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西印度櫻桃C(萃取液)

Introduction:

Also known as Barbados Cherry, West Indian Cherry, Cereza, Cerisier and Semeruco. 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.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. Phenolic 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.3 In addition, anthocyanins and other phenolic compounds have been used successfully in the characterization of fruits and juices.

Due 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 13C 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 elucidation2, but these are very expensive techniques which are not readily available.5 Due to simplicity of methodology and low cost, techniques like paper chromatography and thin-layer chormatography have been applied routinely in many laboratories.2,6-8 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 665 Phenolic Compounds in Acerola Fruit (Malpighia punicifolia, L.) 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.9 However, 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.

Material and Methods:

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.

The solvents used were of HPLC grade obtained from Tedia Co. 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. An integrator model C-R6A 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 physicochemical 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) 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.11 UV-Vis spectroscopy was carried out in the fractions separated by paper chromatography that were dissolved in methanolic 0.01% HCl. UVVis

spectra were obtained with a Beckman DU 650 spectrophotometer

, in the range of 200-600 nm, with the absorption spectra registered before and after the addition of 3 drops of a solution of the aluminum chloride

salt in absolute ethanol (5% m/v).8,12-14 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.8,13,14 The amount of total anthocyanin in the

acerola skin was estimated based on the calculation described by Lees and Francis15 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.17 For 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.at 325 nm.18 In both cases, a Lichrospher-100 RP-18 column 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 Figure1. 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, 14.9min 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 Figure 1. Chemical structure of phenolic compounds identified in acerola.

their antioxidant properties.9,22-24 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)

In conclusion, the phenolic compounds detected in acerola may be classified in two categories; phenolic anthocyanin pigments and non-anthocyanin phenolics. The pigments detected were a 3,5-diglycosilated malvidin, a 3-monoglycosilated cyanidin and pelargonidin. Nonanthocyanin phenolic compounds identified were pcoumaric acid, ferulic acid, caffeic acid, chlorogenic acid,

kaempferol and quercetin.

However, in the presence of acerola cherry extract, both soy and alfalfa extracts potently inhibited the formation of LDL-. These findings show that acerola cherry extract can enhance the antioxidant activity of soy and alfalfa extracts in a variety of LDL oxidation systems. The protective effect of these extracts is attributed to the presence of flavonoids in soy and alfalfa extracts and ascorbic acid in acerola cherry extract, which may act synergistically as antioxidants. It is postulated that this synergistic interaction among phytoestrogens, flavonoids, and ascorbic acid is due to the "peroxidolitic" action of ascorbic acid, which facilitates the copper-dependent decomposition of LDL peroxides to nonradical products; this synergy is complemented by a mechanism in which phytoestrogens stabilize the LDL structure and suppress the propagation of radical chain reactions. The combination of these extracts markedly lowers the concentrations of phytoestrogens required to achieve significant antioxidant activity toward LDL.

Acerola, also known as Barbados cherry or West Indian cherry, is grown to a minor extent in the frost-free regions of Florida and in Hawaii, primarily in home gardens (Miller et al. 1965). This plant is most noted for the extremely high ascorbic acid (vitamin C) content of its fruit, with 10 to 40 mg/g of edible fruit, far more than any other known fruit. By comparison, The juice retains its cherry-red color and flavor if it is processed and frozen immediately. The development of a chemical method of producing vitamin C has reduced the need for acerola.

The acerola is believed to originate from the Yucatan and can be found growing in the sandy soils of Mexico, Central America, northern South America (Venezuela, Surinam, Columbia) and throughout the Caribbean (Bahamas to Trinidad). Acerola has now been successfully introduced in sub-tropical areas throughout the world (Southeast Asia, India, South America), and some of the largest plantings are in Brazil.

The acerola is a deciduous tree typically found in dry woodlands. It has poor cold tolerance, with young plants typically killed at temperatures below 30°F - trees can survive brief exposure to 28°F, but will lose leaves. They are also sensitive to wind as they have shallow root systems, but are drought tolerant, and will adopt a deciduous habit.

The acerola tree is a large, relatively fast growing bushy shrub or small tree it can grow up to 15 feet). The branches are brittle, and easily broken. Acerola leaves are dark to light green and glossy when mature. They are obviate to lanceolate, with minute hairs which can be irritating to some people. The flowers are small, pink to white in colour and have five petals. Acerola fruit are round to oblate and cherry-like, but with 3 lobes. They are bright red (rarely yellow-orange) with thin skin, and are easily bruised. The pulp is juicy, and quite acidic with a delicate flavour, and apple notes.

The fruit of the Acerola Cherry tree, Malpighia punicifolia L. is rich in Vitamin C and carotenoids, with the cherry-like fruits being one of the richest known natural sources of vitamin C. The fresh fruit can contain up to 4000mg Vitamin C per gram of fresh weight (although typically, it is around 1500mg). Oranges provide 500 to 4,000 parts per million Vitamin C or ascorbic acid, while Acerola assays in the range of 16,000 to 172,000 parts per million. Green fruits have twice the Vitamin C level of mature fruits. Fruits develop to maturity in less than 25 days.

