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 1: Percentage yield of the crude extract of the collected plants:
Name of the plant
|
% (wt/wt)
|
Punica granatum
|
0.30
|
Opuntia ficus- indica
|
0.06
|
Preliminary phytochemical screening
The air-dried powders of all plants under study were subjected to the phytochemical tests and the results are combined in Table 2.
Preliminary phytochemical screening revealed that the air dried, powdered plant peels under study contain carbohydrates and glycosides, sterols and triterpenes, and flavonoids. 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.
Table 2: Results of preliminary phytochemical screening of the air dried powdered plant peels
Test
|
P. granatum
|
O. ficus -indica
|
Sterols &/or Triterpenes
|
+
|
++
|
Carbohydrates &/or glycosides
|
+
|
+
|
Anthraquinones
|
-
|
-
|
Flavonoids
|
+
|
+
|
Coumarins
|
-
|
-
|
Alkaloids&/or nitrogenous Compounds
|
-
|
-
|
Proteins
|
+
|
+
|
Tannins
|
+
|
-
|
Saponins
|
-
|
-
|
(++): Appreciably present
Determination of the total phenolics and flavonoids
The total amount of phenolics was calculated as gallic acid equivalent while total flavonoids were as expressed as mg of catechin equivalent (CE) per g. The results are summarized in Table 3. Total phenolic and flavonoid concentrations of P. granatum peels were much higher than that of O.ficus-indica (27.600 mg GAE/g and 1.195 mg CE/g), respectively.
Table 3: Identification of total phenolics and flavonoids in P. granatum and O.ficus-indica peel extracts
Sample
|
Total phenols (mg GAE/g)
|
Total flavonoids (mg CE/g)
|
P. granatum
|
27.600
|
1.195
|
O. ficus-indica
|
5.820
|
0.350
|
HPLC Identification and quantification of flavonoids and phenolics
The analysis revealed that P. granatum peels composed mainly of 16 flavonoidal and 18 phenolic compounds, while O. ficus-indica comprised of 18 flavonoids and 10 phenolics. The most predominant flavonoid was hesperidin (31885.85 mg/100g), while the major phenolic compounds were pyrogallol (116278.78 mg/100g), catechein (41864.38 mg/100g), and gallic acid (13396.79 mg/100g). On the other hand, HPLC analysis of O. ficus-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).
Table 4: Identification and quantification of flavonoids of the ethanolic extracts of P.granatum and O. ficus-indica by HPLC analysis
Flavonoids
|
Concentration (mg/100g)
|
P.granatum
|
O. ficus-indica
|
Apigenin -7- glucoside
|
287.87
|
295.23
|
Apigenin -6- arabinose-8- galactoside
|
1182.32
|
752.21
|
Apigenin -6- rhamnose-8- glucoside
|
936.16
|
452.32
|
Apigenin -7-O-neohespiroside
|
376.45
|
211.23
|
Naringin
|
2489.85
|
231.21
|
Hesperidin
|
31885.85
|
114.65
|
Isorhamnetin 3-O-rutinoside
|
----
|
1738.24
|
Isorhamnetin 3-O-galactoside
|
----
|
864.51
|
Quercetin 3-O-rutinoside
|
----
|
498.25
|
Quercetin 3-O-glucoside
|
----
|
291.35
|
Rutin
|
1504.15
|
425.35
|
Kaempferol-3,7-dirhamnoside
|
1981.26
|
2919.31
|
Quercetrin
|
1069.09
|
258.35
|
Acacetin-7- neo hesperside
