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Biochemistry & Analytical Biochemistry
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Antioxidant and Anticancer Activities of Different Constituents Extracted from Egyptian Prickly Pear Cactus (Opuntia ficus-indica) Peel

Faten M. Abou-Elella and Rehab Farouk Mohammed Ali*

Department of Biochemistry, Faculty of Agriculture, Cairo University, 12613, Giza, Egypt

*Corresponding Author:
Rehab Farouk Mohammed Ali
Department of Biochemistry
Faculty of Agriculture
Cairo University, 12613, Giza, Egypt
Tel: 002-01282959994
E-mail: [email protected]

Received Date: September 29, 2014; Accepted Date: October 13, 2014; Published Date: October 20, 2014

Citation: Abou-Elella FM, Ali RFM (2014) Antioxidant and Anticancer Activities of Different Constituents Extracted from Egyptian Prickly Pear Cactus (Opuntia ficus-indica) Peel. Biochem Anal Biochem 3:158. doi: 10.4172/2161-1009.1000158

Copyright: © 2014 Abou-Elella FM et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Different extracts and their fractions of the Opuntia ficus-indica peel were evaluated for anticancer and antioxidant activities. The anticancer activity was tested using trypan blue technique against Ehrlich ascites carcinoma cells (EACC). Antioxidant activity was determined using the, α- diphenyl- β-picrilhydrazyl radical-scavenging assay system, and compared with those of the positive controls of butylated hydroxytoluene (BHT). Chloroform and ethanol extracts exhibited the strongest antioxidant and anticancer activity in the assay system. The ethanolic extract was further separated into nine fractions, designated as (E1-E9), by TLC. In most cases, E8 and E9 fractions have the strongest antioxidantandanticanceractivitiesthereforethesetwofractionswereidentifiedusingdifferent spectroscopic tools (MS, IR, 1H-NMR and 13C-NMR). The compounds were chemically identified as 17-hydroxy betanin and betanin. The total phenolic contents, tannins, flavonoids, betanins and reducing powers of the different extracts, and their fractions, were also measured. The antioxidant and anticancer potency showed significant relationship with the total phenolic content.

Keywords

Prickly pear cactus (Opuntia ficus-indica); Natural phytochemicals; Phenolic compounds; Antioxidant; Anticancer; 17-hydroxy betanin; Betanin

Introduction

Several species of cactus pear plants belonging to the Cactaceae family are originated from Central America (Mexico). These species became widespread in semi-arid regions of the world, including Egypt. They are of special interest because they are one of the few crops that can be cultivated in areas which offer very little growth possibility for common fruits and vegetables (Han and Feker [1]). About 1500 species of cactus are in the genus Opuntia and many of them produce edible and highly favored fruits. In addition, Mexicans have used Opuntia leaves and fruits for their medicinal benefits, such as treating arteriosclerosis, diabetes, and gastritis. Numerous studies have evocated the analgesic and anti-inflammatory actions of the genus Opuntia by using their fruit extract. Opuntia fruits and young stems have been traditionally used in folk medicine to treat diabetes, hypertension, asthma, burns, edema, and indigestion. In addition to their nutritional and medicinal properties, these plants contain compounds which have several commercial applications. One such compound is betalain, a water-soluble nitrogen-containing pigment and found in high concentrations in cactus pear plants (Castellar et al. [2]) in the form of betalamic acid. This acid is the chromophore common to all betalain pigments (Strack et al., [3]) andused as natural food colorants (Stintzing et al., [4]). The most important biocoumpounds detected also in cactus fruit are phenolic compounds (Kuti, [4]), betacyanins, and betaxanthin pigments, all of which have potent antioxidant properties (Zakharova and Petrova [5]). Phenolic compounds can be defined as substances possessing an aromatic ring, carrying one or more hydroxyl groups, including their functional derivatives (Andres- Lacueva et al., [6]). Their chemical structure and concentration are quite variable and depend on the variety, ripening stages, and the kind of plant tissue (Wallace, [7]). From these groups are glycosylated flavonols, dihydroflavonols, flavonones and flavonols which have been found in Cactaceae plants and fruits (Kuti, [4]). They have antioxidant properties of which make the fruit take important product for preventing human health against degenerative diseases such as cancer, diabetes, hyperglycemia (Galati et al., [8]) hypercholesterolemia, arteriosclerosis and gastric diseases (Galati et al., [9]). As reported other flavonol-rich foods showed active role against those diseases (Jacob et al., [10], Lampila et al. [11], Trejo-Gonzalez et al., [12], Galati et al., [9]). The antioxidant activity of cactus fruit is twice as high as pears, apples, tomatoes, bananas, white grapes, and is comparable to red grapes and grapefruit (Butera et al., [13]). Kuti, [14] reported that the antioxidant effects are due to the major flavonoids encountered in cactus fruits. Flavonoids are more efficient antioxidants than vitamins, since phenolic compounds are able to delay pro-oxidative effects in proteins, DNA, and lipids by the generation of stable radicals (Shahidi, et al. [15]). Polyphenolic compounds in O. ficus indica have been shown to induce a hyperpolarization of the plasma membrane and to raise the intracellular pool of calcium in human Jurkat T-cell lines (Aires et al., [16]).

