Bee Pollen attenuates Rifampicin and Isoniazid in Combination induced Oxidative Stress in Testis of SD Rats

 

Umesh Bharti1, Neelima R. Kumar2, Jaspreet Kaur3

1Department of Zoology, PG Govt. College for Girls, Sector-11, Chandigarh

2Department of Zoology, Panjab University, Chandigarh

3Department of Biotechnology, UIET, Panjab University, Chandigarh

*Corresponding Author E-mail: dbhv90@gmail.com

 

ABSTRACT:

Objective: Analysis of shielding potential of bee pollen in rifampicin and isoniazid drugs in combination induced toxicity in testis of SD rats.

Materials and Methods: SD rats (180±20 g body weight) were divided into four groups, i.e. group C (Control), group O (Bee pollen 100mg/kg body weight/day orally), group D12 (Rifampicin 100mg/kg body weight/day and isoniazid 50mg/kg body weight/day orally), group D12O (rifampicin 100mg/kg body weight, isoniazid 50 mg/kg body/day and bee pollen 100mg/kg body orally) under hygienic conditions.

Results: Aqueous extract of bee pollen (100 mg/kg body weight) when fed orally showed normal level of MDA, SOD, GR, GPx, GST, CAT and GSH representing no harmful effect caused by bee pollen in the rats. The anti-tuberculosis drugs (rifampicin and isoniazid in combination) and bee pollen treated groups showed significant decrease in the level of lipid peroxidation while the activity of SOD, GR, GPx, GST, CAT and GSH were elevated representing reduced oxidative stress in the drug treated with bee pollen groups along with near normal histological alterations in testis of rat when compared with drug treated rats.

Conclusion: Bee pollen has shown the protective effect against damage and oxidative stress induced by anti-tuberculosis drugs (rifampicin and isoniazid in combination) in rat testis.

 

KEYWORDS: Bee pollen; testis; oxidative stress; rifampicin; isoniazid.

 

 


INTRODUCTION:

Bee pollen exhibit tremendous nutritional and biological activities including antioxidant, antimicrobial, anti-inflammatory and anti-carcinogenic due to the presence amino acids, proteins, hormones, enzymes, minerals, vitamins, flavonoids, phenolic acids, anthocyanins, tannins and phytochemicals1,2,3,4.Bee pollen has been used for several centuries to cure burns, wounds and gastrointestinal disorders5,6. The aim of the present study was to investigate the modulatory effects of bee pollen against rifampicin and isoniazid in combination drug induced testis damage. The study was conducted in SD rats as four groups. 

 

First group (C) was kept as control and was fed with saline water. To the second group (O) aqueous extract of bee pollen (100mg/kg/day body weight) were given orally and third group (D12) was administered with rifampicin (100mg/kg/day body weight) and isoniazid (50 mg/kg/day body weight) in combination for thirty days. The fourth group (D12O) was fed with bee pollen extract along with anti-tuberculosis drugs (rifampicin and isoniazid in combination). The protective effect of the bee pollen was monitored by his to pathological imaging, and antioxidant parameters (CAT, GR, LPO, GST, GSH, SOD and GPx) from testis samples of the rats. Rifampicin and isoniazid drug induced testis damage and antioxidant stress markers were significantly normalise near control with the bee pollen extract treated groups. The results demonstrated that the bee pollen protects the testis from the oxidative stress and promotes the healing of the damage to testis induced by rifampicin and isoniazid in combination induced toxicity.

MATERIAL AND METHODS:

Drugs:

Rifampicin and Isoniazid drugs (Himedia)

 

Bee Pollen:

Pollen samples were obtained from the apiary near old Panchkula, Haryana, India.

 

Experimental Animals:

Male SD rats (180±20 gm body weight) were obtained from the central animal house of Panjab University, Chandigarh vide letter no PU/IAEC/2013/18.All the principle laid down by the Institutional Animal Ethical Committee (IAEC), Panjab University were strictly followed in all the experiment.

 

Experimental Protocol:

The rats were divided into four groups randomly (n=6) Group C received only the normal pallet diet, group O was fed with bee pollen (100mg/kg body weight) along with normal pallet diet, group D12 received rifampicin (100mg/kg body weight/day)and isoniazid (50mg/kg body weight/day) along with normal pallet diet and group D12O received rifampicin (100mg/kg body weight/day), isoniazid (50mg/kg body weight/day) and bee pollen (100mg/kg body weight/day) along with normal pallet diet.

