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Polyphenols from Adansonia DigitataExtraction, antioxidant analysis and total phenols contentSILVIA VERTUANI1,2*, EMANUELA SCALAMBRA1, SONIA MOLESINI2, LISA BUZZONI2, ELISA DURINI1, GIANNI SACCHETTI3, STEFANO MANFREDINI1,2**Corresponding authors1. University of Ferrara, Dept. of Pharmaceutical Sciences, via Fossato di Mortara 17-19, Ferrara, 44100, Italy2. University of Ferrara, Ambrosia lab, via Mortara 171, Ferrara, 44121, Italy3. University of Ferrara, Dept. of Biology and Evolution, Sect. Agro-technological and Pharmaceutical Resources (Agri Unife), C.so Ercole I d’Este 32, Ferrara, 44100, Italy

Peer-reviewed scientific article

Stefano ManfrediniSilvia Vertuani

KEYWORDS: Adansoni Digitata, antioxidant activity, polyphenols, red fibre.

ABSTRACT: With the aim to confirm important nutritional properties, we here report the results obtained from baobab plant tissues analysis (ORAC, DPPH, FRAP). The micronized red fibre display the highest antioxidant activity, among the tested samples in all performed assays; methanolic extracts of this fibre (ME and HME) were prepared and tested in comparison to selected plant extracts by ORAC, DPPH, FRAP and PCL assays, furthermore the total phenols content of ME and HME was measured by Folin-Ciocalteu analysis and characterized by RP-HPLC. HME extract revealed higher antioxidant activity than Blueberry extract (1 percent) in all performed texts and comparable antioxidant activity to that of Pomegranate extract (40 percent) in ORAC and DPPH assay; these results are probably due to the high total phenols content (708 ± 14.2mgGAE/g) of the extract.

INTRODUCTION

$QWLR[LGDQWV�� EHFDXVH� RI� WKHLU� EHQHÀ�FLDO� HIIHFW� RQ� KHDOWK�issues (1-8).and their involvement in prevention of oxidation reactions of foods, pharmaceuticals and cosmetics, represent an interesting study target. Currently to counteract oxidation reaction in foods and cosmetics synthetic compounds, like butylatedhydroxyanisole (BHA) and butylatedhydroxytoluene (BHT), are commonly used. However, with regard to the safety of these synthetic antioxidants the literature, even GRFXPHQWLQJ� FRQÁ�LFWLQJ� RSLQLRQV�� RIWHQ� UHSRUWV� VLGH� HIIHFWV�(9-10). These evidences along with the attention of the consumers, increasingly focused on safe and possibly natural antioxidants, have fostered the research on plant sources. In this context baobab (Adansonia Digitata L.) a deciduous tree, of Bombacaceae family, native of central Africa or other tropical climates, has attracted our interest since the year 2000. Baobab produces a nut-type fruit internally splitted into VPDOO�Á�RXU\��GHK\GUDWHG�DQG�SRZGHU\�VOLFHV�LQFOXGLQJ�PXOWLSOH�VHHGV� DQG� À�ODPHQWV�� WKH� UHG� À�EUH�� ,Q� $IULFD�� EDREDE� IUXLW� LV�used as food or natural refreshing drink; leaves, bark and fruits of baobab are differently used for medicinal purposes (11, 12). ,Q�������DOVR�EDVHG�RQ�RXU�HIIRUWV�RQ� WKLV� À�HOG�� WKH�(XURSHDQ�Union has approved baobab fruit as a food ingredient under WKH� (8·V� IRRG� OHJLVODWLRQ�� UHFRJQL]LQJ� EHQHÀ�W� RI� EDREDE�consumption (13). Studies prompted from the ethno-pharmacological usage of baobab, suggest that antioxidant compounds in baobab have a pivotal role in determining EHQHÀ�FLDO�HIIHFWV�RQ�KXPDQ�KHDOWK�����������,Q�WKLV�UHJDUG��ZH�have recently reported the preliminary results of a study in which several baobab constituent have been evaluated by photochemiluminescence (PCL) for their radical scavenging properties against superoxide anion, in comparison with common fruit species with recognized antioxidant power (16). Almost all the investigated baobab products were featured of high integral antioxidant capacity (IAC), and in case of pulp fruit also of high vitamin C concentration (16). The observation that, notwithstanding the high ascorbic acid content, when the fruit was left opened to the air the pulp remained whitish IRU�ORQJ�WLPH��ZKHUHDV�WKH�SDOH�EURZQ�À�EUH�QHWZRUN�LQ�WKH�QXW�

became rapidly reddish, suggested us the presence of other VDFULÀ�FLDO�FRPSRQHQWV� WKDW�PD\�DFW�DV�QDWXUDO�SUHVHUYDWLYHV�toward ascorbic acid oxidation. Taking these suggestions, we started the present investigation with the aim to provide further evidences to the antioxidant properties of the plant by the application of PCL complementary analysis methods such are, DPPH, ORAC and FRAP assays. After that, we directed RXU� DWWHQWLRQ� WR� WKH� VWXG\� RI� UHG� À�EUH�� EHFDXVH� RI� LWV� KLJK�DQWLR[LGDQW� DFWLYLW\�� 0HWKDQROLF� H[WUDFWV� RI� WKLV� À�EUH� ZHUH�prepared and next assayed by Folin-Ciocalteu method and DQWLR[LGDQW�DQDO\VLV��À�QDOO\�WKH�GLIIHUHQW�SRO\SKHQROV�SUHVHQW�LQ�WKH�H[WUDFWV�ZHUH� LGHQWLÀ�HG�E\�53�+3/&�FRXSOHG�ZLWK�GLRGH�array detector and quadrupole mass spectrometer.

MATERIALS AND METHODS

6DPSOHV�RI�%DREDE�IUXLW�UHG�À�EUH�ZHUH�VXSSOLHG�IURP�%DREDE�Fruit Company Senegal, Thies, Senegal. Other study samples were supplied by A.C.E.F., Fiorenzuola d’Arda, Italy. Reactants, solvents and standards samples were purchased from Sigma-Aldrich, Milan, Italy and Carlo Erba Reagenti, Milan, Italy. PCL Kits (ACL) were purchased from Analitik Jena AG, -HQD�� *HUPDQ\�� 7UROR[� >�6��������K\GUR[\���������WHWUDPHWK\O�chroman-2-carboxylic acid] (no. 39,192-1) was purchased from Sigma-Aldrich, Taufkirchen, Germany.Samples absorbance measurement (DPPH, FRAP and Folin-Ciocalteu assays) were carried out on a UV-VIS spectrophotometer ThermoSpectronic Helios g, Cambridge, United Kingdom.

Extraction proceduresMethanolic Extract (ME)�� ��� J� RI� PLFURQL]HG� UHG� À�EUH� ZDV�PL[HG�ZLWK�����P/�RI�PHWKDQRO�XQGHU�DUJRQ�DWPRVSKHUH�IRU���K�DW� URRP�WHPSHUDWXUH��7KH�VDPSOH�ZDV�WKHQ�À�OWHUHG�DQG�the pellet subjected to two further extractions. The obtained supernatants fractions were combined dried under vacuum DW� ORZ� WHPSHUDWXUH� ��� ���&��� 7KH� DFKLHYHG� PHWKDQROLF�H[WUDFW��0(��DPRXQW�ZDV��RQ�DYHUDJH��WKH�������SHUFHQW�RI�the starting material.

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concentration and the absorbance was measured by a VSHFWURSKRWRPHWHU�89�9,6�DW�����QP��,&�� values expressed DV�ǍJ�P/�ZHUH�GHWHUPLQHG�E\�OLQHDU�UHJUHVVLRQ�DQDO\VLV�RI�the results obtained at different sample concentrations.