From literature reports there are few data about opuntia ficus peel for their bioactive compounds [2]. Therefore, our goal in the present study is the separation and chemical characterization of Opuntia ficus peel active compounds which have anticancer and antioxidant activities.

Materials and Methods

Chemicals and reagents

Folin-Ciocalteu reagent, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), sodium carbonate and aluminum chloride were purchased from Sigma Chemical Co., Ltd (St. Louis, MO, USA). Gallic acid, chloroform, ethyl acetate, ethanol, acetone, and methanol were purchased from E. Merck Co. (Darmstadt, Germany), and were distilled before use. All other reagents were of analytical grade.

Plant extraction

Prickly pear fruits (Opuntia ficus indica) in the ripe stage were obtained from the local market, Giza, Egypt. Fresh and mature prickly pear fruits were carefully hand-peeled, and the peels (with a thickness of about 3-4 mm) were cut into small pieces and dried. The air-dried parts were powdered then sample was sequentially extracted with 500 ml each of ethanol 80%, chloroform, ethyl acetate, acetone, methanol and distilled water. Each fraction was separately evaporated using a rotary vacuum evaporator at 45˚C. The obtained extracts were kept in light-protected containers at ˗18˚C until further use.

Phytochemical analysis

Determination of total polyphenols: Total phenolic content of the all extracts was determined using the Folin-Ciocalteu assay system (AOCS, [17]). The results were expressed as µg of gallic acid equivalent per 100 g DW.

Determination of Betalain Content: The content of betaxanthins and betacyanins in the extracts was determined spectrophotometrically at 538 nm and 480 nm respectively using UV–Vis spectrometer, according to the methods of (Ravichandran et al., [18]). The betalain content (BC) was calculated from the equation BC (mg/L) = [(A×DF×MW×1000) / (e)], where A is the absorption, DF the dilution factor and. For quantification of betacyanins and betaxanthins, the molecular weights (MW) and molar extinction coefficients (e) are as follow (MW=550 g/mol; e= 60,000 L/mol cm in H2O) and (MW=308 g/mol; e=48,000 L/mol cm in H2O) were applied.

Determination of total Flavonoids: The total flavonoid content was determined using the Dowd method (Meda et al., [19]). The total flavonoid content was determined using a standard curve with catechin (0-100 mg/L) as a standard. Total flavonoids content is expressed as mg of catechin equivalents (CE)/100 g DW.

Determination of total Tannins: The total tannin content in the extracts was determined by modified method of [20]. The total tannin in the extract was expressed as the equivalent to tannic acid (mg TE/100 g DW).

Viability of Ehrlich Ascites Carcinoma Cells (EACC)

Cancer cells were incubated with various extracts of opuntia ficus indica peels and saline as control for 2 h then the viability was determined by the modified cytotoxic trypan blue-exclusion technique.

