Mutagenic and antimutagenic potentials of fruit juices of five medicinal plants in Allium cepa L.: Possible influence of DPPH free radical scavengers

Mutagenic and antimutagenic activities of freeze dried fruit juices (FDFJ) of Morinda elliptica Ridl. (Rubiaceae), Morinda citrifolia L. (Rubiaceae), Averrhoa bilimbi L. (Oxalidaceae), Phyllantus acidus (L.) Skeels (Phyllantaceae) and Myristica fragrans Houtt. (Myristicaceae) in Allium cepa L was evaluated. Testing the mutagenic activity, onions were suspended in solution of different concentrations of FDFJ alone in tap water for 48 h. Thereafter, root tips were prepared and observed for dividing cells and chromosomal aberrations using a light microscope. Antimutagenicity screening was similar to the mutagenic evaluation, except that the solution of FDFJ was combined with 0.1% cylophosphamide-CP. Free radicals (2,2-diphenyl-1-picrylhyrazyl) scavenging activity of the FDFJ was tested using butylated hydroxylanisole (BHA) and butylated hydroxytoluene (BHT) as the standards and their phenolic contents were evaluated by comparing with gallic acid equivalents (GAE). The free radicals scavenging power of M. fragrans at 1 mg/ml was almost similar to that of BHA and BHT and its phenolic content was 21 ± 6.0 mg GAE/g, the highest among the tested juices. A. cepa cell division was inhibited in a dose-dependent manner by the FDFJ of M. fragrans, while the induced chromosomal aberrations were non dose dependent. The cytotoxicity and chromosomal aberrations of CP were suppressed throughout the tested concentrations of M. fragrans, unlike the effects of other four juices. These results suggest that the observed activities of FDFJ of M. fragrans may be due to the quantity and quality of phenolic compounds with antioxidant activity, suggesting its use in preventing the DNAdamaging effects of mutagens.