|
78.54
|
238.25
|
Quercetin
|
124.73
|
----
|
Naringenin
|
25.11
|
61.25
|
Hespirtin
|
218.68
|
21.20
|
Kaempferol
|
13.13
|
----
|
Luteolin-7-O-glucoside
|
-----
|
152.21
|
Rhamentin
|
67.45
|
----
|
Apigenin
|
17.68
|
69.25
|
Table (5): Identification and quantification of phenolic compounds of the ethanolic extracts of P. granatum and O. ficus-indica by HPLC analysis
Phenolic compounds
|
Concentration (mg/100g)
|
P. granatum
|
O. ficus-indica
|
Gallic acid
|
13396.79
|
157.58
|
Pyrogallol
|
116278.78
|
----
|
4-Amino benzoic acid
|
618.40
|
----
|
Protocatchuic acid
|
3049
|
251.32
|
Catechein
|
41864.38
|
625.25
|
Chlorogenic acid
|
8787.60
|
-----
|
Catechol
|
5656.81
|
452.23
|
Caffeine
|
1543.62
|
----
|
ρ-Hydroxy benzoic acid
|
12391.13
|
----
|
Caffeic acid
|
321.36
|
851.32
|
Quinic acid
|
----
|
4825.71
|
Malic acid
|
----
|
3527.14
|
Vanillic acid
|
1149.96
|
----
|
ρ-Coumaric acid
|
261.38
|
125.31
|
Ferulic acid
|
581.26
|
247.15
|
Iso-Ferulic acid
|
496.96
|
185.21
|
Ellagic acid
|
3418.32
|
----
|
Benzoic acid
|
5066.72
|
----
|
3,4,5- Methoxycinnamic acid
|
170.58
|
----
|
Cinnamic acid
|
49.29
|
----
|
Isolation of P. granatum compounds (Fig. 1)
Triterpenes from chloroform fraction
α-Amyrin acetate (Compound 1) was isolated from the column by petroleum ether: chloroform (60%:40% v/v), purification was performed on TLC plates by benzene-ethyl acetate (19:1 v/v) as a solvent system. It was in a form of white needles, melting point 226 °C. The 1H NMR spectrum (400 MHz, CDCl3, (ppm) revealed signals at δ 0.89 to δ 1.12 (m, 18H, 6 xCH3), δ 1.22 to δ 1.45 (m, 18H, 9 xCH2). At δ 1.89 to δ 2.44 (8H, methine protons), δ 3.61 (1H, CHOH), δ 5.36 (1H, vinylic proton), δ 5.02 and δ 5.14) that's a differentiating from β-amyrin olefinic protons that exist at δ 5.16. Mass spectrum showed M+ at m/z 468 for molecular formula C32H52O2 and 218 (100%) abundance. The other major fragments appeared at m/z: 408, 393, 365, 273, 249, 203, 189. These data are in accordance with [5] which previously isolated this compound from P. granatum leaves.
Friedelin (Friedelan-3-one) (Compound 2) was isolated from petroleum ether: chloroform (50%:50% v/v), then purified on TLC by benzene-ethyl acetate (8:2), the melting point of the white crystals was 263oC. FT-IR: peak at 2965 and 2859 cm-1 for C-H stretching, 1710 cm-1 for C=O stretching, 1442 cm-1 for C-H bending. Mass spectrum showed m/z at 426 [M]+ for molecular formula C30H50O, in addition to other major peaks at 411, 302, 273, 218, 205, 163, 44 (100).1H NMR (400 MHz, CDCl3) (δ ppm): 0.75 (3H, s, H-24), 0.89 (3H, s, H-25), 0.90 (3H, d, H-23), 0.98 (3H, s, H-30), 1.00 (3H, s, H-26), 1.03 (3H, s, H-27), 1.08 (3H, s, H-28), 1.21 (3H, s, H-29), 1.24 (3H, s, H-30), 1.99 (1H, m, H-1a), 2.29 (2H, m, H-2b, H-4), 2.42 (1H, m, H-2a), 1.27-1.79 (m, rest of the protons), These data are in accordance with that mentioned in [25]. This compound was previously isolated from P. granatum leaves by [26].
