Isolation and Identification of the Five Novel Flavonoids from Genipa americana Leaves

Genipa americana is a medicinal plant popularly known as “jenipapo”, which occurs in Brazil and belongs to the Rubiaceae family. It is a species widely distributed in the tropical Central and South America, especially in the Cerrado biome. Their leaves and fruits are used as food and popularly in folk medicine to treat anemias, as an antidiarrheal, and anti-syphilitic. Iridoids are the main secondary metabolites described from G. americana, but few studies have been conducted with their leaves. In this study, the aim was to chemical approach for identify the main compounds present at the extract of G. americana leaves. The powdered leaves were extracted by maceration with EtOH: water (70:30, v/v), following liquid-liquid partition with petroleum ether, chloroform, ethyl acetate and n-butanol. A total of 13 compounds were identified. In addition three flavonoids were isolated from the ethyl acetate fraction: quercetin-3-O-robinoside (GAF 1), kaempferol-3-O-robinoside (GAF 2) and isorhamnetin-3-O-robinoside (GAF 3) and, from n-butanol fraction more two flavonoids were isolated, kaempferol-3-O-robinoside-7-O-rhamnoside (robinin) (GAF 4) and isorhamnetin-3-O-robinoside-7-rhamnoside (GAF 5). Chemical structures of these five flavonoids were elucidated using spectroscopic methods (MS, 1H and 13C-NMR 1D and 2D). These flavonoids glycosides were described for the first time in G. americana.


Extraction and TLC Analysis
Despite being a class of secondary metabolites that have shown promising biological activities in various studies [26], only our study described the presence of flavonoids in G. americana [24]. Therefore, the phytochemical investigation of this work was conducted to isolate and purify the flavonoids of G. americana leaf extract. Initially, the extract (HE) was fractioned and TLC analysis of the HE and fractions, with specific spray reagents, indicated the presence of flavonoids and iridoids [7,27]. The phytochemical screening of the HE, revealed with vanillin sulfuric acid, showed characteristics yellow spots (R f = 0.33 until 0.90), suggesting the presence of flavonoids as observed in our previous work [24]. Afterwards, a liquid-liquid extraction was carried out, affording the following fractions: petroleum ether (PE), chloroform (CHCl 3 ), ethyl acetate (EtOAc), n-butanol (n-BuOH) and the residual aqueous fraction (RA). The PE fraction showed no spot in the TLC plate after spraying with vanillin sulfuric acid; CHCl 3 fraction showed two purple spots (R f = 0.93 and 0.83), suggesting the presence of iridoids; the EtOAc fraction showed only one zone more evident (R f = 0.81 -purple), that indicated the presence of iridoids. When EtOAc fraction was revealed with Natural Product (NP) Reagent (UV 365 nm) a yellow zone was observed (R f = 0.55), suggesting the presence of flavonoids. TLC analysis of the n-BuOH fraction showed four yellow and orange spots (R f = 0.21 until 0.50-NP Reagent/UV 365 nm), suggestive of flavonoids [8].

UHPLC-DAD Characterization
The Research Group on Bioactive Natural Products (PNBio) has been studying the leaves of G. americana and in a recent publication [24] the presence of two new iridoids for its leaves has been identified, showing the chemical potential of the species.
As described earlier, most of the studies have described the presence of iridoids in the genus Genipa, being considered a chemotaxonomic marker [2,24]. Only our study identified flavonoids in leaves [24]. Furthermore, only one article described the presence of leucoanthocyanidins, catechins, and flavanones in the fruits of G. americana by HPLC analysis [28], and another study described its presence in fruits by colorimetric methods [13], demonstrating that the presence of flavonoids in leaf extract was not described previously.
Extract and fractions were analyzed by ultra-performance liquid chromatography (UHPLC). The chromatogram of the HE, recorded in 254 nm, showed four main peaks (3.5 min, UV: 246, 265 and 350 nm; 3.9 min, UV: 255 and 354 nm; 16.6 min, UV: 242 and 287 nm; 17.2 min, UV: 243 nm). Accordingly, the UV spectra of each peak observed in the chromatogram can suggest the presence the flavonoids and iridoids in HE of G. americana leaves ( Figure 1).
Molecules 2018, 23, x FOR PEER REVIEW 3 of 14 As described earlier, most of the studies have described the presence of iridoids in the genus Genipa, being considered a chemotaxonomic marker [2,24]. Only our study identified flavonoids in leaves [24]. Furthermore, only one article described the presence of leucoanthocyanidins, catechins, and flavanones in the fruits of G. americana by HPLC analysis [28], and another study described its presence in fruits by colorimetric methods [13], demonstrating that the presence of flavonoids in leaf extract was not described previously.
Extract and fractions were analyzed by ultra-performance liquid chromatography (UHPLC). The chromatogram of the HE, recorded in 254 nm, showed four main peaks (3.5 min, UV: 246, 265 and 350 nm; 3.9 min, UV: 255 and 354 nm; 16.6 min, UV: 242 and 287 nm; 17.2 min, UV: 243 nm). Accordingly, the UV spectra of each peak observed in the chromatogram can suggest the presence the flavonoids and iridoids in HE of G. americana leaves ( Figure 1).

