Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524128EnglishN-0001November30HealthcareLOW MOLECULAR WEIGHT CHITOSAN AS A VEHICLE FOR SOLUBILIZATION AND AMORPHIZATION OF NON STEROID ANTI-INFLAMMATORY DRUG FOR A NEW GUAR GUM-BASED COLON DELIVERY FORMULATION
English0724Elkhodairy Kadria English Barakat NahlaEnglish Alanazi FarsEnglishThe solubilization and amorphization of the poorly water soluble nonsteroidal antiinflammatory drug, Etodolac (ETD) with a hydrophilic polymer, low molecular weight chitosan (CHT), have investigated. Phase solubility studies were carried out to obtain an insight on the nature of a possible interaction between ETD and CHT in
solution. Binary systems of varying drug polymer ratios were prepared using different techniques namely physical mixing, cogrinding and kneading. Drug–polymer interactions were investigated in solid stateby differential scanning calorimetry, powder X-ray diffractometry and scanning electron microscopy. The results obtained indicated loss of drug crystallinity. Dissolution rate studies revealed that the drug dissolution was improved with increasing the polymer concentration in the mixture in the following order kneading> co-grinding> physical mixing > pure drug. The prepared capsules containing the binary mixture prepared by
kneading in the drug-polymer ratio of 1:19 were coated with guar gum (a film coating material) and their dissolution rates were tested in comparison with commercial capsules present in the Egyptian market. A coating of 10 layers of guar gum prevented the release of etodolac in the acidic pH and permitted its release in colonic environment.
EnglishEtodolac, Binary mixtures, Chitosan, Film coating, Guar gum.Introduction
Oral colon-drug delivery system (CDDS) has been developed as one of the sitespecific drug delivery system. This delivery system, by means of combination of one or more controlled release mechanisms, hardly releases drug in the upper part of the gastrointestinal (GI) tract, but rapidly release drug in the colon following oral administration. The necessity and advantage of CDDS have been well recognized and reviewed [1]. In view of CDDS specifically delivering drug to the colon, a lot of benefits would be acquired in terms of improving safety and reducing toxicity when treating local or systemic chronic diseases, i.e. ulcerative colitis, Crohn?s disease, carcinoma and infections, the optimal colonic drug delivery system, should selectively deliver drug to the colon, but not to the upper GI tract (1). For this reason, the drug concentration was significantly lessened in the upper GI tract, while increased considerably in the colon, resulting in alleviated GI side effects [1]. In addition, CDDS would be advantageous when delay in absorption is desirable from a therapeutical point of view, as for the treatment of diseases that have peak symptoms in the early morning and that exhibit circadian rhythms, such as nocturnal asthma, angina and rheumatoid arthritis [2, 3]. The majority of colorectal cancers (CRCs) are thought to arise as the result of a series of molecular changes that transform normal colonic epithelial cells to adenomatous polyps and ultimately, to invasive cancers [4, 5]. The traditional non-steroidal antiinflammatory drugs (NSAIDs) and selective cyclooxygenase-2 (Cox-2) inhibitors potentially inhibited polyp development and tumour incidence [6]. Cox-2 over expression is thought to play an important role in colon carcinogenesis, as it has been found to be elevated in 40% of colonic adenomas and up to 90% of sporadic CRC [7, 8]. Its pharmacological inhibition by NSAIDs is the central event in the chemoprevention of colon cancer [6, 9-11]. A study showed that treatment with etodolac (ETD) significantly reduced the occurrence of neoplasia, suggesting that this Cox-2 inhibitor has chemopreventive activity against colitisassociated tumorigenesis [12]. Additionally, many researches emphasized the use of ETD for the treatment of prostate cancer [13]. Recent evidences indicated that Cox-2 selective inhibitors may also synergise with new chemotherapeutic agents [6]. It has been reported that ETD when combined with carboplatin, can enhance an action of the anticancer drug through the suppression of FAP-1 expression [14]. Long-term etodolac treatment effectively reduced metachronous cancer development in patients with extensive metaplastic gastritis [15]. Etodolac exhibited antitumor activity and induced E-cadherin expression in bladder cancer cells and might be useful for the clinical treatment and prevention of bladder cancer, especially in poorly differentiated bladder cancer with high COX-2 and low E-cadherin expression [16]. Therefore, many researches intended to formulate ETD as a colon drug delivery for the treatment of colorectal diseases as well as for the management of osteoarthritis [16]. Etodolac is a selective Cox-2 inhibitor, nonsteroidal anti-inflammatory drug. It is used in the management of osteoarthritis, postoperative pain, fever and inflammation. It is chemically designated as 2- (1, 8- Diethyl-4,9-dihydro-3H-pyrano[3,4-6] indol-1-yl) acetic acid. It is poorly soluble in water [17]. For poorly water soluble drugs, the rate of oral absorption is often controlled by the dissolution rate in the gastrointestinal tract. Therefore, the solubility and /or dissolution rate of a drug are key determinants of its oral bioavailability [18]. Thus, increasing the aqueous solubility and dissolution rate of ETD is of therapeutic importance. Several attempts have been made to improve the ETD dissolution properties via solid dispersion technique [19], complexation with cyclodextrin [20, 21] and selfemulsifying technique [22]. The present study was carried out to investigate the feasibility of chitosan enhancing the dissolution of ETD. Chitosan was selected because of its interesting properties, mainly oral biocompatibility, antiulcerogenic activity, direct compression property as well as its colon targeting potency [23]. Chitosan has been used to enhance the dissolution and bioavailability of a number of poorly water soluble drugs [24-27]. Low molecular weight chitosan can function as drug release enhancers for poorly water soluble drugs due to an improvement in wettability resulting from the solubility of low molecular weight chitosan in water [28]. The final goal of our work is to prepare a colon drug delivery system containing the chitosan: etodolac mixture that gave promising results using guar gum. Guar gum was selected as it is a specific colon drug delivery carrier as well as a free film former [29]. In vitro drug release studies have shown that guar gum in the form of compression coat applied over indomethacin core tablets protected the drug from being released under conditions mimicking mouth to colon transit [30] The same results were obtained when guar gum was used as a compression coat for metronidazole and 5- fluorouracil [31,32]. Based on these results, a trial was made to formulate coating solution of guar gum applicable for capsule coating. Solvent costs, environmental pollution and operator safety have driven the move from organic solvents to aqueous film coating. But the replacement of organic solvents has increased the complexity of process. So to take the advantage of both solvent and aqueous coating processes and overcome their limitations, a combined formulation was developed comprising the major portion of solvent as water and minor portion of solvent as organic solvent. This combination of solvents improves the drying rate which is the most critical parameter in aqueous film coating [33]. It has been reported that guar gum solution in a mixture of isopropanol and water in the ratio of 3:7 could be used as a film coating for tablets [33]. Capsules filled with the appropriate amount of the selected mixture of ETD/CHT were coated using guar gum solution with different coat thickness in an attempt to find out the optimized coat thickness that would inhibit ETD release in the acidic environment.
