Phenolic Assesment of Uncaria tomentosa L. (Cat’s Claw): Leaves, Stem, Bark and Wood Extracts

The phenolic composition of extracts from Uncaria tomentosa L. from different regions of Costa Rica was studied using advanced analytical techniques such as UPLC/TQ-ESI-MS and 13C-NMR. Samples from leaves, stems, bark and wood (n = 22) were subjected to extraction to obtain phenolic and alkaloid extracts, separately. Comparatively, higher values of total phenolic content were observed for leaves, stems and bark (225–494 gallic acid equivalents/g) than for wood extracts (40–167 gallic acid equivalents/g). A total of 32 non-flavonoid and flavonoid compounds were identified in the phenolic extracts: hydroxybenzoic acids (benzoic, salicylic, 4-hydroxybenzoic, prochatechuic, gallic, syringic and vanillic acids), hydroxycinnamic acids (p-coumaric, caffeic, ferulic and isoferulic acids), flavan-3-ols monomers [(+)-catechin and (−)-epicatechin)], procyanidin dimers (B1, B2, B3, B4, B5, B7 and two other of unknown structure) and trimers (C1, T2 and one of unknown structure), flavalignans (four unknown structures pertaining to the cinchonain family) and propelargonidin dimers (four unknown structures, reported for the first time in U. tomentosa). Additionally, alkaloid extracts obtained from the plant residue after phenolic extraction exhibited a content of tetracyclic and pentacyclic alkaloids ranging between 95 and 275 mg/100 g of dry material for bark extracts, and between 30 and 704 mg/100 g for leaves extracts. In addition, a minor alkaloid was isolated and characterized, namely 18,19-dehydrocorynoxinoic acid. Our results confirmed the feasibility of U. tomentosa as a suitable raw material for obtaining phenolic- and alkaloid-rich extracts of potential interest.


Introduction
Uncaria tomentosa L., also known as cat's claw, is a creeper plant typical of the rainy tropical forest that belongs to the Rubiaceae family.It is naturally distributed from Peru to Belize, and traditionally used as a medicinal plant.In the last two decades, around 50 compounds have been isolated from U. tomentosa, from which around 35 can be considered exclusive to this species.These compounds include alkaloids, terpenes, quinolic acid glycosides and phenolic compounds, among them coumarins [1].The scientific interest for this plant has increased after the confirmation of its immunomodulatory properties mainly attributed to its alkaloid content [1].Moreover, there is a certain body of scientific evidence that demonstrates a wide spectra of bioactivities for U. tomentosa extracts, including immunomodulatory, antioxidant, antiviral, anti-inflammatory [2], also suggesting that these effects are due to synergism with other chemical compounds present in the plant [3].In fact, some studies indicated that anti-inflammatory activity of U. tomentosa extracts is independent from their alkaloid contents [4,5].
Other studies suggest that phenolic compounds could be responsible for some pharmacologic effects of U. tomentosa, for instance, its antioxidant properties [6,7].However, in general, phenolic composition of this plant has been scarcely studied and there are no reports on phenolic determinations using modern analytical techniques such as mass spectrometry.The presence of non-flavonoids, mainly hydroxycinnamic acids such as caffeic and chlorogenic acids, in U. tomentosa has been described [8][9][10].Flavonoids present in this plant mainly include flavonols (i.e., quercetin and miricetin derivatives) and flavan-3-ols [6,8,9].In relation to this latter group, the monomers (+)-catechin and (´)-epicatechin, and the dimers procyanidins B1, B2 and B4 have been described in different preparations from U. tomentosa [9,11,12].In addition, and of great interest is its content of flavalignans, i.e., epichatechins substituted with phenylpropanoids, such as cinchonains 1a and 1b [13], which are not that common in nature but are present in medicinal plants such as Eriobotrya japonica [14].In this paper, we have obtained extracts from different parts (leaves, stems, bark and wood) of U. tomentosa plants collected in different zones of Costa Rica, and we have characterize them using UPLC-DAD-ESI-TQ MS techniques complemented with 13 C-NMR.Additionally, alkaloids extracts were prepared from the residues of U. tomentosa plants after phenolic extraction, and they were characterized in detail.