Lup-20(29)-en-3β-ol(compound 3) was in a form of colorless amorphous powder, eluted with petroleum ether: chloroform (40%:60% v/v), 1H NMR (400 MHz, CDCl3) (δ ppm): 4.73 (brs, 1H, H2-29), 4.60 (brs,1H, H2-29 ), 3.18 (dd, 1H, H-3 ), 2.99 (brs, 1H, H-19), 1.68 (brs, 3H, Me-30), 1.36 (brs, 3H, Me-23), 1.25 (brs, 3H, Me-25), 0.92 (brs, 3H, Me-24), 0.89 (brs, 3H, Me-26), 0.84 (brs, 3H, Me-28), 0.75 (brs, 3H, Me-27). 26), 14.67 (C-27), 20.82 (C-28), 109.66 (C-29), 22.66 (C-30). Mass spectrum show m/z at 426 [M]+ for molecular formula C30H50O, in addition to 411, 408, 396, 393, 381, 363, 286, 272, 258, 236, 222, 219(100), 207, 205, 191 (41.0), 189. By comparing these data with previous literature [27] and on the basis of spectral data analyses, this compound was identified as lup-20(29)-en-3β-ol.
Flavonoids from ethyl acetate fraction
Quercetin-3,4'-dimethyl ether-7-O-α-Larabinofuranosyl β-D-glucopyranoside (Compound 4) was isolated in the form of yellow powder, m.p. 176 °C. It gave a positive test for flavonoids. Mass spectrum m/z: 625 [M + H]+, 493 [M + H-arabinosyl]+, m/z 331 was attributed to the aglycone moiety, and two diagnostic peaks at m/z 153 and 149 generated through retro-Diels Alder fragmentation were consistent with the presence of two hydroxy groups in ring A and one hydroxy, one methoxy in ring B. An intense peak at m/z 287 (aglycone – CH3CO]+ provided evidence for aglycone moiety being a quercetin 3,4'dimethyl ether. 1H NMR (400 MHz, CD3OD, δ ppm) : 12.73 (5-OH), 7.60 (1H, d), 7.46 (1H, dd, 6'-H), 6.94 (1H, d, 5'-H), 6.75 (1H, d, 8-H), 6.42 (1H, d, 6-H), 5.06 (1H, d) was assigned to a glucosyl anomeric proton and suggested that the glycosidic bond had a β-linkage, 4.74 (1H, d, 1-H) was assigned to the arabinosyl anomeric proton with an α- linkage, The singlet 3.80 (3H, s, 3-OCH3), 3.71 (3H, s, 4'-OCH3) were indicative of a methoxyl group attached to the B and C rings. The above spectral data approved that this compound had both the diglycoside moiety and methoxy groups. This compound was previously isolated from the ethyl acetate fraction of P. granatum bark by [28].
Punicaflavanol named 5, 6, 7, 8, 2', 3', 5'- heptahydroxy -4'-methoxy flavanone (compound 5) was obtained as pale yellow crystals from chloroform–methanol (9:1). It responded positively to the test of flavonoids. The mass spectrum exhibited a molecular ion peak at m/z 366, pointing to the molecular formula C16H14O10. The retro-Diels-Alder fragmentation of ring C yielded the diagnostic peaks at m/z 184 and 182, supporting the presence of four hydroxyl groups in ring A and three hydroxyl and one methoxyl groups in ring B, respectively. The generation of important ion fragments at m/z 140, 188, and 155, 211 also supported the substitution pattern. The ion fragments at m/z 338, 323, and 351 [M – Me]+ arose from the removal of the carbonyl group and the methyl group from the molecular ion peak. The 1H NMR spectrum (400 MHz, CD3OD, δ ppm) showed a one-proton broad signal at δ 7.31 attributed to H-6 suggesting the 2,3,4,5-tetraoxygenated pattern of ring B. δ 5.32 (dd), 2.97 and δ 2.81 (mm) was characteristic of H-2β of the flavanone moiety. A three-proton broad signal at δ 3.62 was attributed to the methoxyl protons attached at C-4'. The 13C-NMR (125 MHz, CD3OD)spectrum displayed important signals for the C-4 carbonyl carbon (δ 192.06) and the flavanone carbon between δ 173.23–37.01. The signals at δ 52.03 confirmed 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-methoxyflavanone and was previously isolated from P. granatum flower [29].