Materials and methods Materials
Etodolac (ETD) was kindly received from Pharco Pharmaceutical Company (Alexandria, Egypt). Chitosan low molecular weight (CHT) was supplied by Sigma Chem. Co. (St. Louis, USA). Guar gum was supplied by Merck Chem. Co. (Bombay, India). All other chemical were of analytical grade and used without further purification.
Methods Phase solubility studies
Aqueous solubility of ETD in presence of CHT was carried out according to the method described by Higuchi and Connors (34). An excess amount of ETD was added to 10 ml of aqueous solutions containing increasing concentration of CHT (0, 0.25, 0.5, 0.75, 1, 1.25, 1.5 and 2% w/v) in screwcapped vials. The suspensions were shaking in a thermostatically controlled water bath (GFL, type 1083, GmbH and Co, Burgweded, W. Germany) at 37 ± 0.5 C° for 48 hrs. After equilibrium has been attained (2 days), aliquots were withdrawn, filtered through 0.45 µm membrane filters, suitably diluted and analyzed for ETD using UV spectrophotometer at 279 nm. Preparation of binary systems ETD-CHT binary systems were prepared using varying drug concentration of 5, 10 and 20 % w/w equivalent to drug: polymer ratios of 1:19, 1:9 and 1:4, respectively. The binary systems were prepared using different methods. Physical mixtures (PMs) of drug and polymer were obtained by simply blending with spatula, co-ground (CG) by co-grinding of drug and CHT for 30 minutes in a ceramic mortar and kneaded mixtures were prepared by kneading the drug-CHT mixture with ethanol-water 6:1 v/v in a ceramic mortar followed by drying in oven for 48 hrs at 40 ºC. The powdered products were sieved (68-125 µm) and were used for subsequent studies. The samples were stored in desiccators till used. Drug content An amount of the prepared powders equivalent to 10 mg of ETD were weighed accurately and triturated with 10 ml of phosphate buffer pH 6.8 and finally the volume was made up to 100 ml with the buffer solution. The solution was filtered through a membrane (0.45 µm). The drug content was analyzed using the filtrate after suitable dilution at 279 nm using UV spectrophotometer (Perkin Elmer, USA). Each sample was analyzed in triplicate.
Particle Size Analysis
The size distribution of physical mixture, co-ground and kneading mixture was measured with a laser diffraction particle size analyzer (SALD-2101 Shimadzu, Japan). The particle size distribution and mean particle size diameter were automatically calculated using the software provided. The size distribution was evaluated with the span value defined as follows. Span = (D90% - D10%) /D50% Where, DN% (N= 10, 50, 90) means the volume percentage of microparticles with diameters up to DN%. The smaller span value indicates the narrower particle size distribution.
Scanning electron microscopy (SEM) The surface morphology of drug, chitosan, PM, CG and KM was examined by means of double sided adhesive tape was placed on an aluminum specimen holder upon which a small amount of powdered samples was deposited. The particles were coated with approximately 10-20 nm gold for 20 S using a sputter coater. Scans were performed at an acceleration voltage of 20 Kv (Jeol, JSM- 6360LV scanning microscope, Tokyo, Japan). The instrument was calibrated for temperature and heat flow using high purity indium and zinc standards.
Differential scanning calorimetry (DSC) DSC
thermograms of pure materials, PM, CG and KM were recorded using Shimadzu differential scanning calorimeter (TA 501 Shimadzu, Japan). Samples (2-4 mg) were placed in sealed aluminum pan was used as a reference. Scanning speed 10 oC min -1 , in the 25–200 oC temperature range. The equipment was periodically calibrated with indium.
X-ray diffractometry
The X-ray diffractograms of pure materials, PM, CG and KM were carried out using a Siemens diffractometer (Siemens D500, Germany) where Cu Kα, radiation was selected by a Ni monochromator. The scanning rate employed was 2°/min over a diffusion angle of 2θ and range of 5-60 °, operated at a voltage of 30 KV and a current of 30 mA, the scan step size was 0.018 (2θ). The analysis was carried out at room temperature under ambient conditions.
In-vitro dissolution study
ETO dissolution study was evaluated using the USP XXIV dissolution rate apparatus II (Pharmatest, Germany) at a stirring rate of 100 ± 2 rpm. Powders samples containing 100 mg of pure drug or its equivalent amount of PM, CG and KM were placed in 900 ml phosphate buffer pH 6.8 at 37± 0.5 ºC for 2 hrs. At predetermined time intervals, 5 ml samples were withdrawn and immediately replaced with an equal volume of prewarmed dissolution medium. All samples were run in triplicate, filtered through 0.45 µm membrane filter and the amount of dissolved ETO was analyzed by spectrophotometer at 272 nm. The In-vitro dissolution study ETO dissolution study was evaluated using the USP XXIV dissolution rate apparatus II (Pharmatest, Germany) at a stirring rate of 100 ± 2 rpm. Powders samples containing 100 mg of pure drug or its equivalent amount of PM, CG and KM were placed in 900 ml phosphate buffer pH 6.8 at 37± 0.5 ºC for 2 hrs. At predetermined time intervals, 5 ml samples were withdrawn and immediately replaced with an equal volume of prewarmed dissolution medium. All samples were run in triplicate, filtered through 0.45 µm membrane filter and the amount of dissolved ETO was analyzed by spectrophotometer at 272 nm. The
Preparation of coating solution
Preliminary experiments were carried out to obtain guar gum solution of appropriate characteristics of viscosity and homogeneity. Solutions of different ratios of water-isopropanol were prepared to find out the optimum ratio for the preparation of 1% w/w gum solution. . The experiment was designed to prepare ratios ranging in the order of: 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9. The weighed amount of guar gum was dissolved in the appropriate amount of water, and then the isopropanol was added dropwise till an almost clear solution was obtained. The solution was mixed thoroughly to effect homogeneity. It was found that the water: isopropanol ratio of 7:3 is the promising ratio for optimum solubility of the gum. This result was in agreement with the work done by Rane and Kale [33].
Preparation of guar-coated capsules
Capsules containing an amount of KM ( 5% w/w) equivalent to 100 mg of ETO was enteric coated with guar gum 1% w/v in mixture of water and isopropanol in the ratio of 7:3 [33]. The enteric coating solution was prepared by dissolving the calculated amount of guar gum in distilled water to prepare 1% w/v solution; Isopropyl alcohol was added drop wise to adjust the viscosity of the coating solution without any precipitation of gum. The prepared coating solution was stirred to effect homogeneity. Coating procedure by dipping method was adjusted to suit the requirements for aqueous coating [34]. For the first three coat the capsule was immersed in the coating solution for 5 seconds and the coating was dried by blowing hot air using hair dryer (drying time was 10-15 minutes), in the subsequent coating, the dipping time was increased to 10 seconds and drying time was reduced to 5-10 minutes. This manipulation in the coating process was essential to avoid the migration of water from coating solution into the capsule core. Capsules of different coat thickness (5, 8 and 10 coats) were prepared and subjected to dissolution studies.
