Antimutagenic and Cytotoxic Potential of Punica Granatum L. And Opuntia Ficus-indica L. Peels

Introduction: This study pointed to validate the phytochemistry, antioxidant and antimutagenic activities of two common plant peels; Punica granatum L. and Opuntia cus-indica L. Material and methods: HPLC analysis was performed for identication of phenolics and avonoids, beside to isolation of some triterpenes and phenolics from both plant peels. Results: P. granatum peels composed of 16 avonoids and 18 phenolics, while O. cus-indica comprised of 18 avonoids and 10 phenolics. α-Amyrin acetate (1), friedelin (2), lup-20(29)-en-3β-ol (3), quercetin-3,4'-dimethyl ether-7-O-α-Larabinofuranosyl β-D-glucopyranoside (4), punicaavanol (5), and two hydrolyzabl tannins (6&7) were isolated from P. granatum peels, while friedelin (8), 24-Methylene-ergosta-5-en-3β-ol (9), apigenin-7-O-glucoside (10), isorhamnetin 3-O-β-D-glucopyranoside (11), and betanin pigment (12) were isolated from O. cus-indica peels. Compounds (1-5 and 8, 10, 11, 12) were isolated for the rst time from both plant peels. P. granatum and O. cus-indica peel extracts have relatively signicant antioxidant, cytotoxic and antimutagenic effects. Discussion and conclusion: The tested plant peel extracts could be a reliable source as natural antioxidant, antimutagenic and cytotoxic agents with a high level of safety. The novelty of this study is the comparison of such activities of the peels under study.


Introduction
The reactive species are highly toxic and mutagenic. They can promote DNA damage and initiate auto oxidation reactions causing cancer, and other degenerative diseases, therefore the intake of natural antioxidants is crucial for free radicals scavenging by different mechanisms, such as active species prohibition or by seizing metal ions, leading to repairing damage [1]. Consequently, many evidences of mutation-related carcinogenesis were established resulting in much detailed studied on mutagenesis [2].
Mechanism of mutagenesis mainly includes the generation of reactive oxygen species. Therefore, the current study focusing on edible plant peels which are rich in avonoids, terpenes, phenolic compounds, and natural pigments which are reported as anticancer, antioxidant and antimutagenic agents [3].
Pomegranate (Punica granatum L.), family Punicaceae, is native to the Mediterranean region and the previous phytochemistry studies of pomegranate fruits revealed the presence of polyphenolic compounds that include avonoids and hydrolyzable tannins beside other main classes [4]. According to literature, many bioactivities of P. granatum L. have been reported such as antioxidant, anti-in ammatory, antimicrobial and others [5; 6]. Although there are a fair number of scienti c studies on P. granatum fruits and peel extracts regarding in vitro and in vivo anticancer activities, insu cient data are available on the antimutagenic effects of P. granatum peels [7; 8].
Prickly pear or cactus pear, Opuntia cus-indica L. belongs to family Cactaceae. The fruits have been widely used in folk medicine to treat many diseases. The majority of studies had focused on cactus fruit as a source of bioactive compounds [9]. Opuntia peels are usually discarded after fruit consumption. However, there are no adequate studies on the peels, although they contain a diversity of signi cant amounts of bioactive constituents with various biological activities [10]. A recent study investigated the prophylactic effect O. cus-indica petroleum ether peel extract against irradiation-induced colitis in rats. The fruit peel extract proved to possess a potential antioxidant and anti-in ammatory activity as well as limiting the colonic complications caused by irradiation. The activities were attributed to the richness of peel extract in fatty acid methyl ester content, terpenes and sterols [11].
Chemical Investigation Plant material Punica granatum L. and O. cus-indica L. (Pink red cultivar) fresh fruits were purchased in January 2019 from the local market, Giza, Egypt. The specimens of fruits were identi ed by Prof. Dr. Gamal Farag professor, Horticulture Research Center, Ministry of Agriculture, Giza, Egypt. The fruits were washed. The peels were collected separately, dried under shade and grinded into powder. Crude ethanolic extracts were prepared by maceration of powdered samples in 80% ethanol. It was then kept at room temperature, shaken every 2 h for one day. Thereafter, it was ltered by using Whatmann lter paper No. 1. and evaporated by rotary evaporator at 50° C till dryness. The crude extracts were kept in the refrigerator for the phytochemical and biological analyses.