A line of Ehrlich Ascities Carcinoma was obtained from National Cancer Institute (NCI) Cairo, Egypt. The tumor cell line was maintained in female Swiss albino mice by weekly intraperitoneal (ip) transplantation of 2.5 × 106 cells/mouse.

Determination of antioxidant efficiency

a) Free radical scavenging activity: The free radical scavenging capacity of various extracts of opuntia ficus peels was measured using α, α- diphenyl-β-picrilhydrazyl (DPPH) assay (Juntachote and Berghofer, [21]). The absorbance of the samples was measured at 517 nm. The radical-scavenging activity was expressed as percentage of inhibition and calculated using the following formula:

% inhibition = [(A control – A sample) / (A control)] × 100.

Where, A is absorbance at 517 nm.

b) Determination of reducing power

The ability of the tested extracts to reduce Fe3+ was assayed by the method of Chou et al. [22]. The absorbance was measured at 700 nm. The results were expressed as µg of gallic acid equivalent per 100 g DW.

Spectroscopic analysis

Thin layer chromatography (TLC): The ethanol (80%) extract of Prickly pear peels (Opuntia ficus-indica) was fractionated using thin layer chromatography (TLC) technique. The extract was applied on silica gel 60 F254 TLC aluminum sheets (20×20) (Merck, Darmstadt, Germany) at one of extremes to separate the different fractions. The ethanol extract was developed with combinations of petroleum ether/ethyl acetate solvents (9:1, 8.5:1.5, 8:2, 7:3, 6:4 and 5:5 v/v) as mobile phase. The last solvent showed to be useful one hence 9 fractions showed the best good resolution. The antioxidant and anticancer activities of these fractions were determined as mentioned while their chemical characterization was analyzed by different spectrophotometric techniques, as follows:

a) Mass spectroscopic (MS) analysis of potent fractions: The potent two fractions of Prickly pear peels were analyzed by Mass spectrum (MS). The mass spectrometer was scanned over the 40 to 400 m/z range with an ionizing voltage of 70 eV. The identification was based on standard mass library of National Institute of Standards and Technology (NIST Version 2.0) to detect the possible fraction structure

b) Fourier transformed Infrared (FTIR) spectra: A Perkin Elmer (Waltham, Massachusetts, USA) was used to obtain Fourier transformed infrared (FTIR) spectra (System 2000) and applied for each pure fraction analysis.

c) Proton magnetic resonance Spectra (1H NMR): The identification of compounds was confirmed by carrying out 1H-NMR analysis using NMR Joel GIM, EX 270 (400 Hz).

d) Carbon magnetic resonance Spectra (13C NMR): The identification of compounds was confirmed by carrying out 13C-NMR analysis using NMR Joel GIM, EX 270 (400 Hz).

Statistical Analysis

The collected data were statistically analyzed in triplicate. Data were analyzed to one-way analysis of variance (ANOVA) and least significant difference (LSD) at p<0.05.

Results and Discussion

In this study different extracts and their fractions of Opuntia ficus indica fruit peel were tested for their anticancer and antioxidant activities. Analysis of natural phytochemicals of different extracts and fractions of Opuntia ficus indica peel were measured. Also identification of active fractions of Opuntia ficus indica peel was carried out by using different spectroscopic techniques.

