SciELO - Scientific Electronic Library Online

 
vol.21 número3Evaluación de la actividad antiproliferativa de extractos metanólicos de plantas de la familia leguminosaeOptimización de las variables de extracción de flavonoides a partir de hojas de Annona muricata L. índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados

Articulo

Indicadores

  • No hay articulos citadosCitado por SciELO

Links relacionados

  • No hay articulos similaresSimilares en SciELO

Compartir


Revista Cubana de Plantas Medicinales

versión On-line ISSN 1028-4796

Rev Cubana Plant Med vol.21 no.3 Ciudad de la Habana jul.-set. 2016

 

ARTÍCULO ORIGINAL

 

Chemical properties and assessment of the antioxidant capacity of native species from the genus Ugni

 

Características químicas y evaluación de la capacidad antioxidante de especies del género Ugni

 

 

Marcia Avello Lorca,I Edgar Pastene Navarrete,I Andrés Barriga,II Magalis Bittner Berner,III Eduardo Ruiz Ponce, III José Becerra AllendeIII

I Faculty of Pharmacy, University of Concepción. PO Box 237, Concepción, Chile.
II Mass Spectrometry Unit, Faculty of Chemical and Pharmaceutical Sciences, University of Chile.
III Faculty of Natural and Oceanographic Sciences, PO Box 160-C, Concepción, Chile.

 

 


ABSTRACT

Introduction: Ugni molinae Turcz., Ugni candollei Barn. (Berg) and Ugni selkirkii (Hook. et Arn.) Berg are Chilean species that share morphological characteristics and they are distributed in geographic locations with very diverse habitats. Its is considered important for the characterization of the Chilean flora to determine if there are similarities in the sort of chemical compounds among species with close morphological relations, growing in different habitats and their consequent biological activity.
Objective: to assess the chemical composition and the antioxidant capacity of leaf extracts from the Chilean species of the genus Ugni, and to compare with the U. molinae characteristics.
Methods: composition of chemical compounds was determined by chromatographic methods (HPLC-ESI-MS). The antioxidant capacity was assessed by DPPH, ABTS and by stabilization of the hydroxyl radical.
Results: as expected, given the great morphological similarity existing among the three species of Ugni that grow in Chile, similarities were found in their chemical composition. Nevertheless, it was also expectable to find variations among them. Thus, U. candollei and U. selkirkii are the species that present greater content and variety of phenolic and terpenic compounds. These species exert greater antioxidant capacity in comparison toU. molinae. U. candollei hightlights for its flavonoid content such as glycosides and quercetin derivatives, and the species U. selkirkii, is important in galotannins. U. molinae is characterized in ellagic acids derivatives.
Conclusion: these data and the morphological characteristics could become a useful toll in order to determine the closeness degree among these species.

Keywords: antioxidant capacity; chemical properties; genus Ugni.


RESUMEN

Introducción: Ugni molinae Turcz., Ugni candollei (Barn.) Berg y Ugni selkirkii (Hook. et Arn.) Berg son especies chilenas que comparten características morfológicas y se distribuyen en lugares geográficos con muy diversos hábitats. Se considera importante para la caracterización de la flora chilena determinar si hay similitudes en el tipo de compuestos químicos entre especies con relaciones morfológicas cercanas, que crecen en diferentes hábitats y su actividad biológica consecuente.
Objetivo:
evaluar la composición química y la capacidad antioxidante de extractos de hojas de las especies chilenas del género Ugni, y compararlas con las características de la especie U. molinae.
Métodos: la composición química se determinó por métodos cromatográficos (HPLC-ESI-MS). La capacidad antioxidante se evaluó por los métodos DPPH, ABTS, y la estabilización del radical hidroxilo.
Resultados:
como era de esperar, dada la gran similitud morfológica existente entre las tres especies de Ugni que crecen en Chile, se encontraron similitudes en su composición química. Sin embargo, también era esperable encontrar variaciones entre ellas. Por lo tanto, U. candollei y U. selkirkii son las especies que presentan mayor contenido y variedad de compuestos fenólicos y terpénicos. Estas especies ejercen una mayor capacidad antioxidante en comparación con U. molinae. U. candollei se caracteriza por su contenido en flavonoides como quercetina glucósidos y sus derivados, y la especie U. selkirkii, por la presencia de galotaninos. U. molinae se caracteriza por contener derivados del ácido elágico.
Conclusión:
estos datos y las características morfológicas podrían convertirse en una herramienta útil para determinar el grado cercanía entre estas especies.

Palabras clave: capacidad antioxidante; género Ugni; propiedades químicas.




