INTRODUCTION:

Cancer is a group of more than 100 different diseases which is characterized by uncontrolled cellular growth, distant metastases and local tissue invasion (Kaufman et al., 1996). Carcinogenesis is a multi-step process that includes initiation phase, promotion phase, conversion phase and finally progression phase.

Choice of cancer treatment is influenced by several other factors, including the specific characteristics of the cancer, the overall condition and whether the goal of treatment is to cure cancer and keep the cancer from spreading or to relieve the symptoms that are caused by cancer.

Cyclophosphamide is probably the most commonly used alkylating agent. Cyclophosphamide administration results in the formation of cross-links within DNA due to a reaction of the two chloroethyl moieties of cyclophosphamide with adjacent nucleotide bases. Cyclophosphamide must be activated metabolically by microsomal enzymes of the cytochrome P450 system before ionization of the chloride atoms and formation of the cyclic ethylenimmonium ion can occur. It is inactive until metabolised in the liver by the P450 mixed function oxidases.

It has a pronounced effect on lymphocytes and can also be used as an immunosuppressant. It is usually given orally or by intravenous injection but may also be given intramuscularly.

Today’s chemoprotective drugs which are usually administered with a specific type of chemotherapy drug; for example, Mesna which is used along with oxazophosphorines class of drugs. Mesna binds to the toxic metabolites of this class of chemotherapy drugs, helping to prevent urogenital problems and bladder inflammation.

Chemoprotective agents are still a new field of study and research will likely yield agents with greater selectivity for normal tissue, as well as the ability to protect a larger number of tissue types. Nanoparticle delivery may also assist in minimizing the side effects of chemotherapy.

METHODOLOGY:

I. Plant extract preparation:

The stem bark was chipped from the collected stems and was dried in a hot air oven at a temperature of 45^oC. This was then ground to fine powder and extracted with 70% methanol as the solvent, using a Soxhlet apparatus. The extract was filtered and evaporated to dryness. The dried extract was re-dissolved in distilled water and used for further studies.

II. Animal grouping and treatment regime:

The animals (with body weights ranging from 25 to 30 gm) were divided into 4 groups of 6 animals each:-

Group I: Normal – Untreated.

Group II: Control – Cyclophosphamide (20 mg/kg b.wt).

Group IV: GALC – Gmelina arborea extract low concentration (250 mg/kg b.wt) and Cyclophosphamide (20 mg/kg b.wt).

Group V: GAHC - Gmelina arborea extract high concentration (500 mg/kg b.wt) and Cyclophosphamide (20 mg/kg b.wt).

The GALC and GAHC groups were orally treated with the extract at doses of 250 mg/kg body weight and 500 mg/kg body weight respectively, for a continuous time period of 20 days, at an administration volume of 0.1 ml for each animal. Within this period, all groups, except normal also received oral administration of cyclophosphamide at a dose of 20 mg/kg body weight, starting from the sixth day upto the fifteenth.

For determining the bone marrow cellularity on the 7^th and 14^th day, 4 animals each were separately stocked for control, GALC and GAHC groups with the above mentioned treatment regime till their sacrifice. On the initial day of the study, 4 animals separately stocked for the purpose were sacrificed from the normal group.

III. Determination of total WBC count and haemoglobin level:

During the course of the study, the haematological parameters of the animals were tracked on every 5^th day. For this, blood was collected from the caudal vein into heparinised tubes and total WBC count and haemoglobin level were checked.

A. Determination of total count:

0.02 ml of blood was added to 0.38 ml of diluting fluid and mixed well. The diluted blood was charged into a Neubauer counting chamber. After 3-4 min, the total number of white blood cells in the four large corner square chambers was counted.

Calculation:

Total WBC = (Number of cells counted x 50) / mm³

B. Determination of haemoglobin (Hb) content:

Cyanmethaemoglobin method (Kit manufactured by Agappe Diagnostics).