1-Natural pyhtochchemicals

Analysis of natural phytochemicals of different extracts and fractions of Opuntia ficus indica peel were measured and the data are given in Table 1. Data shows the total phenolics, flavonoids, tannins, total betalains (Betacyanins and Betaxanthins) contents and yield of different extracts of Opuntia ficus indica peels. Total phenolics of different extracts of Opuntia ficus indica peels are ranged from 221.3 to 1501.7 µg gallic acid/100 g DW. The recovery of polyphenols from plant materials is influenced by the solubility of the phenolic compounds in the solvent used for the extraction process (Niciforovic et al., [23]). Ethanol and methanolic extracts had significantly (p<0.05) the highest amounts of total phenolics (1501.7 and 1078 µg gallic acid equivalents (GAE)/100 g DW, respectively). The phenolic compounds values of water extracts was 612.10 µg GAE/ 100 g DW. These results showed that higher amount of phenol compounds was obtained with solvent which has high polarity. These findings may be due to the fact that phenolics are often extracted in higher amounts in more polar solvents such as aqueous methanol/ethanol as compared with absolute methanol/ethanol (Sultana et al., [24]). On the contrary, the lowest levels of phenolics were estimated inchloroform, ethylacetate and acetone extracts of Opuntia ficus indica peels. Total flavonoid contents of Opuntia ficus indica peels, extractes are given in Table 1. The flavonoids contents varied in the different extracts which ranged from 74.12 to 472.33 µg catechin equivalents (CE)/100 g. Ethanolic and methanolic extracts showed significantly (p<0.05) the highest amounts of total flavonoid content. This observation may be due to the fact that ethanol-water mixtures are highly polar and thus show greater efficacy in the extraction of polar phytochemicals such as phenolics and flavonoids (Shabir et al., [25]). However, the lowest amounts of flavonoids were measured in chloroform and acetone extracts of Opuntia ficus indica peels. The level of flavonoids in plant extracts depends on the polarity of solvents used in the extract preparation (Min and Chun-Zhao, [26]). Non-polar solvents are unsuitable for extraction of polar phenolics and flavonoid compounds (Khoddami et al., [27]). Less polar flavonoids (e.g., isoflavones, flavanones, methylated flavones, and flavonols) are extracted with chloroform, dichloromethane, diethyl ether, or ethyl acetate, while flavonoid glycosides and more polar aglycones are extracted with alcohols or alcohol–water mixtures. Glycosides have increased water solubility and aqueous alcoholic solutions are suitable (Marston and Hostettmann, [28]).

Parameters μg/ml
Plant extracts Yield % Total phenols Flavonoids Tannins Betacyanins Betaxanthins
Ethanol 64.74 1501.7 472.333 72 0.733 1.045
Chloroform 4.8 352.26 127.23 26.09 0.412 0.431
Ethyl acetate 6.18 221.26 90.5 13.42 0.085 0.072
Acetone 2.5 241.2 74.3 19.75 0.1785 0.147
Methanol 50.7 1078 357 49 0.247 0.595
Water 36.12 612 194.5 29.85 2.94 2.05
LSD at 0.05 - 4.12 12.80 4.29 0.119 0.22

Table 1: Phytochemical analysis of Opuntia ficus indica extracts.

Tannins are another major group of polyphenols in our diets. Their contents in extracts obtained from opuntia ficus indica peels using various solvents are given in Table 1. Significant (p ≤ 0.05) differences were observed between tannin contents of different extracts. Indeed, ethanol showed significantly (p ≤ 0.05) the highest level of tannins 72.14 µg tannic acid/100 g DW, followed by the methanolic and ethanolic extracts with values of 48.91 and 47.31 µg tannic acid/100 g DW, respectively. On the other hand, extracts obtained with acetone and ethyl acetate had significantly the lowest tannin contents 19.75 and 13.42 µg tannic acid/100 g DW, respectively. These findings confirm the fact that polar compounds dissolve in polar solvents and non-polar compounds in non-polar compounds. Betalains consist of two sub-classes: betacyanins (red-violet pigments) and betaxanthins (yellow-orange pigments), (Delgado-Vargas et al., [29], Stintzing and Carle, [30]). They have antimicrobial and antiviral effects (Strack et al., [3]) and also can inhibit the cell proliferation of human tumor cells (Reddy et al., [31]).

