Sei sulla pagina 1di 14

Journal of Functional Foods 27 (2016) 503–516

Journal of Functional Foods 27 (2016) 503–516 Available online at ScienceDirec t journal

Available online at

ScienceDirec t

journal homepage: www. el s e vi e r. com/lo c a t e/j ff Comprehensive
journal homepage: www. el s e vi e r. com/lo c a t e/j ff Comprehensive

Comprehensive characterization and antioxidant activities of the main biflavonoids of Garcinia madruno : A novel tropical species for developing functional products

A novel tropical species for developing functional products Luis Carrillo-Hormaza a , * , Ana M.

Luis Carrillo-Hormaza a, *, Ana M. Ramírez a , Camilo Quintero-Ortiz a , Marlon Cossio a , Sonia Medina b , Federico Ferreres b , Angel Gil-Izquierdo b , Edison Osorio a, *

a Grupo de Investigación en Sustancias Bioactivas, Facultad de Ciencias Farmacéuticas y Alimentarias, Universidad de Antioquia UdeA, Calle 70 No. 52-21, 050010 Medellín, Colombia

b Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), P.O. Box 164, Campus University Espinardo, 30100 Murcia, Spain


Article history:

Received 28 April 2016

Received in revised form 20

September 2016

Accepted 4 October 2016

Available online


Garcinia madruno


Metabolic profiling

Structure–antioxidant activity



The aim of the study was to determine the qualitative and quantitative plant secondary

metabolite profile of Garcinia madruno by LC-MS n and to evaluate the structure–activity re-

lationship (SAR) with its antioxidant properties. A total of 21 biflavonoids and 3 organic acids

were identified. The leaves were the most promising source of biflavonoids, and the epicarp

was the richest source of morelloflavone type biflavonoids ( > 10%). Morelloflavone and

fukugiside were the major biflavonoids found in all samples as well as the compounds re-

sponsible for antioxidant activity. The inter-flavonoid bond did not increase the antioxidant

activity by single electron transfer (SET) mechanism but increased the activity by single proton-

loss electron transfer (SPLET) mechanism. The results suggest that G. madruno contains

potentially useful amounts of bioactive biflavonoids; in particular, the leaves and epicarp

may be a potential source of functional products.

© 2016 Elsevier Ltd. All rights reserved.



Garcinia is considered the most diverse and bountiful genus of the Clusiaceae family (Jamila, Khairuddean, Khan, & Khan, 2014 ). Phytochemical studies have reported the isolation and identification of biflavonoids, flavonoids, benzophenones, xanthones, and organic acids. As a chemotaxonomic particu- larity, some species of Garcinia overexpress specific metabolites

from one of the mentioned groups. For instance, the benzo- phenones are the most representative secondary metabolites from Garcinia xanthochymus , G. mannii , G. staudtii , and G. subelliptica ( Acuña, Dastmalchi, Basile, & Kennelly, 2012; Hemshekhar et al., 2011); xanthones are found in the fruits of G. mangostana (Wittenauer, Falk, Schweiggert-Weisz, & Carle, 2012); organic acids (specially hydroxycitric acid) are found in the fruits of G. cambogia (Semwal, Semwal, Vermaak, & Viljoen, 2015); and biflavonoids are found in the seeds of G. kola (Ayepola,

* Corresponding authors. Grupo de Investigación en Sustancias Bioactivas, Sede de Investigación Universitaria, Universidad de Antioquia, Carrera 53 # 61-30 Lab 229. 050010 Medellin, Colombia. Fax: + 57 (4) 2196590. E-mail addresses: (L. Carrillo-Hormaza); (E. Osorio)

1756-4646/© 2016 Elsevier Ltd. All rights reserved.


Journal of Functional Foods 27 (2016) 503–516

Cerf, Brooks, & Oguntibeju, 2014 ), the fruits of G. brasiliensis ( Gontijo et al., 2012 ) and the cortex of G. hombroniana (Jamila et al., 2014 ). A compilation of scientific data shows that many of those species have been exploited in pharmaceutical and food fields. In fact, G. mangostana, G. cambogia and G. kola are the most recognized Garcinia species used in nutraceutical, dietary supplements or functional foods products. The use of these species have been reported for the prevention or treat- ment of multiple symptoms and diseases such as ulcers, diarrhoea, hypertension, obesity, inflammatory disorders, hepatic damage, among others, and it has been related mainly to the content of biflavonoids, hydroxycitric acid, xanthones

and benzophenones (Adegbehingbe et al., 2008; Farombi, 2011; Hemshekhar et al., 2011; Saiyed et al., 2015; Tang et al., 2009; Udani, Singh, Barrett, & Singh, 2009; Vasques et al., 2014). Mean- while, G. madruno (Kunt) Hammel (known as “madroño”) is a tropical tree of Central and South America characterized by its exotic yellow fruit with edible pulp of mangosteen-like bitter- sweet flavour (Cury, Abela, Bravo, Peñarrieta, & Rendón, 2012). Previously, we isolated and identified from the leaves some known biflavonoids: amentoflavone, morelloflavone, volkensiflavone, fukugiside and espicataside, as well as madrunoudeaside, a novel acetylglucoside of morelloflavone (Osorio, Londoño, & Bastida, 2013; Osorio, Montoya, & Bastida,

2009 ).

Biflavonoids are characterized as the covalent union of two monomeric units of flavonoids through C—C or C—O—C bonds between flavanone–flavone, flavone–flavone, flavanone– flavanone or flavone–flavanonol ( Ferreira, De Carvalho, & Da Silva, 2012 ). At the same time, those monomers may present several substitutions, giving rise to glycosylated, methylated, sulphated or isoprenylated biflavonoids, among others (Acuña et al., 2010; Ito et al., 2013; Yang et al., 2010). Therefore, a theo- retical number of biflavonoids could exist. However, unlike some of their monomeric constituents, biflavonoids are character- ized by presenting a distribution among nature restricted to some species, highlighting the Ginkgo biloba and some species from the genera Garcinia and Selaginella ( Ferreira et al., 2012; Kim, Park, Son, Chang, & Kang, 2008). The Garcinia biflavonoids generally lead to 3 8 biflavanones or 3 8 flavanone– flavone and carry at least one stereogenic centre but also show atropisomeric behaviour due to restricted rotation about the central axis ( Li et al., 2002; Osorio et al., 2013). Nonetheless, the majority of the reports regarding these biflavonoids involve laborious pre-treatment and methodological approaches based on chromatographic isolations and identification by spectro- scopic techniques. Thereupon, the results obtained from these methodologies, while confirming the presence of a series of metabolites, their limitations and the lack of quantitative strat- egies have not allowed determination of the metabolic profiles in relation to the expression and content of biflavonoids, preventing us from performing qualitative–quantitative com- parisons intra- and inter-species, as well as establishing the main natural sources of these compounds. A number of studies have shown that biflavonoids gener- ally possess a high level of antioxidant activity and relevant pharmacological activities such as being antimicrobial, anti- allergenic, anti-inflammatory, hepatoprotective and antiviral (Ferreira et al., 2012). In relation to the G. madruno biflavonoids, previous studies have shown that these compounds have the