Dissolution studies of coated capsules
The dissolution studies of the prepared coated capsules were performed as previously mentioned. The capsule dissolution study was performed for the first 2 hrs in pH 1.2 then the pH of the medium was render 6.8 by the addition of calculated amount of trisodium hydrogen phosphate. The dissolution study in pH 6.8 was carried out for further 3 hrs.
Results and discussion Phase solubility studies
In order to gain insight into the nature of a possible interaction between ETD and CHT in solution, phase solubility experiments were performed. The phase solubility profiles for the ETD- CHT systems were presented in Figure 1. The diagram showed that the aqueous solubility of the drug is increased linearly as a function of CHT concentration. The solubility curve was regarded as an AN type of phase solubility diagram. These results are consistent with the formation of weak soluble complexes between ETD and CHT [35]. The anionic nature of the drug and the strong positive charge of the polymer at pH < 6.5 [24] favored an electrostatic interactions in addition stabilizing complexation may be assumed. The negative curvature of type AN diagram was probably due to self association phenomena of CHT molecules at higher concentration [35]. The slope of the initial straight line part of the curve, demonstrated the relative affinity of the drug for the polymer [36]. Approximate 15 fold increase in drug solubility was observed in the presence of 2% w/v chitosan. The results of phase solubility study were found in accordance with the established formation of soluble complex between CHT and poorly soluble drugs [25].
Drug content of the prepared binary systems
The drug content of the prepared binary systems were found to be in the range of 99.2 ± 0.35 to 101.1 ± 0.29 % indicating that the present methods for the preparationof solid systems can be applied with high content uniformity.
Micromeritic properties
It is known that in glass, solid solutions and amorphous dispersions, the particle size is reduced to a minimum level. After carrier dissolution, the drug is molecularly dispersed in the dissolution medium resulting in an enhanced dissolution rate [37]. The micromeritic behaviors (mean particle size, span) of pure etodolac and different ETD-CHT binary mixtures are shown in Table I. The data of the table revealed that ETD has larger mean particle size than all other etodolac simples obtained via grinding or kneading technique. The smallest mean particle size was obtained for the kneaded binary mixture containing 5 % w/w of the drug. It is obvious that as the ratio of CHT decreased from 95% w/w to 80% w/w, the particle size of the drug increased from 81.50 to 150 µm in the kneaded mixtures. The results confirm that kneading method is more effective than the co-grinding method in amorphization of ETD. The smaller span values indicating narrower particle size distribution. No significant difference between the different kneading mixtures (P 0.05), while physical mixture differs significantly from co grinding and kneading mixture (P 0.05).
Scanning electron microscopy
The SEM images for pure ETD, 1:19 ETDCHT PM, CG and KM are shown in Figure 2 A-E, respectively. Pure drug image showed prismatic-like structure crystals. The crystalline ETD form was confirmed bySEM, and the CHT presented a regular shape. The PM showed characteristics related to ETD and CHT, On the other hand, particles obtained by different methods as grinding or kneading presented particular morphologies, with reduced sizes when compared to raw materials. The micrograph of ETD-CHT KM did not show the original prismatic crystalline structure of ETD, but showed very small particles thus indicating the amorphization of the drug.
Differential Scanning Calorimetry (DSC) The DSC
thermogram of ETD (Figure 3) exhibited a typical of a crystalline anhydrous substance, showing a sharp endothermic peak (T= 150 °C) corresponding to the melting point of ETD. The DSC trace of CHT was typical an anhydrous amorphous compound. Physical mixture and co grinding mixture revealed the presence of the drug peak with slight shift in the melting temperature of the drug along with significant decrease in the endothermic peak. Kneading mixture showed complete disappearance of the endothermic peak of the drug indicating a change in the nature of ETD from crystalline to amorphous one. The disappearance of the ETD fusion peak observed may be related to a chemical or physical interaction between the drug and CHT or the possible formation of an amorphous system.
X-ray diffraction studies The XRD analysis
was performed to confirm the results of the DSC study. Diffraction spectra of pure ETD, ETD-CHT physical mixture, co- grinding mixture and kneading he XRPD pattern of the etodolac (Figure 4 B) showed sharp peaks at diffraction angles of 2θ 9.4, 13.9, 14.7, 18.9, 23.2, 27.7, 31.8, 34 and 40.2. The diffractoghrams of the physical mixtures and co grinding mixture were similar to those obtained for pure drug, but the peak size was reduced (Figure 4Cand 4D). Whereas, ETD-CHT kneading mixture (Figure. 4E) did not show these peaks indicating a transition of ETD from a crystalline to an amorphous state. These results, taken together with DSC data, indicate a formation of amorphous ETD:CHT system.mixture are shown in Figure 4. In vitro release studies The effects of varying drug/chitosan ratio and preparation methods of drug: polymer mixture on etodolac dissolution rate is shown in Figure 5. In general, drug dissolution progressively improved with increasing the polymer proportion in the mixture and reached the highest values at the 1:19 w/w drug: polymer ratio. The slight increase in drug dissolution shown by simple physical mixtures could be due to a reduction of the interfacial tension between the hydrophobic drug particles and the dissolution medium, owing to the presence of the hydrophilic polymer, as well as to a local solubilizing effect acting during the early stages of the dissolution process in the microenvironment surrounding the drug particles [36]. The high rate of drug dissolution shown by kneaded mixtures and coground mixtures could be attributed to the intimate physical content between ETD and hydrophilic carrier, to particle size reduction brought about by the mechanical treatment and to a decrease in drug crystallinity during cogrinding with the amorphous carrier [38- 41]. These finding were in agreement with the results of solid state studies, confirming that the best dissolution performance of kneaded products is mainly ascribable to the almost complete drug amorphization achieved in these systems. At this stage of the study, it can be concluded that chitosan can favorably enhance the etodolac dissolution property. Generally, dissolution rate of ETD increased significantly with increasing the polymer concentration in the order of KM> CG> PM> pure drug. Table II illustrates the percent drug dissolved at 30 min, dissolution efficiency at 120 min and relative dissolution rate at 60 min of pure ETD, PM, CG and KM. The data demonstrated that CG and KM exhibited a higher relative dissolution rate at 60 min than PM, when compared to the pure drug. The analysis of release data (Table II) confirms that the dissolution efficiency (DE) was statistically different (P Englishhttp://ijcrr.com/abstract.php?article_id=2221http://ijcrr.com/article_html.php?did=2221. Yang L, Chu JS, Fix JA. Colon-specific drug delivery: new approaches and in vitro/in vivo evaluation. Int J Pharm 2002; 235: 1-15
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Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524128EnglishN-0001November30HealthcareSTRENGTHENING AND UPGRADING OF SUB-CENTERS IS ESSENTIAL IN THE EXISTING PUBLIC HEALTH CARE SYSTEM: A STUDY CONDUCTED IN A BLOCK OF WARDHA DISTRICT
English2532Abhay Bhausaheb MudeyEnglish Mehiliquea SEnglish Ramchandra C. GoyalEnglish Vasant V WaghEnglishBackground: Indian Public Health Standards (IPHS) have prescribed certain standards providing basic primary health care services to the community. Sub-centers are still snowed under certain problems like non-availability of required staff, inadequate physical infrastructure and insufficient quantity of drugs and lack of community participation. Hence the present study was undertaken with the objectives to assess the availability and adequacy of paramedical and supporting staff, infrastructure facilities and their extent of utilization
. Methods: This cross-sectional multistage sampling study which was carried out in 31 sub-centers of a Block of Wardha district, Maharashtra. IPHS assessment tool was used to collect information/data by interviews and observations technique. Face-to-face interviews were conducted with all the staff. Observations and responses were recorded in the prescribed IPHS format.
