Protective effect of Eucalyptus citriodora extract against cyclophosphamide induce damage in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg 9

doi:10.4103/2229-5119.102753

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ORIGINAL ARTICLE

Year : 2012 | Volume : 3 | Issue : 2 | Page : 101-104

Protective effect of Eucalyptus citriodora extract against cyclophosphamide induce damage in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg ⁹

Yasir H Siddique, Smita Jyoti, Falaq Naz
Department of Zoology, Drosophila Transgenic Laboratory, Section of Genetics, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, Uttar Pradesh, India

Date of Web Publication

20-Oct-2012

Correspondence Address:
Yasir H Siddique
Department of Zoology, Drosophila Transgenic Laboratory, Aligarh Muslim University, Aligarh - 202002, Uttar Pradesh
India

DOI: 10.4103/2229-5119.102753

Abstract

Background: Hsp70 has been considered to be one of the candidate genes for predicting the cytotoxicity against environment chemicals. The fruit fly Drosophila melanogaster is a well-established model for the study of antigenotoxic effects of various natural compounds and plant extracts because of its well-documented genetics and developmental biology. In the present study, the effect of Eucalyptus citriodora extracts was studied against the cyclophosphamide induce cytotoxic damage in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg⁹. Materials and Methods: The leaf extract was prepared by soaking the sample (2 g of dry weight) in 300 ml of acetone for eight hours at 60°C in the Soxhlet's apparatus. The extract concentrations of 0.25, 0.50 and 1.0 μl/ml of food were established. The same concentrations were established along with 0.025 and 0.100 μl/ml of cyclophosphamide (CP), and the third instar larvae were allowed to feed on it for 24 and 48 hours. The hsp70 expression was quantified by O-nitrophenyl-β-d-galactopyranoside (ONPG) assay. Results: The doses of Eucalyptus citriodora extract given along with 0.025 and 0.100 μl/ml of CP showed a dose dependent significant decrease in the hsp70 expression for both the durations (24 and 48 hrs) of exposure. Conclusion: The results are suggestive of the protective role of E. citriodora extract against CP induced toxicity in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg⁹ .

Keywords: Cyclophosphamide, Drosophila melanogaster (hsp70-lacZ)Bg⁹ , Eucalyptus citriodora

How to cite this article:
Siddique YH, Jyoti S, Naz F. Protective effect of Eucalyptus citriodora extract against cyclophosphamide induce damage in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg ⁹. J Nat Pharm 2012;3:101-4

How to cite this URL:
Siddique YH, Jyoti S, Naz F. Protective effect of Eucalyptus citriodora extract against cyclophosphamide induce damage in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg ⁹. J Nat Pharm [serial online] 2012 [cited 2013 May 22];3:101-4. Available from: http://www.jnatpharm.org/text.asp?2012/3/2/101/102753

Introduction

Cyclophosphamide (CP) is an alkylating agent. ^[1] It is used as a chemotherapeutic agent to treat various types of cancer. ^[2] The use of CP has limitation as it causes damage to the normal tissues and disrupts various biological and physiological processes. ^[3] The study on anticancerous drugs is of special interest as they may induce secondary cancers in cancer patients. ^[4] Hence any modulatory agent that reduces its toxicity may reduce the possibility of secondary cancers during the therapy. In India, plant extracts are being used to treat a number of ailments such as skin diseases, diarrhea, and leprosy. ^[5] In this context the effect of Eucalyptus citriodora extracts was studied against the toxic effects of CP on third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg⁹ .

Materials and Methods

Fly strain

A transgenic Drosophila melanogaster line that expresses bacterial β-galactosidase as a response to stress was used in the present study. ^[6] The flies and larvae were cultured on standard Drosophila food containing agar, corn meal, sugar and yeast at 24±1°C. ^[7]

Experimental design

CP at 0.025 and 0.100 μl/ml of food concentration alone and with 0.25, 0.50 and 1.0 μl/ml of E. citriodora extract were established. The third instar larvae were allowed to feed on them for different durations (24 and 48 hrs).

Preparation of leaf extract

The leaves of Eucalyptus citriodora were collected from the Aligarh Muslim University Campus and were identified by Dr. Badaruzzaman Siddiqui, Associate Professor, Department of Botany, AMU, Aligarh (Specimen Voucher No. 47771). The leaves were air dried and grinded to fine powder. Extraction was performed by soaking samples (2 g of dry weight) in 300 ml of acetone for eight hours at 60°C in Soxhlet's apparatus. After filtration, the extract concentrations of 0.25, 0.50 and 1.0 μl/ml of food were established. ^[8],[9]

Soluble-O-Nitrophenyl-β-d-Galactopyranoside (ONPG) assay

The expression of hsp70 gives a measure of cytotoxicity. ^[10] Briefly, after washing, the larvae in the phosphate buffer, the larvae were taken through the micro-centrifuge tube (20 larvae/tube; 5 replicates/group), permeabilized for 10 min in acetone and incubated overnight at 37°C in 600 μl of ONPG staining buffer. Following incubation, the reaction was stopped by adding 300 μl Na ₂ CO ₃ and the extent of reaction was quantified by measuring the absorbance at 420 nm. ^[11]

Statistical analysis

Statistical analysis was carried out by one-way analysis of variance (ANOVA) using commercial Software Statistica Soft Inc. (2007).