INTRODUCCIÓN

Genus Ugni Turczaninow belongs to the family Myrtaceae and comprises between 19 and 21 taxa located from Mexico, Central America, Venezuela, to Chile and Argentina.1 The species of this genus can be shrubs or small trees, with perennial leafs and cultivated for both their ornamental importance and the attractiveness of their fruits. From all species, three grow in Chile: Ugni molinae Turcz, Ugni candollei (Barn.) Berg and Ugni selkirkii (Hook. et Arn.) Berg. These plants are shrubs distributed in Central Chile from the Maule Region to the Chiloe Island, including Juan Fernandez Archipelago.

Ugni molinae Turczaninow. This native plant is widely known by its local name "murtilla". It is distributed from the Maule Region to the Chiloe Island, including Juan Fernandez Archipelago and is also found in Río Negro and Neuquén, in Argentina. This species grows mainly near the coast, in both wet and dry environments, in the edge of forests or in rocky areas. U. molinae is an evergreen shrub, small in drought conditions and it can reach 2 m in zones with high rainfall. Fruits of U. molinae are useful to relief circulatory disorders and for improving visual acuity, especially in the night. U. molinae leaves have been used by aboriginal people as stringent for treating diarrheas and dysenteries, as infusions.2

Ugni candollei (Barnéoud) Berg. This endemic species is known as "white murta". It is commonly distributes from Valdivia to Chiloe and it has also been described for Maule Region. It normally grows in the coastal zone. U. candollei is an evergreen shrub that can reach up to 2 m in height. No popular uses are known for this shrub.2

Ugni selkirkii (W. J. Hooker et Arnott) Berg. This species is endemic from Robinson Crusoe island (Masatierra Island), in Juan Fernandez Archipelago. It can reach 2 m height and it is pubescent in branches and young shoots3. No popular uses are known for this shrub. The current population trend is marked by a notorious declining, mainly due to the strong competition with expanding species that cover its habitat.3-5

Although no evolutionary studies on the group are known, given their morphological similarity it seems that these species are closely related. Because of such relation, they could share similar chemical compounds. There is only knowledge on the chemical composition of U. molinae, species assessed respect to its biological activity.6-9 These authors have described the presence of phenolic substances such as phenolic acids and flavonoids, compounds with renowned antioxidant capacity, as well as compound of terpenic kind, such as pentacyclic triterpenic acids.

As U. molinae shares morphological characteristics with species of the genus that grow in Chile, U. candollei and U. selkirkii, probably share chemical properties and consequently develop similar biological activity. Therefore, the aim of this study is to assess the chemical composition and the antioxidant capacity of the leaf extracts of the Chilean species of the genus Ugni, U. candollei and U. selkirkii, and to compare with the U. molinae characteristics.10

 

Methods

1. Vegetal material

Biological material from the native species of the genus Ugni was collected in blooming season (November-March 2008-2009): U candollei, Los Ríos Region (Valdivia, Oncol Park, Oncol hill); U. selkirkii, Juan Fernandez Archipelago (Robinson Crusoe Island, Selkirk viewpoint, Portezuelo hill). Species were identified by the taxonomist Dr. Roberto Rodríguez, at the Faculty of Natural and Oceanographic Sciences, University of Concepción. A specimen from each species was recorded at the CONC herbarium (125465 and 162345, respectively).

2. Preparation of extracts

Biological material was dehydrated in the shade, at room temperature and the size was reduced in blade mill. For obtaining the extracts, 50 grams of the grinded sample in a Soxhlet apparatus were processed. The following solvents were successively used: hexane, chloroform, ethyl acetate and methanol, until exhaustion of vegetal material. The relation mass solvent was 1:6. Every extract was concentrated in evaporator and taken to dryness in liophilizer. Recognition reactions of secondary metabolites were carried out for every extract (Shinoda reaction for flavonoids recognition, FeCl3 reaction for tannin recognition and foam test for saponin recognition). Samples were stored in a dry place and protected from light until their utilization. For chemical and biological determinations, the extracts obtained with methanol were selected, due to their content of phenolic compounds and saponins.

