Diabetes mellitus is a group of syndrome characterized by hyperglycaemia, altered metabolism of lipids, carbohydrates and proteins, and an increased risk of complications from vascular diseases. Diabetic patients generally experience sexual abnormalities such as sexual dysfunction, impotence and infertility. Low testosterone levels, testicular dysfunction and spermatogenic disruption in the testis have been observed in diabetic men and experimental animals, which lead to erectile dysfunction and a reduced sperm motility, sperm count and semen volume. Therefore, diabetes has a significant impact both directly and indirectly on the fertility of men with this disease.[3, 4] Enhanced oxidative stress (OS) and changes in antioxidant capacity are considered to play an important role in the pathogenesis of chronic diabetes mellitus. Reactive oxygen species (ROS) is considered as a strong stimulus for the release of cytokines. Recent studies have been showed that the over-expression of pro-inflammatory cytokines such as transforming growth factor-β1 (TGF-β1), interleukin-1β (IL-1β) and serine/threonine protein kinase (Akt) play a crucial role in the testis during pubertal maturation, in regulation of steroidogenesis, spermatogenesis and functional development of male secondary reproductive organs in men and animals with diabetes. These inflammatory factors not only raise diabetes-associated testicular inflammatory stress but also disturb systemic immune functions. Antioxidants may also be useful in the treatment of male infertility.
Current studies have also been indicated that diabetes-mediated OS can induce apoptosis of germ cells. Apoptosis, known as programmed cell death, is a form of cell death that serves to eliminate dying cells in proliferating or differentiating cell populations. Apoptosis control is critical for normal spermatogenesis in the adult testes. Previous findings suggested an increased apoptosis in the seminiferous tubule of streptozotocin (STZ)-induced diabetic mice and rats.[11, 12] Furthermore, there was an evidence that over-expression of wild type or activated Akt can rescue cells from apoptosis induced by various stress signals.
Ferulic acid (4-hydroxy-3-methoxy cinnamic acid) is a ubiquitous phenolic compound of plant tissues and thus constitutes a bioactive ingredient of many foods. Ferulic acid is rich in rice bran, whole grain foods, citrus fruits, banana, beet root, cabbage, spinach and broccoli. Ferulic acid exhibited antioxidant, antihypertensive, antihyperlipidemic, antimicrobial, anticarcinogenic, antidiabetic, anti-inflammatory, hepatoprotective, neuroprotective and radioprotective properties and also demonstrated therapeutic potential as a hormetin for age-related diseases.[14, 15]
Prompted by the evidence, our recent study was designed to investigate the effect of ferulic acid on post-diabetes testicular damage involving the sperm parameters, serum biochemical estimation, antioxidant defence systems, histological architecture changes and cell damage biomarkers correlates with apoptotic events.
MATERIALS AND METHODS
Male albino Wistar rats (125 ± 25 g) were procured from the Indian Institute of Chemical Biology, Kolkata, India, and were used for the experiment. Rats were fed a standard rat chow and tap water ad libitum. In the windowless animal quarter, automatic temperature (22 ± 2 °C) and lighting controls (light on at 7:00 h and off at 21:00 h, 14 h light/10 h dark cycle) was performed. All experimental procedures were conducted in conformity with Institutional Animal Ethics Committee CPCSEA (CPCSE Reg. No.1458/PO/a/11/CPCSE) for the care and use of animals and were strictly followed throughout the study.
Rats after acclimatization were randomly divided into four groups with ten animals each: Group I – normal control (NC), Group II – diabetic control (DC), Group III – ferulic acid (50 mg kg−1 alternative day)-treated diabetic rats (DF1), Group IV – ferulic acid (50 mg kg−1 daily)-treated diabetic rats (DF2).
The doses of ferulic acid were selected on the basis of previously reported protective and antioxidant properties of this compound in rats. These doses have been found to suppress STZ-induced Type I diabetes in rodent model. Ferulic acid was dissolved in water and administered orally to all the treated groups.