Results

The results of the present study reveal that the exposure of 0.025 and 0.100 μl/ml of CP to the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg⁹ for the duration of 24 and 48 hrs showed an increase in expression of hsp70 [Table 1]. The exposure of the third instar larvae to 0.025 μl/ml of CP along with 0.25, 0.50 and 1.0 μl/ml of E. citriodora extract for 24 hrs results in the reduction of the expression of hsp70 [Table 1]. Similarly, the exposure of third instar larvae to 0.100 μl/ml of CP along with 0.25, 0.50 and 1.0 μl/ml of E. citriodora extract for 24 hrs results in reduction of the expression of hsp70 [Table 1]. Similar results were obtained for 48 hrs of treatment of CP along with various doses of E. citriodora extract [Table 1]. Regression analysis was also performed to study the dose effects of E. citriodora on hsp70 expression induced by 0.025 and 0.100 μl/ml of CP for the exposure to different durations [Table 2]. The exposure of third instar larvae for 24 hrs to 0.025 μl/ml of CP along with 0.25, 0.50 and 1.0 μl/ml of E. citriodora extract was associated with the β-coefficient of -0.9960 (F=123.93; P<0.0078) [Figure 1]; [Table 2]. For 48 hrs the β-coefficient value was -0.999 (F=4173.87; P<0.00065) [Figure 2]; [Table 2]. The exposure of third instar larvae for 24 hrs to 0.100 μl/ml of CP along with 0.25, 0.50 and 1.0 μl/ml of E. citriodora extract was associated with β-coefficient value of -0.999 (F=1526.25; P<0.0020) [Figure 3]; [Table 2] and for 48 hrs the β-coefficient value was -0.9040 (F=4.47; P<0.032) [Figure 4]; [Table 2].

Figure 1: Effect of Eucalyptus citriodora extract on the third instar larvae exposed 0.025 μl/ml of cyclophosphamide (CP) for 24 hrs of duration (the dose of extract is in μl/ml)

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Figure 2: Effect of Eucalyptus citriodora extract on the third instar larvae exposed 0.025 μl/ml of cyclophosphamide (CP) for 48 hrs of duration (the dose of extract is in μl/ml)

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Figure 3: Effect of Eucalyptus citriodora extract on the third instar larvae exposed 0.100 μl/ml of cyclophosphamide (CP) for 24 hrs of duration (the dose of extract is in μl/ml)

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Figure 4: Effect of Eucalyptus citriodora extract on the third instar larvae exposed 0.100 μl/ml of cyclophosphamide (CP) for 48 hrs of duration (the dose of extract is in μl/ml)

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Table 1: β-galactosidase activity measured in the transgenic Drosophila melanogaster (hsp70-lacZ)Bg9 third larvae exposed to different concentrations of Eucalyptus citriodora leaf extract and cyclophosphamide

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Table 2: Regression analysis for the dose effect of Eucalyptus citriodora leaf extract on the larvae exposed to different doses of cyclophosphamide for 24 and 48 hrs of duration

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Discussion

The results of the present study reveal that the leaf extracts of the E. citriodora reduced the toxic effects induced by CP. The CP is converted into 4-hydroxycyclophosphamide by mixed function of oxidase enzymes in liver. Hydroxycyclophosphamide exists in equilibrium with its tautomer, aldophosphoramide. The major portion of the aldophosphoramide is oxidized by the enzyme aldehyde dehydrogenase (ALDH) to carboxyphosphoramide, but the minor portion is converted into phosphoramide mustard and acrolein. ^[12] Phosphoramide mustard has been reported for the toxicity of CP forming DNA cross links. ^[13] In our earlier study the tissue damage was observed in the midgut tissues of the third instar larvae because the midgut tissues have the high concentration of cytochrome species and microsomal oxidase activity. ^[7],[14] Therefore, the metabolic activation of the CP takes place in the midgut tissues. The extract of E. citriodora has compounds that are potent enough to reduce the toxic effects of CP. The study of the toxic effects of anti-cancerous drugs is of special significance because of the possibility that they may induce secondary tumors. It has been well established that the secondary cancers are complications of traditional treatments with chemotherapy. ^[4] For these reasons, utilization of anti-carcinogenic nutrients could play a vital role in protecting against chemotherapeutic drugs. ^[15] Medicinal plants and their products have been used for centuries to cure various ailments. ^[5] E. citriodora or lemon-scented eucalyptus is a good source of eucalyptus oil. ^[16] The oil is regarded as non-toxic and has been reported to have pharmaceutical properties. ^[17] The main component of extract is citronella. However, it also consists of cineole, citranellol, limonene, linalool and α-terpinene. ^[18] It has been reported that various components of the eucalyptus oil act synergistically to show the overall activity. ^[19] A wide range of the leaf extracts was studied on third instar larvae of Drosophila melanogaster (hsp70-lacZ)Bg⁹ . The dose of 1 μl/ml was found to be non-cytotoxic. ^[7] For the activity of any plant extract, it has been suggested that the property of any plant extract should be studied as a whole because since antiquities various plants extract were/are being used to cure various ailments as a whole in Unani and Ayurveda. ^[20] According to the National Toxicological Programme guideline's development and validation of alternative models are necessary to obtain reliable and sensitive results. ^[21] Drosophila model in toxicological evaluation is time and cost-effective in comparison to rodents has been used to study the effects of various natural plant's products/extract. ^[22]

Conclusion

The results of the present study reveal that the leaf extracts of E. citriodora is potent in reducing the toxic effects of CP, in third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg⁹ . The hsp70 expression in response to various compounds is a potential indicator of non-target toxicity. The present study also supports the use of hsp70 expression to study the protective effect of natural plant products/extracts.

Acknowledgment

We express our sincere regards and gratitude to the Chairman, Department of Zoology, AMU for providing laboratory facilities. We are also grateful to Dr. D. Kar Chowdhuri, Scientist F and Head Embryo Toxicology, IITR, Lucknow, U.P., India for providing Bg⁹ Drosophila strain.

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