HPLC-ESI-IT-MS/MS Characterization
Through the TLC and UHPLC-DAD, we can suggest the presence of iridoids and flavonoids in leaves extract from G. americana. Therefore, it was decided to perform an HPLC-MS/MS study to allow the identification these compounds.
In this way, we selected of HE, Fr.EtOAc, and Fr.n-BuOH to analyze. The HPLC-MS/MS revealed the presence of several majority peaks and allowed suggest the structures of 13 compoundsincluding five flavonoids (subsequently isolated), using the program DataAnalysis 4.2 (Bruker, Billerica, MA, USA) ( Table 1).

HPLC-ESI-IT-MS/MS Characterization
Through the TLC and UHPLC-DAD, we can suggest the presence of iridoids and flavonoids in leaves extract from G. americana. Therefore, it was decided to perform an HPLC-MS/MS study to allow the identification these compounds.
In this way, we selected of HE, Fr.EtOAc, and Fr.n-BuOH to analyze. The HPLC-MS/MS revealed the presence of several majority peaks and allowed suggest the structures of 13 compounds-including five flavonoids (subsequently isolated), using the program DataAnalysis 4.2 (Bruker, Billerica, MA, USA) ( Table 1).
It is a iridoid already identified in fruit of Gardenia jasminoides Ellis (Rubiaceae) [19,22,31,32] and Wendlandia formosana Cowan leaves [33].  [31,34], but the first time is described in the Genipa genus. Table 1 This is an iridoid glucoside, and it has already been isolated in Hedyotis tenelliflora Blume (Rubiaceae) [2], but is also described for the first time in the Genipa genus. The present study contributed to describe a new phytochemical approach for Genipa americana, considering that until this moment most of the studies reported only the presence of iridoids [2,4,24].

Isolation of Major Compounds
In order to verify the positions of the sugars of the O-glycosides present in the flavonoids identified by HPLC-MS/MS (Section 2.3), the major constituents of the EtOAc and n-BuOH fractions were isolated and identified.
Based on the chromatographic profile observed by TLC and UHPLC analysis, EtOAc fraction (3.27 g) was chosen initially to be fractionated by vacuum liquid chromatography (VLC) and then fractions 8 and 9 were gathered and chromatographed by classical column using silica gel (0.063-0.200 mm) and Sephadex LH-20 gel, which yielded subfractions 134. Then subfraction (61-111) was gathered and isolated by preparative HPLC and three well-separated peaks were obtained in the chromatogram. Thereby, the following flavonoids were purified: Since the n-BuOH fraction (5.0 g) also had a flavonoid rich phytochemical profile ( Figure 1) and higher yield, it was submitted to fractionation by VLC with silica gel, which yielded 11 subfractions. The fraction 4 (1.07 g) was isolated by classical column chromatography, and its sub-fraction 4-E of chromatography (1.0 g) was dissolved in methanol and chilled to 2 • C, for five days, which promoted the crystallization of a part of the sample, allowed its separation. These fractions were named fraction 4-E.1 (crystallized) and fraction 4-E.2 (not crystallized). The fraction 4-E.1 was submitted to preparative HPLC and two flavonoids were further separated:

Isolation of Major Compounds
In order to verify the positions of the sugars of the O-glycosides present in the flavonoids identified by HPLC-MS/MS (Section 2.3), the major constituents of the EtOAc and n-BuOH fractions were isolated and identified.
Based on the chromatographic profile observed by TLC and UHPLC analysis, EtOAc fraction (3.27 g) was chosen initially to be fractionated by vacuum liquid chromatography (VLC) and then fractions 8 and 9 were gathered and chromatographed by classical column using silica gel (0.063-0.200 mm) and Sephadex LH-20 gel, which yielded subfractions 134. Then subfraction (61-111) was gathered and isolated by preparative HPLC and three well-separated peaks were obtained in the chromatogram. Thereby, the following flavonoids were purified: Since the n-BuOH fraction (5.0 g) also had a flavonoid rich phytochemical profile ( Figure 1) and higher yield, it was submitted to fractionation by VLC with silica gel, which yielded 11 subfractions. The fraction 4 (1.07 g) was isolated by classical column chromatography, and its sub-fraction 4-E of chromatography (1.0 g) was dissolved in methanol and chilled to 2 °C, for five days, which promoted the crystallization of a part of the sample, allowed its separation. These fractions were named fraction 4-E.   In the study, a simple and effective procedure allowed the isolation of flavonoids from the leaves of G. americana. Previously only two papers described the detection of flavonoids in fruits [13,28] and one study published by our group identified these compounds in leaves [24] from G. americana. It is important to mention that this is a new phytochemical approach concerning the leaves of this species. Flavonoids are products of secondary metabolism in plants and are of interest to the pharmaceutical and food industries because of their reported wide range of biological effects [26], and they have long been associated with good health benefits, which could be attributed to their antioxidant capabilities [26].   In the study, a simple and effective procedure allowed the isolation of flavonoids from the leaves of G. americana. Previously only two papers described the detection of flavonoids in fruits [13,28] and one study published by our group identified these compounds in leaves [24] from G. americana. It is important to mention that this is a new phytochemical approach concerning the leaves of this species. Flavonoids are products of secondary metabolism in plants and are of interest to the pharmaceutical and food industries because of their reported wide range of biological effects [26], and they have long been associated with good health benefits, which could be attributed to their antioxidant capabilities [26].

Kaempferol-3-O-robinoside-7-O-rhamnoside (robinin) [45] (GAF 4,
Only after the isolation was it possible to analyze the compounds by 1 H-and 13 C-NMR and to differentiate the types of sugar present, being possible to identify the final portion as galactose. In addition, it can be confirmed that the sugars are present in positions-3 (GAF 1-5) and-7 (GAF 4-5).
Compound GAF 1 exhibited in vitro inhibitory activities against leukemia K562 cells in different extents [47], and cytotoxic activity against HepG-2cells.The hydroxylation pattern of the C-rings of the flavonoid compounds like quercetin aglycone, play an essential role in their cytotoxic activities, especially the inhibition of protein kinase antiproliferation activity [48].
The isorhamnetin 3-O-robinoside (GAF 3) showed a protective effect against lipid peroxidation induced by H 2 O 2 and antigenotoxic potential on human chronic myelogenous leukemia cell line K562 [49].
Flavonoids are considered as a class of natural products of high pharmacological potency but, unfortunately, many of them have a low solubility in water. We have also isolated one flavonol glycosides very soluble in water: robinin (GAF 4). Similar compound have been previously isolated from leaves of Atropa belladonna [38] but your structure have not been fully elucidated. Robinin (GAF 4) displayed a marked activity, inhibiting edema (38.8%) at a concentration of 0.0027 mmol/kg of body weight, four hours after injection of carrageenin [47]. GAF 4 was also able to inhibit lymphocyte proliferation to a greater extent (IC50 ∼ = 25 µg mL −1 ) and were twice more active than crude extract of Alternanthera brasiliana [37].
No reports about pharmacological activities were found for GAF 5.
This work described the first time the isolation and identification of flavonoids in leaves at G. americana. This is an important finding for subsequent studies aimed at the standardization of leaf extracts.

Plant Material
The

Extraction and TLC Characterization
The leaves of G. americana was evaporated at 40 • C in a circulating air oven and powdered leaves (600 g) were extracted by maceration with ethanol:water (70:30, v/v) for five days (plant:solvent, 1.5:10, w/v; 4 L; at room temperature). Then, the organic solvent was evaporated under reduced pressure in rotary evaporator (temperature below 45 • C) and water residue was freeze-dried, obtaining the hydroethanolic extract (HE).
In the phytochemical screening the HE and fractions were analyzed by TLC using aluminum sheets, coated with silica gel F254 as absorbent and chromatographed with ethyl acetate:formic acid:water: methanol (10:1.6:1.5:0.6, v/v/v/v) as mobile phase. The TLC was analyzed under 254 and 365 nm ultraviolet (UV) light and then sprayed with vanillin sulfuric acid (4%) or natural product reagent (0.5%)-NP reagent.