INTRODUCTION
in the main functions of their chemical constituents. In the recent years, investigations into phytochemicals used as food or to treat illnesses have increased, because juices are often suspected to contain constituents that are harmful or/and protective to the genetic system (nucleic acids) of living organisms. The damaging effects of chemical compounds on the genetic system of organisms have been reported to elicit several human diseasessuch as cancer, type II diabetes, Alzheimer's disease, coronary heart disease and aging, through the generation of reactive oxygen species (ROS), free radicals as well as oxygen anions (O 2 ) in the cells (Odin, 1997;Ajith and Janardhanan, 2002;Gupta et al., 2008;Rosidah et al., 2008;Zahin et al., 2010). However, antimutagenic compounds prevent and protect the genetic system from any forms of damage that may be caused by either ROS generated through metabolic activity of chemicals in drugs and food substances or through radiation. Consequently, degenerative diseases that occur as a result of assaults done to the macromolecules can therefore be averted. Phenolic compounds are plant metabolites widely spread throughout the plant kingdom (Gülcin et al., 2010). Sulaiman et al. (2011) reported that phenolic compounds have protective role against oxidative damage diseases, perhaps due to their reputed antioxidant activity, made possible by their synergistic effectiveness as hydrogen donors and reducing effects, also as free radical scavengers. Reports have shown that certain phytochemical compounds can induce and prevent DNA damage (Barcelos et al., 2007). Therefore, an investigation into the mutagenicity and antimutagenicity of plant constituents would be beneficial in providing information concerning possible dangers associated with the consumption of plant parts. It could also serve as basis of chemotherapy, especially for cancer, whose occurrence has DNA damage as a principal step to carcinogenesis.
Among the plants that are being used for food and medicinal purposes in Asia and some other countries in the world are; Morinda elliptica (L.) Ridl, Morinda citrifolia (L.), Averrhoa bilimbi (L.), Phyllantus acidus (L.), Skeels and Myristica fragrans (Houtt.). Often the use of these plants as food or medicines may involve any one or combinations of different parts such as leaves, stem roots and fruit juices. In traditional pharmacopoeia, the fruit of M. citrifolia, also known as noni (Hawaiian name) is used to prevent and cure several diseases such as cancers, arthritis, diabetes, high blood pressure, muscle aches and pains, menstrual difficulties, headaches, heart disease, gastric ulcers, mental depression, poor digestion, atherosclerosis, blood vessel difficulties and drug addiction (Chan-Blanco et al., 2006;Liu et al., 2007). These beneficial effects of M. citrifolia may result from certain compounds like scopoletin, nitric oxide, alkaloids and sterols, as well as due to the antioxidants potential. Morinda elliptica is employed for the treatment of diarrhea, headache and haemorrhoids, as well as to increase appetite. Anthraquinones (AQs), the secondary metabolite compounds isolated from M. elliptica had antiviral, antimicrobial, cytotoxic, antitumour-promoting properties and antioxidant activities (Chong et al., 2005). Fruit and leaves extracts of A. bilimbi were observed to reduce blood glucose and food intake in diabetic rats (Pushparaj et al., 2000). Semi purified fractions of A. bilimbi possessed hypoglycemic and hypolipidemic potencies in Type I diabetic rats that were fed with these fractions orally and intraperitoneally (Tan et al., 2005). Acidic fruit like bilimbi is utilized as a substitute for Akeem et al. 10521 common sanitizers in Malaysia. The fruit of A. bilimbi is used to wash seafood, especially freshwater fish, to remove any foul smell (Norhana et al., 2009). Myristica fragrans has been used as an aromatic stimulant, abortifacient, antiflatulent and as a means to induce menses for many years (Donald, 2008). Argenteane, a dilignan and powerful antioxidant like vitamin E, was isolated from the mace of M. fragrans (Calliste et al., 2010). Different parts of Phyllantus acidus are being used to treat oxidative stress related diseases in Malaysia, South America and Africa (Eldeen et al., 2011). The fruit of Phyllantus acidus contains 40 mg/100 g ascorbic acid, the acidic contents of lemon and grapefruits are similar to that of P. acidus (Duangpron and Siripong, 2009). P. acidus was reported to be rich in adenosine, and lacks cytotoxic effects (Sousa et al., 2007). However, there are limited reports on the possible toxicity of the juices of these plants in eukaryotic cells, coupled with the fact that there are no known published data on their cyto-geno toxicities in an in vivo genetic system as being reported presently. Allium cepa (L., 2n = 16) assay is a popular and reliable test for cyto-geno toxicities screening of chemical compounds. It is an established plant bioassay, validated by the international programme on chemical safety (IPCS, WHO), as an efficient and standard test for chemicals screening and in situ monitoring of environmental substances genotoxicity (Kumari and Mukherjee, 2009). Its acceptability for testing the effects of both water-soluble and non-soluble substances on cell division and chromosome is due to the similarities in its results compared with those from animal bioassays (Yildiz et al., 2009). A. cepa test permits the study of different, but complementary aspects of food contamination and could be helpful to better evaluate cancer hazards related diets (Feretti et al., 2007).
Thus, this study sought to investigate the possible mutagenic and antimutagenic activities of fruit juices of these five plants, as affected by their DPPH free radicals scavenging effectiveness. citrifolia and nuts extracted from M. fragrans, after which the fruits were ground separately to fine paste using an electric blender. Juice was squeezed out of the individual fruit paste and filtered through a white muslin cloth. The filtered juice was concentrated at 40°C under rotavapor (R-125) and kept in a deep freezer at -80°C for 24 h. Frozen juice was freeze-dried at -50°C under vacuum pressure in a Labconco freeze dryer (Lyph-lock 6) and kept at 4°C for screenings.

Antioxidant test
The freeze dried fruit juices (FDFJ) were tested for their ability to scavenge free radicals from 2, 2-diphenyl -1-picryl-hydrazyl (DPPH) following the described method (Rosidah et al., 2008), but with some modifications. Briefly, 2 mg/ml of each of the FDFJ was weighed and dissolved in HPLC grade methanol. To 100 µl solution of the juice prepared in triplicate at each concentration, 200 µl of 0.1 mM DPPH was added in a 96-well microplate. The blank reaction at each concentration contained 100 µl FDFJ + 200 µl HPLC MeOH. The plate was wrapped with aluminium foil and incubated at root temperature for 30 min. Absorbance of the reaction between FDFJ and DPPH solution was measured at 517 nm (wavelength) in a microplate reader (power wave × TM). Butylated hydroxy anisole (BHA) and butylated hydroxy toluene (BHT) prepared at 1 mg/ml served as the reference standards. The percentage free radical scavenging activity of the FDFJ and standards were calculated as shown below (Ak and Gülcin, 2008;Gülcin, 2009).
Where, A control is the absorbance at 517 nm of the control reaction containing all reagents except the test compound; A sample is the absorbance at 517 nm of reaction containing test compound (Gülcin, 2009;Talaz et al., 2009).