3. Chromatographic analysis

The analyses of leaf extracts from the Ugni species were carried out by means of HPLC, according to Cho et al.11. For the analysis, a LC-MS system (Agilent Technologies Inc., Palo Alto, CA, USA) was used. This system is equipped with binary pumps, an online degasifier, automatic injector and a UV-VIS detector. UV traces were registered at 280 nm. Separation of phenolic compounds was carried out by means of a Zorbax Eclipse column XDB-C18 150 × 4.6 mm, 5 µm and 80 Å (Agilent Technologies Inc., CA-USA). Injection volume was 20 µL, with a flow of 1.0 mL/min. Solvent system was composed of the solvent A (double distilled water containing 0.1% formic acid v/v) and the solvent B (acetonitrile containing 0.1% formic acid). The gradient system was as follows: 0-5min, 5% B; 5-50 min, 100% B; 50-55 min, 100% B; 55-57.5 min, 100-5% B; and 57.5-60 min, 5% B. LC/MS detection was carried out immediately after the UV-VIS measurements. Analyses were carried out by means of Bruker Esquire 4000 (Bruker Daltonik, GmbH, Germany) ions trap ESI-IT mass spectrophotometer, operating under the following ion optics: capillary temperature, 225 ºC; capillary voltage, 5.7 kV; cone voltage, 35 V and voltage spray 2.8 kV. Nitrogen was used as nebulizer gas (pressure: 30 psi, temperature, 35 ºC) and drying gas (10 L/min). Products from mass spectra were recorded in a range of m/x 50-1500 in both positive and negative mode. Data were collected by means of the Esquire Control 5.2 software and processed by means of Data Analysis 3.2 software (Bruker Daltonics Esquire 5.2, Bruker Daltonik GmbH). Instrument parameters were optimized in a routine prior to the analysis of extracts.

4. Antioxidant capacity

Was carried out according to Joyeux et al.12, stabilization of the DPPH radical; Ghiselli et al.13, stabilization of the ABTS radical; Halliwell et al.14, stabilization of the hydroxyl radical (OH). The antioxidant capacity was expressed as IC50, which is defined as the final required concentration of the sample to reach 50% of the inhibition of the radical. As standard, gallic acid and Trolox® (Merck, Germany) were used. Three repetitions per extract were carried out.

5. Statistical analysis

All determinations were carried out in triplicate. Mean values and standard deviations (±SD) were calculated. Statistical tests were carried out in order to analyze correlations between values. Variance analysis (ANOVA) was used and differences were considered as significant at P < 0.01.

 

Results

U. candollei and U. selkirkii are species with high concentration of total polyphenols in the extracts obtained with methanol. U. candollei presents the greatest levels, including total flavonoids (0.070 ± 0.002 g EQ/g dry matter). With respect to the content of total saponins, no significant differences among species were observed, reaching a maximum of 0.015 g saponins/g dry matter in U. selkirkii.

Composition by chromatographic analysis

Tentative identification of the main phenolic and triterpenic compounds in the extracts of U. candollei (Uc) and U. selkirkii (Us) was carried out by means of HPLC-ESI-MS. The allocation of peaks was carried out by means of the fragmentation pattern analysis and its comparison with mass spectra from both standards and literature. Retention times and m/z in negative polarity are presented for the respective compounds in Table 1, and the chromatograms in Figure 1. Spectral properties were obtained from those signals considered more intense and pure. The positive mode was also used to confirm the identification of some compounds.

Methanolic extracts (table 1, Fig 1).

 

 Table 1. HPLC-MS of methanolic extracts

Peak

tR

(min)

lmax (nm)

[M-H]-

MS/MS Ions

Tentative identification

Species

 

1

2

3

 

4

 

5

6

7

8

 

9

 

10

11

12

 

13

 

14

15

16

 

17

18

 

19

 

20

21

22

23

 

24

25

26

 

27

28

29

30

31

32

 

33

34

35

36

37

38

39

40

41

42

43

44

45

46

 

47

48

 

2.4

2.8

3.1

 

4.7

 

4.9

5.1

5.4

5.5

 

12.3

 

12.8

13.1

13.6

 

13.9

 

14.0

14.1

14.3

 

14.5

14.6

 

14.9

 

15.1

15.2

15.6

15.9

 

16.1

16.1

16.4

 

16.6

16.9

17.9

20.7

20.8

23.2

 

23.6

25.9

27.1

29.1

29.1

31.2

31.6

33.8

34.7

35.2

36.0

36.4

40.9

41.3

 

41.6

41.8

295,272,240

 

280

 

280

 

 

280

 

 

326,295,240

 

326,295,240

 

272

326,295,240

 

 

 

280

 

 

280

 

280

374,254

 

 

260

 

 

 

356,300,267

 

355,255

260

 

 

 

374,254

356,300,267

 

 

280

 

 

 

260

 

355,254

355,254

 

355,254

 

 

367,256

 

317,253

 

317,253

 367,256

 367,256

260

 356,280

 260

365

 354,254

198

 194

 198

 198

198

194

 194

 194

 198

194

194

194

194

194

194

 

 

343.2

271.1

633.0

 

633.3

 

353.7

665.2

451.4

706.5

 

887.3

 

635.3

635.3

927.3

 

943.5

 

615.4

871.5

943.6

 

449.2

615.3

 

787.4

 

927.4

927.3

595.4

595.5

 

867.6

551.3

551.4

 

895.4

898.8

521.2

301.1

595.5

329.3

 

696.3

1007.9

813.7

555.2

975.3

487.3

679.6

649.9

749.0

701.6

1268.5

951.7

748.9

685.2

 