Phytochemical screening
The powdered plant peels were subjected to the phytochemical screening for carbohydrates, sterols and / triterpenes, tannins, avonoids, alkaloids and nitrogenous compounds as mentioned in [12].
HPLC analysis HPLC analysis were carried out according to Matilla [13] using Agilent Technologies 1100 series liquid chromatography equipped with an auto sampler and a diode-array detector. The analytical column is Eclipse XDB-C18 (150 X 4.6 µm; 5 µm) with a C18 guard column (Phenomenex, Torrance, CA). The mobile phase consisted of acetonitrile (solvent A) and 2% acetic acid in water (v/v) (solvent B). The ow rate was kept at 0.8 mL/min for a total run time of 70 min and the gradient program was as follows: 100% B to 85% B in 30 min, 85% B to 50% B in 20 min, 50% B to 0% B in 5 min and 0% B to 100% B in 5 min. The injection volume was 50 µL and peaks were monitored simultaneously at 280 and 320 nm for the benzoic acid and cinnamic acid derivatives, respectively. All samples were ltered through a 0.45 µm Acrodisc syringe lter (Gelman Laboratory, MI) before injection. Peaks were identi ed by congruent retention times and UV spectra and compared with those of the standards.

Determination of total phenolic and avonoid contents
The total phenolic content was determined according to the Folin-Ciocalteu procedure. The total phenolic content was determined by means of a calibration curve prepared with gallic acid, and expressed as milligrams of gallic acid equivalent (mg GAE) per gram of sample. While the total avonoid content was expressed as mg of catechin equivalent (CE) per g of sample according to [14].
Isolation of the main compounds from P. granatum and O. cus-indica peels The total extract of both plant peels was concentrated and partitioned with chloroform targeting triterpene isolation, the residue was fractioned with ethyl acetate for isolation of phenolics in both plant peels. While further fractionation step with butanol was performed for P. granatum peels for isolation of hydrolysable tannins. The chloroform soluble fraction was concentrated and loaded on a silica gel column, eluted with various ratios of petroleum ether and chloroform. The fractions were tested for the presence of triterpenes using Liebermann-Burchard reagent, then they were spotted on TLC plate and sprayed with 10%H 2 SO 4 reagent [15]. The positively tested fractions were puri ed on TLC silica gel plates using benzene:ethyl acetate solvent system (19:1 & 8:2 v/v). Furthermore, the ethyl acetate fraction was concentrated and puri ed on TLC silica gel chromatography using CHCl 3 : CH 3 OH (9:1 v/v). The obtained bands were examined under UV at 254 and 366 nm. The presence of avonoid constituents was con rmed by spraying with 1% ethanolic solution of AlCl 3 [16]. The isolated compounds were further puri ed several times with preparative TLC and their structure elucidation were con rmed by different spectral analyses.
On the other hand, the butanol fraction of P. granatum was subjected to silica column chromatography using acetone: water solvent system at different concentrations with increasing order of polarity, similar fractions was collected puri ed on sephadex column chromatography aiming for isolation of tannins.
Based on the spectral analyses (FT-IR, 1 H-NMR, 13 C-NMR and Mass spectroscopy) of the isolated compounds, the structures of the compounds were interpreted.
Isolation and characterization of betanin pigment from O. cus-indica peels Fresh peels of O. cus-indica were carefully chopped and extracted with ethanol: water (80%), under continuous mechanical stirring for 1 h in the dark. The pH of the extraction solvent was adjusted to 4.5 for more stability of the extracted pigment. The extract was centrifuged at 15000 g for 30 minutes at 4˚C. Then it was subjected to silica gel column chromatography for puri cation of betanin with the elution solvent system of methanol: water: glacial acetic acid (9:0.3:0.7). Puri cation was performed using TLC silica gel plates and developed using methanol : 5% aqueous acetic acid (1:1) v/v, visualized under short and long UV wavelengths (254, 366 nm), then sprayed with vanillin reagent mixture. Similar bluish red bands with the same R f values were collected and concentrated under vacuum using the rotatory evaporator and kept in freezer till further spectral investigation. The con rmation and structure elucidation of betanin was done by UV-visible, FT-IR, and 1 HNMR spectroscopy [19].