Water extracts showed significantly (p<0.05) the highest contents of total Betacyanins and Betaxanthins 2.94 and 2.06 µg /ml, respectively. This finding may be due to the fact that betalains are water-soluble nitrogen-containing pigments, which are synthesised from the amino acid tyrosine and classified into two structural groups: the red-violet betacyanins and the yellow-orange betaxanthins. (Azeredo, [32] and Ravichandran et al., [18]), followed by these values of ethanol extract were 0.73 and 1.04 µg/ml, respectively. Moderate amounts of betalains were shown for chloroform, and methanol extracts. However, the lowest amounts of betalains were measured in ethyl acetate and acetone extracts of Opuntia ficus indica peels. One of the most important physicochemical properties of betalain plant pigments are their significant polarity and ionization (dissociation, zwitter-ionic behavior) in aqueous solutions. The increased polar character of betacyanins and betaxanthins results in insolubilities in any of the popular organic polar or semi-polar solvents except of water and the mixtures with low-molecular alcohols (Stintzing et al., [30], Wybraniec et al., [33]). The yields of extracts obtained from opuntia ficus indica peels using various solvents are given in Table 1. The yield is ranged from 2.5 to 64.74 µg/100 g of opuntia ficus indica peels. Opuntia ficus indica peels extracted with ethanol had the highest yield of extract (64.74%) followed by methanol extracts (50.7%). In many studies, yield of phenolics extraction showed a strong correlation with the polarity of the solvent used; high polarity solvents being the best for extraction (Lopez et al., [34]). However, the lowest values (4.8 and 2.5 µg/100 g) of extraction yield were recorded for chloroform and acetone extracts, respectively.

Anticancer activity against Ehrlich ascities carcinoma cells

In the present work, trypan blue assay was used for evaluation of the anticancer activity of the different extracts in addition to nine fractions derived from ethanol 80% extract of Opuntia ficus indica peel. This assay has been widely used to investigate the ability of plant extracts and fractions and or pure compounds of those, to act as anticancer. The percent anticancer activities of Opuntia ficus indica extracts were summarized in Table 2. The highest percent of Anticancer activity was observed by chloroform and ethanol extracts (96% and 83%, respectively), followed by acetone while aqueous residue showed lower anticancer activity. At all concentrations tested, all extracts exhibited dose-dependent anticancer activity. Detailed analysis for the values listed in Table 2 and the results suggested that extracts obtained by chloroform and ethanol represented good anticancer agents.

% Dead cells
Plant extract 20μg/ml 50μg/ml 100μg/ml
Control 0.0 0.0 0.0
Ethanol 22 30 83
Chloroform 18 27 97
Ethyl acetate 23 10 0.0
Acetone 9b 33 24
Methanol 20 11 0.0
Water 24 0.0 8
LSD at 0.05 13.2 2.74 14.3

Table 2: Anticancer activity of opuntia ficus indica extracts

Nine fractions (E1-E9) were isolated using TLC were tested for their anticancer activity and the data are given in (Table 3). Two fractions (E8-E9) gave high anticancer activity as 51.70%, respectively therefore these two fractions chemically identified using spectroscopic analysis. The other seven fractions [2,6,13,16,17,32,35] came in the second rank which gave low anticancer activity (Table 3). The highest anticancer activity of the extracts may be due to the presence of betanin pigments which identified in ethanolic fractions (Figure 1).

Fractions % Dead cells/100μg/ml
Control 0.0
E1 4
E2 24.5
E3 9
E4 12
E5 4
E6 4
E7 9
E8 76
E9 51.5
LSD at 0.05 5.8

Table 3: Anticancer activity of opuntia ficus indica fractions

biochemistry-analytical-biochemistry-structure

Figure 1: Chemical structure of betanin (A), chemical structure of 17-decarboxy betanin (B)

These results are in agreement with other studies which suggested that the cactus 8 pear fruit extract inhibits the proliferation of skin and lung cancer (Kapadia et al., [36]). The antiproliferative effect of betanin, isolated from the fruits of Opuntia ficus indica, was evaluated on human chronic myeloid leukemia cell line (K562) and the results show dose and time dependent decrease in the proliferation of K562 cells treated with betanin (Sreekanth et al., [37]). Also this study revealed the apoptotic characteristics such as chromatin condensation, cell shrinkage and membrane blebbing DNA fragmentation pattern of cells treated with betanin. Flow cytometric analysis of cells treated with 40 mM betanin showed 28.4% of cells in sub G0/G1 phase. Betanin treatment to the cells also induced the release of cytochrome c into the cytosol, down regulation Bcl-2, and reduction in the membrane potentials. These studies demonstrated that betanin induces apoptosis in K562 cells through the intrinsic pathway (Loro et al., [38] and Sreekanth et al., [39]).