Hydrolysable tannins from butanol fraction
In the current study, two major hydrolysable tannins were isolated and identified 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 Rf value was 0.35 in water: acetic acid (3:2)v/v solvent system and gave bluish-black color with 5% FeCl3 spraying agent. The 1H NMR spectrum (400 MHz, CD3OD, δ ppm) showed the presence of signals at 4.6 (proton of anomer of glucose), 2.00–4.9 (protons of glucose moiety), 6.68–7.21 (protons of gallagyl moiety), 7.50–10.00 (protons of hydroxy group). 13C-NMR (125 MHz, DMSO): 88.50 & 97.15 (α and β-anomeric carbon of glucose), 63.15–75.31 (other carbons of glucose), 168.21 & 169.54 (C=O associated with gallayl moiety), 158.00 & 158. 12 (carbon associated with lactone moiety), 109.84–148.62 (carbon associated with gallagyl moiety). FT-IR: Peak at 2 900 cm−1 for C-H stretching, 3 455 cm−1 indicated the presence of the O-H stretching, 1 720 cm−1 for C=O stretching, 1 355 cm−1 for C-H bending, 1170 cm−1 for C-O stretching), 1600 cm−1 represented C = C benzene ring.
punicalagin (compound 7) was in the form of yellow amorphous powder, melting point 251°C (247-250°C, 30]. Its Rf value 0.43 in water: acetic acid (3:1)v/v solvent system. On the other hand, the H1 NMR spectrum (400 MHz, CD3OD, δ ppm) showed the presence of signals at δ 5.02 for the anomeric glucose proton, signals at 4.91, 5.11, 4.81, 3.21 & 4.18 (other protons of the glucose moiety), at 7.01 & 6.52 for gallagyl moiety protons, while signals values at 6.593 & 6.596 represented the hexa hydroxy diphenoyl moiety protons. Hydroxy group protons appeared at 8.094. The 13C NMR (125 MHz, DMSO) specrtal data were as following: peaks at 90.12 & 93.90 assigned for α and β-anomeric carbon of glucose, 66.91–75.56 for other glucose carbons, while peaks at 169.12 & 168.01 represented the C=O associated with hexa hydroxy diphenoyl moiety. Gallagyl moiety C=O appeared at 168.62 & 169.25. The lactone carbon showed peak at 156.91 & 157.82. Finally peaks at 108.81-146.90 referred to the hexa hydroxy diphenoyl & gallagyl moiety carbon. FT-IR spectra: peak at 2930-2730 cm−1 represented the C-H stretching, while peak 3445 cm−1 assigned for O-H stretching, 1732 cm−1 indicated the carbonyl group stretching, C- H bending represented by peak at 1350cm−1, peaks at 1175–1 181 cm−1 associated with the C-O stretching, and the aromatic conjugation showed at 1620 cm−1.
It is to be mentioned that compounds (1-5) were isolated and identified for the first 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. ficus-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.
24-Methylene-ergosta-5-en-3β-ol (compound 9) was isolated as white powder, EI-MS, m/z 398 [M+] (100%) for the molecular formula C46H28O. Other main fragments were m/z 383 [M-CH3]+, 365 [M-CH3-H2O]+, 314 [M-C5H9-CH3]+, 299 [M-C7H13-2H]+, 281 [M-C7H13-H2O-2H]+, 271 [M-side chain-2H]+ , while 1H-NMR (400 MHz; CDCl3, d ppm) showed δ ppm 0.69 (s, H18), 0.98 (d, 6.6, H21), 1.00 (s, H19), 1.04, 1.06 (d, 6.8, H26, H27), 3.55 (m, H3), 4.67 (s, H28), 4.73 (s, H28), 5.36 (d, 4.9, H6). This compound was previously isolated from O. ficus-indica peels by [11].