0.02 ml of fresh whole blood was mixed with 5 ml of the cyanmeth reagent. The optical density was measured at 546 nm against blank after 5 min incubation at room temperature. The OD of standard solution corresponding to 60 mg/dl haemoglobin at 546 nm was also read against reagent blank.

Calculation:

Haemoglobin (g/dL) = (OD of treated x 60 x 0.251) / OD of standard

IV. Estimation of body weight:

The body weights of all the animals were measured, starting from the first day, and repeated every fifth day, until sacrifice. On the 21^st day of the study, the animals were sacrificed and the blood and tissue parameters, relative organ weights as well as the bone marrow cellularity were checked.

V. Determination of relative organ weights:

After sacrifice, the liver, kidney, spleen, heart and brain were excised and weighed. The percentage weight of each organ, relative to the total body weight was determined.

VI. Determination of bone marrow cellularity:

Bone marrow cells from both femurs were flushed out with phosphate buffered saline. The number of bone marrow cells were determined using a haemocytometer and expressed as total live cells (×10⁶)/femur.

VII. Evaluation of biochemical parameters:

1. Serum parameters:

A. Estimation of Aspartate aminotransferase (AST)/ SGOT:

2,4-DNPH (Reitman and Frankel Method):

Kit manufactured by Span diagnostics Ltd. (Cogent) was used for this estimation.

The reaction systems used for this study included blank, standard, test (for each serum sample) and control (for each serum sample). 0.25 ml of buffered aspartate was added into all the test tubes. This was followed by the addition of 0.05 ml of serum into the test group tubes and 0.05 ml of working pyruvate standard into the standard tubes. After proper mixing, all the tubes were kept for incubation at 37^oC for 60 minutes, after which 0.25 ml each of 2,4- DNPH reagent was added into all the tubes. Then, 0.05 ml of distilled water and 0.05 ml of each serum sample was added to the blank and the serum control tubes respectively. The mixture was allowed to stand at room temperature for 20 minutes. After incubation, 2.5 ml of solution I was added to all test tubes. Mixed properly and optical density was read against purified water in a spectrophotometer at 505 nm within 15 minutes.

The enzyme activity was calculated as:-

AST (GOT) activity in IU/L) =

[(Absorbance of test - Absorbance of control)/ (Absorbance of standard - Absorbance of blank)] x concentration of the standard

B. Estimation of Alanine Aminotransferase (ALT)/ SGPT:

2,4-DNPH (Reitman and Frankel Method):

Kit manufactured by Span diagnostics Ltd.(Cogent) was used for this estimation.

The reaction systems used for this study included blank, standard, test (for each serum sample) and control (for each serum sample). 0.25 ml of buffered alanine was added into all the test tubes. This was followed by the addition of 0.05 ml of serum into the test group tubes and 0.05 ml of working pyruvate standard into the standard tubes. After proper mixing, all the tubes were kept for incubation at 37^oC for 60 minutes, after which 0.25 ml each of 2,4- DNPH reagent was added into all the tubes. Then, 0.05 ml of distilled water and 0.05 ml of each serum sample was added to the blank and the serum control tubes respectively. The mixture was allowed to stand at room temperature for 20 minutes. After incubation, 2.5 ml of solution I was added to all test tubes. Mixed properly and optical density was read against purified water in a spectrophotometer at 505 nm within 15 minutes.

The enzyme activity was calculated as:-

ALT (GPT) activity in IU/L) =

[(Absorbance of test - Absorbance of control) / (Absorbance of standard - Absorbance of blank)] x concentration of the standard

C. Estimation of Alkaline phosphatase:

Kind and King’s method:

Kit manufactured by Span diagnostics Ltd.(Cogent) was used for this estimation.

The working solution was prepared by reconstituting one vial of buffered substrate with 2.2 ml of water. 0.5ml of working buffered substrate and 1.5 ml of purified water was dispensed to blank, standard, control and test. Mixed well and incubated at 37⁰C for 3 minutes. 0.05 ml each of serum and phenol standard were added to test and standard test tubes respectively. Mixed well and incubated for 15 minutes at 37⁰C. Thereafter, 1 ml of chromogen reagent was added to all the test tubes. Then, added 0.05 ml of serum to control. Mixed well after addition of each reagent and the O.D of blank, standard, control and test were read against purified water at 510nm.

