INTRODUCTION:

Cyclophosphamide (CP) with trade name cycloblastin, Endoxan is an alkylating agent widely used in cancer chemotherapy [1,2]. It is used also as an immunosuppressive agent for organ transplantation, and a medicine used to treat severe inflammatory illnesses such as complicated system, Systemic lupus, Erythematosus (SLE/Lupus) polyositis (muscle inflammation) scleroderma and vasculitis (inflamed blood vessels). [3].

The cytotoxic effects of CP resulted from chemically reactive metabolites that alkylate DNA and protein producing crosslink [4]. It has been also reported that CP killed dividing cells in the body, including cancer cells and injury of normal cells which include red blood cell, which gives rise to numerous side effects [5,6]. Several studies has been reported that oxidative stress mediated disruption of redox balance after CP exposure generates biochemical and physiological disruptions [7].

Cell cycle impairment induced after acute CP treatment could be mediated by G2/M activated in response to DNA damage [8]. Also after exposure to CP may alteration in hematological parameters and impair the functional activities of liver and kidney of male Rattus norvegicus by interfering enzymatic metabolic activities and protein synthesis [9], and development of sinusoidal obstruction syndrome (veno-oculusive disease) and total serum bilirubin levels [10].

Herbal drugs play a major role in treatment of many common diseases and the natural products have recently gained acceptance and have continued to gain grounds therapeutics due to their acclaimed efficacy in management of many ailments with little or no side effects when used appropriately. Many natural products have been widely reported to ameliorate at varying degrees the side effects of oxazophosphorines e.g. CP [11].

Capparis pinosa L. (Cappariaceae) is common permeate shrub and a favored plant for restoring vegetation on dry regions in west or central Asia. It is particularly widely grown in the Mediterranean basin. From ancient time Greeks and Romans were used for medicinal inflammatory, hypotensive and spasmolytic effects [12,13,14]. Capparis spinosa (caper) source of natural disinfections, vermifuges, infusions and decoctions. Caper root bark have been traditionally used for dropsy, anemia, arthritis and gout. Caper contain considerable amounts of the antioxidant, alkaloids, glycosides, trepans, steroids, essential oils, phenols and bioflavonoid [15,16,17].

The current study aimed to focused on the protective efficacy of ethanolic extract and aqueous extract of leaf bud of C. spinosa against side effects of cyclophosphamide induced blood parameter, liver and kidney toxicity in the mice.

MATERIAL AND METHODS:

Animals:

Twelve weeks old adult male mice(Mus musculus L.) were used In this study, weighing 25±2 gm and acclimatized and maintained at (25±2C° ) With a 12 hours light- dark cycle, food and water were given ad libitum.

Chemicals:

Cyclophosphamide (CP) Trade name – Endoxan, 50 Drag/Grageas purchased from Germany Allemagne Alemania. A dose of CP was 25mg/Kg b.w. ip. The Kit for the estimation of aspartate transaminase (AST), and alanine transaminase (ALT) were purchased from Rondox, Biomericux. The chemical compounds used for analytical grade is ethanol, Bouin solution for fixing and hematoxylin- eosin for staining.

Plant material:

Leaf bud of Capparis spinosa L. were collected from Abu- Al Kaseeb region in Basrah southern of Iraq, cleaned with cold distilled water, dried in the shadow at room temperature, ground powdered and kept until of use. The plant was taxonomies in Biology department, at college of Education for pure Sciences in Basrah University.

Preparation of extract:

A: Powdered leaf bud (100g) of C. spinosa L. were extracted exhaustively with 90 % ethanol, after filtration with using Whatman No.1 filter paper, the extract was concentrated and dried using Rotary flask Evaporator, which stored in refrigerator for further use.

B: Powdered leaf bud (100g) of C. spinosa L. were extracted with water, after filtration with using Whatman No.1 filter paper, the extract was concentrated and dried using rotary flask evaporator, which stored in refrigerator for further use.

Treatments:

Twenty four male mice used In this study (6 mice each)were randomly assigned to different treatment the experimental groups were as follow:

· Group 1: Control group received 0.2ml. normal saline

· Group 2:CP group received normal saline for one week then 25mg/kg B.W. CP i. p. was given twice week for 3 weeks.

· Group3: Ethanolic extract of C. spinosa L. leaf bud (200mg/kg B.W.) was given orally (gavages) by gastric intubation using a once daily for month .

· Group4: Aqueous extract of C. spinosa L. of leaf bud (200mg/kg B.W.) was given orally (gavages) by gastric intubation using a once daily for month.