Diabetes was induced by a single intra-peritoneal injection of STZ (50 mg kg−1, freshly dissolved in 5 mmol l−1 citrate buffer and pH 4·5). STZ-injected animals were given 5% glucose for 24 h to prevent initial STZ-induced hypoglycaemic mortality. A period of 2 days after STZ treatment, development of diabetes was confirmed by measuring blood glucose levels in blood samples. Rats with blood glucose levels of 250 mg dl−1 or higher were considered to be diabetic and used for the experiment. Experiment was conducted up to 10 weeks. After 10 weeks of the experiment, blood samples were collected for biochemical estimations, rats were then sacrificed and testes were isolated for antioxidant status, histopathological, immunohistochemical and apoptotic studies.
Ferulic acid, STZ, 3,3′-diaminobenzidine tetrahydrochloride (DAB), proteinase K and bovine serum albumin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). In situ apoptosis kit was purchased from Takara Inc., Ltd (Japan), and rat anti-mouse IL-1β, TGF-β1 and Akt antibodies were purchased from Biolegend Pvt., Ltd (USA). Horseradish peroxidase-conjugated secondary goat anti-rabbit IgG was purchased from Bio-Genei (Bangalore, India). Double-antibody RIA kit was purchased from Immunotech, Beckman Coulter Co. (Los Angeles, CA, USA), and Insulin RIA kits were obtained from Immunotech (France). All other chemicals and reagents used were of analytical grade and purchased in purest form, available from local firms.
Assay of glucose and insulin
Plasma level of glucose was assayed using commercially available kits provided by Autospan (Mumbai, India). Serum insulin level was measured by immunoradiometric (RIA) assay by using kits provided by Immunotech.
Serum testosterone analysis
Blood samples were collected, and the sera were separated and kept at −20 °C until testosterone concentrations were measured by means of radioimmunoassay technique. Serum concentrations of total testosterone were measured by using a double-antibody RIA kit. The assay sensitivity per tube was 0·025 ng ml−1.
Epididymal spermatozoa were collected by cutting the cauda region of the epididymis into small pieces in 2 ml of normal saline pre-warmed to 37 °C. Sperm was forced out of the cauda epididymis with fine forceps by putting pressure on the lower region of the cauda epididymis, not forcing out excess material, i.e. immature cells. In this study, sperm motility, count and viability were evaluated by using conventional methods. Progressive sperm motility was performed immediately after the collection of sperms. The number of motile spermatozoa was calculated per unit area and expressed as sperm motility percentage. Sperm counts were carried out using a hemocytometer, and the results were expressed as millions per millilitre of suspension. Sperm viability was performed using eosin and nigrosin stain. The dead sperm took up the stain. A hundred sperm cells were counted to obtain the percentage of live/death ratio.
Determination of antioxidant enzymes in testis tissues
The lipid peroxidations in tissue homogenate of control and all treated groups of animals were measured by the quantification of thiobarbituric acid-reactive substances. Super-oxide dismutase (SOD) and catalase activity were estimated by the process described by Kakkar. Reduced glutathione (GSH) was determined by the method of Balaraman.
A period of 10 weeks after the experiment, animals from each group were randomly selected. A portion of the testis is excised from ether-anesthetized rat fixed in 10% formalin and processed for histological studies. Tissues are dehydrated through 70%, 90% and 100% alcohol and embedded in low-melting-point paraffin wax. Sections of 5 µm thickness were cut and placed serially on glass slides. The sections were deparaffinized in xylene and rehydrated through 100%, 90% and 70% alcohol. Three continuous sections were made from each testis tissue and stained with hematoxylin and eosin for histological evaluation using light microscopy.
For the immunostaining of TGF-β1, IL-1β and Akt, immunohistochemical detection of TGF-β1, IL-1β and Akt protein in cold acetone-fixed paraffin-embedded testis section was performed by the avidin–biotin–peroxidase complex method. The 5 µm thin sections of lysin-coated slides were deparaffinized and rehydrated through 100%, 90% and 70% alcohol. For the immunolabelling of TGF-β1, IL-1β and Akt, antigen retrievals were facilitated by heating the sections in citrate buffer pH 6·0, for 20 min. Endogenous peroxidase activity was blocked with 1% H2O2 in 0·1 M Tris–NaCl (pH 7·6), for 30 min. After incubation in 5% normal goat serum, sections were then separately incubated overnight at 4 °C with the primary rat anti-mouse TGF-β1, IL-1β and Akt antibody. Sections were then incubated with a secondary goat anti-rabbit IgG for 30 min at 37 °C with 1:100 dilution. This was followed by incubation with H2O2 and methanol (1:4) for 1 h. After incubation, 100–400 µl of DAB reagent was added to each section; as soon as sections turn brown, the slides were immersed in double-distilled water (ddH2O). Then, slides were counter-stained with Harris hematoxylin for 2 min. The tissue sections were washed in ddH2O for 5 min each. The sections were dehydrated and mounted with distyrene plasticizer xylene and served as a positive control. The percentage of immunopositive cells was counted under a light microscope.