Calculation:

Serum alkaline phosphatase activity in KA units =

[(O.D. Test-O.D. Control) / (O.D. Standard- O.D. Blank)] x 10

D. Estimation of Creatinine:

Jaffe’s Kinetic method:

Kit manufactured by Asritha Diatech India (Euro) was used for this estimation.

Working reagent (WR) was prepared by mixing equal volumes of R1 and R2. The reaction systems consisted of standard and test solutions. 1 ml of WR was dispensed to all the tubes, followed by the addition of 0.05 ml of creatinine standard and 0.05 ml of serum into the standard and test sample tubes respectively. Mixed well and read the absorbance of standard and test against distilled water at 520 nm after 30 sec (A₀) and 90 sec (A₁)

ΔA for standard (S) and Test (T) was determined as:-

ΔA_s = A_S1- A_S0

ΔA_T= A_TI-A_TO

Serum creatinine (mg/dl) = (ΔA_T/ΔA_s) x 2

E. Estimation of Serum Urea:

Estimation was done by Berthelot Enzymatic method for the determination of urea in serum.

Working reagent was prepared by the addition of 25 ml of distilled water to R1. From this 1ml of solution was pip petted out into all the test tubes. 0.01 ml of distilled water was added into the blank test tube followed by 0.01 ml of standard into the standard tube and 0.01 ml of serum into the test tubes. Mixed well and incubated for 5 minutes at 37⁰C followed by addition of 1 ml of R2 into all the test tubes. Mixed well and incubated for 5 minutes at 37⁰C. At the end of incubation absorbance of standard and test was measured against blank at 578nm.

Urea mg/dl = A_t /A_s × 40

F. Estimation of Serum Total Protein:

Biuret method:

Kit manufactured by Asritha Diatech India (Euro) was used for this estimation.

1 ml Biuret reagent was added into all the test tubes followed by 0.01 ml of total protein standard into the standard tube and 0.01 ml of serum into the test groups. Mixed well and incubated at 37⁰C for 5 minutes.Measured the absorbance of standard and test sample against blank at 555nm within 60 minutes.

Calculation:

Total protein (g/dl) = (O.D. test / O.D. std.) x 6 g/dl

2. Tissue anti-oxidant parameters:

Preparation of Tissue Homogenates (Liver and Kidney):

On 21^st day, animals were sacrificed. Liver and kidney were excised and rinsed thoroughly in ice cold saline to remove the blood. They were gently blotted between the folds of thoroughly in ice-cold saline to remove any blood traces. They were then gently blotted between the folds of a filter paper and weighed in an analytical balance. 25% of homogenate was prepared in 0.05M phosphate buffer (pH 7) using a polytron homogenizer at 4˚C. A part of this homogenate was used for the determination of total protein and lipid peroxidation (LPO). Rest of the homogenate was centrifuged at 10,000 rpm for 30 minutes in a cooling centrifuge for removing the cell debris, unbroken cells, nuclei, erythrocytes and mitochondria. The supernatant was used for the estimation of superoxide dismutase (SOD), total protein, glutathione peroxidase (GPx) and glutathione (GSH).

A. Estimation of Tissue Superoxide Dismutase (SOD) Activity:

For each sample to be assayed, the amount of enzyme added to the medium was kept below 1 unit of enzyme activity. Incubation medium contained 0.1ml of tissue sample, 0.2ml EDTA/NACN, 0.1ml NBT, and 0.05ml riboflavin and phosphate buffer to give a total volume of 3ml. The tube without animal sample was kept as control. Riboflavin was added after the tubes were brought to room temperature. Then the tubes were placed in a bright box where they received uniform illumination for 15 minutes. The optical densities were measured at 560nm. One unit of enzyme activity is defined as amount of enzyme giving 50% inhibition of the reduction of NBT and expressed as units/mg. protein.