CP was injected (25mg/kg B.W. group 3,4), twice in week for 3 weeks. after one week of received the extract.

After 24 h., from last treatment, the mice of all groups were scarified by cervical dislocation under ether anesthesia and venous blood samples were collected by direct heart puncture. The blood samples were divided for measurement of some blood parameters (HB,PCV, RBC, PLT, total WBC and deferential WBC), and the other was centrifuged, serum was recovered, and diagnostic Kits were used to determine the serum activity of AST and ALT, in addition, the organs liver and kidney were removed for histopathological examinations.

Hematological test:

The blood samples, immediately upon collection was analyzed using an automated dialyzer machine.(Mindray BC-2800Vet)

Histopathological Examination:

Five µm thick paraffin section of Bouin solution fixed liver and kidney samples were stained with hematoxylin - eosin (H&E) for photo microscopic observation of liver and kidney histological architecture of control and treated mice.

Statistical analysis:

The result was analyzed statistically by using statistical program (SPSS). One way ANOVA Analysis of variance were done to evaluate the significant difference between treatments and control groups under probability p≤0.05.

RESULTS:

The Hematological parameters of present study were found in (Table 1). The RBC, HB, PCV and PLT in Cp treated group were significantly decreased compared to the control. Whereas, PCV and PLT in Cp+ ethanolic and aqueous extracts of C. spinosa L treated groups were non significantly differ (p≤0.05) compared to the control, except the RBC of group CP+ ethanolic extract of C. spinosa showed the significantly decreased compared to the control.

The total leucocytes and percentage differential leucocytes count were found in (Table 2). There was a significant decrease in the total leucocytes in CP group when compared to control, on the other hand there was no significantly differ in total leucocytes when treated the mice with ethanolic and aqueous extract of C. spinosa L. There was a significant decrease in the percentage of granulocyte in group CP compared with control and other groups. But the percentage of lymphocytes of CP group show significant decrease when compared with control and other groups. Whereas the monocytes show no change in all groups.

The biochemical parameters (ALT and AST) in blood of mice after exposed of CP and pretreated with ethanolic and aqueous extract of C. spinosa L. leaf bud were found in (Table 3). There was a significant increase in the ALT and AST of CP group compared to control. While in group treated with aqueous extract of C. spinosa + CP showed no significantly difference in ALT and AST when compared with control, But the CP+ ethanolic extract group a slight increase in AST when compared with control and CP group. But the ALT was not significantly differ compared with control.

Table 1: Effects of Ethanolic extract and aqueous extract of C. spinosa L. leaf bud on hematological parameters of mice peripheral blood in CP – induced toxicities, (n=6)

Parameter Control CP CP + EE CP + AE

Dose(mg/kg) 0.2ml/25g 25mg/kg 200mg/kg 200 mg/kg

RBC ×10¹²/L 8.58 ± 0.7^a 7.36 ±1.0^b 7.58 ± 0.3^b8.18 ± 0.5^a

HB (g/L) 122.3 ± 7.1^a 96.8± 10.1^b 121.0± .5.1^a 122.5± 5.9^a

PCV% 44.4± 6.0^a 31.6± 9.7^b 38.6± 3.05^a 42.93± 3.5^a

PLT^* 1294.1± 925.0± 1380.3± 1061.8±

510.2^a109.8^b288.1^a329.0^a

Values are the mean ± SD. Similar letters: non-significant. Different letters: significant differ among groups P≤ 0.05. EE=Ethanolic extract of C. spinosa. AE= Aqueous extract. CP= Cyclophosphamide.

*:× 10¹²/L

Table 2: Effects of ethanolic and aqueous extract of C. spinosa L. leaf bud on the number and percentage of leukocytes in blood of mice in CP – induced toxicities. (n=6)

Parameter control CP CP+EE CP+ AE

Dose (mg/kg) 0.2ml/25g 25mg/kg 200mg/kg 200 mg/kg

WBC ᵡ 10⁹/L 4.4±0.4^a 3.3±0.7 ^b 4.7± 0.7^a 4.2± 1.0^a

Granu.% 35.1± 72.7± 34.2± 44.7±

10.9^a11.6^b 10.3^a12.3^a

Lymph.% 57.9± 26.6± 61.5± 51.6±

12.3^a10.7^b16.9^a17.7^a

Mono. % 6.9± 2.4^a 4.8± 2.4^a 4.9± 0.9^a 4.6± 12.3^a

Values are the mean ± SD. Similar letters: non-significant P≤ 0.05. Different letters: significant differ among group P≤ 0.05. EE=Ethanolic extract of C. spinosa L. AE= Aqueous extract. CP= Cyclophosphamide.