Apoptosis by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end-labelling (TUNEL) assay
Apoptotic cells in testis tissue sections were determined by TUNEL assay as described by Guneli and his co-workers with slight modification. The sections were digested with proteinase K (20 µg ml−1) and were kept for 15 min at room temperature. Slides were then quenched by 2% hydrogen peroxide for 5 min at room temperature and incubated with terminal deoxynucleotidyl transferase (TdT) buffer followed by TdT reaction solution containing TdT and deoxyuridine triphosphate for 90 min at 37 °C. Then, the sections were washed with 2% standard saline citrate for 10 min at room temperature. Further, the slides were washed with phosphate buffer saline for 5 min and incubated with anti-digoxigenin peroxidase for 30 min at room temperature. Colour was developed using 0·05% DAB in 0·01% H2O2 diluted with Tris–HCl (pH 7·5), then lightly counterstained with 3% methyl green. Sections were then washed, dehydrated and mounted. Apoptotic cells were identified by chromogen-generated dark-brown stain over the nuclei.
Apoptotic index (AI) = (number of labelled cells/total number of cells counted) × 100.
The results were expressed as mean value ± standard error mean. Statistical comparisons were carried out by analysis of variance (ANOVA) followed by post hoc Tukey's multiple-comparison test. Differences are statistically significant when P < 0·05 and P < 0·01.
Testis weight and body weight
The body weight and testis weight of the rats are presented in Table 1. The final body weight and testis weight of diabetic rats were significantly lower than those of NC rats. Both these weights were significantly improved by the administration of ferulic acid compared with those of the diabetic rats. The effect was much significant with daily dose of ferulic acid treatment as compared with the effect with alternative dose (P < 0·01).
Table 1. Effects of ferulic acid on the body weight, testis weight and biochemical parameters (blood glucose, plasma insulin and serum testosterone levels)
Normal control (NC)
Diabetic control (DC)
Ferulic acid 50 mg kg−1 alternative day (DF1)
Ferulic acid 50 mg kg−1 daily (DF2)
Results are analysed by one-way analysis of variance (ANOVA) confirmed by Tukey's post hoc multiple-comparison test.
Values are presented mean ± standard error of the mean of ten animals for each experimental group.
P < 0·05, significant differences compared with diabetic control rats.
P < 0·01, significant differences compared with DF1 rats.
Blood glucose levels in STZ-induced Type I diabetic rats were statistically significantly higher compared with those of the NC rats, as shown in (Table 1). Treatment with ferulic acid significantly lowered the blood glucose level as compared with the treatment with DC. Treatment with ferulic acid daily (50 mg kg−1) showed a pronounced effect (P < 0·01) in maintaining the blood glucose level as compared with the treatment with alternative dose (50 mg kg−1).
Serum insulin and testosterone levels
The serum insulin and testosterone levels of DC rats were significantly lower than the levels in the normal rats (Table 1). Conversely, diabetic rats treated with ferulic acid showed a marked increase in the serum insulin and testosterone levels in comparison with the DC rats. The effect was much pronounced with ferulic acid daily treatment group in comparison with the effect with alternative dose (P < 0·01).
The STZ-induced diabetic group showed a significantly decreased sperm count, motility and viability as compared with the NC groups. Values of sperm count in 106 ml−1, motility percentage and vitality percentage were given in Figure 1. The administrations of ferulic acid 50 mg kg−1 alternative day and daily for 10 weeks significantly increased sperm motility, viability and count in the diabetic treatment group. Interestingly, ferulic acid daily treatment has shown better efficacy (P < 0·01) than alternative day treatment.
The administrations of daily and alternative doses of ferulic acid to diabetic rats resulted in a significant decrease of thiobarbituric acid-reactive substances to near normal levels, which indicates improved oxidative status in testis of diabetic rats. The effect was more pronounced (P < 0·01) with ferulic acid daily-dose treatment (Figure 2).