Phenolic Yield and Total Phenolic Content in U. tomentosa Extracts
The purification of the polar fraction with solvents of low and medium polarity allowed us to obtain phenolic enriched fractions with yields as shown in Table 1.Leaves gave the higher yields for all plants with an average of 11.7%, followed by bark with an average of 5.4% while wood and stems had a similar lower yield average of 3.2% and 2.9%, respectively.The total phenolic contents of the different extracts resulted in lower values for wood extracts (40-167 gallic acid equivalents/g) and higher values for leaves, stems and bark (225-494 gallic acid equivalents/g) being values varying depending on plant origin, for instance bark samples in the case of the location of Sarapiqui (S1), leaves samples in Palacios (P1) and stem samples in Asomat (AS).UPLC/TQ-ESI-MS analysis was performed in the 22 extracts obtained from U. tomentosa, as described in the experimental section.A total of 32 phenolic compounds were determined, including hydroxybenzoic acids (benzoic, salicylic, 4-hydroxybenzoic, protocatechuic, gallic, vanillic and syringic acids), hydroxycinnamic acids (p-coumaric, caffeic, ferulic and isoferulic acids), flavan-3-ols (monomers, procyanidin dimers and trimers, and propelargonidin dimers) and flavalignans (cinchonains) (Tables 2 and 3).Table S1 (Supplementary Materials) reports their MS/MS parameters and, as an example, Figure 1 shows the MRM (Multiple Reaction Monitoring) transitions for flavan-3-ols monomers [(+)-catechin and (´)-epicatechin, m/z 289/245], procyanidin dimers (m/z 577/289), propelargonidin dimers (m/z 561/289), procyanidin dimers (m/z 865/577) and cinchonains (m/z 451/341) found in an extract from U. tomentosa leaves.At least four different propelargonidin dimers (with retention times of 4.43, 5.01, 5.65 and 9.27 min) were detected in all the U. tomentosa extracts, with the only exceptions of the wood extracts from Asomat (AS-M) and Fortuna 1 (F1-M) (Table 3).To our knowledge, our results reveal for the first time the presence of propelargonidins in U. tomentosa.

Molecules 2015, 20, page-page
To summarize the results from these analyses, a statistical Principal Component Analysis (PCA) was performed for the 22 extracts taking into consideration 33 variables (individual phenolic compounds + total phenolic content) (Figure 2).Two components (PC1 and PC2) were obtained: PC1 represented 39% of variance and showed a negative correlation (loadings > 0.7) with the total phenolic content, procyanidins B1, B3, B7, and with an unknown procyanidin (RT = 5.47 min), as wells as positive correlation with syringic acid.PC2 represented 16% of variance and was negatively correlated with procyanidin C1.In the plane represented by the two components, leaves extracts were grouped separately from the rest of the samples.Wood extract presented the highest values in PC1, and were also separated in another homogeneous group.On the other hand, bark and stems showed a high variability and overlapping in the planes of both components, indicating that phenolic composition of these parts can be strongly affected by geographical origin or location.

13 C-NMR Analysis of U. tomentosa Extracts
As in agreement with the UPLC-MS/MS results, the 13 C-NMR analysis of U. tomentosa extracts showed characteristic signals for procyanidins and propelargonidins, respectively reported by Czochanska [15] and Fu [16] for other substrates.As an example, Figure 3 illustrates the 13 C-NMR spectra of an extract from U. tomentosa leaves.Signals in the region between δ 160 and 150 ppm corresponded to carbons C5, C7 and C8 of the A ring from both procyanidins and propelargonidins (Figure 4).Distinctive signals characteristic of procyanidins were found at δ 145 ppm corresponding to C3′ and C4′ of the B ring and at δ 132.6 ppm corresponding to C1′.In addition, signals characteristic of propelargonidins were found at δ 130.1 ppm attributed to C1′ and at δ 129.4 ppm corresponding to carbons C2′ and C6′ of the B ring.Other characteristic signals appear at δ 119 ppm (C6′) for procyanidins and the cluster of peaks between δ 115 and 117 ppm that correspond to C2′, C5′ (procyanidins) and C3′, C5′ (propelargonidins).The signals between δ 95 and 96 ppm are assigned to C6 and C8 from the A ring, when this unit does not have a substituent.Furthermore, the signal at