Biological investigations
Median lethal dose (LD 50 ) Adult albino mice, of Sprauge Dawely strain 25-30 g (6 animals/ group) were obtained from the animal house colony of National Research Centre, Cairo, Egypt. They were kept under the same hygienic conditions and well-balanced diet and water. Doses of the two extracts were calculated according to [20], and were administered orally. The median lethal doses (LD 50 ) of the ethanolic extracts of P. granatum and O. cusindica fruit peels were determined according to the method described by [21]. The mice were observed for 24 hours, symptoms of toxicity and mortality rates in each group were recorded and LD 50 was calculated for each extract.

In vitro antioxidant activity
Free radical scavenging capacities of the sample were determined on the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) using Trolox as a standard. The nal concentration was 200 µM for DPPH and the nal reaction volume was 3 mL. The absorbance was measured at 517 nm against a blank of pure methanol after 60 min of incubation in a dark condition. Then the absorbance was taken at 517 nm using the spectrophotometer. The standard curve was prepared using Trolox. Results were expressed as mg Trolox equivalents (TE)/g sample [14].
In vitro cytotoxic activity Potential cytotoxic activity of the two ethanolic extracts was tested using the method of [22] against HEPG2 (liver carcinoma cell line), PC3 (prostate Cancer), MCF7 (breast carcinoma cell line, HCT116 (colon carcinoma cell line), and A549 (lung cancer). The potency was compared with reference drug (Doxorubicin). Statistical analysis for cytogenetic analysis, the signi cance of the results from the negative control data and between plant extracts and CP group comparing to CP alone was calculated using t-test.
Antimutagenicity study (chromosome evaluation in somatic and germ cells) Laboratory-bred strain Swiss albino male mice of 10-12 weeks old with an average weight of 25-30 g were housed in six groups (5 animals/ group) and maintained under standard conditions.
Group I was the non-treated group (negative control). Group III was treated by repeated oral administration with the dose of 150 mg/kg b.wt of each extract for 7 days.
Groups IV, V, VI was treated by repeated oral administration with the doses of 50,100 and 150 mg/kg b.wt of each extract for 7 days with i.p. injection of (CP) 20 mg/kg body weight for 24 h.
For somatic and germ cell preparations, animals from the different groups were injected i.p. with colchicine, 2-3 h before sacri ce.
Chromosome preparations from bone marrow (somatic cells) carried out according to the method of [23] methods for spermatocyte cells. One hundred well spread metaphases were analyzed per mouse. Metaphases with different abnormalities in somatic and germ cells were recorded with a 100X magni cation light microscope (Olympus, Saitama, Japan).
Evaluation of the effect of the two plant extracts to inhibit DNA damage induced by CP was carried out according to [24] equation as follows: Statistical analysis for cytogenetic analysis, the signi cance of the results from the negative control data and between the plant extracts plus CP comparing to CP alone was calculated using t-test.

