Effect of ethanolic fruit extract of Cucumis trigonus Roxb. on antioxidants and lipid peroxidation in urolithiasis induced wistar albino rats.

Urolithiasis was induced using ethylene glycol in wistar albino rats, the formation of calcium stones in the kidney results with the damage of antioxidant system. Ethanolic extract of Cucumis trigonus Roxb fruit of family Curcurbitaceae was used to treat urolithiasis. On this course, the extract also repairs the changes that happened in the enzymatic, non enzymatic antioxidants and lipid peroxidation in liver and kidney of urolithiasis induced rats. The results obtained from the analysis were compared at 5% level of significance using one way ANOVA. The results show that the ethanolic fruit extract has repaired the levels of antioxidants and malondialdehyde to their normal levels.

Introduction

The use of traditional medicine and medicinal plants in most developing countries, as a normative basis for the maintenance of good health, has been widely observed (Falodun A et al., 2006). Plants are utilized as therapeutic agents since time immemorial in both organized (Ayurveda, Unani) and unorganized (folk, tribal, native) form. The healing properties of many herbal medicines have been recognized in many ancient cultures (Rajeshwari sivaraj et al., 2011).

Cucumis trigonus Roxb of family Cucurbitaceae is distributed throughout India and found in areas of Ceylon, Afghanistan, Persia and Northern Australia. It is used for various ailments in Indian Traditional System of Medicine (Naveena, B.M. et al., 2004) Fruit and roots have medicinal value. The fruits are used in flatulence, leprosy, fever, jaundice, diabetes, cough, bronchitis, ascites, anaemia, constipation, other abdominal disorders and amentia. (Naik, V.R. et al., 1981). In addition, fruit pulp is bitter, acrid, thermogenic, anthelmintic, liver tonic, cardio tonic, appetizer, expectorant and intellect promoting (Kirtikar, K.R. et al., 2009). The title plant is reported to possess analgesic, anti-inflammatory and diuretic activity. Recently it's proteolytic and serine protease activity has been reported (Naik, V.R. et al., 1980).

Urolithiasis refers to the solid nonmetallic minerals in the urinary tract. Among the several types of kidney stones, the most common are calcium oxalate. The formation of these stones involves several physicochemical events, beginning with crystal nucleation, aggregation and ending with retention within the urinary tract (Purnima et al., 2010). It has been described as the third most common affliction of human urinary tract (Atmani et al., 2004). Some common causes are inadequate urinary drainage, foreign bodies in the urinary tract, microbial infections, diet with excess oxalates and calcium, vitamin abnormalities, viz. vitamin A deficiencies, vitamin D excess, metabolic diseases like hyperparathyroidism, cystinuria, gout and intestinal dysfunction (Tiselius et al., 2008).

Lipid peroxidation represents oxidative tissue damage caused by hydrogen peroxide (H2O2) superoxide anion (O2)and hydroxyl radicals (OH), resulting in structural alteration of membrane with release of cell and organelle contents, loss of essential fatty acids with formation of cytosolic aldehyde and peroxide products. Malondialdehyde is a major end product of free radical reaction on membrane fatty acids. Although the cell is endowed with several antioxidant systems to limit the extent of lipid peroxidation, under certain conditions protective mechanism can be overwhelmed, leading to elevated tissue levels of peroxidation products. Antioxidant can be classified as preventive and chain breaking antioxidants. Antioxidant vitamins such as alpha tocopherol (vitamin E), vitamin A and ascorbic acid (vitamin C) belong to the second category. Such compounds can intercept free radical induced chain reaction and prevent further oxidation (Kato et al., 2007). Many studies on the etiology of stone diseases have focused on the properties of urine that effect crystal nucleation and growth. Crystal adherence to the surface of injured renal epithelial cells is considered initiating events in the genesis of urolithiasis. Factors leading to initiation of calcium oxalate formation are still not known. However the oxidant (free radical production) and antioxidant imbalance may be one of the major factors leading to the process of crystal deposition in renal tissues (Kato et al., 2007).

The present study involves the activity of the ethanolic fruit extract of C.trigonus towards the enzymatic antioxidants such as, superoxide dismutase (SOD), Catalase (CAT) and glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST), glucose-6-phosphate dehydrogenase (G-6-PD), non-enzymatic antioxidants such as total reduced glutathione, vitamin C and vitamin E and the activity of lipid peroxidation in liver and kidney of control and urolithiasis induced rats.