The anticancer effect of ethanolic extract may be due to presence of Betanin, which, effectively inhibited lipid peroxidation and heme decomposition, suggesting that these pigment may provide protection against certain oxidative stress-related disorders as reported by Kanner et al., [39]. Also the highest anticancer activity of ethanolic extract may be due to presence of higher percent of poly phenols that play an important role in antioxidant activity (Wu et al., [40]) and have evidently shown antiproliferativeactivity or cytotoxicity to humanoral cancer cells (Seeram et al., [41]), melanoma cells (Rodriguez et al., [42]), and lung metastasis induced by B16F10 melanoma cells (Menon et al., [43]).

Antioxidant activity

DPPH radical scavenging activity of various extracts of Opuntia ficus indica peels and synthetic antioxidant (BHT) are given in Table 4. In DPPH scavenging assay, the antioxidant activity was measured by the decrease in absorbance at 517 nm. The DPPH radical received an electron or hydrogen radical from an antioxidant compound to become a stable diamagnetic molecule (Juntachote and Berghofer, [21]). All extracts of Opuntia ficus indica peels exhibited appreciable scavenging activity ranging from 13.04% to 91.7% (Table 4). The free radical scavenging activities of extracts depend on the ability of antioxidant compounds to lose hydrogen and the structural conformation of these components (Aksoy et al., [35]). The DPPH radical-scavenging activity was dependent on the concentration of extracts (Table 4). Ethanol, water and methanol extracts had significantly (P<0.05) the highest DPPH radical scavenging activity (Table 4). As the concentration of phenolic compounds or degree of hydroxylation of the phenolic compounds increases DPPH radical scavenging activity increases, (Sultana et al., [24]). The antioxidant activity of phenolic compounds is mainly due to their redox properties, which can play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides (Javanmardi et al., [44]). On the other hand, the lowest DPPH radical scavenging activity values were observed for ethyl acetate and acetone extracts. No significant differences in radical-scavenging activity were observed between BHT as synthetic antioxidant and ethanol extracts of Opuntia ficus indica peels at concentrations of 60 and 100 µg/ml. DPPH radical scavenging activity of methanol and water extracts of opuntia ficus indica peels at concentration of 40, 60 and 100 µg/ml was also significantly higher than that of BHT (Table 4).

Antioxidant activity %
Plant extracts 20 μg/ml 40 μg/ml 60 μg/ml 100 μg/ml
Ethanol 80.6 90.8 91.7 90.6
Chloroform 19.29 32.82 44 58.4
Ethyl Acetate 13.04 18.92 17.42 21.34
Acetone 13.6 23.01 30.7 36.53
Methanol 62.72 87.4 90.49 90.4
Water 68.77 81.08 86.02 86.48
BHT 53.9 65.9 77.1 79.06
LSD at 0.05 6.49 4.2 5.3 6.27

Table 4: DPPH radical-scavenging activities of opuntia ficus indica extracts

Among the tested extracts, the ethanol was found to exhibit the highest phenolic, flavonoids and Tannins content, antioxidant and anticancer activities. Therefore, this extract was fractionated using TLC and the nine different fractions are separated. These fractions screened for their antioxidant capacity using DPPH radical scavenging activity (Table 5). Fractions 1, 2, 8 and 9 constituted are the major fractions of ethanol extract which gave different antioxidant activity (18,18,14 and 13% respectively). As shown in Table 5, all the fractions showed noticeable DPPH scavenging activities and the scavenging activity increased in a dose dependent. Among of the nine fractions, 1 and 2 fractions showed significantly (P<0.05) the highest DPPH radical scavenging activity at every concentration. These findings reveal that the fractions from ethanol extract are good free radical scavengers and able to react with the DPPH radical, which might be attributed to their electron donating ability.