Flavonoids from ethyl acetate fraction
Apigenin-7-O-glucoside (Compound 10) was in the form of yellow crystal, melting point 227 °C, 1 H NMR (400 MHz, CD3OD, δ ppm) 7.81 (2H, d, H-2'/6'), 7.36 (2H, d, H-3'/5'), 6.65 (1H, s, H-3), 6.55 (1H, d, H-6), 6.88 (1H, d, H-8), 5.01 (1H, d). Mass spectrum showed peak at m/z M+ 432 for molecular formula C21H20O10 , other observed fragments at 431 (100%), and 269 after the loss of an hexose moiety from the parent ion. By comparing the resultant data with the available literature [16], this compound was identified. It was previously isolated from O. ficus-indica fruits by [17].
Isorhamnetin 3-O-β-D-glucopyranoside (compound 11) was isolated as pale yellow amorphous powder, mass spectrum m/z: M+ 478(100) for molecular formula C22H22O12, 316 [M-Glc]+, 285, 271. 1H NMR (400 MHz, CD3OD, δ ppm): 7.95 (1H, d, , H-2'), 7.42 (1H, dd, H- 6'), 6.85 (1H, d, H-5'), 6.41 (1H, d, H-8), 6.19 (1H, d, H-6), 5.48 (1H, d, H-1''), 3.80 (3H, s, O-CH3), 3.71 (1H, m, H-4''), 3.60 (1H, m, H-2''), 3.52 (1H, m, H-6''a), 3.41 (1H, m, H-5''), 3.38 (1H, m, H-3''), 3.36 (1H, m, H-6''b); 13C NMR (125 MHz, DMSO, δ): 176.4 (C-4), 164.4 (C-7), 162.2 (C- 5), 155.1 (C-9), 157.4 (C-2), 149.5 (C-4'), 147.1 (C-3'), 134.1 (C-3), 123.6 (C-6'), 122.1 (C-1'), 114.9 (C-5'), 112.9 (C-2'), 103.8 (C-10), 102.3 (C-1''), 99.2 (C-6), 94.6 (C-8), 76.1 (C-3''), 72.9 (C-5''), 71.2 (C-2''), 68.3 (C-4''), 61.1 (C- 6''), 55.8 (O-CH3). These data are in accordance with that previously illustrated in [18].
Characterization of the isolated betanin pigment (compound 12)
The lyophilized pigment exhibited an absorbance at UV spectroscopy with significant 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 C24H26N2O13. 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. ficus-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 identified 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 beneficial impact of betanin as a significant antioxidant and anti-inflammatory 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 first time from the fruit peels in the current study whereas, compound 9 was previously detected in O. ficus indica peels by [11].
Biological activities
Median lethal dose (LD50)
The results of median lethal dose (LD50) of the ethanolic extracts of both plant peels P. granatum and O. ficus-indica are illustrated in Table 6.
The tested extracts were safe showing LD50 5.5 g/kg b.wt. for P. granatum extract, while (7.1 g/kg) for O. ficus-indica ethanol extract.
Table (6): LD50 of total extracts of the two plants:
Plant Peels Extract
|
LD50 (g/kg b.wt.)
|
P. granatum
|
5.5
|
O. ficus-indica
|
7.1
|
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 reflects 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. ficus-indica peels. This result could be attributed to their richness and diversity of many phytochemical classes such as flavonoids, 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].
Table 7: Antioxidant activities of the ethanolic extracts of the two tested plant peels:
Peel extract
|
DPPH (mg /g)
|
P. granatum
|
142.373
|
O. ficus-indica
|
110.800
|
Cytotoxic activity
Cytotoxic activities of the ethanolic extracts of P. granatum and O. ficus-indica peels against different cell lines are illustrated in Table 8.