Materials and Methods

Collection of the plant material

Cucumis trigonus Roxb. fruits were collected from Kovanur area of Coimbatore district [Latitude: 10N 85’48.86”, Longitude: 76N 97‘02.15’], Tamil Nadu, India during the month of September to November,2009. The plant was identified and authenticated by taxonomist Dr.K. Arumugasamy, Assistant Professor, Department of Botany, Kongunadu Arts and Science College, Coimbatore, Tamilnadu, India. Voucher specimen was deposited herbarium centre, Department of Botany, Kongunadu Arts and Science College, Coimbatore.

Preparation of ethanolic fruit extract for in vivo studies

Fruits of the plants were washed, shade dried, powdered and stored in tight containers under refrigeration. 100g of C. trigonus powder was taken in a conical flask. To this 500ml of 99% ethanol was added. The content of the flask was kept in the shaker for 48hr. and the suspension was filtered and residue was resuspended in an equal volume of 99% ethanol for 48hr. and filtered again. The two filtrates were pooled and the solvents were dried in an oven at 37°C and a crude residue was obtained. The yield was 18g, and the residue was suspended in water and administered orally to the experimental rats.

Selection of animals for toxicity studies

Healthy adult male wistar albino rats weighing about 150 to 200 g were collected from Animal Breeding Centre, Kerala Agricultural University, Mannuthy, Thrissur, Kerala, India. The rats were kept in properly numbered large polypropylene cages with stainless steel top grill having facilities for pelleted food. The animals were maintained in 12 hr. light and dark cycle at 28° C ± 2° C in a well ventilated animal house under natural conditions in large polypropylene cages and they were acclimatized to laboratory conditions for 10 days prior to the commencement of the experiment. The animals were fed with standard pelleted diet supplied by AVM foods, Coimbatore, Tamilnadu, India. All animal experiments were performed according to the ethical guidelines suggested by the Institutional Animal Ethics Committee (IAEC).

Experimental design of animals for in vivo studies

The experimental design of animals is given in table 1 for in vivo studies.

Table 1

Experimental design of animals for in vivo studies

The experimental animals in group II, III and IV were induced with ethylene glycol for 28 days to develop urolithiasis and the group II animals were sacrificed once after the induction. Group III and IV animals underwent treatment with fruit extract and the standard drug respectively for 28 days after the induction of urolithiasis (29th to 56th day). Group III and IV animals were sacrificed after the treatment on day 57. Serum, liver, kidney and urine samples were collected and subjected to analysis of marker enzymes, biochemical parameters, antioxidants and parameters related to urolithiasis.

Collection of liver and kidney samples

The experimental animals were sacrificed, liver and kidney were removed immediately, washed with ice cold saline and their weights were recorded. Small pieces of liver and kidney tissues were collected in 10% formalin and used for histopathological studies.

Preparation of tissue homogenate

A 10% tissue homogenate was prepared by homogenizing 1.0g of chopped liver or kidney tissue in 10ml of 0.1M tris HCl homogenizing buffer at pH 7.5. The homogenate was used for assaying the antioxidants and lipid peroxidation.

Estimation of Superoxide Dismutase (SOD)

The method[10] involves generation of superoxide radical of riboflavin and its detection by nitrite formation from hydroxylamine hydrochloride. The nitrite reacts with sulphanilic acid to produce a diazonium compound which subsequently reacts with naphthylamine to produce a red azo compound whose absorbance is measured at 543nm.

Estimation of Catalase (Cat)

Catalase causes rapid decomposition of hydrogen peroxide to water.

The method[14] was based on the fact that dichromate in acetic acid reduced to chromic acetate when heated in the presence of H2O2 with the formation of perchloric acid as an unstable intermediate. The chromic acetate thus produced was measured colorimetrically at 610nm.

Estimation of Glutathione Peroxidase (Gpx)

Glutathione (GSH) was measured by the method[13], in which the sample reacts with DTNB to give a compound that absorbs at 412nm.

Estimation of Glutathione Reductase (GR)

Glutathione reductase catalyses the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) and is assayed by measuring the decrease in absorbance at 340nm[13].

Estimation of Glutathione -S- Transferase (GST)

The enzyme was assayed by its ability to conjugate GSH with CDNB, the extent of conjugation causing a proportionate change in the absorption at 340nm[11].