ability to inhibit the lipid peroxidation of the human LDL and to stabilize the DPPH• radical ( Osorio et al., 2009, 2013 ). Morelloflavone has also been associated with hypocholes- terolemic (Tuansulong, Hutadilok-Towatana, Mahabusarakam, Pinkaew, & Fujise, 2011), anti-inflammatory ( Otuki et al., 2011) and atheroprotective (Decha-Dier & Hutadilok-Towatana, 2008; Pinkaew et al., 2009; Pinkaew, Hutadilok-Towatana, Teng, Mahabusarakam, & Fujise, 2012 ) activities. The chemical and antioxidant profiles of G. madruno biflavonoids may point to a better understanding of the role of these substances in physi- ology processes. However, although the functionality of biflavonoids is highly related to their antioxidant capacity, no clear comparison has been made to understand the structural differences among biflavonoids with the antioxidant activity or that compares the activity between biflavonoids and their respective flavonoids monomers. Therefore, the aim of the present study was to provide a comprehensive antioxidant ac- tivity and a qualitative and quantitative characterization of the G. madruno biflavonoids using a metabolomics approach based on HPLC–DAD–MS n and a SAR analysis.

2. Material and methods

2.1. Chemical and reagents

All solvents used were analytical or HPLC grade (Merck Chemi- cals, Darmstadt, Germany). Deionized water was obtained with

a Mill-Q water purification system (Millipore, Bedford, MA, USA). Standard compounds (±)-6-hydroxy-2,5,7,8-tetramethylchromane-

2-carboxylicacid (Trolox), naringenin, apigenin, luteolin, luteolin-

7- O -glucoside, quercetin and ( + )-catechin were obtained

from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The reference standard amentoflavone and biapigenin were pur- chased from TCI Chemical Co. (Tokyo, Japan) and Phytolab (Vestenbergsgreuth, Germany), respectively; whereas morello- flavone, fukugiside and volkensiflavone were isolated from

a biflavonoid fraction previously obtained (Osorio et al., 2013)

using an HPTLC methodology (Camag-Linomat 5, Switzer- land). The chromatography purity of the isolated standard compounds was >97% for each compound. The reagents 2,4,6- tris (2-pyridyl)-s-triazine (TPTZ), fluorescein and 2,2-azobis (2- methylpropionamidine) dihydrochloride (AAPH) were obtained from Sigma Chemical Co. (St. Louis, MO, USA); Na 2 HPO 4 and KH 2 PO 4 were purchased from Carlo Erba reagents (Milan, Italy); and sodium acetate and FeCl 3 were obtained from J.T. Baker (Xalostoc, México).

2.2. Plant material

Five different G. madruno samples were used: (1) leaves, (2) stems, (3) epicarp, (4) mesocarp and (5) seeds. All samples were collected in February of 2014 from trees located on the Antioquia University campus. Samples 1 and 2 were dried at 40 °C for five days in a drying oven, and samples 3, 4 and 5 were frozen with liquid nitrogen and freeze-drying (EYELA Freeze Dried FDU- 1200). All dried samples were mechanically blended until obtaining a homogeneous particle size. Finally, all the pow- dered samples were stored at room temperature, protected from light and moisture.

Journal of Functional Foods 27 (2016) 503–516


2.3. Extraction procedure

For the qualitative, quantitative and antioxidant activity, the powdered G. madruno samples (100 mg) were extracted in a temperature-controlled sonication bath (Elma P60H, Singer, Germany) at a fixed power of 180 W, a frequency of 37 kHz, and an acoustic energy density (AED) of 36 W L 1 . All extractions were carried out at 30 °C using 1.3 mL of an ethanol/water (74:26) mixture for 60 minutes. After extraction, the mixture was cen- trifuged for 5 min at 13.000 rpm at 4 °C. The supernatant was decanted and transferred to a volumetric flask, and the pellet was washed with an additional 500 L of extraction solution. Samples were centrifuged, decanted and transferred to the same volumetric flask. The volume was finally adjusted with water to 2.0 mL. This extract was later diluted, filtered through a 0.45- m nylon membrane and stored in the dark at 20 °C until further analysis.

2.4. HPLC–DAD–ESI–MS n qualitative analysis of

G. madruno samples

Chromatographic analyses were carried out on an Agilent Zorbax SB RRHT ® (Rapid Resolution High Throughput) C 18 (50 mm × 4.6 mm, with a 1.8 m particle size) column. The mobile phase consisted of two solvents: water with 0.1% formic acid (A) and acetonitrile (B). The following linear gradient was used: 0 min, 10% B; 9 min, 28% B; 19 min, 38% B; 22 min, 70% B; 30 min, 90% B; 35 min, 90% B and finally a reconditioning cycle of the column over 5 min with the initial settings for the next analysis. The flow rate was 800 L min 1 , and the injec- tion volume was 5 L. Spectral data from all peaks were accumulated in the range of 240–400 nm, and chromato- grams were recorded at 280 and 335 nm. The HPLC–DAD–ESI/ MSn analyses were carried out in an Agilent HPLC 1200 series equipped with a diode array detector and mass detector in series (Agilent Technologies, Palo Alto, CA, USA). The HPLC con- sisted of a binary pump (model G1376A), an autosampler (model G1377A) refrigerated at 4 °C (G1330B), a degasser (model G1379B), and a diode array detector (model G1315D). The HPLC system was controlled by ChemStation software (Agilent, v. B.01.03- SR2). The mass detector was a Bruker ion trap spectrometer (model HCT Ultra) equipped with an electro-spray ionization interface and was controlled by LCMSD software (Agilent, v. 6.1).The ionization conditions were adjusted at 350 °C and 4.0 kV for capillary temperature and voltage, respectively. The nebu- lizer pressure and flow rate of nitrogen were 65.0 psi and 11 L min 1 , respectively. The full scan mass covered the range from m/z 150 up to m/z 1100. Collision-induced fragmenta- tion experiments were performed in the ion trap using helium as the collision gas, with voltage ramping cycles from 0.3 up to 2 V. Mass spectrometry data were acquired in the negative ionization mode. MS n was carried out in the automatic mode on the more abundant fragment ion.