 

These treatments were given daily for 30 days in normal hygienic conditions. After 30 days, rats were sacrificed under light anesthesia and cervical dislocation. Testes were removed and weighed for preparing tissue homogenate. Testis samples were washed with ice-cold saline and 10% testis homogenates were prepared in ice cold phosphate buffer saline (pH 7.4) using homogenizer for 2 mins at 3000 rpm in ice. The homogenate were centrifuged at 10,000 g for 20 min at 4°C for preparing post mitochondrial supernatant.

 

Biochemical Analysis:

The protocol used for determining catalase (CAT) activity was by Luck7, glutathione peroxidase (GSH-Px) by Pagila and Valentine8, glutathione-S-transferase (GST) activity by Habiget al.9, GR activity by Carlberg and Mannervick10, lipid peroxidation product by Begue and Aust11, reduced glutathione (GSH) by Sedlak and Lindsay12and superoxide dismutase (SOD) activity was by Kono13.

 

Histopathologic Analysis:

Testis tissues from the rats were fixed in 10% formalin solution overnight. After fixation, the sections were subjected to routine histologic tissue preparation and dehydrated with ethanol series (30%, 50%, 70%, 90% and 100%), cleaned with xylene and embedded in paraffin wax. Paraffin blocks were sliced to 0.5 mm thickness with a microtome and after hematoxylin and eosin (H&E) staining they were analysed under a light microscope

 

Statistical Analysis:

All data were expressed as the mean± standard deviation. Data were analyzed by one-way analysis of variance and means were compared. A probability value of P ≤0.05 was considered statistically significant.

 

RESULTS AND DISCUSSION:

Antioxidant Parameters:

Malondialdehyde (MDA), a lipid peroxidation product, was measured in PMS of testis of the treated animals to analyze the damage caused by oxidative stress induced via rifampicin and isoniazid in combination treatment. The increased amount of MDA in testis tissue serves as an indicator of testis injury due to generation of reactive species. Our results clearly showed that bee pollen extract are significantly effective in the prevention of lipid peroxidation (p≤0.001), and hence oxidative damage, which was induced by rifampicin and isoniazid in combination (Plate1.Fig.1).

 

GSH level was estimated from tissue homogenate and the level of GSH in control group was 1.774±0.090 with no significant difference from pollen treated group (1.713±0.081).The level of GSH was significantly reduced to 50.3% in rifampicin and isoniazid in combination treated group whereas in drugs plus bee pollen treated group GSH level was found to be 1.360±0.073a1b1with 54. 37% increase with respect to drugs treated group (Plate1.Fig.2).GSH is also an essential component of defense system against oxidative stress in the body which prevents cellular damage caused by reactive oxygen species.GST activity was observed to be 0.568±0.049a1 µmoles of CDNB conjugate/min/mg protein in D12 group with a significant reduction of 53.9% when compared with the control group. On administration of bee pollen with rifampicin and isoniazid in combination the GST activity increased to 0.848±0.051a1b1µmoles of CDNB conjugate/min/mg protein (Plate1.Fig.3). When both the drugs rifampicin and isoniazid were given in combination in D12 group, GR activity was 0.101±0.008a1 µmoles of NADPH oxidised/min/mg protein with significant reduction when compared with control rats (Plate1.Fig4). CAT activity in D12O groups was recorded as 0.120±0.007a3b1 µmoles of H2O2 decomposed/min/mg protein with significant increase (p≤0.001) when compared with respective drug treated groups (Plate1.fig.5). In D12 groups, GPx activity was found to be 1.445±0.071a1µmoles of NADPH oxidized/min/mg protein which also showed significant decrease (p≤0.001) with respect to control. In groups D12O, GPx activity was found to be 1.878±0.047a2b1. Bee pollen in these three drug treated groups caused significant recovery of GPx activity when compared with respective drug treated groups, but was still significantly less than the GPx activity of control rats (Plate1.Fig.6). Superoxide dismutase (SOD) protects the organism from the harmful effects of superoxide radicals formed as a result of oxidative stress. SOD activity was decreased in the rifampicin and isoniazid in combination exposed groups. SOD activity was increased in bee pollen treated groups, possibly, owing to the healing of testiculocellular damage (Plate1.Fig.7).


