pereirae. Regarding anthocyanins, malvidin has already been identified in T. lepidota, T. grandiflora, T. semidecantra, and T. urvelleana, while petunidin has been described exclusively in T. granulosa (Table 1). Although the acylation of anthocyanins has already been reported for the Melastomatoideae [4], this is the first characterisation of anthocyanins (i.e., malvidin and petunidin derivatives) in T. pulchra.
Although in Melastomataceae, many compounds have been isolated and identified by extensive spectrometric analyses. However, considering the size of the family, the number of studied species is still low. The most commonly found natural products in this family are terpenes, simple phenolics, quinones, lignans and flavonoids, as well as a vast range of tannins, mainly hydrolysable ones [57].
The results obtained for T. pulchra in the present study describe, for the first time, the presence of five compounds in this family:  [58]. Kaempferol aglycone (29) was also found in Medinilla magnifica Lindley and Centradenia floribunda Planch [57]. Regarding anthocyanins, malvidin p-coumaroylhexoside acetylpentoside (24) was the major anthocyanin in T. pulchra, which agrees with the proposition of malvidin as the most common anthocyanin nucleus in Melastomataceae [57]. This was the first description of petunidin p-coumaroylhexoside acetylpentoside (22) in T. pulchra and in Melastomataceae, since previous studies had described pelargonidin, cyanidin, peonidin, delphinidin, and malvidin glycosides or acylglycosides [57] in this species and family. Further NMR studies of T. pulchra anthocyanins are necessary to underpin the identification performed here by MS and UV.
The large number of flavonols identified in the pink stage of T. pulchra flowers might be an effect of co-pigmentation. It is known that this class of substances is related with white colour and co-pigmentation in coloured tissues. Co-pigmentation can be defined as the formation of noncovalent complexes involving an anthocyanin or anthocyanin-derived pigment and a co-pigment (in the presence or absence of metal ions), as well as subsequent changes in optical properties of the pigment. There are over ten thousand compounds of different classes of phenolic compounds (e.g., hydrolysable tannins, flavonoids, and phenolic acids) that help to stabilise the colour of flowers and increase colour intensity. In addition, glycosylation and acylation enhance the brightness of anthocyanin colours [50].
In conclusion, the Melastomataceae, and in particular, Tibouchina taxa are poorly characterised chemically. Here, out of the seventeen compounds described for the first time in T. pulchra, five of them are reported in the family: 13, 16, and 19 (we isolated two isomers); 17 (only kaempferol-(2 -O-methyl)-4 -O-α-D-glucopyranoside); 23; and 27. Moreover, a novel flavonol was identified as kaempferol 4 -O-(2 -methyl)-α-D-glucopyranoside. Recent advances in spectrometric techniques offer a unique opportunity to improve our knowledge about the chemical structure of natural products. Studies about flower anthocyanins are scarce, and the understanding of their structure, biosynthesis, and the regulatory mechanisms involved in their accumulation pattern helps to improve our knowledge about plant secondary metabolism-as well as the relationship between flower colour and the attraction of pollinators-and brings new insights for future biotechnological applications.

Extraction and Analysis by UPLC-PAD-ESI-QTOF-MS
Phenolic compounds were extracted from 100 mg of petal powder twice with 1.5 mL of 0.2% hydrochloric acid (HCl) in methanol (MeOH). The samples were sonicated for 10 min and centrifuged at 10,000 rpm for 10 min. The extract was filtered (0.45 µm) and analysed by UPLC-PAD-ESI-QTOF-MS. The MS/MS analysis was performed with a Broadband Collision Induced Dissociation (bbCID) detector (Bruker, Bremen, Germany). Separation was achieved by using a C18 column at a flow rate of 0.3 mL min −1 and 4 µL of injection volume. The column temperature was 45 • C, and the solvent system was composed of 1% formic acid in water (A) and 1% formic acid in acetonitrile (B). Gradient elutions were as follow: 5 to 25% of B (0-40 min), 25 to 100% of B (40-42 min), 100% of B (42.0-42.5 min), 100 to 5% of B (42.5-43.0 min), and 5% of B (43-46 min). Separated compounds were first monitored using a photodiode array detector (PAD) (200 to 600 nm), and then MS scans were performed in positive ion mode (MS + ) in the range m/z 75-1250, under the following conditions: capillary voltage set to 4500 V, end plate offset at −500 V, nebulizer at 2 Bar, dry gas flow of 12 L min −1 and dry gas temperature at 200 • C. The MS signal was calibrated using sodium formate. All data were processed using data analysis.

Isolation by Preparative HPLC and Identification by NMR
Acylated flavonoids were isolated from 10 g of pink petal powder by extracting four times with 200 mL of 0.2% HCl in MeOH. Samples were sonicated for 15 min, pillowed for 10 min, and vacuum filtered and concentrated using a rotary evaporator. The crude extract was diluted to approximately 250 mg/mL and analysed by preparative HPLC with a PAD. Separation was achieved on a C18 column at a flow rate of 20 mL min −1 using 1 mL of injection volume and a solvent system composed of 1% formic acid in water (A) and 1% formic acid in acetonitrile (B). Gradient elution were as follow: 10% of B (0-3 min), 10 to 15% of B (3-30  An aliquot was resuspended in 0.2% HCl in MeOH to check the purity by UPLC-MS. For the isolated compounds, the dried sample was dissolved in deuterated dimethyl sulfoxide (DMSO-d 6 ) for NMR analysis. 1 H and 13 C NMR spectra were obtained using an AVANCE III HD spectrometer operating at frequency of 800.182 and 201.2 MHz, respectively, and equipped with a 5 mm TCI CryoProbe. Analyses of HMBC and heteronuclear single quantum coherence (HSQC) were also performed. All data were processed using MestreNova.