3) Oxygen Radical Absorbance Capacity (ORAC ) assay. This test was carried out on a Fluoroskan FL® ascent �7KHUPR� )LVKHU� 6FLHQWLÀ�F�� ,QF��:DOWKDP��0$�� IROORZLQJ� D�SURFHGXUH�PRGLÀ�HG�E\�XV� �����DQG�EDVHG�RQ�D�ZRUN�RI�+RQJ�HW�DO�� ������%ULHÁ�\�� LQ� WKH�À�QDO�DVVD\�PL[WXUH������P/�WRWDO�YROXPH���Á�XRUHVFHLQ�VRGLXP�VDOW�����Q0��ZDV�XVHG�DV�a target of free radical attack with 2,2’-azobis(2-amidino-propane) dihydrochloride (AAPH) as a peroxyl radical generator. Trolox was used to achieve a calibration curve. The tested compounds were dissolved in a phosphate buffer solution (pH=7,4) and prepared immediately before WKH�H[SHULPHQWV��7KH�Á�XRUHVFHQFH�PHDVXUHPHQWV��FDUULHG�RXW�DW����&�ZHUH� UHFRUGHG�DW���PLQ� LQWHUYDOV�XS����PLQ�after the addition of AAPH. The ORAC values, calculated as difference of the areas under the quenching curves RI� Á�XRUHVFHLQ� EHWZHHQ� WKH� EODQN� DQG� WKH� VDPSOH�� DUH�expressed as µmol of Trolox per gram of product.

4) Ferric reducing ability of plasma (FRAP) assay. The ferric UHGXFLQJ�DELOLW\�ZDV�PHDVXUHG�DFFRUGLQJ�WR�D�PRGLÀ�HG�protocol described by Guihua and co-workers (23). Samples were dissolved in the selected solvent (water and/or methanol). The reagent for analysis was freshly prepared by mixing the following solutions in the reported ratio 10/1/1 (v:v:v) i) 0.1 M acetate buffer pH 3.6, ii) TPTZ 10 mmol/L in 40 mmol/ HCl, iii) ferric chloride 20 mmol/L. To a 1.9 mL of reagent were added 0.1 mL of sample extract or solvent when blank was performed. Readings at the DEVRUSWLRQ�PD[LPXP������QP��ZHUH�WDNHQ�DIWHU����PLQ��using a UV-VIS spectrophotometer. Trolox solution was used to perform the calibration curves. The FRAP values DUH�H[SUHVVHG�DV�ǍPRO�HTXLYDOHQWV�RI�7UROR[�SHU�JUDP�RI�product.

Statistical evaluationsThe analysis were carried out in three replicates, average and VWDWLVWLFDO� VLJQLÀ�FDQFH� �6WXGHQW·V� W� WHVW�� 3������� ZHUH� JLYHQ� IRU�all data collected. One-way ANOVA and LSD post hoc Tukey’s KRQHVW� VLJQLÀ�FDQW� GLIIHUHQFH� WHVW�ZHUH� XVHG� IRU� FRPSDULQJ� WKH�bioactive effects of different samples. All computations were made using the statistical software STATISTICA 6.0 (StatSoft Italia srl).

HPLC AnalysisA Shimadzu (SCL-10Avp) liquid chromatography system equipped with diode array detector (DAD) SPD-M10Avp and quadrupole mass spectrometer (Shimadzu LCMS QP8000a) with an APCI interface was used. Separation was performed on a ����[�����PP�����ƒ��3KHQRPHQH[�+\GUR�53�FROXPQ�DW����&��$�JUDGLHQW� FRQVLVWLQJ� RI� VROYHQW� $� �DFHWLF� DFLG� ����� SHUFHQW� LQ�ZDWHU��DQG�VROYHQW�%��PHWKDQRO��ZDV�DSSOLHG�DW�D�Á�RZ�UDWH�RI�

HydrolizedMethanolic Extract (HME)��� J� RI�0(�ZHUH� WUHDWHG�with 100 mL of sodium hydroxide solution (4 N) and keep on stirring for 60 minutes at room temperature. The mixture was WKHQ�DFLGLÀ�HG�ZLWK�K\GURFKORULF�DFLG����SHUFHQW�XQWLO�S+���DQG�then extracted for three times with ethyl acetate. The organic fractions were combined, dried on magnesium sulphate DQK\GURXV�D�À�QDOO\�WDNHQ�WR�GU\QHVV�XQGHU�vacuum. Hydrolized PHWKDQROLF�H[WUDFW��+0(��DPRXQW�ZDV��RQ�DYHUDJH��WKH�������percent of the starting material.