Results: The study reveals that
adequate Immunization, Ante-natal and Family planning services were available at all Sub-centers. Health Workers (female) were available (100%), Voluntary worker (51.61%) whereas Health Workers (male) were not available at any of the sub-centre. In majority of Sub-centers Drugs (40%), Equipments (48.78%), Furniture (44.66%), Basic sanitation and Infrastructure are inadequate and was fall short of IPHS expectations.
Conclusion:
Hence balancing Infrastructure, pooling resources, Intersectoral co-ordination, optimization of health manpower, decentralization of Health Programmes is necessary to fulfill the criteria of IPHS of sub-centers in each Block.
EnglishNRHM, IPHS, Sub- Centers, Block of District, Public health infrastructure.Introduction:
It was recognized that in both developed and developing countries the standard of health services that the public expected was not being provided [1]. At the Joint WHO-UNICEF International Conference in 1978 at Alma-Ata (USSR) proclaimed Primary Health Care as a way to achieve the goal of Health for All. The Alma-Ata Declaration called on all governments to formulate national policies, strategies and plan of action to launch and sustain primary health care as part of a National Health System[2]. In India the first formal National Health Policy was formulated in 1983 and since then there has been marked changes in the determining factors relating to the health sectors and health status.
India is the second largest country in terms of population. In the public sector of rural India, health care services are provided through a network of Sub-Health Centers (sub-centers), Primary Health Centers (PHC), and Community Health Centers (CHC). The Health Care System in India has expanded considerably over the past few decades. There are 1, 44,988 Subcentre, 22,669 PHCs and 3,910 CHCs functioning in the country [3]. The subcentre provides interface with the community at the grass root level providing primary health care services [3].
Though such a vast network of health centers in rural areas looks impressive, sub-center area is still plagued by certain problems like non-availability of required staff and even if they are posted there is always, lack of training programmes in skilled works like family planning and immunization, inadequate physical infrastructure and facilities, insufficient quantity of drugs, and lack of community participation. Upgrading Public Heath Infrastructure in rural areas to measurable standards of quality is being introduced in order to improve the quality of services in these health care centers is a key strategic intervention under the National Rural Health
Mission (NRHM)[4]. A task group under the chairmanship of Director General of Health Services, Government of India, was constituted to recommend the standards to be called as Indian Public Health Standards (IPHS). The overall objective of IPHS is to provide health care that is quality oriented and sensitive to the needs of the community. These are a set of standards envisaged to improve the quality of healthcare delivery in the country under the National Rural Health Mission[5].
Sub-centre are accountable for Assured services like MCH and Family Planning services in which Ante-natal, intranatal, post-natal, new born and child care including immunization, contraception, adolescent health care, assistance to school health services, Janani Suraksha Yojana, treatment of minor ailments and First aid services whereas the specific services of SC includes visit of the doctor at Sub-centre at least once in a month at fixed day and time, Visit of the Health Assistant (male) or LHV to the Sub-Centre at least once a week, facility for referral of complicated cases of pregnancy / delivery available at Sub centre for 24 hours, the ANM/any trained personnel accompany the woman in labour to the referred care facility, the facility for Peripheral blood smear in fever cases and availability of DOTs at the sub-centre. As sub-centers are the first contact point between the health facility and community, the success of any country would depend on well functioning of sub-centers providing services of acceptable standard to the people. Hence it was felt necessary to assess the availability and adequacy of Paramedical staff, support manpower, infrastructure facilities and their extent of utilization at various Sub-centers in a Block of Wardha district, as per IPHS.
Material and Methods The present study was carried out with the aim of an assessment of Public Health Infrastructure at various Subcenters in a selected block of Wardha district using Indian Public Health Standards stated under National Rural Health Mission.
This is a cross-sectional study which was carried out during January 2008 to March 2008 at Sub-centers of Six Primary health care centers (PHC) of one Block of a Wardha district (MS) of Central India, which is also a field practice area of research institute and a medical college.
There are eight blocks in this district from which the study block was selected by simple random sampling. The selected block contains six PHCs with cumulative population of 1, 83,583. All the 31 sub-centers of these six PHCs were further selected for study by Purposive sampling.
An assessment of Sub-Centers was done against Indian Public Health Standards (IPHS) recommended by Director General of Health Services, Government of India. At all sub-centers data was collected by observations and face to face interview with Health Workers. Observations and responses were entered in the prescribed IPHS format for sub-centre[3].
Study tool: Indian Public Health Standards (NRHM) assessment tool/ check list was used to conduct the interviews at the facility level. The check lists were comprised mainly of location, average daily OPD attendance, services availability, staffing pattern, physical infrastructure, equipments, water supply, sewerage, waste disposal, electricity, communication facilities, drugs, equipments and furniture of subcenters.
Data entry and analysis: The collected data was entered into Microsoft office excels for analysis. Mean as a statistical technique was used to calculate the average percentage of the services, staffing pattern, physical infrastructure equipments and drugs in Sub-centers.
Results Assured services like adequate immunization, ante-natal, and family planning services were available at all Sub-centers. All sub-centers of five PHCs were existed in designated government building except one sub-centre (16.66%) existed on shared basis with Anganwadi and one sub-centre (16.66%) existed on shared basis with Zilla Parishad building . The five subcenters (83.34%) of the 6th Waifad PHC were existed in designated-government building except one sub-centre (16.66%) which was on rent.
At fifteen sub-centers (48.38%) the ANM do not reside at the sub-centre staff quarters and at six sub-centers (37.5%) ANMs, who resides at subcentre quarter has occupied even the clinical space for their residential use. Health Worker (female) was available at all the 31 sub-centers, whereas Health Worker (male) was not available at any of the sub-centre. Voluntary worker to keep the Sub-Centre clean and assisting ANM was not available at sixteen sub-centres (51.61%). Equipments available at Sub-centers of five PHCs were between 49-52% except at sub-centers of Deoli PHCs where availability is (41.81%). Furniture availability at sub-centers of Giroli PHC was (52.7%) whereas in the other PHCs sub center it was less than 50%. Drugs Kit A was available (50%) at sub-centres of Deoli PHC; (83.33%) of Giroli PHC; (50%) of Vijaygopal PHC; (50%) of Nachangaon PHC, (83.33%) of Gaul PHC and (66.66%) of Waifad PHC. Drug Kit B was partially available at all sub-centers of all six PHCs. Contraceptive Nirodh and oral pills were available at 18(58.06%) sub centers whereas Copper T was available at only 8(25.80%) sub centers. Emergency contraceptive pills were not available at any sub-centre. Toilet facility was not available at twenty nine sub-centers (93.54%).