712.9

685.2

190.9; 168.8

168.9; 124.9 

300.8; 274.9; 248.9

 

300.8; 275.0; 249.0

 

190.8

352.9; 190.8

405.0; 168.8

352.9; 190.8

 

442.9; 300.8; 270.8; 248.9

464.9; 312.9

465.0; 482.9; 313.2

462.9; 316.9; 298.9; 270.9

470.9; 330.9; 270.8

 

462.9; 300.9

434.9; 303.0; 285.0

471.0; 331.0; 270.9

 

316.0

462.9; 300.9

 

643.9; 617.1; 465.0; 447.9

463.0; 316.0

463.0; 301.0

432.9; 300.9

432.8; 300.8; 178.9

 

433.0; 300.1

533.0; 507.1; 312.9

532.9; 438.9; 270.8

 

447.0; 300.9; 271.0

450.9; 340.9; 300.7

330.9; 270.8; 210.9

272.8; 178.8; 150.8

330.8; 270.8; 211.0

293.0; 229.0; 210.9; 171.0

649.3; 487.1; 315.9

503.2

633.3

487.2

485.3; 439.2

455.2

633.3

631.3; 605.7

712.5

655.3; 633.4

951.0; 633.7

633.6

712.5

639.3; 617.4

 550.4; 532.3

639.2; 617.3

Galloylquinic acid

Galloyl glucopiranose derivative

Hexahydroxydiphenoil galloyl glucose

Hexahydroxydiphenoil galloyl glucose isomer

Caffeoilquinic acid

Caffeoliquinic acid derivative

Gallate derivative

Caffeoliquinic acid

derivative

Hexahydroxydiphenoil galloyl

glucopiranóside derivative

Trigalloyl glucose

Trigalloyl glucose isomer

Myricetin

Myricetin monomethylether derivative

Quercetin hexose galate

Quercetin pentoside

Myricetin monomethylether derivative isomer

Myricetin pentoside

Quercetin hexose galate isomer

 

Tetragalloyl glucose

 

Myricetin deoxihexoside

Quercetin hexoside

Quercetin hexosidopentóside

Quercetin hexosidopentoside isomer

Pentoside ellagic acid

Methoxy flavone derivative

Metoxi flavona derivative isómero

Rhamnoside ellagic acid

Elagitannin

Myricetin monomethyl ether

Quercetin

Myricetin monomethyl ether

Decursin coumarin

 

Rhamnetin derivative

Madecassic acid

Oleanolic glycoside acid

Asiatic acid derivative

Madecassoside

Trihydroxyursenoic acid

Oleanoic acid glycoside

Saponin derivative oleanolic acid

Oleanolic acid glycoside

Oleanolic acid glycoside

Prosaponin oleanane

Prosaponin oleanane

Oleanolic acid glycoside

Cumaroilic derivative  of maslinic acid

Oleanolic acid glycoside

Cumaroil derivative of the maslinic acid isomer

Uca, Usb

Us

Us

 

Us

 

Uc

Uc

Us

Uc

 

Us

 

Us

Us

Uc

 

Us

 

Uc

Uc

Us

 

Us

Uc

 

Us

 

Us

Uc

Uc

Uc

 

Uc

Us

Us

 

Uc

Uc

Uc

Uc

Us

Uc

 

Uc

Us

Us

Uc

Us

Uc

Us

Us

Uc

Uc

Us

Us

Us

Us

 

Uc

Us

                                                                                            aUc: Methanolic Extract U. candollei.
                                                                         bUs: Methanolic Extract U. selkirkii.

 Tannins: It must be taken into account that the most important compounds of certain species from the genus Ugni (U. selkirkii and U. molinae) derive from gallic and ellagic acids. Phenolic acids derived from quinic and caffeic acids are also reported for the species U. candollei and U. selkirkii.

Peak 1 (Uc, Us, tR = 2.4 min), was tentatively identified as galloylquinic acid, because it presented [M-H]- de m/z 343.2 and its fragmentation provided ions of m/z 190.9 and 168.8, corresponding to quinic acid and gallic acid, respectively.

Peak 2 (Us, tR = 2.8 min), was tentatively identified as a derivative from galloylglucopiranose, resulting a [M-H]- of m/z 271, a ionic fragment observed when a cross-ring fragmentation of the glucose ring is observed in the galloyl glucopiranose derivative. Ion fragments in MS2 in m/z 168.9 and 124.9 confirm the sequential loss and decarboxylation of the gallic acid.

Peaks 3 and 4 (Us, tR = 3.1 min, 4.7 min), they have [M-H]- of m/z 633, which are dissociated in order to form m/z 302 hexahydroxidiphenoil (HHDP) galloyl glucose (ellagitannin), through the loss of 332 amu, which indicated the presence of a unit of galloyl glucose. This compound is known as sanguine H4 or sanguine H5, depending where the galloyl unit is attached.