13 C-NMR Analysis of U. tomentosa Extracts
As in agreement with the UPLC-MS/MS results, the 13 C-NMR analysis of U. tomentosa extracts showed characteristic signals for procyanidins and propelargonidins, respectively reported by Czochanska [15] and Fu [16] for other substrates.As an example, Figure 3 illustrates the 13 C-NMR spectra of an extract from U. tomentosa leaves.Signals in the region between δ 160 and 150 ppm corresponded to carbons C5, C7 and C8 of the A ring from both procyanidins and propelargonidins (Figure 4).Distinctive signals characteristic of procyanidins were found at δ 145 ppm corresponding to C3 1 and C4 1 of the B ring and at δ 132.6 ppm corresponding to C1 1 .In addition, signals characteristic of propelargonidins were found at δ 130.1 ppm attributed to C1 1 and at δ 129.4 ppm corresponding to carbons C2 1 and C6 1 of the B ring.Other characteristic signals appear at δ 119 ppm (C6 1 ) for procyanidins and the cluster of peaks between δ 115 and 117 ppm that correspond to C2 1 , C5 1 (procyanidins) and C3 1 , C5 1 (propelargonidins).The signals between δ 95 and 96 ppm are assigned to C6 and C8 from the A ring, when this unit does not have a substituent.Furthermore, the signal at δ 108 ppm correspond to C6 and C8 when the A ring is bonded to another monomer unit through C4ÑC8 and C4ÑC6 type bonds, in agreement with the different isomers detected by UPLC-MS/MS analysis.On the other hand, the region between δ 90 and 70 ppm is useful to determine the stereochemistry of the C ring.In fact, two sets of signals are found at δ 83 and 76 ppm for C2 indicating the presence of both isomers, 2,3-trans and 2,3-cis, respectively, for the two different type of proanthocyanidins, corresponding to catechin and epicatechin units (procyanidins) and afzelechin and epiafzelechin units (propelargonidins).Finally, the broad signal between δ 74 and 73 ppm correspond to C3 extended units (both for isomers cis and trans) while peaks between δ 65 and 64 ppm correspond to C3 terminal units; and C4 corresponding to extended units for both cis and trans isomers appear as a broad peak between δ 38 and 36 ppm while C4 from terminal units showed several signals in the region between δ 29 and 27 ppm (Figure 3).
Molecules 2015, 20, page-page δ 108 ppm correspond to C6 and C8 when the A ring is bonded to another monomer unit through C4→C8 and C4→C6 type bonds, in agreement with the different isomers detected by UPLC-MS/MS analysis.On the other hand, the region between δ 90 and 70 ppm is useful to determine the stereochemistry of the C ring.In fact, two sets of signals are found at δ 83 and 76 ppm for C2 indicating the presence of both isomers, 2,3-trans and 2,3-cis, respectively, for the two different type of proanthocyanidins, corresponding to catechin and epicatechin units (procyanidins) and afzelechin and epiafzelechin units (propelargonidins).Finally, the broad signal between δ 74 and 73 ppm correspond to C3 extended units (both for isomers cis and trans) while peaks between δ 65 and 64 ppm correspond to C3 terminal units; and C4 corresponding to extended units for both cis and trans isomers appear as a broad peak between δ 38 and 36 ppm while C4 from terminal units showed several signals in the region between δ 29 and 27 ppm (Figure 3).Molecules 2015, 20, page-page δ 108 ppm correspond to C6 and C8 when the A ring is bonded to another monomer unit through C4→C8 and C4→C6 type bonds, in agreement with the different isomers detected by UPLC-MS/MS analysis.On the other hand, the region between δ 90 and 70 ppm is useful to determine the stereochemistry of the C ring.In fact, two sets of