Antioxidant activity%
Fractions 20 μg/ ml 40 μg/ ml 60 μg/ ml 100 μg/ ml
E1 13.3 25.3 26.1 29.4
E2 10.99 17.5 18.35 22.6
E3 8.76 8.94 10.34 10.092
E4 8.39 9.28 11.09 12.86
E5 7.83 9.69 10.9 13.89
E6 6.7 7.36 9.5 11.36
E7 6.43 6.89 11.8 12..39
E8 7.45 9.59 9.72 12.02
E9 5.12 7.36 7.82 11.64
LSD at 0.05 0.57 1.43 1.05 0.54

Table 5: DPPH radical-scavenging activities of fractions

Reducing power

The reducing power assay is often used to evaluate the ability of an antioxidant to donate an electron (Yildirim et al., [45]). In reducing power assay, antioxidants cause the reduction of the Fe3+ into Fe2+, thereby changing the solution into various shades from green to blue, depending on the reducing power of the compounds (Ferreira et al., [46]). Results of reducing power activity of various extracts and fractions of Opuntia ficus indica peels and synthetic antioxidant (BHT) are given in Table 6. The results are expressed as gallic acid equivalent using the following equation X= y /0.003 resulting from standard curve of gallic. The reducing power is ranged from 213 to 462 µg GAE/100 g DW. Ethanol, water and methanol extracts showed significantly (P<0.05) the highest reducing power activity (453.413, 403 µg GAE/100 g DW, respectively). However, the lowest reducing power activity was recorded for chloroform extract (213 µg GAE/100g DW). It has been reported that the reducing power of substances is probably due to their hydrogen donating ability (Irshad et al., [47]). Ethanol, water and methanol extracts of opuntia ficus indica peels might, therefore, contain higher amount of reductones than other extracts of opuntia ficus indica peels. Hence, ethanol, water and methanol extracts may act as electron donors and could react with free radicals to convert them into more stable products and then terminate the free radical chain reactions. All extracts of opuntia ficus indica peels (except chloroform extracts) had higher reducing power activity than that of BHT. These findings indicate that the antioxidant activity is well correlated with the amount of phenolics constituent found in the extract. Therefore, phenolic compounds of opuntia ficus indica peels are good electron donors and could terminate the radical chain reaction by converting free radical to more stable products.

Reducing power as Gallic
Plant extracts GAE (μg/g) Fractions GAE (μg/g)
Ethanol 453 E1 400
Chloroform 213 E2 440
Ethyl Acetate 407 E3 333
Acetone 363 E4 307
Methanol 403 E5 104
Water 413 E6 107
BHT 260 E7 107
    E8 63
    E9 53
LSD at 0.05 66.29   28.01

Table 6: Reducing power extracts and fractions of

Also the reducing power of nine fractions are determined (Table 6) and the data show that the highest reducing power values were 400 and 440 µg GAE/100 g DW for fractions 1 and 2, respectively. The trend of reducing power activity of fractions 1 and 2 for did not vary markedly from their DPPH free radical scavenging activities (Tables 4 and 6). It may be concluded from the DPPH and reducing power assays that these fractions have high levels of antioxidants.

Spectroscopic results of Opuntia ficus indica

This study deals with the isolation and identification of bioactive compounds from the ethanol (80%) extract of opuntia ficus indica peel which have anticancer and antioxidant activities. The ethanolic extract of opuntia ficus indica was first fractionated using TLC technique and nine fractions (E1-E9) were separated. The percentage of these fractions on TLC were 18,18, 5, 5, 8, 8, 11,14 and 13%, respectively which are scratched and eluted with the same mobile phase, filtered, evaporated, weighed and then used in antioxidant and anticancer bioassays. The results revealed that fractions E8-E9 gave the highest anticancer and antioxidant activities so they were analyzed by different spectrophotometric methods. The chromatographic and spectroscopic analysis suggested two active compounds are as follows: first compound is 17-hydroxy betanin with molecular weight 507Dalton chemical formula C23H26N2O11 and the second compound is betanin with molecular weight 551Dalton and chemical formula C24H26N2O13 and the both are alkaloids.