Table 8 revealed that the ethanolic extract of P. granatum peels exhibited cytotoxic effect on all tested cancer cell lines. The most significant effect was recorded against HEPG2 (IC 50 =17µg/mL). As for the ethanolic extract of O. ficus-indica peels showed potent cytotoxic activity against colon cancer cell lines (HCT-9) at IC50 =14 µg/ml followed by liver cancer cell lines (HEPG2) at IC50 =18.5 µg/mL, while it has no activity on other cancer cell lines at the tested different concentrations. The American National Cancer Institute assigns a significant cytotoxic effect of any promising anticancer product for future bio guided studies if it exerts an IC50 value < 30 μg/mL [36].
Table 8: Cytotoxic activity against different cancer cell lines
cell lines
|
IC 50 (µg/mL)
|
P. granatum
|
O. ficus-indica
|
liver (HEPG2)
|
17.0
|
18.5
|
prostate (PC-3)
|
17.6
|
---
|
breast (MCF7)
|
22.5
|
---
|
colon (HCT)
|
19.0
|
14.0
|
lung (A549)
|
21.1
|
---
|
Antimutagenicity study (chromosome evaluation in somatic and germ cells)
1. 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-significant in comparing to the control group.
Punica granatum and O. ficus-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 significant 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. ficus-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. ficus-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.
Table 9: Percentage of inhibition of abnormalities in bone marrow cells after treatment with different doses of P. granatum and O. ficus-indica peel extracts.
Groups/Treatments
(mg/kg b.wt)
|
Total abnormal metaphases
|
No. of different types of abnormal metaphases
|
Inhibitory index
|
No.
|
Mean(%) ± SE
|
G.
|
Frag. and/or Br.
|
Del.
|
C.F.
|
M.A.
|
Polyp.
|
I. Negative control
|
20
|
4.00±0.70
|
9
|
8
|
3
|
0
|
0
|
0
|
-
|
II. CP (20)
|
151
|
30.20±0.66 a
|
17
|
63
|
15
|
5
|
48
|
3
|
-
|
III. P. granatum (150)
|
21
|
4.20±0.80
|
10
|
8
|
3
|
0
|
0
|
0
|
-
|
O. ficus-indica (150)
|
27
|
5.40±0.45
|
9
|
15
|
2
|
0
|
0
|
1
|
-
|
IV. P. granatum (50) + CP
|
136
|
27.20±0.58ab
|
18
|
49
|
8
|
0
|
58
|
3
|
12
|
O. ficus-indica (50) + CP
|
154
|
30.80±0.50a
|
11
|
71
|
8
|
4
|
56
|
4
|
4
|
V. P. granatum (100)+ CP
|
125
|
25.00±0.89ab
|
19
|
47
|
3
|
0
|
56
|
0
|
20
|
O. ficus-indica (100) + CP
|
140
|
28.00±0.58 ab
|
17
|
36
|
4
|
0
|
51
|
5
|
19
|
VI. ) P. granatum (150)+ CP
|
118
|
23.60±0.87ab
|
14
|
49
|
5
|
4
|
46
|
0
|
25
|
O. ficus-indica (150) + CP
|
122
|
24.40±0.38ab
|
14
|
52
|
3
|
3
|
48
|
2
|
14
|
Number of examined metaphases=500 (100 metaphase/animal, 5 animals/ group); G.: Gap; Frag. : Fragments;
Br.: Breaks; Del.: Deletions; C.F.: Centric Fusions; M.A.: Multiple Aberrations; Polyp: Polyploidy.
a: Significant compared to negative control (p<0.01); b : Significant compared to CP treatment (p<0.01); t-test.
2. Chromosome evaluation and percentage of inhibition of abnormalities in germ cells (spermatocyte cells):
Different number and percentage of abnormalities in spermatocyte cells of all treated groups after treatment with different doses of both plant ethanolic extracts with CP treated group are shown in Table (10).