Chemical investigation
The air dried powder of the two investigated plants, were separately extracted by maceration with 80% ethanol until complete exhaustion. Percentage yield of the plants are illustrated in Table 1.  Table 2.
Preliminary phytochemical screening revealed that the air dried, powdered plant peels under study contain carbohydrates and glycosides, sterols and triterpenes, and avonoids. It can be noticed also that anthraquinones, and saponins appeared to be absent from all tested plants, while proteins and tannins present in all tested plant material, while tannins present only in P. granatum peels. Determination of the total phenolics and avonoids The total amount of phenolics was calculated as gallic acid equivalent while total avonoids were as expressed as mg of catechin equivalent (CE) per g. The results are summarized in Table 3. Total phenolic and avonoid concentrations of P. granatum peels were much higher than that of O. cus-indica (27.600 mg GAE/g and 1.195 mg CE/g), respectively. On the other hand, HPLC analysis of O. cus-indica ethanol extract revealed that the main compound was kaempferol-3,7-dirhamnoside (2919.31 mg/100g) followed by isorhamnetin 3-O-rutinoside (1738.24 mg/100g). Nevertheless, the main phenolic compounds were quinic and malic acids (4825.71and 3527.14 mg/100g), respectively (Tables 4and 5).  . The signals at δ 52.03 con rmed the existence of one methoxyl group in the molecule. On the basis of spectral data analyses and comparing with literature, the structure has been elucidated as 5,6,7,8,2,3,5-heptahydroxy-4-methoxy avanone and was previously isolated from P. granatum ower [29].

Hydrolysable tannins from butanol fraction
In the current study, two major hydrolysable tannins were isolated and identi ed from P. granatum butanol fraction.
Punicalin (compound 6) was isolated as yellow amorphous powder, m.p. 247°C in accordance to that mentioned in [30]. Its R f value was 0.35 in water: acetic acid (3:2)v/v solvent system and gave bluish-black color with 5% FeCl 3 spraying agent. The 1 H NMR spectrum (400 MHz, CD 3  It is to be mentioned that compounds (1)(2)(3)(4)(5) were isolated and identi ed for the rst time in this study from the fruit peel while, compounds 6 and 7 were previously isolated from P. granatum peels by [30].
Isolation of O. cus-indica compounds (Fig. 2) Triterpenes from chloroform fraction Friedelin(Friedelan-3-one) (compound 8). This compound was previously isolated and characterized from the O. dillenii stems [31]. The spectral data are as fore mentioned in compound 2. cus-indica peels by [11]. Characterization of the isolated betanin pigment (compound 12) The lyophilized pigment exhibited an absorbance at UV spectroscopy with signi cant single peak at 538 nm which expressed to λmax of betanin as reported. The mass calculated by ESI-mass m/z for M + 551 (89%) for molecular formula C 24 H 26 N 2 O 13 . The spectrum also showed base peak at m/z 389 [betanidin] + aglycone that was produced by fragmentation of the parent ion of m/z of 551 assigned to glucose loss of betanin. Our results are in accordance with that reported by [17]. FT-IR (KBr/cm -1 ) showed peak at 2920 cm -1 for C-H stretching, 3420 cm -1 indicated the presence of the O-H stretching, the NH stretching appeared 3250 cm -1 , 1700 cm -1 for C=O stretching, 1250 cm -1 for C-H bending, 1162 cm -1 for C-O stretching), 1638 cm -1 represented C = C of benzene ring.
This compound was previously detected in O. cus-indica peeled fruits using LC-MS analysis in a study performed by [17]. By comparing these spectral data with previous research studies [19], this compound could be identi ed as betanin (Fig. 2). Only a few fruits and vegetables contain betalains and the best known is beetroot (Beta vulgaris), an important food colorant and Opuntia spp. fruits (prickly pear). Several investigations have also reported bene cial impact of betanin as a signi cant antioxidant and antiin ammatory factor, cancer cells suppressor, lipid peroxidation and in heme disintegration [32; 17; 33].
It should be noted that compounds (8, 10,11,12) are isolated from the rst time from the fruit peels in the current study whereas, compound 9 was previously detected in O. cus indica peels by [11].

