Chemoprotective potential of Coccinia indica against cyclophosphamide-induced toxicity.

OBJECTIVE: Although cyclophosphamide (CP), an alkylating agent, is used in the treatment of cancer owing to its broad-spectrum efficacy, its metabolites exhibit severe undesired toxicities in normal cells. The present study was aimed to investigate the chemoprotective potential of Coccinia indica against CP-induced oxidative stress, genotoxicity, and hepatotoxicity.
MATERIALS AND METHODS: Rodents were orally pre-treated with Coccinia indica extract (200, 400, and 600 mg/kg) for five consecutive days. On 5th day, these animals were injected with CP (50 mg/kg i.p) and sacrificed after 24 hrs. for the evaluation of oxidative stress, hepatotoxicity, micronucleus formation, and chromosomal aberrations.
RESULTS: We found that the CP significantly increased malondialdehyde (MDA) and decreased catalase and glutathione (GSH) levels in brain, and it was significantly reversed by Coccinia indica extract (400 and 600 mg/kg). Further, pre-treatment with Coccinia indica extract (200, 400, 600 mg/kg) significantly and dose-dependently reduced micronuclei formation and incidence of aberrant cells. We also found that the CP-induced increase in the serum biomarker enzymes like alkaline phosphatase (ALP), alkaline aminotransferase (ALT), and aspartate aminotransferase (AST) were significantly reduced by Coccinia indica extract.
CONCLUSION: Thus, the present results indicate the protective effect of Coccinia indica extract against CP-induced oxidative stress, genotoxicity, as well as hepatotoxicity.

Introduction

Cancer is one of the most common fatal diseases in global society. Chemotherapeutics are often used to inhibit the proliferation of cancer cells. Cyclophosphamide (CP), a cytotoxic bifunctional alkylating agent, belongs to the class of nitrogen mustard. It is extensively used for the treatment of various cancers as well as immunosuppressant in organ transplantation, rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis.[1] However, application of CP as an effective chemotherapeutic agent is often restricted because of its wide adverse side-effects that includes nausea, vomiting, alopecia, hemopoetic suppression,[2] nephrotoxicity,[3] hepatotoxicity, urotoxicity, cardiotoxicity,[4] immunotoxicity, mutagenicity,[5] carcinogenicity, and teratogenicity.[6] It is observed that anti-neoplastic effect of CP is associated with active metabolite phosphoramide mustard, while the acrolein causes toxic side-effects.[7] Acrolein interferes with the tissue antioxidant defense system, produces highly reactive oxygen-free radicals, and is mutagenic to mammalian cells.

Natural products have been shown to be an excellent and reliable source for the development of new drugs. Flavonoids and phenolic compounds are found to exhibit diverse biological properties, including hepatoprotective, anti-bacterial, and anti-cancer activities. The advantages of phenolic compounds are generally thought to be due to their antioxidant and free radical scavenging properties.[8] Coccinia indica (Cucurbitaceae), commonly known as little gourd and locally known as ‘Kovai,’ grows abundantly and wildly all over India. Indigenous people use various parts of the plant to get relief from diabetes mellitus. The plant has also been extensively used in Ayurvedic and Unani practice in the Indian subcontinent. Earlier scientific investigation showed that crude extract of Coccinia indica exhibits hepatoprotective,[9] antioxidant,[10] anti-inflammatory and anti-nociceptive,[11] anti-diabetic,[12] hypolipidemic,[13] and anti-bacterial[14] activities. In the present investigation, we have investigated the beneficial effects of Coccinia indica against CP-induced oxidative stress and genotoxicity as well as hepatotoxicity.

Materials and Methods

Animals

Swiss albino male mice (20-25 g) and Wistar rats (200-220 gm) were separately group housed in ambient room temperature (25 ± 2°C) and relative humidity (50 ± 5%), maintained at 12:12 h dark–light cycle. Food and water were available ad libitum. All protocols were approved by Institutional Animal Ethics Committee of Pinnacle Laboratories Pvt. Ltd. Bhopal (M.P.) India and carried out under strict compliance with Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment and Forests, Government of India.