signals are found at δ 83 and 76 ppm for C2 indicating the presence of both isomers, 2,3-trans and 2,3-cis, respectively, for the two different type of proanthocyanidins, corresponding to catechin and epicatechin units (procyanidins) and afzelechin and epiafzelechin units (propelargonidins).Finally, the broad signal between δ 74 and 73 ppm correspond to C3 extended units (both for isomers cis and trans) while peaks between δ 65 and 64 ppm correspond to C3 terminal units; and C4 corresponding to extended units for both cis and trans isomers appear as a broad peak between δ 38 and 36 ppm while C4 from terminal units showed several signals in the region between δ 29 and 27 ppm (Figure 3).In addition, 13 C-NMR spectra allowed comparison of the relative abundance of procyanidins and propelargonidins.Figure 5 shows amplifications of 13 C-NMR spectra for the four different parts of U. tomentosa.Spectra corresponding to leaves extracts clearly showed stronger signals at δ 129-130 ppm corresponding to C2 1 , C6 1 and C1 1 of propelargonidins in respect to the signal at δ 145-146 ppm characteristic of C2 1 and C3 1 of procyanidins, which was in agreement with the results from the UPLC-MS/MS (Table 2).Extracts from stems and bark samples had less preponderance of propelargonidins in respect to procyanidins (Figure 5b,c, respectively).Finally, the spectra corresponding to U. tomentosa wood (Figure 5d, P1-M) showed almost no significant signals in the studied region, which was also in agreement with the results from the UPLC-MS/MS (Table 3).
Our study proved the usefulness of both UPLC-DAD-ESI-TQ MS and 13 C-NMR techniques in the characterization of the phenolic composition of U. tomentosa.One relevant contribution of this study was the identification of four propelargonidin dimers, although their complete structures remain to be elucidated.In addition, it has clearly showed the particularities of the different parts (leaves, stems, bark and wood) of the U. tomentosa plant concerning their phenolic content and profile.All of these add new valuable information to the existing literature concerning this topic [8][9][10][11][12][13].
Molecules 2015, 20, page-page In addition, 13 C-NMR spectra allowed comparison of the relative abundance of procyanidins and propelargonidins.Figure 5 shows amplifications of 13 C-NMR spectra for the four different parts of U. tomentosa.Spectra corresponding to leaves extracts clearly showed stronger signals at δ 129-130 ppm corresponding to C2′, C6′ and C1′ of propelargonidins in respect to the signal at δ 145-146 ppm characteristic of C2′ and C3′ of procyanidins, which was in agreement with the results from the UPLC-MS/MS (Table 2).Extracts from stems and bark samples had less preponderance of propelargonidins in respect to procyanidins (Figure 5b,c, respectively).Finally, the spectra corresponding to U. tomentosa wood (Figure 5d, P1-M) showed almost no significant signals in the studied region, which was also in agreement with the results from the UPLC-MS/MS (Table 3).
Our study proved the usefulness of both UPLC-DAD-ESI-TQ MS and 13 C-NMR techniques in the characterization of the phenolic composition of U. tomentosa.One relevant contribution of this study was the identification of four propelargonidin dimers, although their complete structures remain to be elucidated.In addition, it has clearly showed the particularities of the different parts (leaves, stems, bark and wood) of the U. tomentosa plant concerning their phenolic content and profile.All of these add new valuable information to the existing literature concerning this topic [8][9][10][11][12][13].