2.5. HPLC–DAD quantitative analysis of biflavonoids

For the quantification of G. madruno biflavonoids, each extract was analyzed on an analytical HPLC Agilent 1200 Series LC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a vacuum degasser, an autosampler, a quaternary pump and

a Diode Array Detector (DAD). Separation of the compounds

was performed using an Agilent Zorbax SB RRHT (Rapid Reso- lution High Throughput) ® C 18 (50 mm × 4.6 mm, with a 1.8 m particle size) column, with a flow rate of 1.0 mL min 1 at 35 °C. The mobile phase consisted of water with 0.1% acetic acid (A) and acetonitrile (B). The following linear gradient was used:

0 min, 22.5% B; 3.5 min, 22.5% B; 12 min, 50% B; 14 min, 90% B; 18 min, 90% B, and finally a reconditioning cycle of the column during 3 min with the initial settings for the next analy- sis. The injection volume was 2 L. The different compounds were monitored using the DAD set at 280 and 335 nm, and the spectra were recorded between 200 and 400 nm. An external standard method was used for the quantifica- tion of fukugiside, morelloflavone, volkensiflavone and amentoflavone. A stock solution of 1 mg mL 1 in methanol was prepared for each analyte. Consecutive dilutions of the stock solutions in the mobile phase (80% A and 20% B) were ob- tained to provide a linear dynamic range of seven concentration levels for each compound. The content of fukugiside, morelloflavone, volkensiflavone and amentoflavone was ex- pressed as milligrams of compound by gram of dried sample (mg g 1 sample). The limit of quantitation (LOQ) was calcu- lated experimentally according to the values of precision and accuracy but was not necessarily fixed as the lower limit of the dynamic range. For the intra-day precision determina- tion, each matrix was analyzed six times on the same day (n = 6) and the corresponding RSD were calculated. To verify the ac- curacy of the analyses, the method was tested against three calibration levels using the standard references of fukugiside, morelloflavone, volkensiflavone and amentoflavone (n = 9). The relative bias was determined for each level and each compound.


Antioxidant activity


Oxygen radical absorbance capacity (ORAC) value

The ORAC assay was adapted from previously described pro- cedures ( Jiménez et al., 2015 ). AAPH was used as a peroxyl radical generator, and Trolox was used as a standard. More- over, fluorescein was used as the fluorescent probe. Fluorescein, AAPH and the samples were prepared in a 75 mM phosphate buffer at pH 7.4. The samples were mixed with 1.6 M fluo- rescein (1:4) in black 96-well microplates for 30 min at 37 °C. Subsequently, 125 mM AAPH in PBS was added at a 1:5 ratio. Fluorescein intensity was measured every two minutes for 120 min at excitation and emission wavelengths of 485 and 520 nm, respectively, in a Spectra Max Gemini EM – Molecu- lar Devices – (Orleans, USA). The relative ORAC values were calculated using the differences of areas under the decay curves, and the results are expressed as mol equivalents per gram of sample ( mol TE g 1 ) based on a calibration curve with Trolox (12.5–100 mol).

2.6.2. Ferric reducing antioxidant power (FRAP) value

The ability of the samples to reduce the TPTZ–Fe (III) complex was measured according to the method reported by Jiménez et al. (2015 ). The FRAP working solution contained 300 mM of

acetate buffer (pH 3.6), 40 mM of TPTZ and 20 mM of FeCl 3 .6H 2 O

in water at a 10:1:1 ratio (v:v:v). Samples and the working FRAP

solution were mixed at a 1:25 ratio for 10 min at 37 °C in the


Journal of Functional Foods 27 (2016) 503–516

dark. The absorbance was read at 593 nm using an UV/Vis PowerWave TM XS2 spectrophotometer, and it was interpo- lated in a calibration curve with Trolox (31.25–1000 M). The results are expressed in mol Trolox equivalents per gram of sample ( mol TE g 1 ).

2.6.3. Total phenol content (TPC)

Total phenol content was determined by the Folin–Ciocalteu method with some modifications (Jiménez et al., 2015). The re- action mixture contained sample or standard 0.2 N of Folin– Ciocalteu reagent and 7.5% sodium carbonate at a ratio of 25:125:100 ( v:v:v ). After 60 min at room temperature and with protection from light, the absorbance was recorded at 764 nm using an UV/Vis PowerWave TM XS2 spectrophotometer (BioTek USA). The results are expressed in mg gallic acid equivalents per gram of sample (mg EAG g 1 ) based on a calibration curve with gallic acid (10–100 g mL 1 ).

2.6.4. Structure antioxidant–activity relationship of

biflavonoids and related flavonoids

For the SAR study of G. madruno biflavonoids and structurally related flavonoids, a total of 11 compounds (Table 4) were evalu- ated through FRAP and ORAC assay at 100 and 10 M, respectively. The flavonoids used were as follows: (i) G. madruno biflavonoids (morelloflavone, volkensiflavone, amentoflavone and fukugiside; (ii) other biflavonoids (biapigenin [3 8 biapigenina]); (iii) flavonoid monomers of G. madruno biflavonoids (apigenin, naringenin, luteolin and luteolin-7- O - glucoside); and (iv) reference flavonoids (catechin and quercetin).

2.7. Statistical analysis

All the experiments were carried out at least in triplicate, and the results are expressed as the mean ± standard deviations. Statistical analyses were performed by using the Graph Pad Prism® version 5.00 for Windows (Graph Pad Software, Inc.- San Diego CA2007) software. Analysis of variance (ANOVA) was carried out for each variable to test the statistical signifi- cance using a p-value at 5% level.

3. Results and discussion

3.1. Metabolic profile of G. madruno By


Several classes of compounds have been identified in the Garcinia genus, including biflavonoids, flavonoids, benzophe- nones, xanthones, and organic acids. However, only morelloflavone and amentoflavone type biflavonoids have been isolated and identified in G. madruno (Li et al., 2002; Osorio et al., 2013 ). The identification by LC–DAD–ESI–MS n of the main sec- ondary metabolites of G. madruno matrices (leaves, stems, epicarp, mesocarp and seeds) is shown in the Table 1.The com- pound number is based on the elution order shown in Fig. 1. The compounds were tentatively identified based on the in- terpretation of their mass spectral behaviour obtained from MS n experiments and by comparison with the literature. Thus, a total of 26 peaks were identified. The proposed structures of

the identified compounds are shown in Fig. 2. Overall, 3 organic acids and 21 biflavonoids were identified and more than 15 peaks structurally associated with known benzophenones of Garcinia species were detected.

3.1.1. Organic acids

Compound 1 was identified as hydroxycitric acid (HCA) based on its m/z 207 and MS 2 fragments. Compound 2 was identi- fied by MS as the hydroxycitric acid lactone (HCAL) ( Rodríguez-Medina et al., 2009 ). Citric acid (compound 3) was found in all matrices and seems to be the major organic acid of G. madruno, and HCA and HCAL were not found in the seeds. HCA and HCAL have not been previously reported in G. madruno, and this fruit may have been overlooked as an important source of hypocholesterolemic and anti-obesity compounds ( Hemshekhar et al., 2011).