3XULÀ�FDWLRQ�RI�H[WUDFWV2 g of the extract (ME or HME) were solubilized in methanol, absorbed on silica gel (Macherey Nagel 0,63-0,20 mm (70-230 PHVK��DQG�SXULÀ�HG�RQ�D�JODVV�FROXPQ���[����FP��À�OOHG�ZLWK�����J�of silica gel. Elution was performed using a linear gradient of dichloromethane/methanol from 10/0 to 8/2, v/v. The most VLJQLÀ�FDQW�IUDFWLRQV��$��%��&��'��(���DV�HPHUJHG�E\�+3/&�DQDO\VLV��were obtained as follows. Fraction A collecting fractions (400 mL) during the elution of 100 percent dichloromethane (10/0 dichloromethane/methanol); fraction B collecting fractions (400 mL) during the elution of the 9.8/0.2 dichloromethane/methanol mixture; fraction C collecting fractions (400 mL) during the elution of the 9.6/0.4 dichloromethane/methanol mixture; fraction D collecting fractions (400 mL) during the elution of the 9/1 dichloromethane/methanol mixture; fraction E collecting fraction (400 mL) during the elution of the 8/2 dichloromethane/methanol mixture.

Folin-Ciocalteu assayAll the extracts were evaluated for polyphenols content by the Folin-Ciocalteu method (17).Gallic acid was used as standard WR� REWDLQ� D� FDOLEUDWLRQ� FXUYH�� $� ���� P/� DOLTXRW� RI� ZDWHU�GLOXWHG�)ROLQ�&LRFDOWHX�UHDJHQW��������ZDV�DGGHG�WR�WKH�GLOXWH�H[WUDFWV�����P/���7KH�PL[WXUH�ZDV�LQFXEDWHG�IRU���PLQ�DW�URRP�temperature and 300 mL of sodium carbonate solution (200 g/L) was added. The mixture was then incubated for further 90 min at room temperature and the absorbance was measured DW� ���� QP� E\� D� 89�9,6� VSHFWURSKRWRPHWHU�� DJDLQVW� D� EODQN�similarly prepared, containing distilled water instead of extract. The results are expressed as milligrams of gallic acid equivalent SHU�JUDP�RI�UHG�À�EUH��PJ�*$(�J�RI�À�EUH���

Antioxidant capacity assaysVegetable materials and extracts were submitted to the following methods in order to determine their antioxidant activity. 1) Photochemiluminescence (PCL) assay. This analysis

involves the photochemical generation of superoxide free radical (O2) combined with chemiluminescence detection (18). The PCL method was carried out as described by Popov and Lewin (19); it can be conducted by two different protocols ACW and ACL, which permit the measurement, respectively, of the antioxidant capacity of the water- and lipid-soluble components; in this study, measurements were conducted using the ACL protocol. An exact quantity of Adansonia Digitata extract or fruit extract samples was accurately solubilized in methanol or methanol/water 1/1 and properly diluted with Reagent 1 of ACL. A 2.30mL portion of reagent 1 (solvent and dilution UHDJHQW��� ����P/�RI� UHDJHQW��� �EXIIHU� VROXWLRQ��� ���P/�RI�UHDJHQW� ��SKRWRVHQVLWL]HU��� DQG� IURP� �� P/� WR� ��� P/� RI�standard trolox solution 0.1mM (to obtain the calibration curve) or sample solution were mixed and measured by means of Photochem®�� 5HVXOWV� DUH� H[SUHVVHG� DV� ǍPRO�equivalents of Troloxper gram of product.

2) 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH) assay. It was performed according to the method described by Wang and co-workers (20). To a DPPH methanolic solution ����� P/�� ZDV� DGGHG� ������ P/� RI� H[WUDFWV� DW� GLIIHUHQW�

����[�����PP�����ƒ��3KHQRPHQH[�+\GUR�53�FROXPQ�DW����&��$�JUDGLHQW� FRQVLVWLQJ� RI� VROYHQW� $� �DFHWLF� DFLG� ����� SHUFHQW� LQ�ZDWHU��DQG�VROYHQW�%��PHWKDQRO��ZDV�DSSOLHG�DW�D�Á�RZ�UDWH�RI�

Baobab.