 

Plate1. Various biochemical parameters of testis of male SD rats (n=6)

*       Units: LPO- nmoles of MDA/mg protein, GSH- µmoles/g protein, GST- µmoles of CDNB oxidized/min/mg protein, GR- µmoles of NADPH oxidized/min/mg protein, CAT- µmoles of H2O2 decomposed/min/mg protein, GPx µmoles of  NADPH oxidized /min/mg protein, SOD -IU/mg protein

 


Previous workers had reported different natural products like Ocimum sanctum14, cimetidine15, Silymarin16, Piper longum17 and propolis18  brought the activities of SOD, catalase, GR, GPx and MDA to near normal level when administered along with anti-tuberculosis drugs in treated animals. Present study revealed that the decreased CAT, GST, SOD, GR, GPx activities of testis  due to rifampicin and isoniazid in combination were brought to near normal values by bee pollen probably due to presence of bioactive elements like caffeic acid phenethyl ester (CAPE), myricetin, kaempherol, isoquercetin, and flavonoids which convert the reactive free radicals to inactive products. These bioactive elements of bee pollen were reported by several previous workers19,20 with antioxidant and anti-inflammatory properties.

 

 

 

 

Histological results:

This study investigated the testiculoprotective potential of aqueous extracts of bee pollen on rifampicin and isoniazid in combination induced testicle damages in rats. Examination of the testis of control rats showed the typical features of normal seminiferous tubules, spermatogenic cells, Leydig’s cells and spermatozoa (Fig. 8C). Animals receiving aqueous extracts of bee pollen showed normal histological structure of the testis (Fig. 9O). Treatment with rifampicin and isoniazid in combination resulted in disruption of the arrangement of spermatogenic layers, decreased height of germ cells,few and scattered sperms in the seminiferous tubules and interrupted intertubular connective tissue (Fig. 10D12). When compared to testis sections of animals exposed to rifampicin and isoniazid in combination, testis sections of animals treated with rifampicin and isoniazid in combination and aqueous extract of bee pollen showed less prominent histopathological alterations (Fig. 11D,12O).


Plate2. Histological micrograph of testis of sections of the untreated and treated rats

The histological micrographs support the modulatory potential of bee pollen extract against toxicity caused by rifampicin and isoniazid in combination in SD rats.

 


CONCLUSION:

Tuberculosis is infectious disease and one of the main causes of death worldwide. One fourth (2.2 million) of the global incident (9.6 million) TB cases occur in India annually21. Rifampicin and isoniazid are the first line drugs for the treatment of tuberculosis. Since the treatment with a combination of drugs has to be given for a long duration of time and the side effects associated with drugs leads to poor patient compliance. Anti-tuberculosis drugs enhance the formation of free radicals, which cause lipid peroxidation at cellular level and leads to organ damages. The aqueous extract of bee pollen contains many bioactive phenolic compounds with high antioxidant properties acts as natural supplements in reducing the damage caused during tuberculosis therapy. In the present work it was reported that the levels of the testicular antioxidants enzymes, superoxide dismutase, catalase, glutathione reductase, glutathione-s-transferase and glutathione peroxidase are greatly diminished upon rifampicin and isoniazid in combination exposure leading to enhancing the generation of ROS-like superoxide ion, hydroxyl radicals and hydrogen peroxide, oxidative stress can result in peroxidation in germ cells and oxidative damage in Leydig cells and bee pollen extract protects the testis from rifampicin and isoniazid in combination induced toxicity as indicated by the restoration of the histological structure and increase in the number of germ cell layers.

 

It is evident from the above mentioned results that the bee pollen extract wields highly beneficial biological activities in the protection of testicular cells from oxidative stress and toxicity induced by rifampicin and isoniazid in combination treatment. Therefore, we conclude that the bee pollen could be used as food additive, which will enhance the inhibition of the oxidative stress in the treatment of tuberculosis testicular pathologies.

 

CONFLICT OF INTEREST:

Authors have no conflict of interest

 

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Received on 22.09.2017         Modified on 01.11.2017

Accepted on 17.11.2017      © RJPT All right reserved

Research J. Pharm. and Tech. 2018; 11(3): 1159-1163.

DOI: 10.5958/0974-360X.2018.00216.0