Acerola also contains the synergistic bioflavonoids - rutin and hesperidin, carotenoids, and other vitamins, minerals and phytonutrients, making it an ideal food based source of nutrients necessary for immune support. Compared to oranges, acerola provides twice as much magnesium, pantothenic acid, and potassium. Other vitamins present include vitamin A (4,300 to 12,500 IU/100g), thiamine, riboflavin, and niacin in concentrations comparable to those in other fruits. One hundred and fifty other constituents have been identified in acerola; the major ones being furfural, hexadecanoic acid, and limonene. Aside from being an excellent source of powerful antioxidants, Acerola cherries are also rich in protein and mineral salts - principally iron, calcium and phosphorus.

Recent research in cosmetology indicates that vitamin C is a powerful antioxidant and free radical scavenger for the skin, and acerola extracts are now appearing in skin care products that fight cellular aging. The mineral salts contained in acerola have also been shown to aid in the re-mineralisation of tired and stressed skin, while the mucilage and proteins have skin hydrating properties, and promote capillary conditioning.

Acerola cherry :

Acerola has been studied in the laboratory and has been found to be a powerful antioxidant and have anti-tumor potential.

Acerola cherry flavor:

Volatile components have been isolated from acerola fruit. One hundred fifty constituents have been identified in the aroma concentrate, from which furfural, hexadecanoic acid, 3-methyl-3-butenol, and limonene were found to be the major constituents. The amounts of esters, 3-methyl-3-butenol, and their various esters are thought to contribute to the unique flavor of the acerola fruit.

Acerola cherry Research Update Antioxidant activity of dietary fruits, vegetables, and commercial frozen fruit pulps. Fruits, vegetables, and commercial frozen pulps (FP):

consumed in the Brazilian diet were analyzed for antioxidant activities using two different methods, one that determines the inhibition of copper-induced peroxidation of liposome and another based on the inhibition of the co-oxidation of linoleic acid and beta-carotene. The anthocyanin-rich samples showed the highest, concentration-dependent, antioxidant activities in both systems. In the liposome system, at both 10 and 50 microM gallic acid equivalent (GAE) addition levels, the neutral and acidic flavonoids of red cabbage, red lettuce, black bean, mulberry, Gala apple peel, jambolao, acai FP, mulberry FP, and the acidic flavonoids of acerola FP showed the highest antioxidant activities (>85% inhibition). In the beta-carotene bleaching system, the samples cited above plus red guava gave inhibition values >70%. On the other hand, some samples showed pro-oxidant activity in the liposome system coincident with a low antioxidant activity in the beta-carotene system. There was no relationship between total phenolics content, vitamin C, and antioxidant activity, suggesting that the antioxidant activity is a result of a combination of different compounds having synergic and antagonistic effects.

Structural and functional characterization of polyphenols isolated from acerola (Malpighia emarginata DC.) fruit:

Two anthocyanins, cyanidin-3-alpha-O-rhamnoside (C3R) and pelargonidin-3-alpha-O-rhamnoside (P3R), and quercitrin (quercetin-3-alpha-O-rhamnoside), were isolated from acerola (Malpighia emarginata DC.) fruit. These polyphenols were evaluated based on the functional properties associated with diabetes mellitus or its complications, that is, on the radical scavenging activity and the inhibitory effect on both alpha-glucosidase and advanced glycation end product (AGE) formation. C3R and quercitrin revealed strong radical scavenging activity. While the inhibitory profiles of isolated polyphenols except quercitrin towards alpha-glucosidase activity were low, all polyphenols strongly inhibited AGE formation.

[Physico-chemical characterization of acerola (Malpighia glabra L)]:

The acerola Malpighia glabra L., originally from the Antillas and North of South America, known by the people as cereja-das-antilhas or cereja-do-para distinguish itself by its high content of vitamin C. The ripe and fresh acerola fruits utilized in experiments, were obtained from farmers of Maringa region, Parana State, Brazil. The fruits were hulled in steel sieve with 25 mesh and the bagasse (seeds and hull) discarded. These physico-chemical analysis were realized in the pulp: vitamin C, moisture, protein, carbohydrate, fiber, lipids and fatty acids composition. We also determined the content of ash and cadmium, calcium, lead, copper, chrome, iron, magnesium, manganese, potassium, sodium and zinc minerals. The average content of vitamin C was 1.79 g/100 g of pulp, it was higher than the one for other fruits, like pineapple, araca, cashew, guava, kiwi, orange, lemon, and strawberry and lower than the camu-camu sylvestral fruit of Amazonia. The contents of moisture, carbohydrate, fiber,