Determination of total phenolic contents
The concentration of phenolic compounds in the FDFJ was determined according to the reported method (Eldeen et al., 2011). Two milligrams of the FDFJ and 1 mg/ml Gallic acid (GA) were separately dissolved in a mixture of HPLC grade methanol:distilled water (5:5 v/v). Different concentrations of the gallic acid solution ranging from 0.004 to 0.5 mg/ml were prepared by two-fold dilution. Each of the FDFJ and GA (20 µl) was mixed with 40 µl Folin-Ciocalteu reagent in a 96 microwell plate and left at room temperature for 10 min. Thereafter, the reaction was terminated by adding 75 µl of 20% sodium carbonate solution, after which 150 µl distilled water was added to the mixture and incubated at 40°C for 30 min. The absorbance was measured at 765 nm using a microplate reader (power wave X TM). The test was carried out in triplicates. The concentrations of phenolic compounds in the FDFJ was extrapolated from a standard curve drawn using the absorbance at different concentrations of GA and expressed as milligrams of Gallic acid equivalents (GAE) per gram of sample (Gülcin et al., 2010).

Preparation of onion bulbs for planting
Onions (A. cepa L. 2n = 16) were purchased from Jusco shopping complex, Penang, Malaysia. They were sun dried for one week, after which only the healthy onions (not infected by fungi) were selected for planting.
The dried scales on the onions were removed carefully without damaging the primordial root ring.

Mutagenic evaluation
The FDFJ was dissolved in tap water (TW) at 1, 2, 4 and 8% concentrations (w/v) and then vortexed to obtain better dissolution. Five onions, per concentration, were suspended in the solution of FDFJ + TW in 50 ml beakers and kept in the dark for 24 h for the root growth to occur. Fresh solution of each juice was prepared and further suspension of the onions in the solution continued for another 24 h. Tap water (TW) served as the negative control.

Preparation of root tips for microscopic observation
For slide preparation, root tips from the onions were cut, fixed in ethanol/acetic acid (3:1, v/v) and then kept at 4°C for 24 h. The fixed roots were hydrolyzed in 1N HCL as described (Akinboro and Bakare, 2007). Thereafter, the roots were rinsed with distilled water before slide preparation, 2 to 3 root tips were homogenized on a glass slide and stained in 2 drops of aceto-orcein for 10 min. Excess stain was removed with filter paper and a cover slip was gently placed on the stained smear, then, carefully pressed with the thumb. The cover slip was gently tapped with the index finger to ensure that the stained cells did not overlap. Slides (5 per concentration) were observed for different stages of mitosis and chromosomal aberrations under oil immersion objective lens (100×) of a Nikon Eclipse E400 microscope. Number of dividing cells as well as chromosomal aberrations in 1000 counted cells on a slide was recorded. Mitotic index (MI) and frequency of chromosomal aberration (CA) were calculated using the following formulae:

Antimutagenic evaluation
The activity of the FDFJ in suppressing the mutagenic action of cyclophosphamide-CP (CAS: C0768) on the chromosomes of A. cepa was tested in similar way to the mutagenic evaluation in this study, except that 0.1% CP (w/v) was added to the solution of the FDFJ at each concentration. The root tips from the onions grown in the mixture of FDFJ + CP were prepared on microscope slides and observed as described earlier. Percentage of mitotic index (MI) and frequency of chromosomal aberrations (CA), recorded at each concentration were calculated. Percentage reduction of CPinduced chromosomal aberrations at different concentrations of FDFJ was calculated as stated below.
Where, '% R' is the percentage reduction of chromosomal aberration (CA); 'A' is the frequency of CA induced by CP alone; 'B' is the frequency of CA induced by the mixture of FDFJ and CP and 'C' is the frequency of CA induced by tap water alone.