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Antioxidants

temperature, the best performance was achieved at room temperature. In fact, at higher temperature, even shorting the times (20 min), a decrease in the amount of total phenolos was observed, probably due to partial degradation of these molecules. Unexpectedly, the use of ultrasounds, in order to VKRUWHQ�H[WUDFWLRQ�WLPH�DQG�WR�HQKDQFH�\LHOG��GLG�QRW�UHÁ�HFW�D�

VLJQLÀ�FDQW�HIÀ�FLHQF\�LPSURYHPHQW��A second extract was then prepared submitting ME to a basic hydrolysis, this extract was also analysed by the Folin-Ciocalteu assay; the result evidenced, as expected, that the total polyphenols in hydrolysed extracts were in higher amount than in non-hydrolysed ones (Table 3 HME: 708 mgGAE/g; MRE: 383 mgGAE/g). The ME and the HME extracts were also analysed for the radical scavenging activity by the ORAC, DPPH, FRAP and PCL assays together with several plant or fruit extracts known for their antioxidant properties. In all performed tests ME and HME extracts showed an antioxidant capacity higher than red vine, blueberry (1 percent anthocyanosides) and black currant extracts (Table 3). Moreover HME and ME extracts showed also appreciable antioxidant capacity in ORAC and DPPH assay if compared to pomegranate extract (standardized in 40 percent ellagic acid) and green tea extract (70 percent polyphenols), both characterized by a well-known radical scavenger activity (Table 3). With the exception of the PCL analysis, HME and ME provided also best results than BHT in all performed assays.

����P/�PLQ�DV�IROORZV������SHUFHQW�%�OLQHDU�IURP���WR���PLQ�������SHUFHQW�%� OLQHDU� IURP��� WR����PLQ��������SHUFHQW�%� OLQHDU� IURP���� WR����PLQ��������SHUFHQW�%� OLQHDU� IURP���� WR����PLQ���������percent B from 30 to 40 min, followed by washing with methanol DQG� UH�HTXLOLEUDWLRQ�� 6DPSOHV� ZHUH� GLVVROYHG� LQ� ����� SHUFHQW�acetic acid in water/ methanol 1:1, and 40 µl of this solution was injected in the column, DIWHU� À�OWHULQJ� E\� D� ����� �P� À�OWHU��+3/&�À�OWHUV�ZHUH�SXUFKDVHG�IURP�Chemtek Analitica, Bologna, Italy. The detection condition was 270 nm. In addition to their 89� VSHFWUD�� WKH� LGHQWLÀ�FDWLRQ�of phenolic compounds was also carried out by mass spectrometry coupled to HPLC. The APCI parameters were as follows: negative mode APCI, full scan, APCI temperature ����&��&'/�WHPSHUDWXUH�����&��&'/�YROWDJH����9���

RESULTS AND DISCUSSION

We recently investigated baobab derivatives by photochemi luminescence (PCL) and all investigated tissues, in particular the fruit À�EUHV�� SUHVHQWHG� DQ� LQWHJUDO�antioxidant capacity (IAC) higher than compared antioxidants rich fresh fruit (16). In order to give more consistences to the obtained data, additional investigations have been here performed by the use of further antioxidant assays. With the exception of the baobab seed powder and baobab seeds peel, all examined baobab derivatives showed good antioxidant potency in the performed tests (Table 1), in particular UHG� PLFURQL]HG� IUXLW� UHG� À�EUH�displayed best results in all performed assays.Based on their widespread presence in vegetables, we hypothesized that polyphenols could play an important role in determining the antioxidant DFWLYLW\�RI�EDREDE�UHG�À�EUH��2XU�efforts were directed to the development of an extraction PHWKRG� WKDW� ZRXOG� HQVXUH� KLJK� HIÀ�FLHQF\� LQ� WHUPV� RI�H[WUDFWHG�SRO\SKHQROV��'XULQJ�WKLV�SKDVH��WKH�LQÁ�XHQFH�RI�WKH�solvent, temperature and the use of sonication equipment were investigated to maximize the yield with the best time-saving procedure (Table 2). The marker employed, to measure H[WUDFWLRQ�HIÀ�FLHQF\�LQ�HDFK�H[SHULPHQW��ZDV�WKH�WRWDO�SKHQROV�amount, determined with the Folin-Ciocalteu method. 'DWD�DQDO\VLV�VKRZHG�WKDW�WKH�PRVW�VLJQLÀ�FDQW�YDULDEOH�ZDV�WKH�NLQG�RI� VROYHQW�HPSOR\HG��0HWKDQRO�� UHVXOWHG�PRUH�HIÀ�FLHQW�than acetone and methylene chloride at the same condition. Considering the time of extraction, the most reasonable option ZDV�WR�VHOHFW����PLQXWHV�EHFDXVH�WKH� LQFUHDVH�RI�HIÀ�FLHQF\�DW� ORQJHU� WLPH�ZDV�QRW� VLJQLÀ�FDQW�� 5HJDUGLQJ� WKH�H[WUDFWLRQ�

Table 1. Antioxidant capacity assays of baobab derivatives.a < LOQ (Limit Of Quantification)

Table 2. Influence of solvent and operative conditions on the extraction yield of phenolic compounds from baobab red fiber samples. Total amount of polyphenols was determined with the Folin-Ciocalteu method on the extracts obtained from a single 1 hour extraction. *GAE= gallic acid equivalent

Table 3. Antioxidant activity and total polyphenols evaluation of ME, HME and selected extracts. 1([WUDFW�VWDQGDUGL]HG�LQ����DQG�����DQWKRF\DQRVLGHV��UHVSHFWLYHO\��2Extract standardized in 70% polyphenols; 3Extract standardized in 40% of ellagic acid; 4< LOQ (Limit Of Quantification)

Figure 1. ME chromatogram by HPLC-DAD at 270 nm.

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and hydroxytyrosol (27). The chromatogram in Figure 3, shows a satisfactory resolution for all standard compounds. Although ME and HME extracts were both analysed E\� +3/&�� WKH� LGHQWLÀ�FDWLRQ� RI� SRO\SKHQROV� ZDV� À�QDOO\�performed only on the HME fractions B and D because of the higher polyphenols content detected and of the better chromatograms resolution. Peak 1 for example (Figure ���ZDV� LGHQWLÀ�HG�DV�JDOOLF�DFLG�E\�FRPSDULVRQ�RI� 5W� ������min), UV spectra (maximum at 270 nm) and MS spectra (a quasi-molecular ion [M-H]+ at m/z 169.2) with those of an authentic sample (Table 4). In the same way the molecular LRQ�DW�P�]��������GHWHFWHG�RQ�WKH�SHDN����ZDV�LGHQWLÀ�HG�DV�homovanillic acid.The Rt, UV/VIS spectra and molecular ions of the peaks 10 and 11 matched those of the polyphenols p-coumaric acid (m/z 163.1) and ferulic acid (m/z 193.1). Peak detected at 37.2 min, resulted corresponding to that of the cinnamic DFLG�VWDQGDUG��)LJXUH����7DEOH�����

In the analysed samples, it was not possible assert or exclude the presence of further polyphenolic molecules such as vanillic acid, o-coumaric acid, tyrosol and pyrocathecol. In fact, for these compounds only two of the three selected parameters (Rt and UV spectra or Rt and MS spectra) matched those of the polyphenolic standards (data not