Peak 5 (Uc, tR = 4.9 min), has a [M-H]- of m/z 353.7, with a MS2 fragment of m/z 190.8. This suggests that the peak corresponds to 3-O-caffeoilquinic.

Peak 6 (Uc, tR = 5.1 min) and Peak 8 (Uc, tR = 5.5 min), have ion fragments [M-H]- of m/z 353.7, with a MS2 fragment of m/z 190.8, suggesting that both peaks are other forms of caffeloil quinic acid derivatives.

Peak 7 (Us, tR = 5.4 min), the presence of an MS2 ionic fragment of m/z 168.8 suggests that this compound is a galloyl derivative.

Peak 9 (Us, tR = 12.3 min), it has an [M-H]- parental peak of m/z 887.3 and ionic fragments of m/z 300.8, suggesting that this compound is a derivative from HHDP-galloyl glucopiranose or an ellagitannin.

Peak 10 (Us, tR = 12.8 min) and Peak 11 (Us, tR = 13.1 min), were identified as trigalloyl glucose derivatives, with [M-H] of m/z 635.3.

Peak 19 (Us, tR = 14.9 min), has an [M-H]- of m/z 867.6 with MS2 ionic fragments of m/z 617.1, 643.9 and 465. They are exactly equal to those reported for tetragalloyl glucose.

Peak 24 (Uc, tR = 16.1 min), exhibited a [2M-H]- of m/z 867.6. The pseudo molecular ion fragment was of m/z 300.1 (loss of a sugar of 132 units). This suggests that this compound is an ellagic acid pentoside.

Peak 27 (Uc, tR = 16.6 min), exhibited a [2M-H]- of m/z 895.4, with a fragment of the pseudomolcular ion of m/z 450.9 (tertgalloic acid) and its deprotonized ion aglycon ion of m/z 300.9 (loss of one sugar of 146 units). This suggests that this compound is an ellagic acid rhamnoside.

Peak 28 (Uc, tR = 16.9 min), exhibits a [2M-H]- of m/z 898.9 with a pseudo molecular ion of m/z 450.9 (tertgalloic acid) and its deprotonized aglycon ion of m/z 300.1 (ellagic acid). Consequently, it was speculated that this compound corresponded to a ellagitannin.

Flavonoids: In the extracts from the three species, the presence of several flavonoids derivatives of myricetin and quercetin were observed. This coincides with the previously reported for the species U. molinae. In our study it must be noted that quercetin derivatives were predominant in the continental species, whereas in the samples from U. selkirkii collected in the Island, myricetin glycosides predominate. Furthermore, by means of HPLC-ESI-MS, we were able to identify galloylated forms of quercetin and glycosylated myricetin. As seen in Table 1, flavonoids from the different species of the genus Ugni elute after 12 minutes.

Peak 12 (Uc, tR = 13.6 min), had a [2M-H]- of m/z 927.3 and the pseudo molecular ion with a m/z 462.9. Its deprotonized aglycon ion of m/z 316.9 (loss of one sugar of 146 units) is consistent with a myricetin deoxyhexoside. Fragmentation pattern and retention time of myricetin rhamnoside (mirictrin) coincides with this compound.

Peak 13 (Us, tR = 13.9) and Peak 16 (Us, tR = 14.3 min), have a [2M-H]- of m/z 943.5 and the pesudomolecular ion of m/z 470.9. The deprotonized aglycon of m/z 330.9 is consistent with a myricetin monomethyl ether derivative.

Peak 14 (Uc, tR = 14.0 min) and Peak 18 (Uc, tR = 14.6) have a [M-H]- m/z of 615.3. MS2 ionic fragments of m/z 426.9 (loss of 152amu from a galloyl unit) and its deprotonized aglycon ion of m/z 300.9 (loss of one sugar of 162 units) are consistent with quercetin hexoside galate isomers.

Peak 15 (Uc, tR = 14.1), has a [2M-H]- of m/z 870 and the pseudo molecular ion of m/z 433. The deprotonized aglycon of m/z 301 (loss of one sugar of 132 units) is consistent with a quercetin pentoside.

Peak 17 (Us, tR =14.5 min), exhibits a [M-H]- of m/z 449.2, with its deprotonized aglycon ion of m/z 316 (with a loss of a sugar of 132 units), suggesting it is a myricetin pentoside (arabinoside).

Peak 20 (Us, tR =15.1 min), exhibits a [2M-H]- de m/z 927.4. Pseudo molecular ion was of m/z 463 with its deprotonized aglycon ion of m/z 316 (loss of one sugar of 146 units), suggesting that this compound is another myricetin deoxihexoside.