3.1.2. Polyisoprenylated benzophenones

The peaks observed in the last part of the chromatographic profile were associated with polyisoprenylated benzophe- nones (PIBs) ( Fig. 1 ). About twenty peaks associated with PIBs from their molecular ion and fragmentation patterns were found, although more than eighty PIBs have been reported from Garcinia genus ( Kumar, Sharma, & Chattopadhyay, 2013). We were not able to identify PIBs of G. madruno due to the low re- sponse and similarities of the molecular ions and MS n fragments. The main PIBs observed in all G. madruno matri- ces were the compounds 25 and 26 eluting at 27.66 and 31.79 minutes, respectively (Fig. 1). According to the Ms n spectrum, both compounds had the same molecular ion at m/z 601 and similar MS 2 and MS 3 fragments, whereas the UV spectrum and the retention time were pretty different (Table 1). Thus, for the molecular ion of compounds 25 and 26, there are 13 possible structures identified in the Garcinia species so far (camboginol, cambogin, eugeniaphenone, ( ) cycloxanthochymol, garci- nialiptone D, garcinol, guttiferone A, E, F, H o M, isogarcinol or isoxanthochymol) (Kumar et al., 2013).Therefore, further studies using high resolution tandem mass spectrometry and some reference standards are required, taking to account the bio- logical relevance of these compounds (Kumar et al., 2013).

3.1.3. Biflavonoids

The biflavonoids were the most abundant secondary metabo- lites found in G. madruno. These compounds were located in the middle part of each UV chromatogram (Fig. 1) between 7 and 23 minutes. In total, 21 different biflavonoids were iden- tified, ten of them as glycosylated and three as sulphate derivatives. Additionally, eight biflavonoid aglycones were found, one of them methylated and another prenylated. According to the classification of biflavonoids, morelloflavone type biflavonoids were the most abundant group with a total of 10 different com- pounds, followed by GB-a type biflavonoids (8 compounds), amentoflavone type biflavonoids (2 compounds) and GB-1 type biflavonoids (1 compound). Morelloflavone type biflavonoids. Compounds 4, 8, 9,

11, 15, 16, 17, 19, 21 and 22 were identified as morelloflavone type biflavonoids (flavanone-(3 8 )-flavone). Compound 19 (TR = 14.89 min) with a [M-H] ion at m/z 555 was identified as morelloflavone (naringenin-(38)-luteolin) according to its MS n


of Functional Foods 27 (2016) 503–516

Table 1 – Metabolic profile of Garcinia madruno samples by HPLC–DAD–ESI–MS n .




Rt (min) UV (nm)

[M-H] (m/z) LC-ESI/MS n (% base peak)

1 Hydroxycitric acid

Leaves, stems, epicarp and mesocarp.



MS 2 [207]: 188.9 (100), 126.9 (10.3) MS 2 [189]: 126.8 (100), 110.8 (82.9) MS 2 [191]: 126.8 (77.1), 110.8 (100) MS 2 [879]: 717.0 (100), 428.9 (9.9) MS 3 [879 717]: 591.0 (30.86), 565.0 (100), 428.9 (46.3) MS 2 [881]: 719.0 (25.5), 593.0 (46.11), 557.0 (22.9), 431.0 (100) MS 3 [881 431]: 412.9 (16.6), 320.9 (15.52), 294.8 (100) MS 2 [865]: 703.0 (96.8), 597 (26.8), 540.9 (25), 415.0 (100) MS 3 [865 415]: 320.9 (51.6), 309.7 (11.9), 294.8 (100), 268.9 (8.3) MS 2 [799]: 719.0 (100), 593.0 (98), 431.0 (36.25) MS 3 [799 719]: 592.9 (26), 557.0 (12.1), 430.9 (100), 294.7 (6.7) MS 2 [797]: 717.0 (100), 590.9 (21.9), 555.0 (21.2), 428.9 (24.7) MS 3 [797 717]: 591.0 (33.1), 554.9 (24.7), 428.9 (100) MS 2 [717]: 591.0 (65.6), 565.0 (100), 428.9 (64.4) 402.9 (20.9) MS 3 [717 565]: 402.9 (100), 444.9 (13.6) MS 2 [719]: 593.0 (84.0), 557.0 (5.5), 430.9 (100) MS 3 [719 431]: 320.9 (5.8), 294.8 (100) MS 2 [717]: 590.9 (16.8), 555.0 (53.3) 428.9 (100) MS 3 [717 429]: 400.9 (100), 386.9 (46.4), 294.8 (36.4), 249.0 (42.9) MS 2 [573]: 446.9 (100), 428.9 (16.8), 418.9 (22.5) MS 3 [573 447]: 429.0 (36.8), 418.9 (100), 402.8 (7.4) MS 2 [637]: 557.0 (30.3), 430.9 (100), 298.8 (2.8) MS 3 [637 431]: 412.9 (15.2), 320.9 (8.1), 294.8 (100) MS 2 [703]: 540.9 (96.7), 414.9 (100) MS 3 [703 414.9]: 320.8 (8.1), 294.8 (100) MS 2 [635]: 554.9 (100), 428.9 (91.36) MS 3 [635 555]: 448.9 (5.7), 428.9 (100), 402.8 (8.2) MS 2 [701]: 575.0 (15.4), 538.8 (100), 444.8 (19.1), 412.9 (30.4) MS 3 [701 539]: 445.0 (18.3), 432.9 (19.3), 412.9 (100) MS 2 [619]: 538.9 (100), 444.9 (3.6), 413.0 (17.7) MS 3 [619 539]:433.0 (11.6), 412.9 (100) MS 2 [557]: 450.9 (13.7), 430.9 (100) MS 3 [557 431]: 320.9 (8.4), 294.8 (100) MS 2 [555]: 403.0 (6.1), 428.9 (100) MS 3 [555 429]: 400.9 (100), 356.9 (15.2), 294.8 (8.4) MS 2 [541]: 446.9 (15.2), 414.9 (100), 295.8 (7.2) MS 3 [541 415]:320.8 (19.0), 294.8 (100) MS 2 [539]: 444.9 (20.9), 433.0 (11.6), 412.9 (100) MS 3 [539 413]: 384.9 (100), 294.9 (2.8), 264.8 (4.0), 184.8 (3.5) MS 2 [569]: 462.8 (10.5), 442.9 (100) MS 3 [569 443]: 427.9 (100), 410.9 (4.8) MS 2 [537]: 442.9 (14.0), 416.9 (20.21), 398.9 (13.2), 374.9 (100) MS 3 [537 375]: 330.8 (100), 306.9 (11.7), 244.8 (6.2), 188.8 (4.5) MS 2 [605]: 510.9 (11.1), 485.0 (17.6), 467.0 (13.2), 442.9 (100) MS 3 [605 443]: 399.9 (44.2), 387.8 (100), 374.9 (72.9) MS 2 [601]: 557.1 (12), 465.0 (100), 447.1 (28), 409.0 (37), 355.0 (52) MS 3 [601 465]: 327.0 (25), 272.9 (100) MS 2 [601]: 557.1 (12), 465.2 (100), 409.0 (19), 327.0 (26.4) MS3 [601465]: 327.0 (11), 272.9 (100)

2 Hydroxycitric acid lactone

Leaves, stems, epicarp and mesocarp.



3 Citric acid

Leaves, stems, epicarp, mesocarp and seeds.



