CHEMOPROTECTIVE ACTIVITY OF OROXYLUM INDICUM VENT. ROOT BARK AGAINST CYCLOPHOSPHAMIDE MEDIATED MYELOSUPPRESSION AND ORGAN INJURY

Seema Menon 1 , Lincy Lawrence 1 , Noby John 2 and Jose Padikkala 1 . 1. Amala Cancer Research Centre (Recognized research centre of University of Calicut), Amala Nagar, Thrissur 680 555, Kerala. 2. The Erode College of Pharmacy and Research Institute, Vallipurathanpalayam P.O., Erode 638112, Tamil Nadu. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

The study investigated the chemoprotective effect of root bark of Oroxylum indicum Vent., against cyclophosphamide (CTX) induced myelosuppression and oragn injury in BALB/c mice. Four groups of 6 animals each-normal, untreated negative control group and animals treated with O. indicum Vent. root bark 70% hydro methanolic extract at low (OIML-200 mg/kg b.wt.) and high doses (OIMH-400 mg/kg b.wt.) respectively for 20 consecutive days were used for the study. Intra-peritoneal injection of CTX at a dose of 20 mg/kg on consecutive days, from day 6 to 15 was given for toxicity induction in all animals except normal. The body weight changes, hemoglobin level, total leucocyte count and bone marrow cellularity of animals in all groups were tracked periodically. After 120 hours of last CTX injection, mice were sacrificed; blood was collected to analyse serum markers of hepatic and renal function. The antioxidant status of liver and renal tissues based on SOD activity, GPx activity, reduced glutathione level and inhibition of lipid peroxidation were studied. The organo-somatic index (relative organ weight) of spleen as well as histological examination of intestine was determined in animals of all groups. Opposed to the devastating effect of CTX in hematological profile, bone marrow cellularity and relative spleen weight of untreated control group, the OIM treated animals showed significant restoration of these parameters into normalcy. OIM treatment also revealed better antioxidant profile and intestinal histological features, in comparison to the untreated control group, revealing its in vivo chemoprotective property.

Grouping of animals
Four groups of 6 animals each were maintained according to the treatment protocol. Group I served as normal reference group. Group II served as the untreated negative control group, exposed to CTX. Groups III and IV were the extract treated groups receiving oral administration of OIM extract at low (OIML-200 mg/kg b.wt.) and high doses (OIMH-400 mg/kg b.wt.) respectively for 20 consecutive days. For each group, three sub-groups with 6 animals were maintained to enumerate the bone marrow cellularity on initial day, 7 th day and 4 th day.

Cyclophosphamide induction
Induction of toxicity was done in all the groups except I, by intra-peritoneal injection of CTX at a dose of 20 mg/kg of animal body weight as i.p. on consecutive days, from day 6 to 15 (Kumar and Kuttan, 2005a).

Parameters studied
The body weight changes of animals in all groups were tracked periodically in every 5 th day of the study. On every 5 th day of the study, blood was collected into heparinized tubes by caudal vein puncture to track the hematological parameters of blood -[hemoglobin level (Drabkin and Austin, 1935) and total leucocyte count (Chesbrough and Arthur, 1972)] periodically. On the initial day, 7 th day, 14 th day and the final day (immediately after sacrifice), bone marrow cells from both the femurs were collected by flushing out using PBS containing 10% fetal bovine serum (FBS). The total live cell count was determined using a hemocytometer and expressed as "n" (×10 6 )/femur.

Evaluation of renal and liver function markers in serum
On day 20 (120 hours after last Cis injection), mice were sacrificed; blood was collected by heart puncture into nonheparinised vials to collect serum. Serum markers of hepatic and renal function -GOT, GPT, ALP, creatinine and urea were analyzed to evaluate the extent of liver and kidney tissue damage.

Evaluation of lipid peroxidation level and antioxidant status in hepatic and renal tissue
10% (w/v) homogenates of renal tissue and hepatic tissues were prepared in ice cold 0.1M Tris-HCl. Lipid peroxidation measured as MDA level in the tissue homogenate was analyzed according to the method of Ohkawa et al. (1979). The cytosolic fraction of the tissue homogenate was used for studying the antioxidant status based on SOD activity (McCord and Fridovich, 1969), GPx activity (Hafeman et al., 1974) and reduced glutathione level (Moron et al., 1979).