Statistical analysis
Data were expressed as percentage of free radicals scavenged, mitotic index, frequency of chromosomal aberration and percentage reduction of CP-induced CA by the FDFJ. Average values of the measured parameters were compared with the mean values of the controls, using Duncan multiple range comparison and Dunnett's tests under One-way ANOVA in SPSS software, 15.0 version. Data with a value of p 0.05 were considered significant. The bars represent percentage of DPPH free radicals scavenged at different concentrations of the FDFJ and reference standards calculated from the average (n = 3) absorbance ( 517 nm) of an experimental reaction (carried out in triplicate) between the juice or standard, and DPPH solution. Error bars represent standard deviation of average percentage DPPH free radical scavenged at different concentrations. 'b' is significantly different from BHA & BHT. Figure 1 shows the percentage of DPPH free radicals scavenged by the FDFJ. Among the evaluated fruit juices, FDFJ of M. fragrans recorded a concentration dependent DPPH free radical scavenging effect. At 1 mg/ml, the percentage of free radicals scavenged by the FDFJ of M. fragrans was 85.7%, amounting to 8.0%, and 7.1% less than the free radicals scavenging activity of BHA and BHT, respectively. Other FDFJ, except the FDFJ of M. fragrans, could not cause 50% inhibition of free radicals from DPPH at the tested concentrations. However, BHA and BHT (synthetic antioxidants) demonstrated higher free radicals scavenging activity than any of the evaluated FDFJ. Table 1 shows phenolic contents of the FDFJ determined by the Folin-Ciocalteau colorimetric method. The least and highest concentrations of phenolics extrapolated from the Gallic acid calibration curve (regression equation y = 0.5209x + 1.1207, r 2 = 0.9315) were in the range of 9.0 ± 1.5 to 21.0 ± 6.0 mg GAE/g for A. bilimbi and M. fragrans, respectively. The FDFJ of M. citrifolia contained 15.0 ± 0.5 mg GAE/g phenolic content, while it was 12.5 ± 1.0 mg GAE/g and 11.5 ± 2.5 mg GAE/g for M. elliptica and P. acidus, individually.

Effects of FDFJ on mitosis
The effect of the freeze dried fruit juices alone and its combination with CP, on cell division in the root tips of onion was evaluated (Figure 2). In the mutagenic test, FDFJ of P. acidus at 1, 2 and 4% induced higher mitotic indices (MI) than that of the negative control. There was a complete arrest of cell division at 8% concentration of M. elliptical and at 4% of A. bilimbi. Mitotic indices induced by the FDFJ of M. elliptica at 1, 2 and 4% were the least, compared with those of M. citrifolia, A. bilimbi and M. fragrans. At 1%, mixture of each of the FDFJ with CP, except for the combination of A. bilimbi with CP, induced MI value higher than that of CP and FDFJ alone. No dividing cell was observed at 4% of A. bilimbi and at 8% of M. elliptica and P. acidus in the antimutagenic test (Figure 2). At 8%, each of the mixtures of M. citrifolia or M. fragrans with CP resulted to 49 and 51% mitotic indices of their negative controls, respectively. Interestingly, inhibition of cell division by the combination of FDFJ of M. fragrans with CP was dose-dependent ( Figure 2).

Morinda citrifolia
15.0 ± 0.5 Averrhoa bilimbi 9.0 ± 1.5 Phyllantus acidus 11.5 ± 2.5 Myristica fragrans 21.0 ± 6.0  fragrans caused chromosomal aberrations that were insignificantly different (p 0.05) from those recorded for their individual negative controls. The potency of the FDFJ to reduce the mutagenic effect of CP on the chromosomes of A. cepa is shown in Table 3. The frequency of chromosomal aberrations (CA) induced by CP alone was the highest, compared with those caused at different concentrations of the FDFJ + CP. At 2% of the FDFJ of M. citrifolia, 8% of A. bilimbi and P. acidus, the percentage reduction of CP-induced CA was negative, suggesting that the mixture of each