Discussion
MCH and Family Planning services like Ante-natal , intra-natal, post-natal, new born and child care including immunization, contraception, adolescent health care, assistance to school health services, Janani Suraksha Yojana, treatment of minor ailments and First aid services were available at all Sub-centers. All sub-centers of six PHCs were existed in designated government building except three sub-centers (09.67%) which are existed on shared basis with Anganwadi, Zilla parishad and one SC was on rent basis whereas Khan M.E et al. (1999)[6] in a situation analysis report of Sitapur district done in 1999 in five block level PHCs found that
out of 54 sub-centers only a few sub-centers were situated in government- owned buildings whereas most of the sub-centers were functioning in a small room with no electricity or toilet facilities.
Toilet facility was not available at twenty nine sub-centers (93.54%), the same findings are noted in Social Audit in NRHM (2007)[7] where not all of the centers are running in a Pakka Makan (brick houses) and most of the centre do not have toilet facility.
At fifteen sub-centers (48.38%) the ANM do not reside at the sub-centre staff quarters and at six sub-centers (37.5%) ANMs, who reside at subcentre Quarter, have occupied even the clinical space for their residential use. Kumar BL (1995) [8] in his study in Gujarat in 1995 has found that about 80% of the sub-centre staff does not stay at the headquarters.
Health Worker (female) was available at all the 31 sub-centers, whereas Health Worker (male) was not available at any of the sub-centers. Voluntary worker to keep the Sub-Centre clean and assisting ANM was not available at sixteen sub-centers (51.61%).but Social Audit in NRHM (2007) [7] revealed that not every Sub-centre has a female health worker.
Furniture availability at sub-centers of Giroli PHC was (52%) followed by Deoli PHC (48%), Waifad PHC (48%), Nachangaon PHC (40%), Vijaygopal PHC (36%) and Gaul PHC (32%).But Foo and Gillian H.C.(1996)[9] in his report on the furniture in four states reported that examination table, chairs for clients, benches for waiting patients were available in all the sub-centers of the states of West Bengal, Bihar, Tamil Nadu and Karnataka.
Equipments available at Sub-centers of five PHCs were between 49-52% except at sub-centers of Deoli PHCs where availability is (41.81%) Agrawal Monica et al (2001) [10] in a study in Lucknow stated that all the centers were well equipped whereas Ramana GNV, Devi R, Praksamma et al (1995) [11] in a baseline survey in Shamirpet PHC of Andhra Pradesh in 1995, it was found that many health workers lacked Stethoscopes, Blood Pressure cuffs or both because of which BP was generally not checked at the subcenters. Weighing scale, examination table and chairs for clients, benches for waiting patients were also not available. Drugs Kit A was available at (83.33%) sub-centers of Giroli and Gaul PHC; (66.66%) of Waifad PHC; (50%) subcenters of Vijaygopal, Deoli and Nachangaon PHC whereas Drug Kit B was partially available at all sub-centers of six PHCs. Gandotra et al (1997)[12] in a case study from Gujrat reported that all sub-centers were always short of medicines and contraceptives. Population Council, Sitapur Report (1995) [5] stated that Autoclaves and sterilizers were seen in 12% of the subcenters whereas examination table, stethoscope, and weighing scale were available at only 5-25% sub-centers It is concluded that majority of subcenters fall short of IPHS expectations. Paramedical staff, basic equipments, furniture, drugs and Infrastructure are inadequate whereas facilities for sanitation and cleanliness are lacking at many centers and where it is present was not up to the mark and satisfactory.
Conclusion and Recommendations
? Hence balancing Infrastructure, pooling resources, Intersectoral Coordination, optimization of health manpower, decentralization of health programmes is necessary to fulfill the criteria of IPHS of sub-centers in each Block.
? Recruitment or contractual appointment of sub-center staff has to be done
? All the Sub-centers should run in government-owned building with adequate space, Drugs, Equipments and furniture should be provided.
? Every centre should maintained cleanliness specially toilets.
? Improvement in drug supply chain is extremely important to improve extent of utilization of services.
Englishhttp://ijcrr.com/abstract.php?article_id=2222http://ijcrr.com/article_html.php?did=22221. Kleczkowski, B.M. „ Health care facilities in developing countries? WHO Chronicle 30: 363-9, September 1976.
2. WHO (1978) Health for All Sr.No.1.
3. Indian Public Health Standards (IPHS) for Sub-centre, Director General of Health Services, Ministry of Health and Family Welfare, Government of India, February, 2007
4. Mission Document, National Rural Health Mission, Ministry of Health and family Welfare, Government of India, April, 2005
5. Population Council, 1995. Situational analysis of Family Welfare Programme in Sitapur district of U.P., New Delhi: Population Council, New Delhi 1995
6. Khan M.E., Patel Bella C. and Gupta R.B., The Quality of Family Planning Services in Uttar Pradesh from the Perspective of Service Providers. : In Improving Quality of Care in India?s Family Welfare Programme edited by Micheal A., Koenig and M.E.Khan. Population Council, 1999; p.238-269
7. Social Audit of NRHM in two states, Empowering the poor for claiming their health rights, a report of Utter Pradesh and Uttarkhand, Centre for Health and Social justice-New Delhi,
8 August 2007 8. Kumar BL. Primary Health Care in Gujrat: Evidence on Utilization, Mismatches and Wastage; GIDR Working Paper No: 149, October 2004
9. Foo, Gillian H-C. A synthesis of research finding on quality of service in Indian Family Welfare Programme, Proceedings from a National Workshop on Operational Research for improving quality of services. Bangalore, Karnataka; 1996
10. Ranjeeta Kumari, MZ Idris, Viddya Bhushan and et al; Study on patient satisfaction in the government allopathic health facilities of Lucknow district: Indian Journal of Community Medicine; Volume: 34; Issue:1; Year: 2009; Page: 35-42
11. Ramana GNV, Devi R, Praksamma et al (1995) Improving Quality of care at Shamirpet Primary Health Center, Andhra Pradesh, Population Manager, Vol.5, 1997
12. Gandotra MM, Khan ME, Shah U et al. Quality of Family Welfare services in India: A case study of Gujarat Population Research Center, Baroda and Population Council, New Delhi, 1997
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524128EnglishN-0001November30HealthcarePHARMACOLOGICAL PROPERTIES OF BIOFOULING ASCIDIAN, POLYCLINUM MADRASENSIS SEBASTIAN, 1952 FROM TUTICORIN COAST OF INDIA
English3344Bragadeeswaran SubramanianEnglish Ganesan KittusamyEnglish Prabhu KolandhasamyEnglish Balasubramanian ThangavelEnglish Meenakshi Kesavan VithiyanathapuramEnglish Y.VenkateshwaraluEnglishAscidians are sessile, conspicuous and important members of shallow benthic communities having rich source of nitrogenous secondary metabolites and of peptides and alkaloids. In the present study, biofoulant ascidian, Polyclinum madrasensis was collected from the pearl oyster cages of Tuticorin coast. The protein content was showed 790 μg/ mL of crude extract and molecular weight determination using SDS page indicated well defined bands ranging from 21 to 90 kDa. The methanol and 1:1 Methanol/ Dichloromethane
extracts exhibited strong hemolytic activity on chicken, goat, cow and human erythrocytes (O, A, B and AB groups). The methanolic extract the highest cytotoxicity in brine shrimp (LC50 = 97 μg/mL). Different polar and non polar solvent extracts were assayed for antibacterial activity against ten human pathogens. The crude compound was characterized by 1H NMR and results represented.