Alkaloids Contents of U. tomentosa Extracts
Although the main objective of the research was to study in detail the composition of phenolic extracts of U. tomentosa, alkaloids-rich extracts were obtained through acid-base extraction from the plant residue after phenolic extraction (Section 3.3).The extraction yields for the alkaloid extracts were acceptable, ranging from to 0.44% to 0.81% for leaves and from 0.20% to 0.35% for bark (Table 4).Following the experimental protocol described in Section 3.7, six known pentacyclic (mitraphylline, isomitraphylline, uncarine F, pteropodine, isopteropodine and speciophylline) and two known tetracyclic (rhynchophylline and isorhynchophylline) alkaloids (Figure 6) were identified through NMR data obtained (Figures S1-S10, Supplementary Materials) and quantified through HPLC in

Alkaloids Contents of U. tomentosa Extracts
Although the main objective of the research was to study in detail the composition of phenolic extracts of U. tomentosa, alkaloids-rich extracts were obtained through acid-base extraction from the plant residue after phenolic extraction (Section 3.3).The extraction yields for the alkaloid extracts were acceptable, ranging from to 0.44% to 0.81% for leaves and from 0.20% to 0.35% for bark (Table 4).Following the experimental protocol described in Section 3.7, six known pentacyclic (mitraphylline, isomitraphylline, uncarine F, pteropodine, isopteropodine and speciophylline) and two known tetracyclic (rhynchophylline and isorhynchophylline) alkaloids (Figure 6) were identified through NMR data obtained (Figures S1-S10, Supplementary Materials) and quantified through HPLC in extracts of leaves and bark (Table 4), in agreement with data reported in the literature [2].The tetracyclic alkaloids (rhynchophylline and isorhynchophylline) were present in almost all the extracts, reaching isorhynchophylline the highest value (509 ˘1 mg/100 g of dry material) in the leaf extract from the location of Palacios (P1-H).For the same plant location, the extracts from leaves showed higher content of rhynchophylline and isorhynchophylline than the extracts from bark (Table 4).On the contrary, the pentacyclic alkaloids mitraphylline, isomitraphylline, pteropodine and isopteropodine tended to be more abundant in the extracts from bark in comparison to leaf extracts, with the exception of the location of Los Chiles (L1-H) for mitraphylline and isomitraphylline, and the location of Asomat (AS-H) for pteropodine and isopteropodine (Table 4).Finally, speciophylline, uncarine F were only present in the bark extract from the location of Asomat (AS-C) (Table 4).Total alkaloid content in the extracts obtained from the different locations ranged from 95.0 to 275 mg/100 g of dry material for bark extracts, and from 330 to 704 mg/100 g of dry material for leaves extracts.For the same location, the leaf extracts were between two-and seven-fold alkaloid-richer than the bark extracts (Table 4).In addition, a minor alkaloid was isolated and identified as 18,19-dehydrocorynoxinoic acid when comparing NMR analyses (Figures S11 and S12, Supplementary Materials) with data published by Yuan [17].
Above all, this study confirmed the applicability of subsequent extractions of the plant material to obtain separate phenolic-and alkaloid-rich extracts in a more rational and environmental-friendly way [18].
Molecules 2015, 20, page-page extracts of leaves and bark (Table 4), in agreement with data reported in the literature [2].The tetracyclic alkaloids (rhynchophylline and isorhynchophylline) were present in almost all the extracts, reaching isorhynchophylline the highest value (509 ± 1 mg/100 g of dry material) in the leaf extract from the location of Palacios (P1-H).For the same plant location, the extracts from leaves showed higher content of rhynchophylline and isorhynchophylline than the extracts from bark (Table 4).On the contrary, the pentacyclic alkaloids mitraphylline, isomitraphylline, pteropodine and isopteropodine tended to be more abundant in the extracts from bark in comparison to leaf extracts, with the exception of the location of Los Chiles (L1-H) for mitraphylline and isomitraphylline, and the location of Asomat (AS-H) for pteropodine and isopteropodine (Table 4).Finally, speciophylline, uncarine F were only present in the bark extract from the location of Asomat (AS-C) (Table 4).Total alkaloid content in the extracts obtained from the different locations ranged from 95.0 to 275 mg/100 g of dry material for bark extracts, and from 330 to 704 mg/100 g of dry material for leaves extracts.For the same location, the leaf extracts were between two-and seven-fold alkaloid-richer than the bark extracts (Table 4).In addition, a minor alkaloid was isolated and identified as 18,19-dehydrocorynoxinoic acid when comparing NMR analyses (Figures S11 and S12, Supplementary Materials) with data published by Yuan [17].
Above all, this study confirmed the applicability of subsequent extractions of the plant material to obtain separate phenolic-and alkaloid-rich extracts in a more rational and environmental-friendly way [18].

