GC-MS analysis of PDSE. Data showed the retention time and the relative abundance of different bioactive phytoconstituents. The peak areas (%) of 11-Dodecen-2-one, 2-methyl-10-undecenal, 1-(cyclopropyl-nitro-methyl)-cyclopentanol, 8, 11, 14-Eicosatrienoic acid, (Z, Z, Z). and Octadecanoic acid, 9, 10-epoxy-18 (trimethylsiloxy), methyl ester was 1.03, 1.14, 2.07, 2.48, and 2.53%, respectively. The peak area of Dodeca-1, 6-dien-12-ol, 6, 10 dimethyl, 12-Methyl-E, E-2, 13-octadecadien-1-ol and 13-Tetradece-11-yn-1-ol were 3.10, 3.39 and 3.91%, respectively. The peak area (%) of Ethyl iso-allocholate, 1-Heptatriacotanol, and 9, 12, 15-Octadecatrienoic acid, 2, 3-dihydroxypropyl ester, (Z, Z, Z) were 5.80, 21.07 and 53.49, respectively (Table 1).
PDSE treatment post-CTX-injection protect against body weight loss. The percentage of change in the total body weight of CTX-treated mice (Gp3) was significantly decreased when compared with the control group (Gp1). Treatment with PDSE alone did not show significant alteration in the body weight gain as compared to Gp1. Treatment with PDSE post-CTX injection (Gp6) protected against the loss of body weights when compared to CTX-injected group (Gp3) (Table 2).
Table 1
Biochemical compounds analyzed by GC-MS of the hydro-alcohol seeds extract of P. dactylifera
No. | RT (min.) | Name | M. F. | M. Wt | Peak area % |
1 | 24.61 | 11-Dodecen-2-one | C12H22O | 182 | 1.03 |
2 | 25.51 | 2-methyl-10-undecenal | C12H22O | 182 | 1.14 |
3 | 26.83 | Dodeca-1,6-dien-12-ol, 6,10 dimethyl | C14H26O | 210 | 3.10 |
4 | 27.46 | 13-Tetradece-11-yn-1-ol | C14H24O | 208 | 3.91 |
5 | 31.04 | 12-Methyl-E,E-2,13-octadecadien-1-ol | C19H36O | 280 | 3.39 |
6 | 31.41 | 1-(cyclopropyl-nitro-methyl) cyclopentanol | C9H15NO3 | 185 | 2.07 |
7 | 31.97 | 8,11,14-Eicosatrienoic acid, (Z,Z,Z) | C20H34O2 | 306 | 2.48 |
8 | 32.61 | Ethyl iso-allocholate | C26H44O5 | 436 | 5.80 |
9 | 33.27 | Octadecanoic acid, 9,10-epoxy-18 (trimethylsiloxy), methyl ester | C22H44O4Si | 400 | 2.53 |
10 | 33.87 | 1-Heptatriacotanol | C37H76O | 536 | 21.07 |
11 | 34.22 | 9,12,15-Octadecatrienoic acid, 2,3-dihydroxypropyl ester, (Z,Z,Z) | C21H36O4 | 352 | 53.49 |
Table 2
Initial body weights, final body weights, % change in body weight of groups under the study.
Groups | I. B. wt. (g.) | F. B. wt. (g.) | % Change in B. wt. |
Control | 21.48 ± 1.1 | 31.3 ± 3.6 | 45.85 ± 4.7 |
Control/PDSE | 21.23 ± 1.2n.s. | 30.97 ± 7.4 n.s. | 45.75 ± 4.9 n.s. |
CTX | 21.3 ± 1.1n.s. | 27.8 ± 6.0 n.s. | 30.26 ± 3.5 * |
CTX/PDSE | 21.5 ± 1.2 n.s. | 29.5 ± 3.1 n.s. | 37.20 ± 4.1 n.s. |
The values represented mean ± SD; I.B.W: Initial body weight; F.B.W: Final body weight. P value < 0.05 was statistically significant. PDSE: Phoenix dactylifera seeds extract, CTX: Cyclophosphamide. |
Treatment with PDSE post-CTX-injections ameliorated hematological alterations.
The results showed that as compared to the control group (Gp1), the total number of red blood cells (RBCs), hemoglobin (Hb) concentration and hematocrit (%) did not show significant alterations in all groups (Table 3). Compared to Gp1, treatment with PDSE (Gp2) and CTX-injected group (Gp3) showed significant increase in the total platelets count. A decrease in the WBCs count was found in the group of mice post a single injection of CTX (Gp3) when compared to Gp1. Treatment with PDSE after CTX injections (Gp4) ameliorated the toxic effect of CTX on WBCs and restores their number close to the normal values (Table 3). Mice were treated with CTX (Gp3) showed a decrease in the total number of lymphocytes and monocytes when compared to the control group (Gp1). Treatment with PDSE post-CTX-injection ameliorated the toxic effect of CTX on these cells and return the numbers close to the normal values.
Table 3
Hematological parameters in different groups of mice treated with PDSE, CTX, or with PDSE post CTX-injection.