Estimation of Glucose-6-Phosphate Dehydrogenase (G-6-PD)

Glucose-6- phosphate dehydrogenase is assayed by measuring the increase in absorbance, which occurs at 340nm[12]. When NADP reduces to glucose-6 phosphate to NADP in the reaction catalysed by glucose-6-phosphate dehydrogenase.

Estimation of Total Reduced Glutathione (GSH)

The method[15] was based on the reaction of reduced glutathione with DTNB to give a compound that absorbs at 412nm.

Estimation of Vitamin C (Ascorbic Acid)

Ascorbic acid was oxidised by copper to form dehydroascorbic acid and diketoglutaric acid. These products were treated with 2, 4-dinitrophenyl hydrazine to form the derivative of bis 2,4-dinitrophenyl hydrazine. This compound, in strong sulphuric acid undergoes a rearrangement to form a product with an absorption band that is measured at 520nm. The reaction[16] was run in the presence of thiourea to provide a mildly reducing medium, which helps to prevent interference from non-ascorbic acid chromogens.

Estimation of Vitamin E (Tocopherol)

Tocopherol can be estimated using Emmerie-Engel reaction[17], which is based on the ferric to ferrous ions by tocopherols, which then forms a red color with 2,2΄- dipyridyl. Tocopherols and carotenes are first extracted with xylene and the extinction read at 460nm to measure carotenes. A correlation is made for these after adding ferric chloride and reading at 520nm.

Estimation of Lipid Peroxidation (LPO)

Malondialdehyde has been identified as the product of lipid peroxidation that reacts with thiobarbituric acid to give a red color absorbing at 535 nm[18].

Results

The activities of enzymic antioxidants like Superoxide dismutase, Catalase and glutathione peroxidase in the liver and kidney of control and experimental rats using the fruit extract is shown in the tables 2 and 3.

Table 2

Effect of ethanolic fruit extract of C. trigonus on SOD, CAT and GPx in liver

Table 3

Effect of ethanolic fruit extract of C. Trigonus on SOD, CAT and GPx in kidney

Experimental design

Group I: Control rats - received normal pelleted diet

Group II: Urolithiasis induced rats - received 0.75% ethylene glycol in water for 28 days

Group III: Plant drug treated rats - urolithiasis induced rats received C.trigonus fruit extract (150 mg / kg body weight) by oral administration for subsequent 28 days at a rate of 1.0 ml / rat / day

Group IV: Standard drug thiazide treated rats - urolithiasis induced rats received thiazide (150 μg / kg body weight) by oral administration for subsequent 28 days at a rate of 1.0 ml / rat / day

Comparison between the groups

‘a’ represents comparison between group II and I

‘b’ represents comparison between group III and II

‘c’ represents comparison between group IV and II

‘d’ represents comparison between group IV and III

*The symbols represent statistical significance P<0.05; ns-not significant

Units

♦ 50% inhibition of nitrate formation / min / mg protein

♦♦ μ moles of hydrogen peroxide decomposed / min / mg protein

♦♦♦ μg of glutathione consumed / min / mg protein

♦ 50% inhibition of nitrate formation / min / mg protein

♦♦ μ moles of hydrogen peroxide decomposed / min / mg protein

♦♦♦ μg of glutathione consumed / min / mg protein

The ethanolic extract of C.trigonus fruit have shown a significant changes in the levels of enzymic antioxidants like glutathione reductase, glutathione - S - transferase, glucose - 6 - phosohate dehydrogenase and the results were expressed at 5% level of significance in table 4 and 5.

Table 4

Effect of ethanolic fruit extract of C. trigonus on enzymatic antioxidants in liver

Table 5

Effect of ethanolic fruit extract of C.trigonus on enzymatic antioxidants in kidney

*The symbols represent statistical significance P<0.05; ns-not significant.

Φ μg of CDNB-GSH conjugate /min/mg protein

ΦΦ 0.01 OD /min/mg protein

ΦΦΦ N moles of NADPH broken /min/mg protein

Φ μg of CDNB-GSH conjugate /min/mg protein

ΦΦ 0.01 OD /min/mg protein

ΦΦ N moles of NADPH broken /min/mg protein

Antioxidant can be classified as preventive and chain breaking antioxidants. Antioxidant vitamins like vitamin E (Tocopherol), Vitamin C (Ascorbic acid) belong to the second category. These compounds can intercept free radical induced chain reaction and prevent further oxidation. The ethanolic fruit extract C.trigonus have a very good potential to rebuilt the levels of this antioxidant vitamins and is experimentally verified and the results were shown in the tables 6 and 7.