Determination of organo-somatic index of spleen
The organo-somatic index (relative organ weight) of spleen was determined in animals of all groups.

Histopathological examination of intestine
After the sacrifice of animals, intestine was excised using sterilized scissors and washed with normal saline (0.9% NaCl) and fixed in 10% neutral buffered formalin. After dehydration and embedding, sections of thickness varying from 3-4 μm were made in a microtome and subjected to hematoxylin/eosin double staining. The slides were observed under light microscope (100 x) and photographed.

Statistical analysis
The values were expressed as mean ± SD of 6 animals per group. Statistical evaluation of the data was done by one way ANOVA followed by Dunnett post hoc test using Graph Pad Instat 3 software. Results were considered statistically significant when p value was <0.05.

Changes in body weight
As seen from figure 1, progressive reduction was noticed in the average body weight of animals injected with cyclophosphamide alone (negative control group), from the initial day of CTX administration (31.08 ± 1.3 g) to the day of sacrifice (26.02 ± 0.8 g). The normal reference group showed no noticeable body weight loss. In the OIML group also, there was a slight decline of average body weight from day 5 (29.34 ± 1.4 g) to 20 (26.4 ± 1.0 g). But, in the OIMH group, only negligible change was recorded in body weight from day 5 (30.84 ± 1.2 g) to day 20 (29.514 ± 1.0). Figure 2 shows the change in hemoglobin level of blood in CTX induced animals over the period of study. The CTX induced control group, having received no pre-treatment, suffered slight reduction of blood Hb level from the day of CTX induction (12.87 ± 0.37 g/dL) until sacrifice (12.36 ± 0.37 g/dL), though statistically non significant. However, the Hb values recorded in OIML (12.67 ± 0.1 to 12.59 ± 0.4 g/dL) and OIMH (12.89 ± 0.1 to 12.69 ± 0.5 g/dL) groups were only within a narrower range after CTX administration up to sacrifice.

Changes in haemoglobin level and total leucocyte count
The tracking of mean total leucocyte count in CTX administered animals is shown in figure 3. When the normal reference group not exposed to CTX showed no significant changes, the untreated negative control suffered drastic and significant decline in WBC count from day 5 (13033/mm 3 ) to day 20 (6290/mm 3 ). OIML group also suffered such decline in WBC count from 12883/mm 3 to 8050/mm 3 . But, treatment with OIM at high dose (OIMH) was demonstrated to withstand the myelosuppressive effect of CTX, as noted from non significant change in the total WBC count recorded from the 5 th day (12633/mm 3 ) to 20 th day (10360/mm 3 ).

Changes in bone marrow cellularity
The effect of OIM extract on resisting the myelosuppressive effect of CTX administration was also analyzed based on the weekly bone marrow cellularity track record. The normal reference group showed no notable change. But the CTX administered untreated negative control suffered reduction in 2 week period after CTX administration, from  Table 1 shows the levels of renal and liver function markers in the serum of CTX administered animals.

Lipid peroxidation level and antioxidant status in hepatic and renal tissue
Treatment with OIM enhanced the antioxidant status of the liver and kidney in CTX induced animals as seen in figure 5 and 6. From figure 5, it can be seen that in CTX induced negative control group devoid of extract treatment, the levels of SOD (0.382 ± 0.04 U/mg protein), GPx (7.15 ± 0.43 U/mg protein) and GSH (13.63 ± 1.42 nmoles/mg protein) in liver were significantly lower than that of normal. Similar trend was observed in the levels of these parameters in the renal tissue (SOD -0.577 ± 0.06, GPx -6.75 ± 0.49 and GSH -15.83 ± 2.12) (figure 6). But in OIM treated high dose group (OIMH), levels of liver SOD (0.571 ± 0.04), liver GPx (9.82 ± 0.51), liver GSH (36.88 ± 2.61), renal SOD (0.72 ± 0.07), renal GPx (10.2 ± 0.52) and renal GSH (27.24 ± 2.03) were elevated significantly higher than untreated control.