Table 3: Effects of ethanolic and aqueous extract of C. spinosa L. leave bud on Biochemical parameters (ALT and AST) in blood of mice in CP –induced toxicities. (n=6)

Parameter Control CP CP+ EE CP+ AE

Dose (mg/kg) 0.2ml/25g 25mg/kg 200mg/kg 200mg/kg

AST(U/L) 142.6± 5.5^a 166.0± 5.0.^b 154.16± 8.2 ^c 145.8± 3.6^a

ALT(U/L) 55.0± 4.38^a 63.16± 3.3^b 51.8± 2. 2^a 56. 3± 4.5^a

Values are the mean ± SD. Similar letters: non-significant P≤ 0.05. Different letters: significant differ among group P≤ 0.05. EE=Ethanolic extract of C. spinosa. AE= Aqueous extract. CP= Cyclophosphamide. (AST)= aspartate transaminase, (ALT) alanine transaminase.

Histopathological studies:

Liver:

Histopathological sections of the liver of control group showed normal size and shape of the hepatocytes, sinusoids and central vein (Fig 1-A). The mice received only CP showed dilated of central vein, swelling of hepatocyte, focal accumulation of inflammatory cells (Fig 1-B) and excessive vacuolated of hepatocytes, glycogenic degeneration with pink material which preparation of pink material into cytoplasm of cells, congested of central vein with sinusoid, and vacuolated of cytoplasm of hepatocyt with irregular nucleolus( Fig 1- C &D), unlike those of test animals that received CP + ethanolic extract of C. spinosa showed a mailed change such as hypertrophy of hepatocytes (Fig1-E), and histopathological changes in the liver were reduced to near normal in the mice treated with aqueous extract of C. spinosa L .(Fig 1-F).

Kidney:

The histological examination of kidney of the control group showed the normal size and shape of the renal glomeruli and renal tubules, as in (Fig 2-A).The mice kidney treated with CP showed degeneration of epithelial cells as foaming lumen,(Fig 2-B). Clear hemorrhagic interstitial some of renal tubules are closure with degeneration of epithelial cells as foaming lumen(Fig 2-C), Congestion of blood vessels, the tissue of kidney showed cloudy swelling of epithial cells and hemorrhages, some of cells were elongated towered lumen, and there were inflammatory cells in interstitial tissue, (Fig 2-D). Kidney of Cp + ethanolic extract and aqueous extract of C. spinosa L. appeared as control.(Fig 2-E&F).

Figure 1: Photomicrograph section of organs from mice A- control Liver showed normal hepatocytes and sinusoids B- Liver treated with Cp showed dilated of central vein (stare), swelling of hepatocyte cells (white arrow), focal accumulation of inflammatory cells (black arrow). C- Liver treated with Cp showed excessive vacuolated of cytoplasm (black arrow), and preparation of pink material into cytoplasm of cells (stare). D- Liver treated with Cp showed congested of sinusoid (black arrow), vacuolated of cytoplasm of hepatocyt with irregular nucleolus (white arrow). E- Liver treated with Cp + ethanolic extract of C. spinosa showing hypertrophy of hepatocytes (head arrow). F- Liver treated with Cp +aqueous extract of C. spinosa showed mailed changes, like control liver structure. E&Hx400.

Figure 2: Photomicrograph section of organs from mice A- control Kidney showed normal renal corpuscles and tubules. B– Kidney treated with C-showed, Clear hemorrhagic interstitial some of renal tubules are congestion of blood vessels, the tissue kidney show cloudy swelling of epithelial cells and hemorrhages (black arrow) some of cells are elongated towered lumen (white arrow) . D- Kidney treated with Cp showed cloudy swelling of epithelial cells and hemorrhages. E&F -Kidney treated with Cp + ethanolic extract and aqueous extract appear as control. (H&E)400.

DISCUSSION:

It was investigated the toxicity of cyclophosphamide (CP) and the possible protective effects of ethanolic and aqueous extract of leave bud of Capparis spinosa L. on hematological parameters of peripheral blood and histotoxicity of liver and kidney in adult male mice.