Table 6

Effect of ethanolic fruit extract on non-enzymatic antioxidants in liver

Table 7

Effect of ethanolic fruit extract on non-enzymatic antioxidants in kidney

¥μg/ mg protein

From the table 8, it is evident that the levels of lipid peroxidation were significantly increased (p<0.05) in liver and kidney homogenate on ethylene glycol intoxication, when compared to control rats. The rats treated with fruit extract (group III) showed a significant decrease in the levels of lipid peroxidation.

Table 8

Effect of ethanolic fruit extract of C. trigonus on LPO in liver and kidney

¥ μg/ mg protein

Discussion

Superoxide dismutase (SOD) is widely distributed in cells with high oxidative metabolism and has been proposed to protect against the deleterious effect of superoxide anion. SOD catalytically scavenges the superoxide radicals and thus renders cytoprotection against free radical damage (Fridovich, 1975). Catalase and glutathione peroxidase are involved in the elimination of hydrogen peroxide. The decreased activity of glutathione peroxidase may be correlated to decreased availability of its substrate reduced glutathione. Lowered levels of reduced glutathione have been reported in urolithiatic rats (Muthukumar and Selvam, 1997).

Further catalase has been shown to be inhibited by the high levels of oxalate ( Selvam and Bijikurien 1991).The accumulation of H2O2 formed by the reactions leads to increased formation of hydroxyl radicals either with iron by Fenton type reaction or with iron in the presence of superoxide by Haber-Weiss reaction. This is supported by the fact that xanthine oxidase produces superoxide anions and is found to be increased in EG-treated rats (Ravichandran and Selvam, 1990).

Group III rats treated with the C.trigonus fruit extract showed a significant restoration of antioxidants in homogenates of liver and kidney when compared to ethylene glycol treated rats (group II), which might be an indication of recovery due to the ethanolic extract of C.trigonus with antioxidant property.

Our results coincide with that of Kim et al. (2006) who showed that Camelia sinensis (Green tea) and Aspalathus linearis (rooibos tea) both has inhibitory effect on kidney stone formation with increased antioxidant activity in rats. Farooq et al. (2004) revealed the antioxidant potential of phycocyanin thereby protecting it as a promising therapeutic agent against renal cell injury associated with kidney stone formation.

This study suggests that C.trigonus could prevent the free radical formation in calcium oxalate induced urolithiatic rats and thus protecting the renal cells from oxidative injury. When C.trigonus fruit extract treated rats (Group III) were compared with thiazide treated rats (Group IV) there was no significant difference between these groups of rats showing the similarity of the fruit extract with the standard drug thiazide. From the above results, it is prevalent that the ethanolic fruit extract of C.trigonus normalized the levels of enzymatic antioxidants in liver and kidney.

From the table 4 and 5, it is evident that the levels of non-enzymatic antioxidants were significantly decreased (p< 0.05) in urolithiatic rats (Group II) when compared to control rats (Group I). The oxidants (free radicals) and antioxidant imbalance may be one of the major factors leading to the process of crystal deposition in renal tissues (Kato et al., 2007). Group III rats treated with the C.trigonus fruit extract showed a significant restoration of antioxidants in homogenates of liver and kidney when compared to ethylene glycol treated rats (group II), which might be an indication of recovery due to the antioxidant property of ethanolic extract of C.trigonus.

Our results coincide with that of Farooq et al. (2004) who showed that the levels of antioxidants significantly increased by the administration of phycocyanin from Spirulina platensis. Veena et al. (2005) showed that the sulfated polysaccharides from edible sea weed Fucus vesiculosus increased the antioxidant levels in experimental hyperoxaluria. Thus from the above results it was evident that the levels of non enzymatic antioxidant were brought back to near normal levels on treatment with C.trigonus extract.

From the Table 8, it is evident that the levels of lipid peroxidation were significantly increased (p < 0.05) in liver and kidney homogenate on ethylene glycol intoxication (Group II) when compared to control rats (Group I).

Group III rats treated with the C.trigonus fruit extract showed a significant decrease in the levels of lipid peroxidation in homogenates of liver and kidney when compared to ethylene glycol treated rats (group II), which might be an indication of recovery due to the administration of ethanolic extract of C.trigonus which possess free radical scavenging activity.[28]