Drugs and Chemicals

CP, 5,5’-Dithiobis-(2-nitrobenzoic acid) and colchicine were purchased from Sigma-Aldrich Co., St. Louis, MO, USA. Serum diagnostic kit was purchased from Span diagnostic limited, Surat, India. While other reagents such as thiobarbituric acid, bovine serum albumin, sodium dodecyl sulfate, trichloroacetic acid (TCA), folin-ciocalteu reagent, and all other chemical were purchased from Merck, Mumbai, India.

Plant material and extraction

Plant material (Coccinia indica) was collected locally in the month of August. Herbarium was prepared, and plant was identified at the Department of Botany, Safia college of science, Bhopal, M.P (voucher specimen-178/Bot/ Safia/10). Plant leaves (1 kg) of Coccinia indica were washed with distilled water to remove dirt and soil, properly dried in shade for 4-6 days. After drying, the plant materials were milled to powder, and then it was defatted with petroleum ether and exhaustly extracted with 70% of methanol by cold medium for 72 hours. The extract was separated by the filtration and concentrated on vacuum evaporator, and a dark semi-solid (greenish-black) material was obtained (yield 9.5% w/w). It was stored at 4°C until used. When needed, the residual extract was suspended in distilled water and used in the study.

Preliminary Phytochemical Screening

Preliminary phytochemical screening for the detection of various constituents was carried out by using standard procedures.[15]

Acute Oral Toxicity Studies

Acute oral toxicity study was carried out in mice as per OECD-423 guidelines. The four fixed dose levels were selected as 5, 50, 300, 2000 mg/kg body weight. The mice were continuously observed for their mortality and behavioral response for 24 hr and thereafter once in a day for 14 days.

Experimental Groups

Different groups (n = 6) of mice were treated with either vehicle (normal saline 10 mg/kg p.o) or Coccinia indica extract in the doses of 200, 400, and 600 mg/kg per oral for five consecutive days alone, or along with a single dose of CP (50 mg/kg i.p). CP was administered on 5th day 1 h after Coccinia indica treatment. All animals were sacrificed 24 h after the treatment of CP. Micronucleus assay and chromosomal aberration tests were performed as mentioned in procedures. The brains were processed for estimation of oxidative stress parameters such as lipid peroxidation, catalase, and glutathione content.

In addition, the effect of Coccinia indica extract on CP-induced hepatotoxicity was observed in rats. Similar treatment regimen of Coccinia indica extract along with CP was followed in rats. The rats were anesthetized with thiopentone sodium (60 mg/kg i.p), and blood was collected by puncture of retro-orbital sinus, then centrifuged at 7000 rpm for 10 min. Serum levels of alkaline phosphatase (ALP), alkaline aminotransferase (ALT), and aspartate aminotransferase (AST) were determined using diagnostic kits.

Lipid Peroxidation Estimation

Lipid peroxide in the mouse brain was measured according to the method described earlier[16] with some modifications. The brain tissue was rinsed in ice-cold physiological saline, minced and a 10% w/v homogenate was prepared in 1M Tris-HCl buffer (pH 7.4). The sample was centrifuged at 3000 rpm for 10 min, and supernatant was used for the determination of lipid peroxidation. The supernatant was added to sodium dodecyl sulfate (8.1%), followed by acetic acid (20%) and thiobarbituric acid (0.8%). The volume was made up to 4 ml with distilled water and heated on a water bath at 95°C for 60 min. After cooling with tap water, further 1.0 ml of distilled water and 5.0 ml of the mixture of n-butanol and pyridine (15: 1, v/v) were added and shaken vigorously. After centrifugation at 4000 rpm for 10 min, the organic layer was taken, and its absorbance at 532 nm was measured. Lipid peroxidation was calculated from the standard curve using malondialdehyde (MDA) and expressed as nM/mg protein.