Antioxidant enzyme activity
Figure 2 represents the levels of SOD, catalase and GSH in testis of control and experimental rats. A significant decrease in the levels of SOD, catalase and GSH were observed in diabetic testis. Treatments with daily and alternative doses of ferulic acid significantly altered the antioxidant status. The effects were more significant (P < 0·01) with daily dose of ferulic acid treatment as compared with alternative dose.
Normal control rats showed the presence of normal testicular architecture and regular seminiferous tubular morphology with normal spermatogenesis and complete seminiferous tubule cell series (Figure 3a). Diabetic rat testis showed alteration of cellular architecture, reduction of seminiferous tubules and complete destruction of spermatogenic cells characterized by loss of embryonic cell and germ cell detachment (Figure 3b). Ferulic acid-treated rats (alternative day-treated and daily-treated rats) have shown great degree of improvement in testicular histology with an increase in the number of spermatogenic cells. Diabetic rats treated with ferulic acid alternative day (Figure 3c) showed minimal improvement in the testicular architecture, whereas the histological structure of the testes of diabetic animals treated with ferulic acid daily dose (Figure 3d) resembled to that of the normal rats.
Effect of ferulic acid on expression of TGF-β1 and IL-1β
The expression of TGF-β1 and IL-1β was analysed by immunohistochemistry (Figure 4); the percentage of TGF-β1 and IL-1β immunopositive cells in testis tissues of various groups of experimental rats is shown in Figure 5. A large amount of TGF-β1 and IL-1β immunopositive cells and were detected in the testis tissue sections of the STZ-induced DC rats (Group II) (Figures 4a and 4d). In contrast, a decrement of TGF-β1 and IL-1β immunopositivity (P < 0·01) was observed upon ferulic acid 50 mg kg−1 alternative day-treated rats (Group III) compared with that upon DC (Figures 4b and 4e). Furthermore, a higher decrement of TGF-β1 and IL-1β immunopositivity was marked (P < 0·01) in ferulic acid 50 mg kg−1 daily-treated rats (Group IV) compared with that in DC group (Figures 4c and 4f). Daily dose treatment showed more significant effect in comparison with that of alternative dose (P < 0·01).
Effect of ferulic acid on expression of Akt
The expression of Akt in testis tissue was examined by immunohistochemistry. Figure 4g shows limited expression of Akt immunopositivity. Ferulic acid 50 mg kg−1 alternative day-treated diabetic rats showed a moderate increase in the percentage of Akt immunopositivity as compared with DC rats (P < 0·01, Figure 4h), whereas ferulic acid 50 mg kg−1 daily-treated diabetic rats showed a profound expression of Akt immunopositivity (P < 0·01), as compared with DC rats (Figure 4i). Daily dose treatment showed more significant effect in comparison with alternative dose treatment (P < 0·01). The percentages of Akt immunopositive cells in testis tissues of various groups of experimental rats are shown in Figure 5.
Apoptosis by TUNEL assay
The rate of TUNEL positive cells was generally very low in control group (Figure 6a). In DC group, an average of 20–23 apoptotic cells were found in a field of about 200 cells (Figure 6b), this number decreased to an average of 11–13 stained cells in the alternative day treatment group (Figure 6c) and 5–6 stained cells in the daily dose treatment group (Figure 6d). The percentage of TUNEL-positive apoptotic cells was denoted as AI shown in Figure 5. Quantitative analysis revealed a marked increase in apoptotic cells in testis of diabetic rats, whereas the effect was completely reversed by alternative day and daily doses of ferulic acid treatment (P < 0·01). The AI value of ferulic acid daily-treated group was significantly lower (P < 0·01) as compared with that of the alternative day-treated group.
Almost 25 centuries ago, Hippocrates, the father of medicine, proclaimed: ‘Let food be thy medicine and medicine be thy food.’ Exploring the association between diet and health continues today. Diabetes mellitus is a chronic disease affecting many tissues and systems of the body. Some of these manifestations were spermatogenic and steroidogenic alterations and affect the male reproductive function. The present investigation demonstrated that the protective potential of ferulic acid against hyperglycaemia-mediated OS in STZ-induced diabetic testicular damage. STZ-induced diabetes in rodents appears to be the most suitable animal model because it reflects the symptoms of diabetes in human.