Plant Material
Uncaria tomentosa samples were collected from different places in Costa Rica, some of them from local communities that grow the plant, namely Asomat (AS), AromaTica-Fortuna (F1 and F2) and Gema (GE) in the northern part of the country as well as Aprolece-Palacios (P1) in the Caribbean; and others growing in the wild in Los Chiles (L1 and L2) in the northern part of Costa Rica and Sarapiqui (S1) in the Caribbean.Vouchers for all plants are deposited in the Costa Rican National Herbarium, under series no.AQ2953, AQ2959, AQ2968, AQ3246, AQ3331, AQ3332, AQ3508 and AQ3510, respectively.The plant material was separated in its different parts (leaves, stem, bark and wood) and then dried in a stove at 40 °C, being turned over every 24 h for a week until totally dry.The dried material was then ground and preserved in plastic recipients.

Plant Material
Uncaria tomentosa samples were collected from different places in Costa Rica, some of them from local communities that grow the plant, namely Asomat (AS), AromaTica-Fortuna (F1 and F2) and Gema (GE) in the northern part of the country as well as Aprolece-Palacios (P1) in the Caribbean; and others growing in the wild in Los Chiles (L1 and L2) in the northern part of Costa Rica and Sarapiqui (S1) in the Caribbean.Vouchers for all plants are deposited in the Costa Rican National Herbarium, under series no.AQ2953, AQ2959, AQ2968, AQ3246, AQ3331, AQ3332, AQ3508 and AQ3510, respectively.The plant material was separated in its different parts (leaves, stem, bark and wood) and then dried in a stove at 40 ˝C, being turned over every 24 h for a week until totally dry.The dried material was then ground and preserved in plastic recipients.

Extraction of Phenolic Compounds from the Different Parts of U. tomentosa
The dried material from each part of the plant (leaves, stem, bark and wood) was first extracted (0.05 g/mL) in a mixture of methyl ter-butyl ether (MTBE) and methanol (MeOH) 90:10 (v/v) at 25 ˝C during 30 min in ultrasound; subsequently, it was left standing for 24 h to obtain a non-polar extract of the samples.The solvent was removed by filtration and the extraction process was repeated once.The extracts were concentrated to dryness and washed with MeOH to extract residual polyphenols.After the non-polar extraction, the residual material (0.05 g/mL) was extracted with MeOH at 25 ˝C during 30 min in ultrasound and then it was left standing for 24 h.The solvent was removed by filtration and the extraction process was repeated twice.These three methanol extracts were combined with the MeOH washings of the dried non-polar extracts.MeOH was evaporated in a rotavapor at less than 40 ˝C, and the dried extract was then washed with hexane, MTBE and chloroform successively to deliver a polyphenolic rich-extract.

Obtention of Alkaloids Extracts
After the phenolic extraction, NH 3 (20% v/v in MeOH) was added to the residual material and left at 25 ˝C during 30 min in ultrasound and subsequently it was left standing for 24 h.The solvent was removed by filtration.Afterwards, extraction with MTBE was performed at 25 ˝C during 30 min in ultrasound and then it was left standing for 24 h.The solvent was removed by filtration and the MTBE extraction process was repeated once.The extracts were combined and a solution of aqueous tartaric acid (20% v/v) was used three times to extract the alkaloids from the organic solvent.Next, the acidic phase was treated with a saturated solution of sodium carbonate to attain pH = 10, filtrated and the basic solution was extracted three times with MTBE.The organic solvent was then dried with anhydrous sodium sulfate, filtrated and concentrated to dryness in a rotavapor at less than 40 ˝C, to deliver an alkaloid rich-extract.