DISCUSSION
Many chronic diseases such as cancers, atherosclerosis, diabetes, aging and other degenerative disorders inhumans can be elicited by overproduction of free radicals causing oxidative damage to biomolecules like lipids, proteins and DNA. Fruits and others plants may contain a vast number of free radical scavenging  molecules like phenolic and nitrogen compounds, vitamins and some other endogenous metabolites, which are rich in antioxidants activity (Cai et al., 2004). The antioxidant activity of the FDFJ in this study could be due to their phenolic compounds. The antioxidant activity of phenolic compounds has been reported (Lopes et al., 2004). Thus, the higher phenolic content recorded for the FDFJ of M. fragrans than in the other FDFJ explains its concentration dependent antioxidant activity (Figure 1). The results of free radical scavenging effects of these FDFJ on DPPH solution are in line with the earlier reports (Wang et al., 2010;Dussossoy et al., 2011;Eldeen et al., 2011). Mitotic index is a parameter that reveals cytotoxic effects of chemical substances on the cells, as high and low mitotic indices may suggest the degree of cytotoxicity that a chemical inflicts on meristematic cells. In this study, the induction of less MI values than the MI of individual negative controls by the FDFJ of M. elliptica, M. citrifolia, A. bilimbi and M. fragrans alone suggests cytotoxicty. This agrees with the earlier reports of Chong et al. (2005). Extracts of the plants have been reported to have anti-bacterial activity based on their ability to inhibit microbial cell growth (Norhana et al., 2009;Chatterjee et al., 2007;Chiang and Abdullah, 2007;Ikeda et al., 2009).This kind of activity is therefore expected to be cytotoxic. In addition, the observed cytotoxic effects of the FDFJ on the cell division in A. cepa may be due to their reported antioxidant properties (Calliste et al., 2010;Ahmad et al., 2005). Some naturally occurring anthraquinones in Morinda species and lignans in M. fragrans scavenge reactive oxygen species (ROS) and free radicals, thereby preventing carcinogenesis. It is expected that their anti-cancer effectiveness would be cytotoxic as a result of suppression of mitosis. We are of the opinion that the mitotic inhibitory effect of the FDFJ of M. elliptica, M. citrifolia and A. bilimbi on the cell division in A. cepa was cytostatic due to the induction of non-dose dependent inhibition of mitosis in A. cepa. This may be connected with the kinds and total contents of free radicals scavenging molecules in form of phenolic compounds in the FDFJ. However, the dose-response inhibition of mitosis by the FDFJ of M. fragrans is an indication of cytotoxicity which might have been caused by phenolic compounds acting as antioxidants. This supports the report that polyphenols such as lignans, flavonoids, which are important classes of plant derived compounds, possess a variety of biological activities such as anti-tumor and anti-mitotic activities (Chatterjee et al., 2007). Inhibition of mitosis may occur due to suppression of DNA/ protein (Histones) synthesis or an arrest in the G 2 phase of the cell cycle, preventing the cell from entering mitosis (Turkoglu, 2008). The induction of higher P. acidus at 1, 2 and 4% implies lack of cytotoxicity. It had earlier been reported that extract of P. acidus was non-cytotoxic (Sousa et al., 2007). This may be in connection with its richness in adenosine, a precursor for DNA synthesis during cell division. Induction of higher MI by the mixture of each of the FDFJ with CP at 1% than the MI obtained for CP alone suggests suppression of CPinduced cytotoxicity. Suppression of CP-induced cytotoxicity was more observed with the FDFJ of M. fragrans, perhaps due to the quantity and quality of phenolic compounds which enhance its free radicals scavenging activity (Figure 1 and Table 1). Cyclophosphamide (CP) is a drug usually employed in treating a wide range of neoplastic diseases as well as some non-malignant ones. The therapeutic function of phosphoramide mustard and the major anti-neoplastic metabolite of CP is cytotoxic (Anderson et al., 1995). Complete arrest of cell division, by the FDFJ alone as well as the mixtures of FDFJ of M. elliptical and P. acidus with CP at 8% suggests occurrence of cell death which Akeem et al. 10527 may be caused through a reaction between components of the FDFJ or/and CP to generate product(s) capable of; interfering with the normal progression of mitosis, preventing cells from entering the prophase, or blocking the mitotic cycle at interphase or/and causing an increase in G 2 and S phases duration (Yildiz et al., 2009). The induction of chromosomal aberrations that were not significantly different (p 0.05) from those induced by the negative controls and non dose-related, suggests weak mutagenic action of the FDFJ of M. elliptica, A. bilimbi and M. fragrans. Similarly, obtainment of non dose-dependent chromosomal aberrations caused by the FDFJ of M. citrifolia and P. acidus can also justify their weak mutagenic effects on the chromosomes of A. cepa. Nevertheless, the observed mutagenic effects of the FDFJ may be due to some phenolic compounds and vitamin C reported as natural occurring antioxidants in these juices (Chan-Blanco et al., 2006;Chatterjee et al., 2007;Norhana et al., 2009). A reaction between phenolic compounds and vitamin C in the presence of Cu 2+ and Fe 3+ may enhance mutagenic activity through generation of reactive oxygen species (ROS) which may interact with DNA directly, facilitate redox cycling or interacting with molecular oxygen to produce oxidative stress and genotoxic ROS, that may cause DNA damage (Frankel et al., 2004).