The uses of natural products as alternative medicine had been encouraged especially because they were relatively cheap and with minimal side effects when compared to modern medicine [18,19,20]

Chemotherapy was the non specification of both tumors cell and healthy cell [21]. The CP toxicity was mainly due to its active metabolites acrolein and phosphoramide mustard, this metabolite was only formed in cells that had low levels of aldehyde dehydrogenase (ALDH). Phosphoramide mustard forms DNA cross links between and within inter strand of DNA strands at guanine N-7 position. There for was lead to cell death. [22,23]. Acrolein also produces oxidative stress resulting in decrease in the activities of antioxidant enzymes and in an increase lipid peroxidation, also acrolein and lipid peroxidation product malondialdehyde (MDA) belongs to the carbonyl compounds, which were reactive and could interact with amino acids of protein, causing structural and functional changes in the enzymes [24,25].

All the observed changes in the current study in RBC, WBC, PLT, HB and PCV were significantly decline in CP group when compared with control groups. the result may suggest that CP suppresses bone marrow ability to produced new one. Many chemotherapeutic agents including CP had been showed damage of erythrocytes, leukocytes and platelet, that mean bone marrow suppression or myelosuppression [26,27]. But when pretreated the mice with C spinosa leave bud ethanolic and aqueous extract for 7 days before Cp administration, the result showed non-significant differences when compared with control. The mechanism by which extract its protective action against Cp. Induced the hematology may be attributed due to the antioxidant effect of plant exctract. [28,29]. The antioxidant assays suggest that C spinosa bud contain constituents that were good free radical scavengers and ion chelators. Also the C spinosa buds, rich in flavonoids quercetin and glycosides, which possess strong effect antioxidant free radical scavenging [30].

Cychlophosphamide may increase reactive oxygen species and caused damage to hepatic tissue [31,32]. The liver and kidney was the richest source of both ALT and AST enzymes. And these were marker enzymes for assessment of liver and kidney functions. Any damage to the liver and kidney cells will result in the increase in the both of these enzymes. [33,34]. In this study the increasing activity of enzymes ALT and AST may be return to toxic effect of CP, and could be attributed to be hepatic damage, and resulting increase and leakage out of these enzymes from the liver into blood stream. These result agreement with [35]. But the pretreatment. of C. spinosa extract for 7 days before Cp administration the result showed non-significant differences when compared with control. C spinosa had been reported to have hepatoprotective activity [36] due to presence of flavonoid like quercetin and rutin. The result agreement with [19], against paracetamol.

In this study, were studied the histopathologic changes in the liver and kidney in different groups. In liver Cp treated group showed dilated of central vein, swelling of hepatocyte cells, focal accumulation of inflammatory cells, excessive vacuolated of hepatocytes glycogenic degeneration with pink material which preparation of pink material into cytoplasm of cells, and irregular nucleolus. This findings reflected the direct and significant toxic effects of CP [37,38,39]. Other studies reported that CP forming free radicals, peroxidation and oxidative stress in rats, and severe tissue damage such as necrosis, hemorrhage and fibrosis [40,41]. Acrolein is responsible for such toxic effects as cell death, apoptosis, necrosis, and hydropic swelling [42,38]. The histopathological changes in the liver were reduced to near normal in the mice pretreated with C. spinosa extract for 7 days before Cp administration. A studies showed that capers used as hepatic stimulator and protectors, improving liver function, hepatic activity, and anti-inflammatory [43,44,45].

The histological examination of mice kidney treated with CP showing degeneration of epithelial cells as foaming lumen, Clear hemorrhagic, Congestion of blood vessels the tissue of kidney show cloudy swelling of epithelial cells and hemorrhages, some of cells are elongated towered lumen, and inflammatory cells in interstitial tissue, but Kidney of Cp + ethanolic extract and aqueous extracts of C. spinosa appear as control. This results to be agreement with the report of [46] who reported that after a single administration of CP in rats the histopathological analysis revealed presence of inflammatory lesions in urinary bladders, and kidney with slight congestive hyperemia. In contrast, C.spinosa extract may have protected the kidney from CP- induced toxicity. The C. spinosa ethanolic and aqueous leaf bud has been also contains flavonoids, rutin, and phenolic compound in the antioxidant properties .in the same time these antioxidants has been cheaper and tend to have minimal side effects [45,20].

CONCLUSIONS:

The results showed the important role of ethanolic and aqueous extracts of C. spinosa L. leaf bud to protective efficacy against Cyclophosphamide and keep normal values of blood parameters and liver enzymes in normal levels as well as keep normal tissues of liver and kidneys especially when mice pretreated before one week of exposure to CP.

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Received on 13.12.2018 Modified on 09.01.2019

Research J. Pharm. and Tech. 2019; 12(7):3245-3250.

DOI: 10.5958/0974-360X.2019.00547.X