Streptozotocin-induced diabetes is characterized by severe loss in body weight, and this is also reflected in the present study. The decreases in body and testis weight in diabetic rats showed the loss or degradation of structural proteins due to diabetes. When diabetic rats were treated with ferulic acid, weight loss was reversed. The capability of ferulic acid to protect body weight loss seems to be a result of its ability to reduce hyperglycaemia.
The overall effect of STZ on β-cells leads to the development of insufficient production of insulin and, consequently, the elevation of blood glucose levels. Several investigations have been reported that ferulic acid in STZ-induced diabetic rats alleviates some signs of diabetes, the most marked one being a normalization of blood glucose level. In our study, the mean blood glucose level of STZ-induced untreated diabetic rats was significantly increased above the normal level, and this elevation in blood glucose levels was approximately constant through the course of diabetes, whereas in ferulic acid-treated diabetic rats, the blood glucose level was markedly reduced and reached near normal level at the end of the study.
Akt is a serine/threonine protein kinase that plays an important role in signalling pathways that regulate cell growth, proliferation and differentiation. A previous report has demonstrated that Akt is an important mediator of biological functions of insulin. One of the major effects of this hormone is the enhancement of glucose uptake in muscle, adipocytes, liver and other tissues. Therefore, it is not surprising that Akt signalling has major impact on glucose metabolism. Earlier studies recognized that PI3K is responsible, at least in part, for insulin stimulation of GLUT4, the major insulin-regulated glucose transporter, from intracellular vesicles to the plasma membrane in insulin-sensitive cells. Therefore, the role of Akt in this process has also been evaluated. Prior studies had shown that there was a significant decrease in the serum insulin level in diabetic rats. Similarly, in the present study, the serum insulin level of diabetic rats was significantly reduced below the normal level. However, the serum insulin level was reversed by the administration of ferulic acid when compared with that of the diabetic group.
Previous findings suggested that there was a significant decrease in the total serum testosterone level in diabetic rats. However, the testosterone level was inverted by the administration of ferulic acid when compared with that of the diabetic group. Thus, our results indicate that ferulic acid recovers testosteronaemia in diabetic rats.
Earlier works had reported that diabetes mellitus had deleterious effects on the male reproductive system and may affect endocrine function and spermatogenesis.[26, 34] Similarly, in the present study, also there was significant reduction in the sperm parameters of the diabetic rats, which was normalized by the treatment of ferulic acid.
In diabetes mellitus, hyperglycaemia increases oxidative stress (i.e. ROS), and it causes DNA damage in all tissues such as retina, renal, brain, myocardium and testis. Therefore, increased lipid peroxidation causes DNA damage in sperm cell.[2, 35] There are certain enzymes, which play an important role in antioxidant defence, to maintain viable reproductive ability, a protective mechanism against OS. These enzymes include SOD, GSH and catalase, which convert free radicals or reactive oxygen intermediates to non-radical products. SOD and GSH are major enzymes that scavenge harmful ROS in male reproductive organs. In the present study, it was observed that there was an increased rate of lipid peroxidation as well as decreased levels of SOD, catalase and GSH activities in diabetic testis, which may represent increased level of OS. The restoration of SOD, catalase, GSH level and lipid peroxidation by the treatment of ferulic acid in the diabetic rats not only signifies its antioxidant property but also protects the rats from OS-induced testicular damage.
Testicular dysfunction in the diabetic group was also revealed in histological examination by the atrophy of the seminiferous tubules, decreased tubule diameters and reduction of the spermatogenic cell series. Seminiferous tubule atrophy and the decrease in spermatogenic cells were morphological indicators of spermatogenesis failure.[12, 37] Cameron et al. defined the increasing tubule wall thickness, germ cell depletion and Sertoli cell vacuolization in diabetic human testicular biopsies and in diabetic rats. Histological findings clearly showed that the normal architecture of the testis tissue was altered because of the administration of STZ, and it released ROS, causing degeneration in seminiferous tubules lined by several spermatogenic cell series, Sertoli cells and Leydig cells. In contrast, rats treated with ferulic acid showed noticeable improvement in histopathological parameters. There were no such studies available on the protective nature of ferulic acid on the testis tissue during experimental diabetes. Hence, this investigation should be considered an innovative assessment for the testicular tissue protective nature of ferulic acid in rats insulted by STZ.