4 Morelloflavone-4, 7 -di- O-glucoside

Leaves, stems, epicarp and seeds


290; 335


5 GB-2a-di-O-hexoside

Leaves, stems, epicarp and seeds


288; 324sh


6 GB-1a-di-O-hexoside




7 GB 2a-O -hexoside-sulphate



284; 330sh


8 Morelloflavone-7-O -sulphate-4- O-glucoside

Stems, epicarp and seeds


274sh, 290,




9 Fukugiside

Leaves, stems, epicarp, mesocarp and seeds.


255sh, 274sh,



288, 346

10 GB-2a-O-hexoside

Leaves, stems, epicarp, mesocarp and seeds. 10.81

292; 320sh


11 Morelloflavone-4- O -glucoside

Leaves, stems, epicarp, mesocarp and seeds. 11.02

255sh, 275sh,



287, 334

12 GB-2

Mesocarp and seeds


286, 320sh


13 GB-2a-sulphate

Leaves, stems, epicarp, mesocarp and seeds. 11.47

286, 320sh


14 GB-1a-O-hexoside

Leaves and epicarp




15 Morelloflavone-7-O -sulphate

Leaves, stems, epicarp, mesocarp and seeds. 11.80



16 Espicataside

Leaves, stems and seeds.




17 Volkenisflavone-7 -O -sulphate

Leaves, stems, epicarp, mesocarp and seeds. 14.13

270sh, 288,




18 GB-2a

Leaves, stems, epicarp, mesocarp and seeds. 14.49

292, 320sh


19 Morelloflavone

Leaves, stems, epicarp, mesocarp and seeds. 14.89

254sh, 274sh,



288, 345

20 GB-1a

Leaves, stems, epicarp, mesocarp and seeds. 17.30


21 Volkensiflavone

Leaves, stems, epicarp, mesocarp and seeds. 17.55

274sh, 288,




22 3- O-methylmorelloflavone




23 Amentoflavone

Leaves and stems


338, 268


24 Prenylated-amentoflavone

Leaves and stems


334, 270


25 Polyisoprenylated benzophenone

Leaves, stems, epicarp, mesocarp and seeds. 27.66

278, 310sh


26 Polyisoprenylated benzophenone

Leaves, stems, epicarp, mesocarp and seeds. 31.79

250, 354sh



Journal of Functional Foods 27 (2016) 503–516

508 Journal of Functional Foods 27 (2016) 503–516 Fig. 1 – HPLC fingerprint of Garcinia madruno

Fig. 1 – HPLC fingerprint of Garcinia madruno (280 nm). Peak: 126 (see Table 1 ).

fragmentation pattern ( Fig. 3a) and the retention time of the respective reference standard. Regarding the fragmentation route of morelloflavone, the MS 2 product at m/z 429 [(M-H)- 126] was the most abundant fragment found and corresponded to the loss of -C 6 H 6 O 3 by the cleavage of the C-ring in the naringenin monomer at position “a” ( Fig. 3a). Meanwhile, the neutral loss of CO in the C-ring of the naringenin monomer at position “c”, following the cleavage of the C-ring in flavo- noid II at position “d”, were the most representative MS 2 cleavage sites in the fragmentation pathway of morelloflavone

and almost all morelloflavone type biflavonoids ( Fig. 3a and Table 1). In contrast, compound 9 (TR = 9.92 min) showed a dif- ferent MS cleavage site conducted mainly by position “b” (Fig. 3a and Table 1 ). According to its MS n spectra and the ref- erence standards available, this compound was identified as fukugiside, the 7-O-glucoside of morelloflavone. However, com- pound 11 with the same molecular ion [M-H] and UV spectra of the fukugiside was found at 11.02 min, suggesting the pres- ence of another morelloflavone–hexoside. In fact, the MS 2 spectra of both compounds differ significantly, whereas fukugiside showed a fragmentation pathway dependent on “b” site cleavage, compound 11 showed the MS 2 product ion at m/z

555 [(M-H)-162] , resulting from the loss of a hexose and the

representative fragmentation pathway of morelloflavone (Fig. 3a and Table 1 ). Thus, based upon the reports of biflavonoids in Garcinia genus, compound 11 could correspond to

morelloflavone-4 ’- O -glucoside (Ferreira et al., 2012 ). The ESI– MS spectrum of compound 4 (TR = 7.16 min) showed a molecular ion at m/z 879, the MS 2 spectrum had a peak at m/z

717 [(M-H)-162] , showing the loss of a hexose, and the MS 3

spectrum corresponded to the MS spectrum of fukugiside ( Table 1 ). Thus, this compound was tentatively identified as morelloflavone-4 ,7 -di- O-glucoside. Compound 15 (TR = 11.8) with a [M-H] ion at m/z 635 was identified as morelloflavone-7-O-sulphate, a compound previ- ously isolated and identified in G. madruno (Li et al., 2002). The MS 2 spectrum had a peak at m/z 555 [(M-H)-80] , showing the

loss of a sulphate (SO 3 ), and the MS 3 spectrum corresponded to the MS 2 spectrum of morelloflavone. Whereas compound 8 (TR = 8.80 min) with a molecular ion at m/z 797 had a MS 2 spec- trum peak at m/z 717 [(M-H)-80] , showing the loss of a sulphate, and the MS 3 spectrum [797 717] corresponded to the MS 2 spec- trum of morelloflavone-4-O -glucoside (compound 11 ). Thus, this compound tentatively was identified as morelloflavone- 7 - O -sulphate-4 - O -glucoside. Additionally, compound 22 (TR = 18.0 min) with a [M-H] ion at m/z 569 was identified as O-methylated morelloflavone derivate.The MS 2 spectrum found was in accordance with the morelloflavone type biflavonoids frag- mentation pathway (Fig. 3a), showing the major MS 2 fragment at m/z 443 [(M-H)-126] , and the MS 3 spectrum had a peak at m/z 428 [(M-H-126)-15] , showing the loss of a methyl (-CH 3 ). There- fore, taking into account the biflavonoids found in Garcinia genus, compound 22 was tentatively identified as 3 - O -methyl- morelloflavone ( Ferreira et al., 2012 ). Following the same fragmentation pathway of morelloflavone, compound 21 (TR = 17.55) was identified as volkensiflavone (naringenin-(38)- apigenin), a morelloflavone type biflavonoid previously identified in this species ( Li et al., 2002; Osorio et al., 2013 ). In the same way, two known G. madruno volkensiflavone derivates were found. Compounds 16 and 17 (RT = 11.90 and 17.55 min, respectively) were identified as espica- taside (volkensiflavone-7 - O -glucoside) and volkenisflavone- 7 - O -sulphate, respectively ( Li et al., 2002; Osorio et al.,