Organo-somatic index of spleen
The impact of CTX administration on immune system was inferred on the basis of the organo somatic index of spleen, which was significantly very low in the untreated control (0.251 ± 0.016), with reference to normal; but was near normal in OIML (0.303 ± 0.016) and OIMH (0.318 ± 0.017) groups.

Histopathology of intestine
The histological sections of the intestine of normal mice showed typical structure, with regular villi and glands (Figure 7). The villi and glands are lined by columnar cells. The muscle layer also appeared normal. But, in the 700 control animal intestine, the mucosal glands and villi showed hyperplasia, with considerable mucosal infiltration of lymphocytes. The muscle layer also showed inflammatory changes. In the OIML group intestine, though the villi appear normal, there were a few damaged areas in the mucosal layer. In the meantime, treatment with OIM extract at high dose had considerable protection on the intestinal mucosa and muscle layer, in spite of simultaneous CTX administration. This is evident from the presence of normal villi and mucosal glands, as revealed through histopathology. The muscle layer also appeared normal.

Discussion:-
The study has revealed the chemoprotective activity of O. indicum Vent. root bark against organ specific toxicity induced by CTX. Antioxidant pool of the liver and kidney reportedly undergoes depletion under the effect of CTX administration, as seen in earlier animal studies (Premkumar et al., 2003, Sheweita et al., 2016). Similar devastating effects on antioxidant defense were also observed in the untreated animals induced with CTX in the current study. But, on treatment with OIM, the antioxidant markers of liver and renal tissue maintained significantly higher levels than the negative control, seemingly resistant to the oxidative stress challenge posed by CTX. Though CTX increases the susceptibility of renal and hepatic tissues to increased lipid peroxidation (Bhattacharya et al., 2003, Stankiewicz and Skrzydlewska, 2003, Ray et al., 2011, plant extracts rich in antioxidants are found to be ameliorative against this effect (Ingale et al., 2013, Khan et al., 2014, Devi and Mazumder, 2016. Concomitant effects were produced by OIM treatment in this study.
By CTX induction, elevation of serum GPT and GOT levels shot up significantly, which were alleviated by OIM treatment. This is suggestive of hepatic injury. Moreover, these two inducers were demonstrated to cause renal tissue damage, as evident from the elevation of serum urea and creatinine levels in the untreated control. Yet, an overall ameliorative effect was observed in these effects by OIM treatment, as revealed from the sera profile of treated animals.
Common side effects associated with cancer chemotherapy are myelosuppression, immunosuppression and anaemia, because rapidly dividing normal cells of the body also form targets for chemotherapeutic drugs, besides cancer cells (Spivak et al., 2009). These effects have also been found true with CTX in experimental animals (Thews et al., 2001). The effects are manifested as drops in Hb and WBC count in blood, reduction in bone marrow cellularity and decrease in the relative weight of spleen (Begum and Anuradha, 2011, Ahmad et al., 2013, Neboh and Ufelle, 2015, Sakthivel and Guruvayoorappan, 2015. In the present study, CTX administration was found to affect WBC count significantly. CTX administration seemingly exerted its myelosuppressive effect on bone marrow of untreated CTX administered animals, evident from a significant and progressive reduction of bone marrow cellularity. Consequently, the relative organ weight of spleen, the secondary lymphoid organ was also found to be notably low in untreated control, in comparison with the normal. Such suppressive changes were evaded by the OIM treated animals, in a statistically significant level.  (Rehman et al., 2012, Warpe et al., 2015b). It is hence, conclusive that the presence of anti-oxidant compounds in the OIM extract may possibly have mediated the chemoprotective effects, through their combined action. Though it is not suggestive of an immediate clinical supplementation in humans undergoing chemotherapy for cancer, the study extends the scope of exploring active fractions from this plant extract for using it as an adjuvant in CTX-mediated chemotherapy, and thereby reduce the damaging side effects of these drugs, without compromising their cytotoxic activity. Values are expressed as mean ± SD for 6 animals; a p< 0.01, b p<0.05 compared to normal; c p<0.01, d p<0.05 compared to control.