Biological activities
Median lethal dose (LD 50 ) The results of median lethal dose (LD 50 ) of the ethanolic extracts of both plant peels P. granatum and O.
cus-indica are illustrated in Table 6.
The tested extracts were safe showing LD 50 5.5 g/kg b.wt. for P. granatum extract, while (7.1 g/kg) for O.
cus-indica ethanol extract. In Vitro antioxidant activity DPPH free radical scavenging assay was carried out on the ethanolic extracts of both plant peels in order to investigate their antioxidant activities using Trolox as a standard. Results were expressed as (mg Trolox equivalent TE/g) in Table 7.
Measuring the antioxidant capacity of the fruit peel total extracts was performed through evaluating their ability to convert the violet color of DPPH to the yellow and using Trolox as a standard [34]. The extent of discoloration re ects the activity of the tested extracts as free radical scavenging agents. Results revealed that P. granatum peel extract has relatively more antioxidant effect than that of O. cus-indica peels. This result could be attributed to their richness and diversity of many phytochemical classes such as avonoids, triterpenes, pigments and tannins. These classes possess their antioxidant ability via neutralizing reactive oxygen species such as hydrogen peroxide. Thus, the ability of phytochemicals to inhibit free radical generation, by restoring the redox state of the internal tissue organs can possibly provide reasonable explanation for their prophylactic role as well [35]. Cytotoxic activity Cytotoxic activities of the ethanolic extracts of P. granatum and O. cus-indica peels against different cell lines are illustrated in Table 8.

Chromosome evaluation and percentage of inhibition of aberrations in bone marrow cells (somatic cells)
Different number and percentage of abnormalities in all treated groups are shown in Table 9. CP treated group (II) induced a high percentage of aberrations (p<0.01). The percentage of aberrations in the animal group treated with 150 mg/kg b. wt of each plant peel extract for 7 days (group III) was nearly close to the negative control group where they were statistically non-signi cant in comparing to the control group.
Punica granatum and O. cus-indica peel extracts exhibited safe effect regarding the total abnormal metaphases comparing to the control group. This proved the safety of the tested extracts on chromosomes of somatic cells.
Pre-administration of CP-treated groups with the tested extracts at the doses 50, 100 and 150 mg/kg b. wt for 7 days (groups IV, V and VI ) reduced the number of abnormalities in a statistically signi cant manner (p<0.01). This reduction of abnormalities is a dose dependent increased with increasing the dose of treatment. The percentage of the inhibitory index of the different plant extracts is listed in Table 9.
The percentage of inhibitory index in chromosome aberrations in bone marrow cells of P. granatum and O. cus-indica peels extracts were greater than the negative control even at the lowest dose of the plant extract (50 mg/ kg b.wt) of P. granatum and O. cus-indica, they recorded (12 and 4), respectively. The obtained results emphasized the antimutagenic effect of the tested extracts as they have the ability to repair the genotoxic effect of the anticancer drug cyclophosphamide.  The percentage of inhibitory index in chromosome aberrations in bone marrow cells of P. granatum and O. cus-indica peels extracts were greater than the negative control even at the lowest dose of the plant extract (50 mg/ kg b.wt) of P. granatum and O. cus-indica, they recorded (26 and 5), respectively, which proves the antimutagenic effect of the tested extracts as their potentiality to repair the genotoxic effect caused by the anticancer drug.
The selected anticancer drug cyclophosphamide CP induced signi cant percentage of chromosomal aberrations. Its cytotoxic effects result from chemically reactive metabolites that alkylate DNA and protein, producing cross-links. The injury of normal tissues is the major limitation of using CP, which gives rise to numerous side effects [37]. Tables 9 and 10 showed the antimutagenic effect of both tested extracts where they have the ability to repair the genotoxic effect of the anticancer drug cyclophosphamide. They possessed safe effect on genetic material (non-genotoxic) in both somatic and germ cells in the examined groups compared to the negative control group. In addition, they achieved antimutagenic activities in comparing to the positive control group and with CP treated groups administered with each extract, a statistically signi cant decrease in chromosomes abnormalities was found in the bone marrow cells and germ cells. The rate of protection was proportionally associated to the dose of the extracts.