UHPLC Characterization
The samples were analyzed by ultra-high performance liquid chromatography coupled with a diode array detector (UHPLC/HPLC-DAD), model UFLC (Shimadzu, Kyoto, Japan), containing a quaternary pump system (LC-20A 3 XR), equipped with a degasser (DGU-20A 3 ), auto-sampler (SIl-20AC XR), column oven (CTO-20AC), and diode-array detectors (SPD-M20A), with Software LC Solution (Shimadzu, Kyoto, Japan) controlled system. A C 18 column Shim-pack XR-ODS 30 × 2 mm, 2.2 µm (Shimadzu, Kyoto, Japan); a temperature of 25 ± 2 • C was used for the analysis and separation of the compounds and was achieved using a solvent system mixture of acetonitrile: acidified water (with 0.3% formic acid) as the mobile phase with a flow rate of 0.3 mL/min, and a detector was set at 254 nm.

HPLC-ESI-IT-MS/MS Characterization
The hydroethanolic extracts and EtOAc and BuOH fractions were analyzed by HPLC-IT-MS/MS. UFLC (Shimadzu, Kyoto, Japan) containing two LC20AD solvent pumps, a SIL20AHT auto sampler, a SPD-M20A detector and a CBM20A system controller, coupled with an ion-trap mass spectrometer (AmaZon X, Bruker, Billerica, MA, USA). LC experiments were performed using a C 18 column (Kromasil-250 mm × 4.6 mm × 5 µm) and the following gradient elution: solvent A: water and formic acid (0.1%, v/v); solvent B: acetonitrile; injection volume of 20 µL, and flow rate of 0.6 mL/min. The ion-trap analysis parameters are as follows: capillary 4.5 kV, ESI in positive mode, final plate offset 500 V, 40 psi nebulizer, dry gas (N 2 ) with flow rate of 8 mL/min and a temperature of 300 • C. CID fragmentation was achieved in auto MS/MS mode using advanced resolution mode for MS and MS/MS mode. The spectra (m/z 50-1000) were recorded every two seconds.
The data obtained were interpreted with the help of the following: Metlin, MassBank and Scienfinder.

Preparative HPLC Optimization and Analyses
In order to obtain the isolate compounds, these fractions were subjected to preparative HPLC for further purification. The preparative HPLC separations were used with a C 18 column (200 mm × 20 mm, 5 µm); and mobile phase was selected based on the polarity of the likely compounds and the analytical HPLC conditions. Several mobile phases composed of acetonitrile (B)-water(A) in various concentrations of acetonitrile (15%, 18%, 20%, 21%, 23%, 24%, 25%, 30%) were tested. The results indicated that the best separation conditions were achieved using acetonitrile in a gradient mode (0-28 min, 21-23%) for the ethyl acetate fraction and isocratic mode (0-30 min, 18%) for n-BuOH fraction and a flow rate of 10 mL/min and a monitoring wavelength of 254 nm, 270 nm and 340 nm, with LabSolutions software (Shimadzu, Kyoto, Japan). Under the above conditions, a satisfactory separation of each the targeted compounds was achieved.

MS/MS of Isolated Compounds
The isolated compounds were analysis by MS-MS in positive and negative modes were by mass of direct infusion the microTOF II-ESI-TOF-Bruker Daltonics (Bruker, Billerica, MA, USA), with a drying gas flow rate o f4 L/min at 180 • C, nebulizer gas 0.4 bar (pressure), internal calibration standard: TFA, and syringe flow: 10 µL/min.

Nuclear Magnetic Resonance Spectroscopy (NMR) of Isolated Compounds
The nuclear magnetic resonance (NMR) spectra were performed on a (Bruker, Billerica, MA, USA) (400 MHz for 1 H and 100 MHz for 13 C) and chemical shifts are given in ppm relative to residual DMSO-d 6 (2.5), and to the central peak of the triplet related to DMSO-d 6 carbon (39.5 ppm).

Conclusions
Through characteristic fragmentation patterns of substances obtained by MS/MS data, 13 compounds were identified. The flavonoids were isolated and identified.
In contrast to literature, which describes mainly the presence of iridoids for the fruit and leaf extracts of G. americana, in this paper the leaves showed to be rich in O-glycosidic flavonoids. The isolation was carried out in few steps and allowed the identification of five flavonoids glycosides not described until this moment to the G. americana leaf extract. These flavonoids GAF 1, 3, 4, and 5 were identified for the first time in the Rubiaceae family and flavonoid GAF 2 is unknown for genus Genipa. All flavonol aglycones have sugars units, attached only at the 3-position. The similarities of the compounds of five chemical structures suggest a common biosynthetic pathway in this species. The present article was conducted to evaluate the chromatograph profiles of the leaf extract from Genipa americana, to be used in future to the quality control for this species. It can also be suggesting that flavonoids identified for this species may be associated, at least in part, to pharmacological properties of the plant.