CP induced a high percentage of aberrations (p<0.01). The percentage of aberrations in the animal groups treated with 150 mg/kg b. wt of both plant peel extracts for 7 days was nearly close to the negative control. This show the safety of all extracts on chromosomes of germ cells.
Pre-administration of CP-treated groups with plant extracts at the doses 50, 100 and 150 mg/kg b. wt for 7 days reduced in a statistically significant manner (p<0.01) the number of abnormalities. This reduction of abnormalities is a dose dependent increased with increasing the dose of treatment.
The percentage of inhibitory index in chromosome aberrations in bone marrow cells of P. granatum and O. ficus-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. ficus-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 significant 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].
Results from 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 significant 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.
Many studies proved that high flavonoid intake may be correlated with a decreased risk of cancer and showed strong antioxidant effect. They provide evidence for the protective roles of flavonoids against cancer [38]. In vitro studies indicate that the anticancer activities of flavonoids 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, flavonoids could be developed as promising antioxidant agents as well as their chemopreventive effect.
Both P. granatum and O. ficus-indica polyphenols have possessed in vitro antioxidant effect, together with curable effects against cancer, and inflammatory diseases [5 & 41].
Table 10: Percentage of inhibition of abnormalities in spermatocyte cells after treatment with different doses of P. granatum and O. ficus-indica peel extracts.
Groups/Treatments
(mg/kg b.wt)
|
Total abnormal metaphases
|
No. of different types of abnormal metaphases
|
Inhibitory index
|
No.
|
Mean(%)±SE
|
XY-uni.
|
Auto.uni
|
XY-uni.+ Auto.uni.
|
Frag.
|
Chain (IV)
|
I. Negative control
|
14
|
2.80±0.73
|
11
|
3
|
0
|
0
|
0
|
0
|
II. CP (20)
|
83
|
16.60±0.50a
|
44
|
30
|
4
|
2
|
3
|
0
|
III. P. granatum (150)
|
16
|
3.20±0.58
|
11
|
5
|
0
|
0
|
0
|
0
|
O. ficus-indica (150)
|
19
|
3.80±0.52
|
12
|
7
|
0
|
0
|
0
|
0
|
IV. P. granatum (50)+ CP
|
65
|
13.00±0.70ab
|
53
|
12
|
0
|
0
|
0
|
26
|
O. ficus-indicia (50) + CP
|
73
|
14.60±0.58a
|
48
|
21
|
1
|
1
|
2
|
5
|
V. P. granatum (100) + CP
|
60
|
12.00±0.70ab
|
46
|
14
|
0
|
1
|
0
|
33
|
O. ficus-indica (100) + CP
|
62
|
12.40±0.52ab
|
49
|
12
|
0
|
0
|
1
|
23
|
VI. P. granatum (150)+ CP
|
56
|
11.20±0.80ab
|
34
|
20
|
0
|
1
|
1
|
39
|
O. ficus-indica (150)+ CP
|
56
|
11.20±0.42ab
|
35
|
17
|
1
|
21
|
1
|
33
|
Number of examined metaphases=500 (100 metaphase/animal, 5 animals/ group); XY-uni: XY- univalent; Auto. uni.: Autosomal univalent; XY-uni.+ Auto. uni.: XY-univalent + Autosomal univalent; Frag.: Fragment. a: Significant compared to negative control (p<0.01); b: Significant compared to CP treatment (p<0.01); t-test.
Discussion
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. ficus-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. ficus-indica peels possessed the cytotoxic mechanism of action by decreasing cell proliferation and apoptosis induction in the cancer cells. This was confirmed 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 significant 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 flavonoids including anthocyanins, catechins and other complex flavonoids 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. ficus-indica peels) on the redox-regulated pathways involved in the cell growth and inflammation 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 H2O2-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 efficient prevention of micronuclei and chromosomal aberrations frequency in bone marrow cells and DNA fragmentation in vivo.