The antioxidant activity of HME, higher in all tests than ME, as well as increased total content of phenols, suggested to us that the basic hydrolysis promotes the release of polyphenols present into the starting material in complexed form. This UHVXOW�ZDV� FRQÀ�UPHG�E\�+3/&�DQDO\VLV�� WKH� FKURPDWRJUDPV�comparison displayed an increase of polyphenols in HME compared with ME extract, together with the improvement of the chemicals detection (Figure 1 and Figure 2). 7KH�FOHDYDJH�RI�WKH�HVWHU�ERQGV�JUHDWO\�VLPSOLÀ�HV�WKH�DQDO\VLV�by reducing the number of derivatives and increases stability and reproducibility of the sample.6LQFH�WKH�LGHQWLÀ�FDWLRQ�RI�SHDNV�LV�D�GLIÀ�FXOW�WDVN�FRQVLGHULQJ�the number of components and complexity of plant samples, different methods were explored to fractionate the crude extracts (ME and HME) as well as to remove undesired LQWHUIHULQJ� FRPSRQHQWV�� 6HYHUDO� SXULÀ�FDWLRQ� PHWKRGV� VXFK�as the liquid-liquid extraction, the solid/liquid extraction, the SXULÀ�FDWLRQ�RQ�63(�FDUWULGJHV�DQG�WKH�HOXWLRQ�WKURXJK�FROXPQ�chromatography have been thoroughly investigated. The resulting mixtures were all analysed by HPLC for qualitative determination. Best results were obtained from column chromatography fractionation and particularly by using a linear gradient of organic solvents (dichloromethane/methanol, 10/0 to 8/2, v/v).The characterization step was carried out comparing the chromatographic data of the obtained mixtures with those of a set of standard polyphenols selected because of their ELRORJLFDO�VLJQLÀ�FDQFH��UHFXUUHQFH�LQ�YHJHWDEOH�H[WUDFWV�DQG�commercial availability (24-26). The polyphenolic structures selected were: trans-cinnamic acid, p-coumaric acid, caffeic acid, ferulic acid, o-coumaric acid, pyrocathecol, p-hydroxybenzoic acid, vanillic acid, gallic acid, protocatechuic acid, syringic acid, tyrosol, homovanillic acid

Figure 2. HME chromatogram by HPLC-DAD at 270 nm.

Figure 3. Fractionament of the standard phenolic compounds by HPLC-DAD at 270 nm. Peak numbers: (1) gall ic acid; (2) hydroxytyrosol; (3) SURWRFDWHFKXLF� DFLG�� ���� S\URFDWKHFRO�� ���� W\URVRO�� ���� S�K\GUR[\EHQ]RLF�acid; (7) homovanillic acid; (8) vanillic acid and caffeic acid; (9) syringic DFLG�� ����� S�FRXPDULQ� DFLG�� ����� IHUXOLF� DFLG�� ����� R�FRXPDULF� DFLG�� �����cinnamic acid. Peak numbers 12, 14 and 16 are degradation products of hydroxytyrosol.

Table 4. Phenolic acids identified by HPLC-DAD/APCI-MS in EDREDE�UHG�ILEUH��njmax= 270 nm).

Figure 4. HME chromatogram after purification by chromatography on silica gel (Fraction D). Peak numbers are referred to Figure 3.

)LJXUH����+0(�FKURPDWRJUDP�DIWHU�SXULILFDWLRQ�E\�FKURPDWRJUDSK\�RQ�VLOLFD�gel (Fraction B). Peak numbers are referred to Figure 3.

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shown). We have speculated that the reasons of this result could be correlated to interference which makes impossible to perform an accurate UV spectra or does not consent compound ionization. Further studies are required to better investigate the presence of WKHVH�DQG�RWKHU�PROHFXOHV�LQ�WKH�EDREDE�UHG�À�EUH�

CONCLUSION

We have comparatively examined several tissues from the baobab plant and, to the best of our knowledge, this is the first report of analysis on the constituents of baobab red fibre using a simple, direct and rapid RP-HPLC method with concomitant evaluation of antioxidant capacity of crude fractions by four different complementary techniques. The results of this study correlate baobab red fibre antioxidant activity to the presences of polyphenols and, in our opinion, also confirms the hypothesis that they may work as sacrificial molecules in preserving pulp integrity upon fruit storage and even after exposure to light and air. It was also possible to obtain red fibre extracts, rich in polyphenols, and with increased antioxidant activity compared to the parent fibre. HME was the most interesting extract exhibiting high antioxidant capacity by the presence of not linked polyphenols, deriving from the hydrolysis process. In addition, this extract showed an antioxidant potency comparable to that of BHT in DPPH and FRAP tests and clearly higher in ORAC test (Table 3). The high antioxidant activity of HME deserves further interest and supports the baobab fruit as a valuable source of functional molecules. Furthermore this data confirm the potential of the fruit and other plant tissues (leafs) as nutrient in food indust ry.

AKNOWLEDGMENTS

Authors wish to thank Alberto Casolari for careful technical assistance. This work was supported by Ambrosialab srl with 2008 and 2009 grants and lab facilities.

REFERENCES AND NOTES

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