The mass spectrum of compound 1 displayed a molecular ion peak +M at m/z 506 which suggesting the molecular formula C24H25N2O13 (+1) for compound 2 the presence of fragment ion peaka m/z= 162 indicated the presence of hexose moiety. The presence of fragment ion peaks at m/z 73, 147, 191, 204 and 205 indicated the presence of glucose. Appearance of all these fragments confirming that the compound is betanin (m/z 551).

The IR Spectrum indicates the compounds functional groups of compound hence, compound 1, showed sharp peaks at around 1047, 3398, 1274, 1082, 879 and 3398 cm-1, which indicate the presence of a carbonyl group, carboxylic acid, sugar unit in pyranose, glycoside bond, β–glycosidic linkage and aromatic amine, respectively. In the IR spectrum of compound 2 the sharp peak at around 1047, 3394, 1087, 1082, 880, 2975 cm-1 indicate of a carbonyl group, carboxylic acid, sugar unit in pyranose, glycoside bond, β–glycosidic linkage and aromatic amine, respectively.

The 1H-NMR spectrum of compound (1) indicates that ,the compound under study had the following type of protons; multiplex signals at δ in ppm 7.2 -8.5, 5, 3.4-4, 2-2.3which is characteristic for aromatic protons, anomeric proton of glucose, other sugar protons and 2- 2.3 were assigned for acid proton, respectively.

The 1H-NMR spectrum of compound (2) indicates that ,the compound under study had the following type of protons; multiplex signals at δ in ppm 7-8.4, 5,3.5-4, 2-2.3which is characteristic for aromatic protons, anomeric proton of glucose, other sugar protons and 2- 2.3 were assigned for acid proton, respectively.

13C-NMR spectrum of compound 1 confirms the presence of the most important carbon signals displayed at δ 174.3, 167.8 ppm which attribute to the carbonyl group (C=O)while multiplex signals at 125-132 in ppm confirm the presence of aromatic ring.

Concerning the two compounds our results are agreement with Lotfi et al. [48] who showed that Bixa orellana L. and Opuntia tuna Mill are two important sources for natural colors. Bixin, betanin, isobetanin and indicaxanthin were qualitatively and quantitatively determined using TLC densitometric analysis. Also Yeddes et al., [49] detected three groups of compounds (flavonols, phenolic acids and betalain pigments) in methanolic extract of opuntia ficus indica. The few edible known sources of betalains are cactus fruits, such as those of Opuntia and Hylocereus genera (Stintzing et al., [50-52] and Castellar et al., [2]). The literature reports few scientific studies regarding the presence of phenols or other antioxidant compounds in cactus pear fruits. The purple cultivar has the highest concentration of total phenols, at approximately 660 mg/l juice (Butera et al., [5], Stintzing et al., [30,53,54]).

Conclusions

This study of O. ficus indica fruit peel indicated the presence of biocompounds with possible commercial applications. These biocompounds include polyphenols, flavonoids, tannins and betalains, all of which have anticancer and antioxidant activities. These findings make Opuntia fruits peel a promising source of biologically active polyphenolic and betacyanins mixtures. The results obtained in the present study clearly demonstrate that the ethanolic and choloform extracts have fairly active compounds for in vitro anticancer and DPPH free radical scavenging activities. Also the fractions E8 –E9 showed high antioxidant and anticancer activities. In addition, our results suggest that phenolic compounds might be major contributors to the antioxidative and anticancer activities of Opuntia ficus indica peel extract. The spectroscopic analysisof promising two fractions E8-E9 suggested that the first compound is decarboxy betanin and the second compound is betanin. Our data may contribute to a rational basis for the use Opuntia ficus indica peel extract in possible therapy of diseases related to oxidative stress, or have potential for developing phenolic –rich extracts and fractions. Our results also indicated that inclusion of antioxidant-rich extracts (or fractions) of Opuntia ficus indica peel as a dietary supplementary has beneficial effects for human health.

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