EnglishPolyclinum madrasensis, biofoulants, protein estimation, hemolysis, cytotoxicity, antibacterial, 1H NMR Study.Introduction
Nature is a large treasure trove of organic molecules that have countless biological functions. Over billions of years, nature has produced these organic molecules, which have varied uses, some of which are yet to be determined (Kita and Uemura 2006a and b). The Ascidians, commonly called sea squirts (Subphylum: Urochordata, Class Ascidiacea) are dominant organisms in many marine communities, having a wide geographic distribution (Seed and O?Connor 1981). Ascidians contain a wealth of interesting pharmacological substances (Rinehart et al., 2006). One of the reasons for this ecological success is the ability of these animals to synthesise secondary metabolites with important defensive roles, including antimicrobial peptides (Lee et al., 1997), cytosine like compounds (Raftos and Nair 2004), lectins (Green et al., 2003) and antileukemic compounds (Takeara et al., 2008). Saclike filter feeder ascidians have been reported to be an important source in drug discovery. Tetrahydroisoquinolone alkaloid „Ecteinascidin 743? from Ecteinascidia turbinata, cyclic depsipeptides „Dehydrodidemnin B? and „Didemnin B? from Trididemnum solidum,
cyclic peptide „Vitilevuamide? from Didemnin cuculiferum and „Diazonamide? from Diazona angulata are a few tunicate compounds in anticancer preclinical or clinical trials (Jain et al., 2008). In the present study, biofoulant ascidian was collected from Tuticorin coast of India for new pharmaceutical and biomedical substances exploration.
Materials and Methods
Ascidian collection
Bulk samples of Polyclinum madrasensis Sebestian, 1952 (548 gms. in wet wt.) was collected from the cement blocks, pilings and oyster cages of Tuticorin coast (Lat. 8 0 47? 20” and Long. 780 09? 70”), Tamil Nadu, India by SCUBA diving at the depth ranging from 4 to 6 m during June, 2008.
Extraction
The extraction method suggested by Malla Reddy et al. (2005) was followed. The freshly collected ascidian was soaked in methanol at the site of collection until workup. The initial methanol extract was decanted and the ascidian material was extracted with 1:1 methanol: dichloromethane (3 x 0.5 L) at room temperature. The combined extract including initial methanol extract was filtered, and the solvent was removed under reduced pressure to give predominantly an aqueous suspension, which extracted into ethyl acetate (3x 0.5 L), and concentrated under reduced pressure to give a dark brown gummy mass of 10.98 gms. For antibacterial activity, extraction method suggested by Chellaram et al (2004) was followed. The freshly collected ascidians each 100 gms in wet weight were soaked in methonal, dichloromethane, ethanol, acetone,chloroform, n-butanol, ethyl acetate and diethyl ether at the site of collection until workup. The initial respective solvent extract was decanted and repeated for three times (3 x 0.2 L) at room temperature. The combined extract was filtered, and the solvent was removed under reduced pressure to give a dark brown gummy mass. This crude extract was further studied for antimicrobial activity.
Protein estimation
Protein content was estimated by the method of Bradford et al. (1976). The Standard protein sample was prepared at 2 mg/ mL of BSA. The assay relies on the binding of the dye Coomassie Blue G250 to the protein molecule measured calorimetrically at 595 nm. Dilutions of protein standards with concentrations of 20, 40, 60, 80 and 100 μg/100 μL were assayed.
Molecular weight determination - SDS - PAGE
Crude protein of Polyclinum madrasensis was subjected to electrophoresis following the method of Laemmli (1970) in 12 % polyacrylamide slab gels. 50 µg of the crude protein in each case was diluted with sample buffer (3:1) and heated at 94 0C for 30 seconds, then loaded. The Molecular weight markers (Fermentas SMO431 14.4 - 116 KDa) used viz., consisted of Lysozyme (14.4), beta - lactoglobulin (18.4), RE Bsp 981 (25.0), Lactate dehydrogenase (35.0), Glutathione S (29.0), Ovalbumin (45.0), Bovine Serum Albumin (66.2) and Phophorylase b (98.0). 10 µl of the marker was loaded in the extreme right wells and the proteins were loaded subsequently. Upon completion of electrophoresis, the gel was washed gently with distilled water to remove excess SDS, stained in Coomassie Blue R250 (Coomassie brilliant blue R250, 1.25 g; Methanol 227 mL; Glacial acetic acid 46 mL; distilled aqueous to make up to 500 mL) for two hours at room temperature and then destained (Methanol 7 mL; glacial acetic acid 7 mL and distilled aqueous to make up to 100 mL) for 48 hours. Protein bands were visualised as dark blue bands on a light blue background. The samples were solublised in reducing sample buffer and equal amount of protein was loaded into 12 % SDS- Polyacrylamide gel and electrophoresis was carried out at constant current (30 mA). The molecular weight of the documented figure was analysed using the Total Lab Package Version 2.01.
Pharmacological study
The extracts of Polyclinum madrasensis was tested for hemolytic, cytotoxicity using brine shrimp lethality and antibacterial activities.
Hemolytic activity
Methanolic and 1:1 DCM/ MeOH extracts of Polyclinum madrasensis was assayed on chicken, goat, cow and human erythrocytes (A, B, AB and O blood groups) following the method of Pani Prasad and Venkateshvaran, (1997). The chicken, goat and cow blood samples were obtained from the nearby slaughterhouse in Parangipettai, while clinically healthy human blood samples were obtained from local hospital using 2.7 % ethylenediaminetetraacetic acid (EDTA) solution as an anticoagulant at 5 % of the blood volume and brought to the laboratory. The blood was centrifuged thrice at 5,000 rpm for five minutes. 1 % erythrocyte suspension was prepared for hemolysis study.