B. Estimation of Glutathione (GSH):

To 0.1 ml of homogenate 125ml of 25% TCA was added to precipitate proteins. The tubes were cooled by keeping on ice bags for 5 minutes and the mixture was further diluted with 0.6 ml. of 5% TCA; centrifuged for 10 minutes and 0.1 ml of resultant supernatant was taken for GSH estimation. The volume of the aliquot was made up to 1 ml with 233ml of 0.2M phosphate buffer (pH 8.0) and 667 ml of freshly prepared 0.6mM DTNB The intensity of yellow colour formed by DTNB addition was read at 412 nm with distilled water as reference. Standard curve of GSH was prepared using concentrations varying from 5-100 nmol in 5% TCA for each assay. Value was expressed as nmol / mg protein.

C. Estimation of Glutathione Peroxidase (GPx) Activity:

The reaction mixture containing 0.1 ml each of tissue sample, GSH, NaN₃ and H₂O₂was made up to a total volume of 2.5 ml using phosphate buffer and was incubated at 37°C for 6 minutes. After addition of 2 ml. of 1.67 % HPO₃, this mixture was centrifuged at 800´g for 15 minutes. 667ml of the supernatant was added to a mixture of 1ml of Na₂HPO₄ and DTNB. After 10 minutes of incubation at 37°C, the absorbance of the reaction mixture was measured at 412 nm, with distilled water as reference. The enzyme activity was expressed as units/mg protein.

D. Estimation of Tissue Lipid Peroxidation:

0.1ml. of the tissue sample (25%) in Tris buffer (pH 7.0) was added to a reaction mixture containing KCl (0.1 ml.), ascorbic acid (0.1ml.), ferrous ammonium sulphate (0.1 ml) and tris buffer (0.1 ml.) mixed into a final volume of 0.5 ml. The reaction mixture was incubated for 1hr at 37°C. The amount of MDA formed was expressed as nmol/mg. protein.

VII. Histopathology:

A portion of small intestine belonging to one animal in each group was preserved in 10% formaldehyde solution for histopathological studies. Hematoxylin and eosin were used for staining; later, the histopathological slides were photographed.

VIII. Statistical Analysis:

The experimental results are presented as Mean ± SD for 5 animals in each group. Statistical evaluation of the data was done by one way ANOVA followed by Dunnet’s t-test. Results were considered statistically significant when p<0.05.

RESULTS:

Characteristics of Gmelina arborea stem bark extract.

Name of extract: 70% Methanolic Extract

Colour: Black

Consistency: Sticky

Yield (% W/W): 9.5

1. Effect of Gmelina arborea stem bark extract on total WBC count:

The results revealed that the control animals treated with cyclophosphamide alone showed lower levels of WBC count, when compared to the normal reference group. On treatment with GA extract, the WBC levels of GALC and GAHC were replenished to its normal states (Fig 1).

2. Effect of Gmelina arborea stem bark extract on haemoglobin level:-

The Cyclophosphamide treated group did not show any significant difference in the haemoglobin level up to the 20^th day, when compared to the normal group. The plant extract treated groups also did not show any notable changes in the level of haemoglobin (Table 1).

Fig :1 Effect of Gmelina arborea on Total WBC Count (Values are expressed as mean ± SD for 6 animals).

Table: 1-Effect of Gmelina arborea on Haemoglobin level

	NORMAL	CONTROL	GALC	GAHC
0^th DAY	12.972±0.931	12.866±0.365	12.725±121.563	13.377±0.447
5^th DAY	12.566±0.424	12.588±0.365	12.563±0.440	13.077±0.345
10^th DAY	13.059±0.543	12.948±0.287	12.049±0.201	12.776±0.842
15^th DAY	12.461±0.6119	12.810±0.832	13.029±0.685	12.478±0.518
20^th DAY	12.910±0.218	12.578±0.268	13.035±0.717	13.035±0.503