Catalase Estimation

Brain tissue homogenate (10% w/v) was prepared in 50 mM phosphate buffer and centrifuge at 15000 rpm for 10 min. The change in absorbance was followed spectrophotometrically at 240 nm after the addition of H2O2 (30 mM) to 100 μl of supernatant in 50 mM phosphate buffer (pH 7). The activity of the enzyme was expressed as U/mg tissue, where U is “μmole of H2O2 reduced/min.”[17]

Glutathione Estimation

The mouse brain homogenate was used for the estimation of reduced glutathione (GSH) content. Tissue was rinsed in ice-cold physiological saline, minced and the homogenate (10% w/v) was prepared in phosphate buffer (pH 7.4). Centrifuged at 3000 rpm for 10 min and the supernatant was used for the determination of GSH. Homogenates were immediately precipitated with 0.1 ml of 25% TCA, and the precipitate was removed after centrifugation. Free SH groups were assayed in a total 3 ml volume by adding 2 ml of 0.6 mM 5,5’-dithiobis (2-nitro benzoic acid) prepared in 0.2 M sodium phosphate buffer (pH 8.0), to 0.1 ml of the supernatant, and absorbance was read at 412 nm. GSH level was expressed as millimoles/gm wet weight.[18]

Micronucleus Assay

A mouse bone marrow micronucleus test was carried out according to earlier method[19] with some modification. The animals were sacrificed by cervical dislocation 24 h after CP treatment. The bone marrow from both the femurs was flushed in the form of a fine suspension into a centrifuge tube containing bovine serum albumin (BSA). The cells were dispersed by gentle pipetting and collected by centrifuge at 1500 rpm for 10 min at 4°C. Cell pellets were re-suspended in drops of BSA, and bone marrow smears were prepared. After 24 h of air-drying, the smears were stained with May-Grunwald/Giemsa stain. In this method, polychromatic erythrocytes were stained reddish-blue, while nuclear materials are stained as dark purple. Six mice were used for each experimental point, and a total of 6000 PCEs were scored for each experimental point for determining the percentage of micronucleated polychromatic erythrocytes. Cytotoxicity of CP was determined from PCEs to NCEs ratio, and total 200 cells per animal were counted.

Chromosomal Aberration Assay

The chromosomal aberration assay was performed as described earlier[20] with some modification. Briefly, mice were treated with colchicine (4 mg/kg i.p) 1.5 h to arrest the metaphase stage, prior to sacrifice, and femur bones were isolated. The bone marrow was flushed out from both femurs using 0.56% (w/v) KCl solution and incubated at 37°C. After centrifugation (1000 rpm, 10 min), the supernatant was discarded and the pellet were re-suspended in cornoy's fixative (3:1 mixture of methanol and glacial acetic acid). The suspension was dropped on the ice-cold slides (previously kept in 90% alcohol in freezer) using pastures pipette, and slides were immediately flamed for few seconds and allowed to dry at room temperature. After drying, slides were stained with phosphate-buffered 5% Giemsa solution. A total of 100 well-spread metaphase plates per animal in each group (total 600 metaphase in each group) were analyzed, and the chromosomal aberrations were observed for fragment, deletion, gap, pulverization, ring, and polyploidy aberrations.

Statistical Analysis

All results were analyzed by One-way analysis of variance (ANOVA), and post-hoc analysis was performed with Bonferroni's test. Value of P < 0.05 was considered to be statistically significant in all the cases.

Results

Preliminary phytochemical screening suggests the presence of carbohydrates, glycosides, alkaloids, tannins, phenolic compounds, and flavonoids in the Coccinia indica extract [Table 1].

Table 1

Preliminary phytochemical screening

Acute Oral Toxicity Study

Coccinia indica extract was found to be non-toxic up to 2000 mg/kg body weight [Table 2]. Finally, the dose of 200 mg/kg, 400 mg/kg, 600 mg/kg were chosen for further studies.