2013). GB-1a type biflavonoids. Compounds 5, 6, 7, 10, 13, 14,

18 and 20 were identified as GB-1a type biflavonoids (flavanone- (38)-flavanone). According to the biflavonoids found in Garcinia genus, compound 18 (RT = 14.49 min) with a molecular ion at m/z 557 could be the biflavonoid GB-2a (naringenin-(3 8 )- eriodictyol) or GB-1 (naringenin-(3 8 )-dihydrokaempferol). Following the proposed fragmentation pathway of morelloflavone type biflavonoids (Fig. 3a), the MS and the MS 2 spectrum of GB- 2a and GB-1 theoretically will not present significant differences,

Journal of Functional Foods 27 (2016) 503–516


Journal of Functional Foods 27 (2016) 503–516 509 Fig. 2 – Chemical structures of the main

Fig. 2 – Chemical structures of the main compounds detected in Garcinia madruno. *The structure showed is tentative. **The garcinol structure was associated to the compounds 25 and 26 .

as well as the UV spectrum. However, the MS 3 spectrum of this peak showed a characteristic ion fragment at m/z 321 that was associated only with the fragmentation of GB-2a and could be explained by the loss of the B ring of the flavonoid II (eriodictyol) [(M-H-126)-110] (cleavage site “c” Fig. 3b). While this cleavage site is commonly reported for the fragmentation of eriodictyol and dihydrokaempferol (Tsimogiannis, Samiotaki, Panayotou, & Oreopoulou, 2007), applying the GB-2a fragmentation pathway

to GB-1, the MS 3 fragment formed will be at m/z 337, 16 m/z units more than the ion observed. Therefore, based upon GB-2a iden- tification, the fragmentation pathway of GB-1a type biflavonoids is shown in Fig. 3b . Overall, there are few significant differ- ences in comparison with morelloflavone type biflavonoids.The MS 2 products of the aglycones are formed mainly by the same cleavage site of morelloflavone type biflavonoids, correspond- ing to the loss of -C 6 H 6 O 3 by the cleavage of the C-ring in


Journal of Functional Foods 27 (2016) 503–516

510 Journal of Functional Foods 27 (2016) 503–516 Fig. 3 – MS n fragmentation pathway proposed

Fig. 3 – MS n fragmentation pathway proposed for morelloflavone (A), GB-1a (B) and amentoflavone (C) type biflavonoids.

Journal of Functional Foods 27 (2016) 503–516


Journal of Functional Foods 27 (2016) 503–516 511 Fig. 4 – Chromatographic separation with a Rapid

Fig. 4 – Chromatographic separation with a Rapid Resolution High Throughput column by HPLC–DAD for the quantitative analysis. (A) Chromatogram of biflavonoid standard compounds. (B) Comparison of the chromatographic profiles of different G. madruno matrices. Compounds 9 , 19, 21 and 23 correspond to fukugiside, morelloflavone, volkensiflavone and amentoflavone, respectively.

naringenin monomer at position “a” (Fig. 3a and b). Nonethe- Other type of biflavonoids. Compounds 23 and 24 were

bi-flavones, as well as for amentoflavone ( Table 1 ). In addi-

less, position “d” was the most representative MS 2 cleavage site

tion, according to its MS n spectrum, it was tentatively identified

in the fragmentation pathway of GB-a biflavonoids instead of


prenylated-amentoflavone because the fragmentation pattern

position “c” of the morelloflavone type biflavonoids (Fig. 3a). Fol-


this compound was the same as the amentoflavone, adding

lowing the GB-1a fragmentation pathway, compound 20 was identified as GB-1a (naringenin-(38)-naringenin). On the other

a prenylated group in the main fragment found (68 m/z units more) ( Table 1 ). Finally, compound 12 (TR = 11.26 min) with

hand, six derivates of those compounds were found. Com-


[M-H] ion at m/z 573 was tentatively identified as

pounds 5, 6, 10 and 14 were identified as GB-2a-di-O-hexoside,

GB-2 (naringenin-(3 8 )-taxifolin), a GB-1 type biflavonoid

GB-1a-di-O-hexoside, GB-2a-O-hexoside and GB-1a-O-hexoside, respectively. The main MS 2 fragments ions of these com-

(flavanone-(38 )-flavanonol).

pounds were obtained by the loss of -C 6 H 6 O 3 [(M-H)-126] , the


Qualitative and quantitative analysis of

loss of one or two hexoside moiety [(M-H-162) or (M-H)-162-

G. madruno biflavonoids

162] and the successive loss of -C 6 H 6 O 3 and one hexoside moiety [(M-H)-126-162] . Whereas compounds 13 and 7 were identi- fied as GB-2a-sulphate and GB-2a- O -hexoside-sulphate, respectively, and both showed a MS 2 major fragment loss of 80 m/z (SO 3 ) in regard to their molecular ion.

For the quantitative analysis of G. madruno biflavonoids, a shorter RP–HPLC–DAD method was developed using the avail- able reference standards. Under optimized conditions, the best separation of fukugiside (9), morelloflavone (19), volkensiflavone ( 21 ), and amentoflavone ( 23 ) was obtained in 10 minutes (Fig. 4a), assuring an adequate separation for a quantitative ap-

identified as amentoflavone type biflavonoids (flavone-(3′→8)- flavone). Compound 23 (TR = 18.74 min) with a [M-H] ion at m/z 537 was identified as amentoflavone (apigenin-(3 ′→ 8 )- apigenin) according to its MS n fragmentation pattern (Fig. 3c), the retention time and the UV spectrum of the respective ref- erence standard. The MS 2 spectrum of this compound showed ions at m/z 443, 417, 399 and 375 (Table 1).These fragments have already been found in other LC–ESI–MS/MS analysis of amentoflavone (Liao et al., 2015; Wang et al., 2014). In all these cases, the main MS 2 fragment was found at m/z 375, corre- sponding to the loss of —C 9 H 6 O 3 [(M-H)-162] by cleavage at position “a” of the C-ring in one of the apigenin monomers ( Fig. 3c). On the other hand, compound 24 (TR = 23.20) with a [M-H] ion at m/z 605 had the UV spectrum characteristic for

proach of all target biflavonoids in each of the G. madruno samples evaluated (Fig. 4b). The analytical performance of the method is shown in the Table 2 . The range selected was fixed according to the chromatographic response for each biflavonoid with the aim to quantify all analytes in each sample using only one dilution. Good linearity was achieved over the concentra- tion range presented; the four calibration curves exhibited linear regressions of at least r 2 > 0.992 and met the homoscedasticity criteria. The LOQ was determined experimentally according to the parameters of precision and accuracy fixed in a coeffi- cient of variation and relative bias (less than 10%). For all analytes, the LOQ were 0.625 g mL 1 (Table 2). In regard to the repeatability and accuracy of the method, the results ob- tained were satisfactory. The RSD were lower than 10% for each


Journal of Functional Foods 27 (2016) 503–516


Accuracy (%RE, n = 9)

High d






Half c





Low b






Epicarp Mesocarp Seeds









Table 2 – Analytical performance of the developed HPLC–DAD method for the quantification of Garcinia madruno biflavonoids.