Values are expressed as mean ± SD for 6 animals

Table : 2- Effect of Gmelina arborea on body weight

	0^TH DAY	5^TH DAY	10^TH DAY	15^TH DAY	20^TH DAY
NORMAL	30.3±1.189	28.44±2.018	26.52±0.929	28.28±1.136	29.22±1.657
CONTROL	31.68±1.294	29.11±1.292	27.84±1.384	28.87±1.634	28.52±1.395
GALC	26.38±1.031	27.57±1.557	26.92±1.939	27.06±1.436	27.06±1.436
GAHC	25.8±1.644	25.48±0.897	26.08±1.109	24.82±1.035	24.08±0.769

Values are expressed as mean ± SD for 6 animals.

Table: 3 - Effect of Gmelina arborea on relative organ weight

	HEART	KIDNEY	LIVER	SPLEEN	BRAIN
NORMAL	0.499±0.095	1.323±0.061	5.576±0.601	1.221±0.18	1.215±0.153
CONTROL	0.538±0.059	1.315±0.89	5.052±0.068	0.409±0.101	1.214±0.159
GALC	0.517±0.017	1.333±0.1	5.482±0.638	0.467±0.08	1.251±0.132
GAHC	0.514±0.034	1.432±0.115	5.056±0.399	0.831±0.12	1.559±0.075

Values are expressed as mean ± SD for 6 animals.

3. Effect of Gmelina arborea stem bark extract on body weight:

Results revealed that, the administration of Gmelina arborea extract does not produce any significant changes in the body weight of the animals during the period of study. The cyclophosphamide alone treated control group has showed a slight decrease in the body weight of the animals (Table 2).

5. Effect of Gmelina arborea stem bark extract on relative organ weights:

The weight of heart, kidney and brain of all animals of all group were found to be in similar range to that of the normal group. Whereas, the spleen weight of control and GALC groups were lowered when compared to the normal. While that of the GAHC group has improved the relative organ weight when compared to the control (Table 3).

6. Effect of Gmelina arborea stem bark extract on bone marrow cellularity (BMC):-

The bone marrow cellularity was found to be lower in the cyclophosphamide treated group compared to normal, GALC and GAHC groups (Table 4). The treatment with low and high doses of GA replenished the BMC to the normal range.

7. Effect of Gmelina arborea stem bark extract on serum parameters:

A) Estimation of aspartate aminotransferase (AST/ SGOT):-

The results revealed that the control animals treated with cyclophosphamide alone showed higher levels of the serum marker GOT (129.165 ± 8.367U/L), compared to the normal reference group (108.234 ± 6.186 U/L). On treatment with GA extract, there was a decrease in the level of SGOT with values recorded as 121.386±6.382 U/L and 115.32±7.82 U/L respectively for GALC and GAHC groups.

B) Estimation of alanine aminotransferase (ALT)/ SGPT:-

This liver specific serum marker GPT showed a steep increase in the control group (82.2±8.833U/L), while the normal group SGPT level was 42.975± 3.465 U/L. This was indicative of liver damage by cyclophosphamide administration in the control animals without any drug supplementation. A lowering of SGPT level was found in the GA treated low and high dose groups where the readings were 72.384±7.212 U/L and 55.54±5.638 U/L respectively.

C) Estimation of alkaline phosphatase (ALP) :-

The ALP concentration was found to be higher in the untreated control group (84.963±6.629 U/L). The GALC and GAHC groups produced mean ALP activities of 76.297 ± 5.729 U/L and 65.83 ± 5.827 U/L respectively, showing moderate restoration towards normal value (60.217±4.739 U/L).

D) Estimation of serum creatinine:- The creatinine concentration was found to be higher in the cyclophosphamide alone treated control group (1.25± 0.061 mg/dl) compared to the normal (0.666 ± 0.028 mg/dl). The treatment with low and high doses of GA showed a slight decrease in the creatinine range, i.e; 1.083 ± 0.026 mg/dl and 1 ±0.031 mg/dl respectively.