Cytotoxicity using brine shrimp lethality assay
To determine the toxic effects on Artemia salina (brine shrimp) the method of Meyer et al (1982) was adopted. The extracts were dissolved in 0.01 mL of DMSO and incorporated into 5 mL of sea water (pH= 8.8 and Salinity = 28 ‰) containing ten Artemia sp. Each concentration (10, 20, 40, 60, 80 100 µg/mL) was tested thrice, and a control DMSO was done each time. The vials were maintained under illumination. Survivors were counted after 24 hrs and the percentage of deaths at each dose and control (DMSO and saline water). The LC50 values of brine shrimp were obtained from counts using the probit analysis method described by Litchfield and Wilcoxon (1941).
Antibacterial Activity
Antibacterial activity was carried out by using standard disc diffusion method (McCaffrey and Erdean, 1985; Murugan and Santhana Ramasamy 2003). The following microorganisms, Staphylococcus aureus, Salmonella typhi, Salmonella paratyphi, Klebsiella oxytoca, Klebsiella pneumonia, Vibrio cholerae, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, and Lactobacillus vulgaris were used. The extracts were applied to 6 mm sterile discs in aliquots of 30 µL of solvent, allowed to dry at room temperature and placed on agar plates seeded with microorganisms. The bacteria were maintained on nutrient agar plates and incubated at 37° C for 24 hrs. Zones of growth inhibitions were measured following incubation. All extracts were tested thrice at a concentration of 30 µg disc-1 .
Preliminary characterisation of the extracted compound by 1H NMR 1H NMR
spectra were performed at Bruker Avance 300 MHz NMR Spectrophotometer operating at 400.24 and 100.614 observations. The crude sample was dissolved in DMSO/CDCl3 as solvents, TMS as internal reference. Spectra were acquired using a 5 mm 1H dual tuned probe. Temperature was maintained at 25 0C.
Results
Protein Estimation
The protein content in Polyclinum madrasensis was 790± 0.5 µg/ mL of crude. This result clearly indicates the high amount of protein and other biochemical components present in their body.
Molecular weight détermination - SDS - PAGE
Upon SDS - PAGE on 12 % gel, Crude protein of P. madrasensis yielded 9 bands ranging from 20 to 97 kDa with well defined bands of 20, 37, 52 and 90 kDa (Plate.1).
Biomedical properties study Hemolytic activity
All blood groups showed promising activity. Chicken, goat, cow and A, B, O and AB of human erythrocytes were vulnerable to lysis. The MeOH and 1:1 DCM: MeOH, extracts on human AB blood group and cow blood showed maximum of 0.16 specific Hemolytic Unit (HU) and minimum of 0.01 specific Hemolytic Unit was recorded in goat blood from 1:1 DCM: MeOH extract (Fig. 1)
Cytotoxicity using brine shrimp lethality assay Polyclinum madrasensis extracts tested at 10, 20, 40, 60, 80 and 100 µg/mL-1 and showed highest cytotoxicity, indicating the presence of cytotoxic compounds. The LC50 values are given in Fig. 2. The different solvent system extracts showed cytotoxic properties against Artemia salina larvae. The maximum cytotoxicity (97 µgmL-1 ) was observed in MeOH extracts and minimum (20 µgmL-1 ) was observed in ethyl acetate extract.
Antibacterial activity
Fig. 3-10 shows the results of in vitro antimicrobial activity against pathogenic bacteria. The crude extracts showed high and moderate antibacterial activity against 10 pathogens assayed. Among pathogens bacteria tested, P. aeruginosa was the most sensitive against DCM extract (15 mm) and minimum of 5 mm were recorded in diethyl ether and n-butanol extracts against Klebsiella oxytoca and P. aeruginosa respectively.
1H-NMR study
The 1H-NMR spectrum was taken in DMSO D6. From the spectral signal at 7.9 indicates the presence of guanidine and thymidine containing alkaloid, however the compound is to be isolated and confirmed.
Discussion
As far as we know, human consumption of ascidians as a table food occurs in Japan and Chile. Some ascidians (e.g. Halocynthia roretzi) is widely enjoyed as food in Japan, particularly in the Hokkaido and Johoku districts because of the high amount of protein, carbohydrate and other essential micronutrients (Nanri et al., 1992). The amount of carbohydrate, protein, lipid and minerals such as phosphorous and calcium in ascidians is previously reported by (Rajesh and Ali 2008). They also found that the concentration of total carbohydrate was higher in the test (tunic) in their body. This corresponds to the higher amount of cellulose and crude fibre. The present investigation shows that Polyclinum madrasensis contain high amount of protein indicating their high food value and thus surpass many marine food sources in terms of value added marine food.
In the present study, the molecular weight of the protein yielded 9 bands ranging from 21 to 87 kDa with well defined bands of 20, 37, 52, 57and 90 kDa. It supports the previous studies i.e., the tunicate, Styela plicata from Sydney harbour, Australia, analysed for molecular weight of the proteins by SDS PAGE revealed that it contained a single protein of approximately 14 kDa (Nair et al., 2001). Green et al. (2003) demonstrated the molecular weight of protein from hemolymph (43 kDa) of the solitary ascidian Styela plicata from Australian waters. Santos et al. (1992) demonstrated the electrophoresis of the sulfated polysaccharides from different species of ascidians. Chemical analysis of polysaccharides from ascidians reveals sulfate ester, high galactone content and small amounts of glucose and hexosamine in proportions that vary among the different species. The sulfate content may account for part of the variation in electrophoresis motilities as observed in Herdmania momus, Ciona oblonga and C. intestinalis. The endoderm specific alkaline phosphate protein with molecular mass of 86 kDa and 103 kDa were reported by Kumaro et al. (1996) from Halocynthia roretzi from Japan. Hemolytic activity of extracts the MeOH and 1:1 MeOH: DCM extracts on human AB blood group and cow blood showed maximum of 0.16 specific Hemolytic Unit (HU), followed by 0.08 HU observed in MeOH extracts on chicken, cow, human A and B erythrocytes. Minimum of 0.01 specific Hemolytic Unit was recorded in goat blood from 1:1 DCM: MeOH extract. Present study supports the previous reports. i.e., Jimenez et al. (2003) revealed the hemolytic activity of ascidians E. vannamei, Euherdmania sp., D. psammatodes, D. ligulum and Polysyncraton sp. and concluded that lytic protein substances present in these animals, invertebrates and prochordates may be involved in immunity or prey capture. Lee et al. (2001) revealed that hemolytic activity of extracts from the ascidian, Halocynthia aurantium showed 21 % lysis against human red blood cells. The hemolytic activity of tunicate, Halocynthia aurantium disrupted 8 % and 16 % of human O and B erythrocytes respectively (Jang et al., 2002). Gouiffes et al. (1988) demonstrated that the Bistramide A, compound derived from the ascidian, Lissoclinum bistratum from UA islet in New Caledonia, France showed cytotoxicity against Artemia salina larvae in less than 1 µg/ mL. Methanolic and crude extract of P.madrasensis are more toxic and they showed LC50 values of 97 and 95 respectively, than digitalin (LC50 = 151 µg/mL-1 ) and Caffeine (LC50 = 306 µg/mL-1 ) and it is correlated with previous works done by Meyer et al. (1982) and Jimenez et al. (2003). The minimum of (LC50 = 21 µg/mL-1 ) was showed in ethylacetate extracts of Polyclinum madrasensis. The species studied in the present work, Polyclinum madrasensis has not been previously investigated for the presence of cytotoxic compounds from Indian water. The epidioysterol is toxic against A. salina larva, derived from morocco ascidian Cynthia savignyi. The LC50 value showed 71 µg/mL. The percentage of death increased at 10, 30, 50 and 100 µg/ mL for 20 %, 25 %, 42 % and 63 % respectively. Three cyclotetrapeptides were isolated from the ascidian Cystodytes dellechiajei (Polycitoridae), only the valine containing peptide had significant cytotoxic effect and it showed LC50 value of 1.5 µg/mL (Aracil et al., 1991). Extracts of Ecteinascidia turbinata (Phelebobranchia, Perophoridae) exert several biologically significant effects; they are more cytotoxic. A new class of metabolites, the Eudistomins based on the β-carboline ring system, have been isolated from several species of Eudistoma (Ploycitoridae). Antibacterial activity has been previously reported from extracts of some ascidians. From present study, the bacteria tested P. aeruginosa is the most sensitive against 1:1 DCM: MeOH extracts (15 mm) followed by MeOH extract against Klebsiella pneumonia (14 mm) inhibitory activity. Minimum of 5 mm were recorded in diethyl ether and n-butanol extracts against Klebsiella oxytoca and P. aeruginosa respectively. Abourriche et al. (2003) evaluated the antibacterial activity against A. tumifaciens, E.coli, P. aeruginosa and S. aureus from the extracts of Moroccan Atlantic sea ascidian, Cynthia savignyi. It shows that, except for the dichloromethane extract, all extracts were active against bacteria. A. tumifaciens was the most sensitive. Activity of hexane and diethyl ether extracts of this ascidian against A. tumifaciens was slightly less, but higher than the activity of Lissoclinum fragile extracts.