G exp a Range ( g mL 1 ) LOQ ( g mL 1 ) Intra-day precision (RSD, n = 6)





Leaves Stems














Cochrans homoscedasticity test, G tables = 0.561. 10.00 g/mL to fukugiside and morelloavone, 1.25 g mL 1 to volkensiavone and amentoavone. 50.00 g/mL to fukugiside and morelloavone, 10.00 g mL 1 to volkensiavone and amentoavone. 200.00 g/mL to fukugiside and morelloavone, 35.00 g mL 1 to volkensiavone and amentoavone.


















Calibration curve r 2

Y = 9.691x 8.254 Y = 6.635x 7.987 Y = 3.671x 0.479 Y = 19.90x 9.850

λ (nm)










analyte in each G. madruno matrix evaluated, and the relative bias was lower than 10% in all levels and compounds evaluated. According to the results, there are significant differences in the chromatographic profiles and content of biflavonoids between the different G. madruno samples (Fig. 1, Tables 1 and 3 ). As a whole, the leaves followed by the stems had a higher diversity of biflavonoids, whereas the mesocarp was the sample with the lowest expression of these compounds. On the other hand, amentoflavone type biflavonoids were not detected in any of the G. madruno fruit samples, and compound 12 was found only in the mesocarp and the seeds. Thus, the pres- ence and absence of those compounds could be identity markers of the G. madruno fruits. Nevertheless, further robust chemometric approaches are required to establish the iden- tity and quality markers between the different G. madruno matrices. Quantitatively (Table 3),morelloflavone and fukugiside were found in higher concentrations than volkensiflavone and amentoflavone in G. madruno samples. Indeed, morelloflavone was the main biflavonoid found in the epicarp, stems and me- socarp, whereas fukugiside was the main biflavonoid found in the leaves and stems. The morelloflavone content ranged between 94.0 ± 4.99 and 1.67 ± 0.14 mg g 1 of the dried samples, decreasing in the order of epicarp > stems > leaves > seeds > me- socarp. In comparison with morelloflavone, the content of fukugiside was lower, ranging from 35.05 ± 0.81 to 6.73 ± 0.29 mg g 1 , decreasing in the order of leaves > epicarp > seeds > stems. In the mesocarp, this compound was detect- able, but the response was lower than the LOQ ( Table 2 ). Volkensiflavone was found in all matrices, and its content ranged from 5.83 ± 0.31 to 0.12 ± 0.01 mg g 1 , with the epicarp and leaves being the main sources of this compound. Finally, amentoflavone was detected and quantified only in the leaves and stems, and its content was the lowest of the biflavonoids evaluated. The sum of the content of biflavonoids was expressed as total percent of biflavonoids ( Table 3). According to this result, the samples ranged from 11.4 to 0.18% (w/w, dry wt.), in which the epicarp was the matrix with the highest content of biflavonoids, fol- lowed by the leaves, stems, seeds and mesocarp. In all cases, the content of morelloflavone plus fukugiside reached values above 90% of the total biflavonoid reported for each matrix. In comparison with the biflavonoid content reported in other species, the quantitative results obtained were highly rel- evant.Within the genus Garcinia, Acuña et al. (2012) determined the content of morelloflavone, volkensiflavone, amentoflavone and fukugiside in fruits of eight different Garcinia species by RP- HPLC-DAD. In general, these authors have reported lower content of biflavonoids. In fact, between the eight species analyzed, G. xanthochymus reached the highest value of biflavonoids (2.5% w/w, dry wt.). Moreover, Anu Aravind et al., using HPTLC, found significant fukugiside content in the leaves of G. travancorica (7% w/w, dry wt.); however, other compounds were not quantified in the study (Anu Aravind, Asha, & Rameshkumar, 2015). In other species of the genus, the information found did not provide quantitative data to be able to make a comparison. Mean- while, in another genus, the scenario was similar. For instance, in Ginkgo biloba, the maximum content of biflavonoids reached was only 1.68% in optimized extracts (Van Beek, 2002); similar

behaviour was found in some Selaginella species (Aguilar et al.,

2013 ) and Hypericum perforatum ( Hosni, Msaâda, Taârit,

Journal of Functional Foods 27 (2016) 503–516


Table 3 – Biflavonoids content, total phenolic content and antioxidant activity of the samples of Garcinia madruno .


Biflavonoids (BF) amount (mg g 1 )









Total BF (%)


26.0 ± 1.05 c

35.00 ± 0.81 d

3.63 ± 0.17 d

2.26 ± 0.10 b

66.96 (6.70) d

44.930 ± 1.41 d 35.076 ± 0.48 c 70.770 ± 0.62 e 11.660 ± 0.13 a

215.40 ± 6.13 d 147.28 ± 3.30 c 327.61 ± 15.20 e 130.74 ± 1.35 b

109,505 ± 2569 d 102,373 ± 1254 c 175,834 ± 6609 e 21,670 ± 1285 a


33.5 ± 1.06 d 6.73 ± 0.29 a 2.83 ± 0.13 c 0.88 ± 0.04 a 43.93 (4.39) c


94.0 ± 4.99 e 1.7 ± 0.14 a 6.6 ± 0.05 b

13.90 ± 0.69 c <LOQ 8.40 ± 0.40 b

5.83 ± 0.31 e 0.12 ± 0.01 a 1.26 ± 0.01 b


113.80 (11.40) e 1.79 (0.18) a





16.29 (1.63) b 19.190 ± 1.24 b 94.053 ± 5.82 a 48,588 ± 373 b

Data are presented as mean ± SD (n = 3). Values within a column followed by the same superscript letters are not significantly different at the 0.05 level according to ANOVA. ND: Not detected.

a Expressed as mg Galic acid equivalents g 1 of sample.


b Expressed as mol Trolox equivalents g 1 of sample.

c Expressed as mol Trolox equivalents 100 g 1 of sample.

Hammami, & Marzouk, 2010). The comparison of these results

with those of previous researchers is difficult due to the differ- ences in the nature of the sample and pre-concentration technologies, extraction systems, and assay methodologies. However, taking to account that the total biflavonoid content found in G. madruno is underestimated by the compounds iden- tified as biflavonoids but not quantified (Fig. 1 and Table 1), our results propose that G. madruno is one of the sources with a high biflavonoid content in nature; specifically, the leaves and the epicarp will reach a biflavonoid content comparable with some species recognized by their high concentrations of flavonoid, such

as the content of total catechins reported in some varieties of

tea (more of 20% of total catechins, w/w, dry wt.).

3.3. Antioxidant activity of G. madruno samples

A single antioxidant method can provide basic information

about antioxidant properties, but a combination of methods can describe the antioxidant properties of the samples in more detail ( Jiménez et al., 2015 ). In this study, a set of anti- oxidant assays was performed to fully characterize the antioxidant potential of G. madruno . Table 3 shows the TPC content and the ORAC and FRAP value of G. madruno matri- ces. The decreasing order obtained was the same for the three

assays (epicarp > leaves > stems > seeds > mesocarp), as well as the total percent of biflavonoids, indicating a high correla- tion between antioxidant activity and biflavonoid content (Table 3). As a whole, epicarp and leaves were the matrices with the highest antioxidant activity. The results obtained in the ORAC assay were higher in comparison with the values re- ported in the database of fruits recognized by its scavenger potential, such as blackberries, blueberries, pomegranate and apples ( Speisky, López-Alarcón, Gómez, Fuentes, & Sandoval-Acuña, 2012 ).