Understanding the mechanisms and signalling pathways underlying diabetes-induced male germ cell apoptosis is essential to the development of a strategy to prevent the loss of spermatogenic cells for diabetic patients. It has previously been shown that diabetes mellitus increases OS in the diabetic male testis. OS is recognized as a strong mediator of apoptosis, and mitochondria play an important role in the apoptotic process. The seminiferous tubules were the sites of spermatogenesis where germ cells develop into spermatozoa in close interaction with Sertoli cells. The Sertoli cell was an important testicular somatic cell, which controls the germ cell environment by the secretion and transport of nutrients and regulatory factors. The mitochondrial dysfunction induced by OS can lead to the release of cytochrome c and then caspase activation, which results in apoptotic cell death. Previous findings suggested that diabetes mellitus induces testicular dysfunction by causing apoptotic cell death.[12, 38] We therefore investigated the active germ cell apoptosis by TUNEL assay in the testis tissue. In the present study, it was seen that STZ-induced diabetic rats showed an increase in the number of TUNEL positive cells than the normal and administration of ferulic acid significantly decreased the number of TUNEL positive cells in the diabetic treated group along with the over-expression of Akt. Our recent study is also in agreement with the previous findings regarding the prevention of apoptosis during growth factor withdrawal and paradigms with OS.
The results so obtained indicate the protective effect of ferulic acid on testicular damage. Normal spermatogenesis requires signalling pathways to be able to regulate a precise balance among cell survival, proliferation and differentiation. The regulation of spermatogenesis involves endocrine, paracrine and autocrine factors that form a complex signalling network.[10, 42] The detailed molecular pathways that define the interactions between germ and Sertoli cells in the testis, however, remain largely unknown. The Sertoli cell is responsible for the overall control of testis development. The interstitial space around the seminiferous tubules contains another somatic cell type, the Leydig cell, which is responsible for testosterone production. TGF-β1 was expressed by the Sertoli cell and may be important for spermatogenesis. Sertoli cell production of TGF-β1 may be targeted to the germinal cell population in part because of the blood–testis barrier. IL-1β was produced in the testis by Sertoli and Leydig cells. These interleukins can regulate Sertoli, Leydig and germ cell growth and differentiation functions. A previous report has suggested that STZ-induced diabetes enhanced the expression of TGF-β1 and IL-1β in the testis and changes in testicular function. In the present investigation, we demonstrated that injection of STZ induces rapid and transient alterations in the levels of pro-inflammatory cytokines in the rat testis. In our recent findings, over-expression of TGF-β1 and IL-1β in STZ control rats was markedly reduced by ferulic acid. On the other hand, STZ-challenged diabetic group showed significantly decreased Akt protein expression in seminiferous tubules in rat testis. The administration of ferulic acid elevated the Akt protein expression in diabetic treated rats.
The cytoprotective and antioxidant roles of ferulic acid have been demonstrated in many experimental systems. In particular, ferulic acid displayed antioxidant and antiapoptotic activities and enhanced cell stress response in the organ of Corti of guinea pigs exposed to noise. In addition with this, Calabrese and his co-workers successfully demonstrated the antioxidant and cytoprotective effects of ferulic acid ethyl ester on human dermal fibroblasts. Our data on the protective effect of ferulic acid on diabetes-induced testicular oxidative damage confirm the paradigm and highlight the potential therapeutic use of this phenolic compound in the prevention of OS-mediated testicular damage.
In conclusion, the present study provides evidence that ferulic acid reduced blood glucose, biochemical parameters, OS, pro-inflammatory cytokines, apoptosis and upregulated serum testosterone, plasma insulin and Akt proteins in diabetes, which may contribute to its protective effects on post-diabetes complications such as testicular damage. These findings suggest that ferulic acid may serve as one useful new therapeutic agent in the testicular damage.
CONFLICT OF INTEREST
The authors declare that there is no potential conflict of interest.
The authors are indebted to the Hari Charan Garg charitable trust for the financial support to carry this work. The authors would like to thank Mr. Jayanta Bhowmick for his assistance in preparing and evaluating histopathological slides and highly acknowledged Mr. Subhash Kumar Manna for technical support and Mr. Lal Mohan Masanta for providing laboratory support.