Peak 21 (Uc, tR =15.2 min), exhibits a [2M-H]- of m/z 927.3. A pseudo molecular ion of m/z 463 and a deprotonized aglycon ion of m/z 301 (sugar loss of 162 units). This suggests that this compound is a quercetin hexoside.

Peak 22 (Uc, tR = 15.6 min) and Peak 23 (Uc, tR = 15.9 min) have a pseudo molecular ion of m/z 595.4. In MS2, peaks produced ionic fragments of m/z 432.9 (loss of sugar of 162 units) and its deprotonized aglycon ion of m/z 302 (loss of sugar of 132 units). This suggests that these compounds are quercetin pentosides hexoside.

Peaks 25 and 26 (Us, tR = 16.1 min, 16.4 min) exhibit a pseudo molecular ion [M-H]- of m/z 551.3. The ionic fragment of m/z 313 also suggests that these compounds are methoxyisoflavones isomers derivatives. The absence of other features does not allow a better allocation.

Peak 29 (Uc, tR = 17.9 min) and Peak 31 (Us, tR = 20.8 min) produced ionic fragments of m/z 331, 271 and 211. These fragments are typical of a mono-galloyl-glycoside derivative. However, the loss of 190 amu from the pseudo molecular ion of m/z 521 in order to form the ionic fragment of m/z 331 could be assigned to the loss of methylglucuronic acid from monoethylether myricetin. The low intensity of the signals does not allow a better allocation.

Peak 30 (Uc, tR = 20.7 min) produced a pseudo molecular ion of m/z 301.1 and MS2 fragments of m/z 272.8 (loss of 28 amu from a carbonyl group), 178.8 (retrocyclation after the fission over the bond 1 and 2) and 150.8 (a ring fragment via RDA) that coincide exactly with those produced by quercetin in negative mode.

Peak 33 (Uc, tR = 23.6 min), produced a pseudo molecular ion of m/z 6963.3. MS2 fragments of m/z 649.3 (loss of a sugar of 162 units) and 315.9 (loss of 170 amu from water and gallic acid) only allow suggesting that this compound is a methyl quercetin (rhamnetin) derivative.

Other phenols

Peak 32 (Uc, tR = 23.2 min) produced a pseudos molecular ion of m/z 329.3 [M-H]-. MS2 ionic fragments of m/z 229 and 210.9 are consistent with coumarin decursin or decursinol angelate.

Glycoside triterpenic: In all studied extracts the presence of pentacyclic triterpenic derivatives was observed by means of HPLC-ESI-MS. Taking into account the main fragments, those compounds are derived from oleanoic, ursenoic acid, asiatic acid, madecassic acid and maslinic acid. The most abundant are those derived from oleanoic acid. Some of these compounds were previously reported in U. molinae. Different isomers and conjugated forms of these compounds elute between 27 and 42 min and in a zone corresponding to a complex mixture of saponins. For these substances, only in a few cases it was possible to assign an identity. In order to elucidate an exact structure, additional experiments (NMR) and the comparison to standards become necessary.

Peak 34 (Us, tR = 25.9 min), produced a [2M-H]- of 1007.8 and a pseudo molecular ion of m/z 503.2, consistent with hydroxyasiatic acid (madecassic acid) or trihtdroxyoleanolic acid.

Peak 35 (Us, tR = 27.1 min), contains the pseudo molecular ion [M-H]- of m/z 813.7, with an MS2 ionic fragment of m/z 633.3 (loss of one hexose and water). Other fragments observed in positive polarity suggest that this compound is a glycoside of oleanolic acid. Peak 39 (Us, tR = 31.6 min), Peak 42 (Uc, tR = 35.2) also presented the MS2 ionic fragment of m/z 633.3.

Peak 36 (Uc, tR = 29.1 min), contains the pseudo molecular ion [M-H]- of m/z 555.2, with a MS2 ionic fragment of m/z 487.2, consistent with a derivative of the asiatic acid, arjunolic acid, trihydroxyoleanolic acid or trihydroxiursenoic acid.

Peak 37 (Uc, tR = 29.1 min), contains the pseudo molecular ion [M-H]- of m/z 975.3, with a MS2 of m/z 485.3 and 439.2. These data and those observed in positive mode suggest that it corresponds to a madecasoside.

Peak 40 (Us, tR = 33.8 min) also contain the fragment m/z 649.9, as well as the pseudo molecular ion of the ion fragments MS2 of m/z 605.7 and 631.3. By means of 1H and 13C NMR and hydrolysis, this structure could be unequivocally identified.

Peak 38 (Uc, tR = 31.2 min), contains the pseudo molecular [M-H]- of m/z 487.3. All data were identical to those from trihydroxiursenoic acid.