Determination of Total Phenolic Content
Total polyphenols were determined by a modification of the Singleton and Rossi method [19], which is based on the oxidation of the hydroxyl groups of phenols in basic media by the Folin-Ciocalteu (FC) reagent (mixture of phosphotungstic and phosphomolybdic acids of yellow colour).Briefly, each extract sample was dissolved in MeOH (0.1% HCl) and mixed with 0.5 mL of FC reagent.Then, 10 mL of Na2CO3 (7.5%) and water were added to complete 25 mL.A blank was prepared similarly but with 0.5 mL of MeOH (0.1% HCl) instead of the sample.After standing in the dark for 1 h, absorbance was measured at 750 nm and absorbance values were extrapolated in a gallic acid calibration curve.Values of total phenolic content were expressed as mg gallic acid equivalents (GAE)/g sample.Analyses were performed in triplicate.
3.6.Analysis by 13 C-NMR 13 C nuclear magnetic resonance (NMR) spectra of the samples (25 mg) were recorded in CD 3 OD (0.75 mL) on a Bruker Ascend 400 MHz instrument.
For preparative separations of alkaloids, the same Agilent HPLC-DAD equipment was used with a semi-preparative Synergi Polar RP-80 column (150 mm ˆ10 um), a pre-column Phenomenex 15 mm ˆ10 µm and a solvent system consisting of A: MeOH, B: CH 3 CN and C: NH 4 OAc buffer (pH 5.0) with a linear gradient of A:B:C from 20:20:60 up to 35:35:30 in 35 min at flow of 1 mL/min.NMR analysis (Supplementary material) were performed on samples dissolved in CDCl 3 in a Brucker Ascend 400 MHz instrument.

Statistical Analysis
Principal component analysis (PCA), from standardized variables, was applied to summarize the phenolic data from leaves, stem, bark and wood extracts from the U. tomentosa plant parts using the STATISTICA program for Windows, version 7.1 (StatSoft.Inc., Tulsa, OK, USA, www.statsoft.com).One-way analysis of variance (ANOVA) followed by Tukey's post hoc test was applied to alkaloids content, and differences were considered significant at p < 0.05.

Conclusions
This paper reports valuable information about the phenolic and alkaloid composition of the plant U. tomentosa.Using advanced analytical techniques such as UPLC/TQ-ESI-MS and 13 C-NMR, a total of 32 phenolic compounds have been identified, including hydroxybenzoic acids, hydroxycinnamic acids, flavan-3-ols monomers, procyanidin dimers and trimers, flavalignans, and propelargonidin dimers; these later were reported for the first time in U. tomentosa.Concerning alkaloids, eight main previously-reported tetracyclic and pentacyclic alkaloids were quantified in the extract, as well as the detection of other minor ones.For both phenolic and alkaloid extracts, the part of the plant used (leaves, stems, bark and wood) conditions their composition.In addition, as expected, some variability was observed in the content of phenolic compounds among the different origins/locations of the plants.
The extraction procedure used was successful in obtaining added-value extracts from U. tomentosa.The phenolic extracts obtained, particularly rich in proanthocyanidins, may exert the health benefits attributed to these compounds, mainly in cardiovascular diseases [24,25] due to its anti-platelet properties and its effect in lipids metabolism and vascular function [26].In addition, their content in flavalignans (cinchonains) might be extra value as recent reports on insulinotropic effects of cinchonain 1b suggest their possible use in the treatment of diabetes type 2 [27].Leaves are particularly the part of U. tomentosa plants with the highest feasibility for use in the elaboration of standardizing phenolic extracts in a sustainable way, thus susceptible to application in the food, cosmetic and/or pharmaceutical industries.

Figure 2 .
Figure 2. Plane defined by the two first principal components (PC1 and PC2) resulting from the PCA analysis of total phenolic content (PT, PRO) and individualized phenolic composition of U. tomentosa phenolic extracts (n = 22).

9 Figure 2 .
Figure 2. Plane defined by the two first principal components (PC1 and PC2) resulting from the PCA analysis of total phenolic content (PT, PRO) and individualized phenolic composition of U. tomentosa phenolic extracts (n = 22).

Table 1 .
Extraction yield and total phenolic content of extracts from U. tomentosa.

Table 2 .
Phenolic composition of extracts from bark and leaves of U. tomentosa.

Table 3 .
Phenolic composition of extracts from stems and wood of U. tomentosa.