Mutagens and carcinogens such as CP may act through the generation of ROS that play a major role as endogenous initiators of degenerative processes, such as DNA damage and mutation and its promotion (Negi et al., 2003). The reduced values of CA observed with the FDFJ + CP compared with those induced by CP alone implies antimutagenic effectiveness of the evaluated FDFJ. This activity may be connected to DPPH free radicals scavenging molecules in the FDFJ, counteracting free radicals from the acrolein component of CP. CP is an alkylating agent that causes chromosomal aberrations and DNA damage through generation of carbonium ions, which react with the electron-rich area of the nucleic acids and proteins (Schneider et al., 1997;De et al., 1995). Among the investigated FDFJ, the FDFJ of M. fragrans scavenged DPPH free radicals in a dosedependent manner. It is possible to say that antimutagenic activity of the FDFJ of M. fragrans against CP-induced mutagenicity may due to antioxidants.
Myricetin (3, 3, 4, 5, 5, 7-hexahyroxylflavone) is a natural flavonoid, found in many fruits, vegetables, herbs and other plants, capable of scavenging ROS and ensures cytoprotective and antimutagenic effects (Wang et al., 2010). Argenteane, a dilignan antioxidant that is powerful like vitamin E was isolated from M. fragrans (Calliste et al., 2010). It is not surprising that the free radicals scavenging activity of the FDJF of M. fragrans was almost the same as that of BHA and BHT at 1 mg/ml in this study. Vitamins A, C and E have been reported to protect DNA from the mutagenicity of oxy radicals and products of promutagen activation of free radical origin (Odin, 1997). Also, DNA protecting role of phenolic com-compounds in M. fragrans has been reported (Chatterjee et al., 2007). These phytochemicals may be responsible for the observed antimutagenic action of this FDFJ against the mutagenicity of CP. To some extents, it is therefore, logical to say that there is a direct relationship between phenolic contents, antioxidant and antimutagenic activities. This is in line with the opinions of some investigators who previously used Ames test to evaluate mutagenicity and antimutagenicity of plant extracts and fruit juices (Zahin et al., 2010;Schneider et al., 1997;Cherdshewasart et al., 2008;Ham et al., 2009;Karekar et al., 2000;Oh et al., 2008;Rauscher et al., 1998;Son et al., 2003). In the other of FDFJ except M. fragrans, induction of negative percentage reduction of CP-induced CA or total arrest of cell division by the mixture of the FDFJ and CP at certain concentrations may be due to prooxidative activity of phenoilc compounds, causing generation of ROS to cause more DNA damages. The induction of CA by CP in this study was possible due to the metabolism of CP to phosphoramide mustard and acrolein constituents. With the breaking down of CP to these two constituents by the A. cepa cellular components, the suitability and reliability of the A. cepa test in evaluating the antimutagenicity of chemical compounds against mutagenicity of an indirect mutagen like CP is convincingly supported by our results. Anticarcinogenic and antimutagenic activity of medicinal and food plants may be due to a variety of mechanisms such as inhibition of genotoxic effects, inhibition of cell proliferation, signal transduction modulation, scavenging of free radicals, induction of detoxification enzymes, induction of cell-cycle arrest and apoptosis, modulation of cytoskeletal proteins playing a key role in mitosis and the inhibition of topoisomerase I or II activity (Zahin et al., 2010). Therefore, it can be concluded from our results that the mechanism of antimutagenic effect of the FDFJ on the mutagenic action of CP may be due to prevention of acrolein free radicals-induced DNA damage in A. cepa cells.

Conclusions
In this study, it was clearly demonstrated that the FDFJ of M. fragrans, among the evaluated fruit juices, possessed consistent cytotoxic and non significant genotoxic effects on the cell division and chromosome structure in Allium cepa.
This FDFJ also had significant antimutagenic activity against CP-induced mutagenicity at all tested concentrations. These activities are not unlikely to be connected with the phenolic compounds, acting as antioxidants in the FDFJ of M. fragrans. It is therefore, logical to say that the type (s) of free radical scavengers present in this juice may be promising candidate(s) for cancer prevention. Nevertheless, further investigations should be geared towards validation of these findings using the animal bioassays.