Peak 41 (Uc, tR = 34.7 min), Peak 45 (Us, tR = 40.9 min) and Peak 47 (Uc, tR = 41.6 min), produced some common fragments of m/z 712.9, 532.3 and 550. Ionic fragment of m/z 457 was observed in positive polarity. These compounds are glycosides from oleanolic acid. Their structure must be elucidated by means of NMR analysis.

Peak 43 (Us, tR = 36.0 min) and Peak 44 (Us, tR = 36.4 min), have in common the ionic fragment of m/z 633. The fragment has been observed in several oleanane prosaponins. A loss of 176 amu (glucuronic acid) forms an ionic fragment of m/z 457. In fact, this fragment is observed in positive mode.

Peak 46 (Us, tR = 41.3 min) and Peak 48 (Us, tR = 41.8 min) produced a pseudo molecular ion of m/z 685.2 and MS2 fragments of m/z 639.3 ND 617.4, suggesting that they are cumaroilic derivatives of the maslinic or alphytolic acid.

 

Scavenging capacity of free radicals

The scavenging capacity of free radicals was investigated in the extracts obtained with methanol from the species. This study was carried out by means of several methods that study the stabilization of free radicals by donation of hydrogen atoms, or by electron transfer with later proton donation. table 2 indicates the scavenging capacity of free radicals. U. candollei capacity is more important, and it is related to the polyphenols and total flavonoids contents (correlation coefficient equal to 0.881, 0.776 and 0.863 for the method DPPH, ABTS and OH, respectively p<0.05).

DISCUSSION

Hitherto, it is interesting to make a relation respect to the collection place of the samples. All species were collected over the sea level, in volcanic soils and close to the sea. U. candollei was collected in the Oncol hill, at 715 m above sea level. The hill belongs to the Oncol Park which is a private conservation park, located in the Municipality of Valdivia, in Los Ríos Region, distant 29 km from the city. This species concentrates the highest levels of both polyphenols and flavonoids. Factors that could contribute to this phenomenon are constituted by exposure to radiation, the high environmental humidity and the constant rainfall15,16. Authors 17,18 have described the participation of these factors on the biosynthesis of phenolic metabolites. Similarly, U. selkirkii was collected at the top of the Selkirk viewpoint, located in the Portezuelo hill, at 565 m above sea level, in Juan Fernandez Archipelago.

Chemical properties of U. selkirkii, collected in Juan Fernandez, are probably due to the weather characteristics, such as high humidity and the volcanic origin of the place19. The high content of iron and aluminum oxyhydroxides, with deficiency in the supply of nitrogen, phosphorous, potassium and sulfur favors the biosynthesis of both phenolic compounds and terpenoids, such as saponins19-21. What differentiates island and mainland soils, also volcanic, is that the latter are special at surface level and proximity to the craters. This would explain more important levels of certain nutrients, as well as the lack of other. Humidity, soil characteristics and radiation could influence on the genetic characteristics that determine the phenotypic characteristics of the species, because the adaptive response to natural habitat conditions makes the functions of these compounds become more important, as described by Vivanco20, Davies & Schwinn21 and Vermerris22. Finally, U. molinae was collected in valleys from the Biobío Region.10

These backgrounds, could explain chemical differences between species. Compounds identified from the extracts such as flavonoles, as well as gallic and ellagic derivatives can exert the scavenging capacity of free radicals through hydroxyl groups and their electronic stability23.

As expected, it was possible to determine similarities and variations in the chemical properties among the species of the genus Ugni that inhabit in Chile; U. candollei and U. selkirkii are the species that present greater content and variety of phenolic and terpenic compounds (pentacyclic triterpenic saponins derivatives from oleanoic acid, mostly). These species exert greater antioxidant capacity in comparison to U. molinae10. On the other hand, U. candollei exceeds because of its flavonoids content, such as glycosides and quercetin derivatives, whereas U. selkirkii is notorious in gallotannins. U. molinae is characterized in ellagic acids derivatives10. These data and the morphological characteristics could constitute as important evidences for evolutionary studies of these species.

 

ACKNOWLEDGMENTS

This work was supported by CONYCIT Grant 2409106; Postgraduate Council, University of Concepción, DI Grant 210.074.043; Ring Project ADI-38. Ring ACT-38; Project Our Flora from Chile, CONAF, Chilean Navy, Oncol Park.

 

REFERENCES BIBLIOGRAPHIC

1. Mabberley DJ. The Plant Book. Cambridge University Press. England; 1997.

2. Hoffmann A. Flora Silvestre de Chile, Zona Araucana. Fundación Claudio Gay. Chile; 1991.

3. Danton PH. Plantas Silvestres de la Isla Robinson Crusoe, Guía de Reconocimiento. Orgraf Impresores. Chile; 2004.

4. Cuevas J, Marticorena A, Cavieres LA. New additions to the introduced flora de of the Juan Fernández islands: Origin, distribution, life history traits and, potential of invasion. Rev Chil Hist Nat. 2004;77(3):523-38.