Table 2

Acute oral toxicity studies

Effect of Coccinia Indica on MDA, Catalase, and GSH Levels in Brain Altered by CP

As shown in Figure 1, CP (50 mg/kg i.p) significantly increased MDA level and decreased catalase as well as GSH levels as compared to control group [F (7, 47) = 9.704, P < 0.0001; F (7, 47) = 3.901, P < 0.0025; F (7, 47) = 6.366, P < 0.0001; One-way ANOVA]. Further, post-hoc comparison by Bonferroni's test showed that Coccinia indica extract 400 and 600 mg/kg (P < 0.05) but not 200 mg/kg (P > 0.05) decreased CP-induced elevation of MDA level and reduced the effect of CP on catalase and GSH levels. However, alone treatment of coccinia indica extract in all the doses did not affect (P > 0.05) the brain levels of MDA, catalase, and GSH.

Figure 1

Effect of Coccinia indica on MDA, catalase, and GSH levels altered by CP in brain. All the value expressed as mean ± SEM (n = 6). **P< 0.001, ***P< 0.0001 Vs control (saline + saline); #P< 0.05, ##P< 0.001, ###P< 0.0001 Vs Saline + CP (50 mg/kg, i.p)

Effects Of Coccinia Indica Against CP-Induced Micronuclei Formation

The effect of Coccinia indica extract on the frequency of MnPCEs formation in bone marrow cells, 24 hr. after CP treatment, is depicted in Table 3. CP (50 mg/kg i.p) induced significant micronuclei formation and P/N ratio as compared to control group [F (7, 47) = 225.1, P < 0.05, F (7, 47) = 22.79, P < 0.05, One-way ANOVA]. Further, post-hoc comparison by Bonferroni's test revealed that Coccinia indica extract (200, 400, 600 mg/kg) dose-dependently and significantly decreased the CP-induced micronuclei formation (P < 0.05). Coccinia indica extract 600 mg/kg dose significantly increased P/N ratio (P < 0.05). However, alone treatment of Coccinia indica extract in all the doses (200-600 mg/kg) did not affect the micronuclei formation (P > 0.05).

Table 3

Protective effect of Coccinia indica on CP (50 mg/kg i.p)-induced MnPCEs and ratio of PCE/NCE in bone marrow

Effects of Coccinia Indica Against CP-Induced Chromosomal Aberration

As depicted in Table 4, CP (50 mg/kg i.p) single dose administration significantly increased incidence of aberrant cells (%) as compared to control group [F (7, 47) = 508.6, P < 0.05, One-way ANOVA]. Further, post-hoc comparison by Bonferroni's test showed that Coccinia indica extract (200, 400, 600 mg/kg) significantly and dose-dependently decreased incidence of % aberrant cells (P < 0.05). However, alone treatment of Coccinia indica extract in all the doses (200-600 mg/kg) did not affect the % of aberrant cells (P > 0.05).

Table 4

Protective effect of Coccinia indica on CP (50 mg/kg i.p)-induced chromosomal aberrations in bone marrow cells

Serum Biochemical Parameters

Effect of Coccinia Indica extract against CP-induced elevated level of ALP, ALT, and AST

As shown in Figure 2, CP (50 mg/kg i.p) significantly increased ALP, ALT, and AST levels as compared to control group [F (7, 47) = 17.21, P < 0.0001; F (7, 47) = 38.19, P < 0.0001; F (7, 47) = 36.78, P < 0.0001; One-way ANOVA]. Further, post-hoc comparison by Bonferroni's test showed that Coccinia indica extract 400 and 600 mg/kg (P < 0.05) but not 200 mg/kg (P > 0.05) decreased CP-induced elevation of ALP level. However, alone treatment of coccinia indica extract in all the doses did not affect the ALP, ALT, and AST levels (P > 0.05).