3.3.1. Structure antioxidant–activity relationship (SAR) of

biflavonoids and structurally related flavonoids

Table 4 shows the results obtained for the SAR study of G. madruno biflavonoids and structurally related flavonoids. The single electron transfer (SET) and the single proton-loss elec- tron transfer (SPLET) were the antioxidant mechanism evaluated through FRAP and ORAC assay, respectively ( Firuzi, Lacanna, Petrucci, Marrosu, & Saso, 2005; Zhang et al., 2013 ). Regarding

the SET mechanism, the FRAP value obtained increased in the following order: naringenin < apigenin < amentoflavone

< biapigenin < volkensiflavone < catechin < morelloflavone

< luteolin-7- O -glucosyde < fukugiside < luteolin and querce-

tin. The compounds naringenin, apigenin, amentoflavone, biapigenin and volkensiflavone had a ferric reducing poten- tial comparable to the reference compound used (Trolox). Meanwhile, catechin, morelloflavone, luteolin-7- O -glucoside, fukugiside, luteolin and quercetin had at least twice the ac- tivity of Trolox. Regarding the flavonoid monomers, quercetin, followed by luteolin and catechin, were the compounds with the highest activity, whereas naringenin had the lowest re- ducing capability. These results are in agreement with other reports and confirm that the combination of the catechol group in the B ring, the 3-hydroxy group, the 2,3-double bond and the 4-oxo function in the C ring are the functional groups that give the major contribution to effectively reducing the complex [Fe-(TPTZ) 2 )] 3+ (Firuzi et al., 2005). On the other hand, the results showed that the activity of the biflavonoids is conducted mainly for just one of its monomers. According to the ANOVA analy- sis ( Table 4 ), the flavonoid–flavonoid binding of biapigenin, amentoflavone and volkensiflavone had a negligible synergis- tic effect in comparison with their respective flavonoid monomers (apigenin and naringenin; p -value > 0.05). Indeed, an antagonistic effect was observed for the naringenin– luteolin binding of morelloflavone, significantly decreasing the activity of this compound in regard to the most active monomer (luteolin, p-value < 0.05). This could be explained by a steric im- pediment promoted by the naringenin monomer, which decreases the effective reducing capability of luteolin against the complex [Fe-(TPTZ) 2 )] 3+ . In the SPLET mechanism, the results obtained were significantly different ( Table 4 ). The ORAC value obtained for all compounds were at least 3-fold more than the activity found by Trolox, in which luteolin-7- O -glucoside was the flavonoid with the lowest activity, followed by volkensiflavone < amentoflavone < apigenin < querce- tin < luteolin < naringenin < morelloflavone < catechin < fukugiside and biapigenin. According to these results, the main struc- tural requirements for an effective reducing capability previously observed does not seem to be the functional groups that give the major contribution and explain the peroxyl radical- scavenging activity of flavonoids and biflavonoids. In fact, under


Journal of Functional Foods 27 (2016) 503–516

Table 4 – Structure–antioxidant activity relationship (SAR) of G. madruno biflavonoids and related flavonoids by SPLET and SET mechanism.


Functional groups


Antioxidant activity

R 1

R 2

R 3

R 4

R 5

R 6

R 7

SPLET ORAC value* SET FRAP value*

Naringenin (Na) Apigenin (Ap) Amentoflavone (Am) [Ap-(3′→ 8 )-Ap] Biapigenin (Bia) [Ap-(38)-Ap] Volkenisflavone (Vo) [Na-(3 8 )-Ap] (+ )-Catechin (Ca) Morelloflavone (Mo) [Na-(3 8 )-Lu] Luteolin 7- O glycoside (Lu 7G) Fukugiside (Fu) [Na-(38 )-Lu 7G] Luteolin (Lu) Quercetin (Q)


H H H Ap (C 8 ) Ap (C 8 ) OH Lu (C 8 ) H Lu 7G (C 8 ) H OH






6.408 ± 0.206 d 5.198 ± 0.359 b 5.063 ± 0.056 b 11.070 ± 0.158 g 4.948 ± 0.302 b 8.226 ± 0.293 e 7.898 ± 0.088 e 2.863 ± 0.056 a 9.511 ± 0.134 f 5.819 ± 0.222 c 5.358 ± 0.224 b

0.942 ± 0.014 a 0.939 ± 0.039 a 0.953 ± 0.017 a 1.027 ± 0.026 ab 1.112 ± 0.021 b 2.181 ± 0.087 c 2.373 ± 0.093 d 2.661 ± 0.085 e 2.914 ± 0.055 f 3.160 ± 0.131 g 3.766 ± 0.013 h




















































Data are presented as mean ± SD (n = 3). Values within a column followed by the same superscript letters are not significantly different at the 0.05 level according to ANOVA. DB = double bond. * Expressed as mol Trolox equivalents/ mol compound.

this mechanism, naringenin instead of luteolin was the flavo- noid monomer of G. madruno biflavonoids with the highest activity found, whereas the biflavonoids were more active than the flavonoid monomers, specifically the flavonoid–flavonoid binding of biapigenin, morelloflavone and fukugiside, which sig- nificantly enhances the activity in regard to its respective monomers, promoting a synergistic effect that was not ob- served by SET mechanism (Table 4). Nevertheless, a structure– activity relationship based just in the functional groups applicable to biflavonoids and related flavonoids could not be performed with our results. Thus, a quantum chemical analysis focused on structural and kinetic parameters of flavonoids and biflavonoids is required for a better understanding of the peroxyl radical-scavenging activity of these compounds by the SPLET mechanism (Zhang et al., 2013).



This study allowed to characterize for the first time the chemi- cal profile of G. madruno by HPLC–DAD–ESI/MS n . The results obtained are the basis for future quality control and identity chemical marker analysis for this species. Undoubtedly, our results propose G. madruno as a prominent species of the Garcinia genus on the expression of highly valuated metabolites. The high content of biflavonoids, antioxidant activity, and the pres- ence of hydroxycitric acid found mainly in the leaves and epicarp could be the starting point for developing functional ingredients for the food and pharmaceutical industries.


The authors thank the Comité para el Desarrollo de la Investigación (CODI) of the University of Antioquia for the fi- nancial support (Cod: CPT-1219). Luis Carrillo-Hormaza is grateful to the National Doctoral Program of COLCIENCIAS (Cod:

647) for provision of a doctoral fellowship.


Journal of Functional Foods 27 (2016) 503–516




Journal of Functional Foods 27 (2016) 503–516