5. Bernadello G, Anderson GJ, Stuessy TF, Crawford D. The angiosperm flora of the Juan Fernández archipiélago (CHILE): Origin and dispersa. Canadian J Botany. 2006;48(1):1266-81.

6. Suwalsky M, Orellana P, Avello M, Villena F. Protective effect of Ugni molinae Turcz against oxidative damage of human erythrocytes. Food Chem Toxicol. 2007;45(1):130-35.

7. Avello M, Valdivia R, Mondaca MA, Ordóñez JL, Bittner M, Becerra J. Actividad de Ugni molinae Turcz. frente a microorganismos de importancia clínica. BLACPMA. 2009;8(2):141-44a.

8. Avello M, Valdivia R, Sanzana R, Mondaca MA, Mennickent S, Aeschlimann V, Bittner M, Becerra J. Extractos a partir de berries nativos para su uso como preservantes naturales en productos cosméticos. BLACPMA. 2009;8(6):479-86b.

9. Rubilar M, Jara C, Poo Y, Acevedo F, Gutierrez C, Sineiro J, Shene C. Extracts of maqui (Aristotelia chilensis) and murta (Ugni molinae Turcz): Sources of antioxidant compounds and α-glucosidase/ α-amylase inhibitors. J Agric Food Chem. 2011;59(5):1630-37.

10. Avello M, Pastene E, Barriga A, Bittner M, Ruiz E, Becerra J. Chemical properties and assessment of the antioxidant capacity of leaf extracts from populations of Ugni molinae growing in continental Chile and in Juan Fernández archipelago. IJPPR. 2014;6(4):746-52.

11. Cho MJ, Howard LR, Prior RL, Clark JR. Flavonoid glycosides and antioxidant capacity of various blackberry, blueberry and red grape genotypes determined by high-performance liquid chromatography/mass spectrometry. J Sci Food Agric. 2004;84(13):1771-82.

12. Joyeux M, Mobstein A, Anton R, Mortier F. Comparative antilipoperoxidant antinecrotic and scavenging properties of terpenes and biflavones from Ginkgo and some flavonoids. Planta Med. 1995;61(1):126-29.

13 . Ghiselli A, Nardini M, Baldi A. Antioxidant activity of different phenolics fractions separated from italian wines. J Agric Food Chem. 1998;46(2):363.

14 . Halliwell B, Gutteridge JMc, Aruoma OI. The deoxyribose method: a simple "test-tube" assay for determination of rate constants for reactions of hidroxyl radical. Anal Biochem. 1987;165(1):215-19.

15. Avello M, Pastene E, Bustos E, Bittner M, Becerra J. Variation in phenolic compounds of Ugni molinae populations and their potential use as antioxidant supplement. SBFgnosia. 2013;23(1):44-50.

16. Ku KM, Choi JN, Kim J, Kim JK, Yoo LG, Lee SJ, Hong YS, Lee CH. Metabolomics analysis analysis reveals the compositional differences of shade grown tea ( Camellia sinensis L.). J Agric Food Chem. 2010;58(1):418-26

17. Oh M, Trick H, Rajashekar C. Secondary metabolism and antioxidants are involved in environmental adaptation and stress tolerance in lettuce. J Plant Physiol. 2009;166:180-91.

18. Kovacik J, Klejdus B, Backor M, Repcak M. Phenylalanine ammonia-lyase activity and phenolic compounds accumulation in nitrogen-deficient Matricaria chamomilla leaf rosettes. Plant Sci. 2007;172(2):393-99.

19. Giorgi A, Mingozzi M, Madeo M, Speranza G, Cocucci M. Effect of nitrogen starvation on the phenolic metabolism and antioxidant properties of yarrow ( Achillea collina Becker ex Rchb.). Food Chem. 2009;114(1):204-11.

20. Vivanco J, Cosio E, Loyola V, Flores H. Mecanismos químicos de defensa en las plantas. Investigación y Ciencia. 2005;341(1):68-75.

21. Davies K, Schwinn K. Molecular Biology and Biotechnology of Flavonoid Biosynthesis in Flavonoids Chemistry, Biochemistry and Applications. U.S: Taylor & Francis Group, LLC. ; 2006.

22. Vermerris W, Nicholson R. Phenolic Compound Biochemistry. Dordrecht. The Netherlands: Springer; 2006.

23. Niki E. Assessment of antioxidant capacity in vitro and in vivo. Free Radical Bio Med. 2010;49(1):503-05.

 

 

Recibido: 22 de noviembre de 2015.
Aprobado: 22 de abril de 2016.

 

 

Creative Commons License Todo el contenido de esta revista, excepto dónde está identificado, está bajo una Licencia Creative Commons