Figure 2

Effect of Coccinia indica on CP-induced elevated level of ALP, ALT, and AST levels. All the value expressed as mean ± SEM (n = 6). ***P< 0.0001 Vs control (saline + saline), ###P< 0.0001 Vs Saline + CP (50 mg/kg, i.p)

Discussion

The present study demonstrated that hydromethanolic extract of Coccinia indica shows chemoprotective effect against CP-induced oxidative stress, genotoxicity, and hepatotoxicity. We and others have found the presence of carbohydrates, glycosides, alkaloids, tannins, phenolic, and flavonoidal compounds in Coccinia indica extract.[914] Flavonoids are a group of polyphenolic compounds, which exhibits biological effects. The presence of high phenolic and flavonoid content has contributed directly to the antioxidant activity by neutralizing the free radicals.[10]

Free-radical-induced lipid peroxidation has been suggested to alter the cell membrane structure and function.[21] An increase in free-radical production mediated by CP metabolites in turn stimulate lipid peroxidation leading to an increase in MDA production.[22] The brain cells are susceptible to oxidative stress induced by CP.[22] The brain has limited access to the bulk of antioxidants produced by the body, hence neurons are first cells to be affected by a shortage of antioxidants.[23]

In the present study, we have found that treatment with CP leads to oxidative stress as evident from significant increase in MDA level and decrease in catalase level. However, pre-treatment of Coccinia indica extract (400 mg/kg) significantly decreased the formation of MDA level as well as enhances catalase level in the brain. Furthermore, CP significantly reduced GSH content in brain cells, and Coccinia indica extract treatment significantly increased GSH enzyme level. It is possible that inhibition of the CP-induced oxidative stress in brain may be due to the antioxidant properties of the extract. Interestingly, the anti-diabetic[12] and hypolipidemic activity[13] have been attributed to its antioxidant property.

Micronucleus assay is well-characterized biomarker of structural and numerical chromosomal damage, which arise from acentric chromosome fragments or lagging whole chromosome(s) that fail to incorporate in to the daughter nuclei after nuclear division. Administration of Coccinia indica extract pre-treatment resulted in inhibition of micronuclei formation and cytotoxicity induced by CP. Chromosomal aberrations analysis shows that the CP (50 mg/kg, i.p) significantly increases the incidence of aberrant cells (%). Coccinia indica extract (200, 400, and 600 mg/kg) pre-treatment significantly and dose-dependently decreased the percentage of aberrant cells as observed from chromosomal aberration assay. Thus, the present study showed that pre-treatment of Coccinia indica protects the genomic damage as evident from micronucleus assay as well as chromosomal aberrations assay in the bone marrow.

It is well established that increased activities of ALP, ALT, and AST enzymes in the serum are known diagnostic indicators of hepatotoxicity. In the present study, CP administration caused significant increase in the serum ALP, ALT, and AST levels in rats. The increased levels of these enzymes and metabolites in the serum could be attributed to the activity of acrolein. Acrolein causes a breach in antioxidant defense system resulting in proliferative production of reactive oxygen species (ROS), which in turn may attack hepatocytes membrane disrupting its structure and function, a leakage of these enzymes into the blood circulation.[24] Earlier studies[212526] also showed that intraperitoneal injection of CP resulted as increase in above serum biomarker enzymes and metabolites for liver function. Hydromethanolic extract of Coccinia indica significantly decreased in the serum ALP, ALT, and AST levels, thus indicating the protective activity against the liver damage. Although further extensive studies are required, however, based on literature, it can be postulated that flavonoid content in Coccinia indica extract may be partly or fully responsible for these activities.

The exact molecular mechanism of chemoprotective effect of Coccinia indica extract is not clear. As mentioned earlier, the chemoprotective effect may be attributed to its antioxidative property. Whether the intervention of antioxidants during cancer chemotherapy influences the efficacy of the treatment or reduces the unwanted side-effect is a subject of intense investigation.

In conclusion, the present studies indicate that Coccinia indica hydromethanolic extract pre-treatment attenuates the CP-induced oxidative stress and genotoxicity in the bone marrow. The chemoprotective potential of Coccinia indica might be due to its antioxidant property. Thus, Coccinia indica has potential as an adjuvant to CP for preventing the side-effects associated with chemotherapeutic applications.