Phenolic Compounds in Acerola Fruit ( Malpighia punicifolia , L . )

Agliconas de antocianinas e outros compostos fenólicos foram identificados na fruta acerola (Malpighia punicifolia, L). As antocianinas, os flavonóides e os ácidos fenólicos foram fracionados e caracterizados através de dados cromatográficos e espectrais. O teor de antocianinas na casca da acerola madura foi 37,5 mg100g. Os pigmentos fenólicos identificados foram a pelargonidina, a malvidina 3,5-diglicosilada e a cianidina 3-glicosilada. Foram também identificados a quercetina, o kaempferol, e os ácidos p-cumárico, ferúlico, cafeico e clorogênico.


Introduction
Phenolic compounds are important for plant metabolism and have also becoming important for humans due to their health characteristics, particularly related to their antioxidant properties. 1 Anthocyanins are one of the most attractive plant phenolic pigments of the group of flavonoids.Their visual impact allied to their health properties make them potentially useful as natural food colorants.
One possible source of anthocyanin pigments is the fruit of acerola (Malpighia punicifolia, L.), a highly productive plant, which provides fruits during long periods of the year and is extensively cultivated in Brazil.The attractive red colour in acerola skin is mainly due to anthocyanins and the abundance of this fruit in Brazil represents a potential source of anthocyanin pigments.
Flavonoids are polyphenolic compounds that occur ubiquitously in food of plant origin and more than 2000 chemically distinct flavonoids have been already reported. 2henolic compounds are also important because of their contribution to the sensory quality of fruits (colour and flavour, including astringency and bitterness) which may be affected during the technological processes used for production of juice and other derived products. 3In addition, anthocyanins and other phenolic compounds have been used successfully in the characterization of fruits and juices. 4ue to the importance of flavonoids, many techniques have been used to identify and quantify these compounds.In the early 1990s, high-performance liquid chromatography (HPLC), with photodiode array detection was used to isolation and quantification. 1H and 13 C NMR spectroscopy are the most powerful techniques for determination of molecular structure.The coupling of HPLC and mass spectrometry methods, such as electrospray, thermospray, or fast-atom bombardment, have been widely used to provide molecular weight and characteristic fragment ions for structural elucidation 2 , but these are very expensive techniques which are not readily available. 5][8] Acerola skin is a byproduct of acerola processing usually discarded in juice and pulp industries and due to its relatively high levels of anthocyanins it could be exploited as a potential source of these pigments.The interest in identifying sources of anthocyanin pigments to be used in food, pharmaceutical and cosmetic industries is based on consumer demand for natural colorants, and on the nutraceutical properties frequently reported for flavonoids. 9However, as far as we know, no studies on characterization of acerola phenolics have been published.Thus, the objective of the present work was to isolate and characterize the phenolic compounds present in acerola and to estimate the amount of anthocyanin pigments found in the acerola skin which is usually a by product from the production of acerola pulp.

Fruit
Acerola fruits (M.punicifolia, L.) were chosen by intensive red colour, firmness and a pleasant acid flavour, which are indicative of an ideal stage of ripeness.The fruits were harvested during June 2000 in a commercial plantation in Rio de Janeiro city (Brazil).After harversting, the fruits were washed, packed in polyethylene bags and stored in the dark at -18 °C for two days before anthocyanin extraction.

Chemicals
Caffeic, ferulic, p-coumaric and chlorogenic acids and quercetin, kaempferol, myricetin, and pelargonidin chloride, were obtained from Sigma Chemical Co (USA).The solvents used were of HPLC grade obtained from Tedia Co (USA).All other chemicals were of analytical grade.

HPLC equipment
A Pharmacia (Switzerland) liquid chromatographic system, equipped with two pumps (LKB model 2248), a controller (LKB model LCC 2252), and an injector motor valve (model PMV-7) with 20 µL loop was used.The system was attached to a UV-VIS detector SPD 10 AV (Shimadzu -Japan).An integrator model C-R6A (Shimadzu -Japan) was used for data processing.

Characterization of anthocyanin aglycones
Anthocyanins were analyzed in the extract from acerola skin where these pigments are concentrated.The skin of ripe acerola fruit (500g) was extracted with 600 mL of acidified methanol (0.1% citric acid, v/v) in the dark at about 15 °C and the extract concentrated under vacuum.Due to the non-availability of standards, anthocyanin characterization was carried out based on several physico-chemical information such as mobility in HPLC, separation by paper chromatography and spectroscopic characteristics.Paper chromatographic separation was performed in the dark using Whatman paper 3 MM Chr (23 x 57 cm) according to the method described by Francis. 10 Analysis of HPLC was performed in a Lichrospher-100 RP-18 column (250 x 4.6 mm, Merck, Germany) using methanolwater (1:1, v/v) acidified with 2 mol L -1 HCl as mobile phase until pH 2.5 and flow rate of 1.3 mL min -1 .Detection was carried out at 530 nm. 11UV-Vis spectroscopy was carried out in the fractions separated by paper chromatography that were dissolved in methanolic 0.01% HCl.3][14] The position of the sugar in the anthocyanin molecule was assigned based on the spectral shifts by calculation of the ratio of absorption at 440 nm to the specific absorption maximum for each pigment. 8,13,14The amount of total anthocyanin in the acerola skin was estimated based on the calculation described by Lees and Francis 15 after measurement of the absorbance of the acidified ethanol extract at 535 nm.