E) Estimation of serum urea:-

The urea concentration was found to be higher in the control group (27.335 ± 3.205 mg/dl) compared to the normal range (17.871 ± 2.268 mg/dl). The GALC and GAHC groups produced mean values of 20.087 ± 3.432 mg/dl and 18.457 ± 3.293 mg/dl indicating gradual restoration to normal values (table 5).

Tab: 4 - Effect of Gmelina arborea on Bone Marrow Cellularity

	NORMAL	CONTROL	GALC	GAHC
INITIAL	1322±72	-	-	-
7^TH DAY	-	1089.22±74	918.75±89	834.5±51
14^TH DAY	-	1234.55±99	1107.32±71	1097.41±67
21^ST DAY	1299.87±63	1216.42±38	1154.86±33	1143.98±56

Values are expressed as mean ± SD for 6 animals.

Table: 5 -Effect of Gmelina arborea on all serum parameters

SERUM PARAMETERS	NORMAL	CONTROL	GALC	GAHC
AST/ SGOT( IU/L)	108.234±6.186	129.165±8.367^a	121.386±6.382	115.32±7.82^c
ALT/ SGPT( IU/L)	42.975± 3.465	82.2±8.833^a	72.384±7.212^d	55.54±5.638^c
ALP(KA)	60.217 ± 4.739	84.963 ± 6.629^a	76.297 ± 5.729^d	65.83 ± 5.827^c
CREATININE(mg/dl)	0.666 ± 0.028	1.25± 0.061^a	1.083 ± 0.026^c	1 ± 0.031^c
UREA(mg/dl)	17.871 ± 2.268	27.335 ± 3.205^a	20.087±3.432^d	18.457±3.293^c

Values are expressed as mean ± SD for 6 animals; a :- p<0.01 and b :- p<0.05 compared to normal ; c:- p<0.01 and d:- p<0.05 compared to control.

8. Effect of gmelina arborea stem bark extract on tissue anti-oxidant parameters:-

A.1) estimation of liver superoxide dismutase (sod) activity:

SOD level was found to be lower in the control group (0.382± 0.039 U/mg). It was markedly replenished to normal state (0.679 ± 0.078 U/mg) by GALC (0.509± 0.051 U/mg) and GAHC (0.58 ±0.046 U/mg) drug regimen respectively.

A.2) Estimation of kidney superoxide dismutase (SOD):-

SOD level was found to be lower in the control group (0.577 ± 0.087 U/mg). It was replenished to normal range (0.805 ± 0.093 U/mg) by the high and low doses of GAHC (0.72± 0.096 U/mg) and GALC (0.68± 0.01 U/mg) drug regimen respectively.

B.1) Estimation of liver glutathione (GSH):

Cyclophosphamide administration in the control group lowered the GSH level in the liver tissue (13.630± 2.403 nmoles/mg protein). GALC treatment also showed increased levels of GSH (27.723± 4.481 nmoles/mg protein), but the high dosage regimen, raised the level to normal (value obtained for the normal group was 37.208 ± 5.419 nmoles/mg protein ) which is clearly evident from the GSH reading of GAHC group, 38.546±5.413 nmoles/mg protein).

B.2) Estimation of kidney glutathione (GSH):

GSH level was found to be lower in the control group (15.825±3.408 nmol / mg protein). It was replenished to normal range (31.244±6.419 nmol / mg protein) by the high and low doses of GAHC (29.758±4.019 nmol / mg protein) and GALC (19.484±3.682 nmol / mg protein) drug regimen respectively).

C.1) Estimation of liver glutathione peroxidase (GPX) activity:-

In normal rats liver Glutathione peroxidase level was 10.044±1.094 U/mg, while cyclophosphamide treatment reduced the synthetic activity of the liver leading to a low GPx value of 7.149±1.036 U/mg in the control group. The impact of cyclophosphamide treatment was reduced in the pre-treated GALC and GAHC groups where the Glutathione peroxidase levels were 8.979±0.903 U/mg and 10.026±0.639 U/mg respectively.