Results from
Many studies proved that high avonoid intake may be correlated with a decreased risk of cancer and showed strong antioxidant effect. They provide evidence for the protective roles of avonoids against cancer [38]. In vitro studies indicate that the anticancer activities of avonoids may be related to inhibiting cell proliferation, adhesion, and invasion, inducing cell differentiation, cell cycle arrest, and apoptosis, etc. [39], while in vivo studies demonstrated their ability to inhibit carcinogenesis by affecting the molecular events in the initiation, promotion, and progression stages [40]. Based on these results, avonoids could be developed as promising antioxidant agents as well as their chemopreventive effect. Both P. granatum and O. cus-indica polyphenols have possessed in vitro antioxidant effect, together with curable effects against cancer, and in ammatory diseases [5 & 41]. Mutagenesis is the of mutations in DNA molecules. Spontaneous mutations are essential to produce genetic variation necessary for natural selection. Contrarily, other mutations that cause changes in the DNA sequence or rearrangement of the chromosomes either as a result of a default in transcription that occur during DNA replication or mitosis or due to environmental exposure to genotoxins [42].
The mechanism of mutagenesis has been reported to be through the generation of reactive oxygen species which mainly act as endogenous promoters for degenerative processes, including DNA damage that probably lead to cancer, heart diseases, aging and others [2].
A plethora of studies proved the antioxidant, anticarcinogenic and other important bioactivities and correlated them to the richness of P. granatum and O. cus-indica peels of a diversity of phytochemicals (such as hydrolysable tannins, polyphenolics, triterpenes and natural pigments) which hindered both mitochondrial signaling pathway modulations and vital carcinogenesis pathways at different stages [11; 43 ; 4]. Nevertheless, methanol extract of O. cus-indica peels possessed the cytotoxic mechanism of action by decreasing cell proliferation and apoptosis induction in the cancer cells. This was con rmed by enhancing the gene Bax expression of pre-apoptosis as well as reducing the gene Bcl-2 expression of anti-apoptosis [44; 6 ; 45].
Phytochemical studies performed on P. granatum peel extract revealed that the high percentage of ellagitannins could be responsible for the signi cant antioxidant and antimutagenic effects. Hence, the reported mechanism of antimutagenic behavior could be attributed to the presence of variety of polyphenolics present in methanol extract such as avonoids including anthocyanins, catechins and other complex avonoids besides hydrolyzable tannins (punicalin, pedunculagin, punicalagin, gallagic acid and ellagic acid esters of glucose) all together play an important role by interacting with the reactive intermediates or interfering with the metabolic activation of the pro-mutagen and consequently, result in different pathways of metabolism of mutagens and carcinogens, and guard the cells against chemically induced mutagenesis [2; 7].
On the other hand, many reports in the literature demonstrated the advantageous effects of betalains (the natural pigment found in O. cus-indica peels) on the redox-regulated pathways involved in the cell growth and in ammation with no noticeable toxic effects in humans [32; 46]. Additionally, another study proved that a cactus pear extract in a 0.1 mg/mL dose reduced the H 2 O 2 -induced DNA damage in human peripheral lymphocytes in the comet assay [47]. Zorgui et al., [48] stated the ability of cactus cladodes to protect against the genotoxicity with an e cient prevention of micronuclei and chromosomal aberrations frequency in bone marrow cells and DNA fragmentation in vivo.

Conclusion
In the present study, employing natural plant wastes, as an important source of natural antioxidants is increasing due to consumers' preference being economic and possess a high level of safety. Punica granatum and O. cus-indica peels have been phytochemically investigated in details and the structure activity relationship was correlated to the anti-mutagenesis and antioxidant activities revealed the high Structure of the isolated compounds from P. granatum