Characterization of flavonols and phenolic acids
Flavonols and phenolic acids are distributed both in the fruit skin and pulp.Consequently, in order to obtain a higher yield, the whole fruit was used for extraction.Frozen acerola fruit (260 g) was blended with 250 mL of cooled distilled water and centrifuged at 3000 x g for 20 min at 4 °C.The extraction and centrifugation procedures were repeated twice.The combined supernatants were filtered through Whatman No.1 filter paper.Selective solvent extractions were then carried out to obtain one fraction containing the flavonol aglycons and other containing the phenolic acids, following the procedure described by Kader et al. 16 Characterization was then carried out using HPLC for separation and identification was done by comparison and spiking with external standards.For the analysis of flavonol aglycons, the mobile phase was methanol containing 0.5% orthophosphoric acid (1:1 v/v) with a flow rate of 1.2 mL/min., and detection at 370 nm. 17or the analysis of the phenolic acids, the mobile phase consisted of a gradient elution with 0.01 mol L -1 tri-sodium citrate solution and 20% methanol adjusted to pH 2.5 with 6 mol L -1 HCl (solvent A) and methanol (solvent B).Elution was performed at a flow rate of 1.0 mL/min with the gradient of 5 min for B, increasing to 19% in 15 min, 20% in 5 min, 30% in 10 min and 50% in 10 min.Detection was at 325 nm. 18In both cases, a Lichrospher-100 RP-18 column (250 x 4.6 mm, 5 µm, Merck, Germany) was used.

Results and Discussion
The main objective of the present study was to characterize the phenolic compounds present in acerola, including the aglycones of phenolic glycosides.The structures of the compounds studied are shown in Figure 1.Phenolic pigments such as anthocyanins are usually found as glycosides in plants, however, when they are ingested as food the sugars are easily hydrolyzed from the aglycones.HPLC analysis of the anthocyanin fraction showed three peaks with retention times of 5.7 min, 14.9 min and 22.1 min (Figure 2).Similarly, paper chromatography revealed three bands with pink, magenta and orange colours, respectively.The typical order of chromatographic elution in reverse phase HPLC of different anthocyanins with similar glycosylation patterns is determined by the polarity of the aglycone.The three peaks observed in the HPLC chromatogram were correspondent to the three bands observed by descending paper chromatography.Peak 1 corresponded to the pink band (Rf = 0.43); peak 2 corresponded to the magenta band (Rf = 0.12) and peak 3 to the orange band (Rf = 0.56).The different colours of anthocyanin pigments reflect the nature of their hydroxylation and methoxylation patterns.An increase in hydroxylation is accompanied by an increase in blue colour while methoxylation enhances the red colour. 1 This would indicate that peak 1 may correspond to a malvidin with one methoxylation and peak 2 to a cianidin with two hydroxylations.Peak 3 indicates one hydroxylation in the molecule which confirms the assignment of pelargonidin that was determined by comparison and spiking with the external standard.
Additional qualitative information was obtained with the aid of the spectral characteristics of the chromatographic bands.The spectral data presented in Table 1 show the maximum absorptions for the chromatographic peaks as 277 and 536 nm for peak 1 (5.7 min); 281 and 527 nm  for peak 2 (14.9 min) and 266 and 523 nm for peak 3 (22.1 min).2]19 No change in colour was observed after addition of aluminum chloride in the fractions corresponding to peaks 1 and 3, but the one corresponding to peak 2 changed from red to blue as a consequence of a shift in the maximum of absorption.This is an indication that peak 2 is an orthodihydroxy anthocyanin belonging to the groups of pelargonidin, peonidin or malvidin. 19This test enables to make a broad differentiation between the derivatives of the cyanidin, delphinidin and petunidin groups, which show a positive colour change, and those of the pelargonidin, peonidin and malvidin groups, which show no colour change. 19These results further support the assignment of peak 3 as pelargonidin and indicate that peak 1 does not belong to the cianidin, delphinidin or petunidin groups.
The ratios of Abs 440 /Abs λmax calculated for peaks 1, 2 and 3 were 11, 24 and 35, respectively.The ratio of absorption at 440nm to the maximum is useful in distinguishing anthocyanins when glycosilated at the 3and 5-positions. 11,13The Abs 440 /Abs λmax ratio is usually about 11 for malvidin, corresponding to 3,5-diglycosides.The Abs 440 /Abs λmax ratio is 24 for cyanidin derivatives substituted at position 3 and is 35 for pelargonidin which is not glycosilated. 11,13he results obtained in the present work are in agreement with literature data 7,13,19,20 for the assignment of a malvidin 3,5-diglycoside as peak 1 and a cyanidin 3monoglycoside as peak 2.
Absence of absorption in the 308-328 nm region of the spectra indicated that none of the acerola pigments were acylated with aromatic organic acid. 20he content of anthocyanins was estimated as 37.5 mg per 100 g of ripe acerola skin.This anthocyanin content is higher than that found in red cabbage (25 mg), plum (2 -25 mg), strawberries (15 -35 mg) and banana bracts (32 mg) per 100g fresh weight. 14,21This indicates that acerola skin which is a by product of the acerola pulp production may be used as a commercial source of natural pigment.
Qualitative HPLC analysis and identification by means of spiking and comparison with external standards were carried out in the fraction containing phenolic acids.Two major peaks were identified as p-coumaric and ferulic acid (peaks 6 and 7).Smaller peaks corresponding to chlorogenic acid (5-caffeoylquinic acid), and caffeic acid (peaks 4 and 5, respectively) were also detected in the fraction (Figure 3).
3][24] The fraction containing flavonol aglycons showed as expected maximum absorbance at 370 nm.Individual peaks obtained by HPLC were assigned after comparison of retention times and coelution with flavonol standards showing the presence of quercetin (Rt 8.2 min) and kaempferol (Rt 13.0 min) (Figure 4).

Figure 1 .
Figure 1.Chemical structure of phenolic compounds identified in acerola.

Table 1 .
Data of anthocyanin pigments from acerola