C.2) Estimation of kidney glutathione peroxidase (gpx) activity:-

GPX level was found to be lower in the control group (6.754±1.090 U/mg). It was replenished to normal range (10.514±1.269 U/mg) by the GAHC (10.212±0.954 U/mg) and GALC (8.176±0.477 U/mg) drug regimen respectively).

D.1) Estimation of liver tissue lipid peroxidation activity:-

The extent of lipid peroxidation in the liver tissue, measured as nmoles of MDA/mg protein, was high in the control group (0.412±0.07 nmoles/mg protien), in comparison to normal (0.257±0.06 nmoles/mg protein). GALC and GAHC pre-treatment evidently reduced the extent of lipid peroxidation in liver tissue. The extent of Lipid Peroxidation in low and high dose groups of GA were 0.352±0.045 nmoles/mg protien and 0.299±0.028 nmoles/mg protien respectively (table 6).

D.2) Estimation of kidney tissue lipid peroxidation :-

The extent of lipid peroxidation in the kidney tissue, measured as nmoles of MDA/mg protein, was high in the control group (0.419±0.056 MDA in nmoles/mg protien), in comparison to normal (0.200±0.051 MDA in nmoles/mg protein). GALC and GAHC pre-treatment evidently reduced the extent of lipid peroxidation in liver tissue. The extent of Lipid Peroxidation in low and high dose groups of GA were 0.268±0.017 MDA in nmoles/mg protien and 0.249±0.047 MDA in nmoles/mg protien respectively.

9. Effect of Gmelina arborea stem bark extract on mice small intestine:

From the histopathological analysis of the intestine of normal mice it was seen that the glands and villi were normal, lined by columnar cells. The muscle layer also appeared normal. But the untreated control group administered with cyclophosphamide alone showed mucosal damage, hyperplasia of the mucosal glands and villi as well as lymphocyte infiltration. The muscle also posses inflammatory changes.

Mean while there was minimum damage to the intestinal architecture in GALC treated groups compared to the control. The intestinal section of GAHC showed normal glands, villi and muscles indicating the protective effect of Gmelina arborea stem bark extract against cyclophosphamide induced toxicity in mice (fig. 2).

DISCUSSION:

Cancer is one of the most common devastating disease affecting millions of people per year around the world. It has been found that cancer is the second leading cause of death in humans.

Chemotherapy drugs are toxic compounds that target cells that are growing rapidly. Chemotherapy can be used to treat many cancers. More than 100 chemotherapy drugs with different mechanism of action are used today, either as single drug or in combination with other drugs or treatments

In this present study cyclophosphamide (CTX) has been used as an anticancer agent. Chemoprotective activity of 70 % methanolic extract of Gmelina arborea stem bark was studied against the toxicity induced by cyclophosphamide administration. Cyclophosphamide is probably the most commonly used alkylating agent. Cyclophosphamide administration results in the formation of cross-links with DNA by the metabolites produced.

There is pharmacologic evidence that the breakdown of CTX into biologically active alkylating compounds takes place principally in the liver (Brock and Hohorst., 1967). As a result toxic metabolites which are produced in the liver leads to the toxicity of the liver. Hepatic damage is always associated with the cellular necrosis, the increase in tissue lipid peroxidation and the depletion in the tissue glutathione (GSH) levels (Sandy and Ben., 1998). These findings are in agreement with those of the present study, in which there was a significant depletion of GSH and a significant increase in lipid peroxides (MDA) in liver and kidney tissue homogenates of cyclophosphamide treated control group when. While the lipid peroxidation level has been significantly reduced in both liver (p<0.05) and kidney (p<0.01) tissues of the Gmelina arborea high dose treated group.

In order to investigate whether the antioxidant activities of G. arborea are mediated by the increase in antioxidant enzyme activity, we measured SOD and GPx activities in different tissues of mice treated with G. arborea stem bark extract. In the present study, treatment of mice with G. arborea stem bark extract significantly (p<0.01) increased the SOD and GPx activities of liver and kidney tissues.