Groups | Hb (g/dL) | RBCs (x106 /µL) | Hct (%) | WBCs (x103 /µL) | Plat. (x103 /µL) |
Control | 11.82 ± 0.3 | 8.82 ± 0.4 | 39.3 ± 3.3 | 8.96 ± 1.5 | 882.8 ± 31.3 |
Control/PDSE | 11.0 ± 0.5 n.s. | 8.7 1 ± 0.1n.s. | 41.9 ± 2.5 n.s. | 8.19 ± 1.8 n.s. | 1589.3 ± 78.2 *** |
CTX | 9.80 ± 0.9 n.s. | 9.03 ± 0.6 n.s. | 38.28 ± 3.6 n.s. | 6.31 ± 2.0 * | 1026.5 ± 78.1 * |
CTX/PDSE | 9.98 ± 0.9 n.s. | 8.15 ± 0.9 n.s. | 35.70 ± 3.7 n.s. | 7.93 ± 2.2 n.s. | 747.8 ± 179.4 n.s. |
The values represented mean ± SD; P value < 0.05 was statistically significant; n.s means non-significant. PDSE: Phoenix dactylifera seeds extract, CTX: Cyclophosphamide. |
Treatment with PDSE improved hepato-renal biochemical dysfunctions induced by CTX
Treatment with PDSE alone did not result in significant changes in the level of AST and ALT when compared to their control. Furthermore, the result showed that there was an increase in the liver transaminases AST and ALT in CTX-injected groups (Gp3), when compared to the control group. Treatment with PDSE post-CTX injections ameliorated the toxic effect of CTX on the liver function as evident by decreased AST and ALT (Fig. 1). Treatment with PDSE alone did not show any significant changes in the levels of urea and creatinine when compared to their control, however, CTX injection led to an increase in. Treatment with PDSE post-CTX injections showed a decrease in urea and creatinine levels when compared to the groups of mice injected with CTX (Fig. 2).
PDSE treatments ameliorated the oxidative stress induced by CTX-injections. Treatment with PDSE (Gp2) alone did not result in any significant changes in SOD, CAT, and MDA levels when compared to the control group (Gp1). CTX-injected groups (Gp3) showed a significant decrease in the hepatic SOD and CAT while significant increase in the level of MDA was observed when compared to Gp1. Treatment with PDSE post-CTX injections mitigated the oxidative stress via enhancing the antioxidant status of liver tissues as evident by the increase of SOD and CAT along with decrease in the level of MDA (Fig. 3).
PDSE treatment improve histopathological alterations in liver and kidney tissues. Examination of liver sections of the control group (Gp1) and the PDSE administered mice (Gp2) showed that hepatocytes radiating from the central vein. The hepatocytes had homogenous granular cytoplasm with centrally located nucleus with prominent envelopes and normally distributed chromatin. Liver strands were alternating with narrow blood sinusoids lined by endothelial cells and distinct phagocytic Kupffer cells (Fig. 4a and b). Liver sections of mice after a single dose of CTX-injection (Gp3) showed noticeable disorganized liver architecture, congested blood vessels with fatty exudate, and many obvious mononuclear infiltrations around damaged cells, also widening of blood sinusoids (Fig. 4c). While the CTX/PDSE-treated mice (Gp4) exhibited improvement of hepatic architecture, hepatic cords radiating from the central vein, regular narrow blood sinusoids network, some hepatocytes show hyper-eosinophilia and activated Kupffer’s cells (Fig. 4d).
Normal histological structure of control mice, renal parenchyma was appeared as the cortex region, normal glomeruli with normal bowman's space, and normal renal tubules (Fig. 5a). The PDSE-treated mice (Gp2) exhibited the cortex that contains glomeruli with normal Bowman’s space and normal appearance of mostly renal tubules (Fig. 5b). The histopathological changes in the kidneys of the CTX-treated mice (Gp3) appeared in the form of destructed, shrunken, and congested glomeruli with irregular bowman's space, most of the renal tubules were damaged, lost their characteristic appearance with intratubular hemorrhage (Fig. 5c). Mice injected with CTX/PDSE (Gp4) showed normal glomeruli and renal tubules, but few numbers of disorganized tubules were observed (Fig. 5d).
Molecular analysis for TGF-β1, NFk-β, and COX-1 genes. Pro-inflammatory cytokines, TGFβ-1, NFk-β, and COX-1 genes expression in liver and kidney tissues were assessed by real-time PCR. Concentration of RNA were determined by Nanodrop, and the results showed that the isolated RNA is pure and with considerable concentrations ranged from 1420 to 2340 ng/µl. The obtained results revealed a significant increase in the expression of the pro-inflammatory genes (TGF-β1, NFk-β, and COX-1) in the liver and kidney tissues of CTX-injected mice. Treatment with PDSE post-CTX injection led to significant decrease in the mRNA expression levels of these genes when compared to their controls (Table 4).
Table 4
Fold changes of the mRNA expression of TGFβ-1, NFκ-β, COX-1 and COX-2 genes in liver and kidney tissues of the different groups.
Tissue/Genes | Groups |
Gp1 (Control) | GP2 (PDSE) | GP3 (CTX) | GP4 (CTX/PDSE) |
Liver | TGFβ-1 | 1.00 ± 0.06 | 1.53 ± 0.19* | 7.11 ± 0.36*** | 4.13 ± 0.27*** |
NFκ-β | 1.00 ± 0.06 | 1.97 ± 0.13*** | 11.72 ± 0.48*** | 7.71 ± 0.34*** |
COX-1 | 1.00 ± 0.06 | 1.43 ± 0.17* | 6.82 ± 0.39*** | 4.23 ± 0.19*** |
Kidney | TGFβ-1 | 1.00 ± 0.06 | 1.23 ± 0.12* | 3.51 ± 0.39*** | 2.11 ± 0.18*** |
NFκ-β | 1.00 ± 0.06 | 1.53 ± 0.15*** | 7.63 ± 0.58*** | 4.27 ± 0.31*** |
COX-1 | 1.00 ± 0.06 | 1.29 ± 0.13* | 4.45 ± 0.42*** | 3.17 ± 0.37*** |
The values represented mean ± SD; P value < 0.05 was statistically significant; n.s means non-significant. |