Liver damage was assessed by biochemical studies such as estimation of serum levels of AST, ALT and ALP. Similarly, Merlin and Parthasarathy, 2011 reported the elevation of serum levels of many biochemical markers like aspartate transaminase (AST), alanine transaminase (ALT), serum alkaline phosphatise (ALP), triglycerides, cholesterol and bilirubin. Treatment of mice with cyclophosphamide increased the serum transaminase level whereas animals pretreated with G. arborea stem bark extract decreased the elevated levels of transaminases (ALT and AST) in a significant manner.

The excretion of both urea and creatinine depends on the glomerular filtration rate. Hence, when damage occurs to the kidney, its values in serum get increased from the normal. The present study revealed that, there is a significant increase in the serum creatinine and urea level on the animals which is treated with cyclophosphamide alone, Whereas the G. arborea high dose treated group showed a decline in the serum levels of Urea and Creatinine in a significant manner (p<0.01).

Changes in the body weight and organ weight of animals were also analysed during the period to find out the toxic effects of cyclophosphamide. Reduction in body weight is observed only in control groups. According to Manson and Kang (1994) and Chahoud et al., 1999, the body weight alterations are usually observed indicative of toxicity in mice. No significant reduction in organ weight was noted except spleen weight. In control group animals, reduction in spleen weight was noted when compared to GALC and GAHC treated groups.

In the histopathological studies of small intestine, G. arborea treated groups showed no significant structural damage when compared to the cyclophosphamide alone treated control group. In this observation it is assumed that G. arborea is highly significant to protect the small intestine form cyclophosphamide toxicity.

These findings clearly demonstrated the chemoprotective activities of G. arborea stem bark extract in experimental mice models.

CONCLUSION:

The evidence obtained from the present study indicates that the treatment with extract of Gmelina arborea can able to protect the liver and kidney against cyclophosphamide induced toxicity. Restoration of serum parameters, tissue antioxidants and haematological parameters to its normal value and the reduction in the level of tissue lipid peroxidation accounts for the chemoprotective activity of 70% methanolic extract of Gmelina arborea stem bark.

There are number of reports evaluating the phytochemical constituents and antioxidant properties of the plant Gmelina arborea. However, the components responsible for the chemoprotective activity are currently unclear. Therefore, further investigations are needed to be carried out to isolate, identify the compounds present in the plant extract and to find out possible mechanism(s) of the protective action of Gmelina arborea.

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Received on 18.08.2014 Modified on 10.09.2014

Research J. Pharm. and Tech. 7(12): Dec. 2014; Page 1420-1428

TISSUE ANTIOXIDANT PARAMETERS		NORMAL	CONTROL	GALC	GAHC
SOD(U/mgprotein)	LIVER	0.679 ± 0.078	0.382± 0.039^a	0.509±0.051^c	0.58±0.046^c
SOD(U/mgprotein)	KIDNEY	0.805 ± 0.093	0.577 ±0.087^a	0.68 ±.01	0.72 ±0.096^d
GSH(nmol/mg protein)	LIVER	37.20 ± 5.419	13.630±2.403^a	27.723±4.481^c	38.546±5.413^c
GSH(nmol/mg protein)	KIDNEY	31.244±6.419	15.825±3.408^a	19.438±3.682	29.758±4.019^c
GPx(U/mg protein)	LIVER	10.044±1.094	7.149±1.036^a	8.979±0.903^d	10.026±0.639^c
GPx(U/mg protein)	KIDNEY	10.514±1.269	6.754±1.090^a	8.176±0.477	10.212±0.954^c
LPO(MDA in nmol/mg. Protein)	LIVER	0.257±0.06	0.412±0.07^a	0.352±0.045	0.299±0.028^d
LPO(MDA in nmol/mg. Protein)	KIDNEY	0.200±0.051	0.419±0.056^a	0.268±0.017^c	0.249±0.047^c