Lissoclinotoxins, on the other hand, are cyclic peptides (lissoclinotoxin A and B), isolated from the tissues of Lissoclinum perforatum. Lissoclinotoxins A and B are powerful antimicrobial agents, with strong activity against a range of bacteria, including ichthyopathogenic strains, such as Aeromonas salmonicida and Vibrio anguillarum, at concentrations between 0.1 to 0.6 µg/mL (Litaudon and Guyot, 1991 and Litaudon et al., 1994). Ascidians have attracted attention as a source of antimicrobial proteins because they are sessile, filter feeding prochordates which hold a phylogenetically strategic position close to the origin of the vertebrate line. They are thus considered to contain antibacterial agents of relevance to either antifouling technology or clinical pharmacology and the tissues of several solitary species have been subjected to broad spectrum screening for bactericidal, antiviral or cytotoxic activity (Rinehart et al., 1983).
Conclusion
All extracts were prepared using the described procedures above and were evaluated for consistency by taxonomic analysis of the ascidians specimens assayed for biomedical properties and preliminary characterisation of the compounds. Much chemical research remains to be done in order to achieve the structural elucidation of the active principles contained in ascidians studied in the present work which demonstrated bioactivity. In these ascidians, the active compounds were present at very low concentrations. Large scale collection will be necessary to obtain sufficient quantities of pure compounds for structural elucidation and further pharmacological evaluation. It is therefore likely that other factors, besides bioactive compounds might play an additional role in the defense mechanisms of the species investigated. Some options for sustainable use of marine resources are chemical synthesis, controlled harvesting, aquaculture of the source organism, in vitro production through cell culture of the macro or microorganism source and transgenic production.
Acknowledgements
Sincere thanks are due to authorities of Annamalai Universities for the necessary facilities provided. The sample processing and NMR spectral studies facilities provided by the Director, Indian Institute of Chemical Technology is acknowledged. Project grant provided by Department of Biotechnology (DBT), New Delhi is gratefully acknowledged.
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9. Jimenez, P. C., S. C. Fortier, T. M. C. Lotufo, C. Pessoa, M. E. A. Moraes, M. O. Moraes and L.V. Costa- Lotufo, 2003. Biological activity in extracts of ascidians (Tunicata, Ascidiacea) from the north- eastern Brazilian coast. J. Exp. Mar. Biol. Ecol., 287: 93- 101.
10. Kitam, M and D. Uemura, 2006a. In Progress in Marine Molecular Biotechnology. Shellfish Poisons. W E G Muller, H G Schroder (ed.) (Berlin, Heidelberg Verlag Press: Springer) 43: 25-32.
11. Kita, M and D. Uemura, 2006b. In Topics in Heterocycles, Bioactive Heterocyclic Alkaloids from Marine Origin. S Eguchi, (ed.) (Berlin, Heidelberg Verlag Press: Springer); 6: 157-163.
12. Kumaro, G., H. Yokosavva and H. Nishida, 1996. Biochemical evidence for membrane- bound endoderm specific alkaline phosphate in larvae of the ascidian Halocynthia roretzi. Eur. J. Biochem., 240: 485-489.
13. Laemmli, U. K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680- 685.
14. Lee, I. H., Y. Cho and R. I. Lehrer 1997. Styelins, broad spectrum antimicrobial peptides from the solitary tunicate, Styela clava. Comp. Biochem. Physiol., 118: 515- 521.
15. Lee, I. H., Y. Lee, C. Kim, L. T. Hong, L. B. Menzel, J. Pohl, M. Sherman, A. Waring and R. Lehrer 2001. Dicynthaurin: an antimicrobial peptide from hemocytes of the solitary tunicate, Halocynthia aurantium. Biochim. Biophys. Acta., 1527: 141-148.
16. Litaudon, M and M. Guyot, 1991. Lissoclinotoxin A, an antibiotic 1, 2, 3 - trithiane derivative from the tunicate Lissoclinum perforatum. Tetrahedron Lett., 32: 911-914.
17. Litaudon, M., F. Trigalo, M. T. Martin, F. Frappier and M. Guyot, 1994. Lissoclinotoxins: Antibiotic polysulphur derivatives from the tunicate Lissoclinum perforatum Revised structure of Lissoclinotoxin A. Tetrahedron Lett., 50:5323- 5334.
18. Litchfield, J. T and P. J. Wilcoxon 1949. Simplified method of evaluating doseeffect experiments. J. Pharmac. Exp. Ther., 96(2):99-113.
19. Malla Reddy S, Srinivasulu M, Satyanarayana N, Kondapi A K and Y. Venkateswarlu, 2005. New potent cytotoxic lamellarins alkaloids from Indian ascidian Didemnum obscurum; Tetrahedran, 61: 9242- 9247.
20. McCaffrey, E. J and R. Erdean, 1985. Antimicrobial activity of tropical and subtropical sponges; Mar. Biol., 89:1- 8.
21. Meyer, B. N., N. R. Ferrigi, J. E. Putnam, L. B. Jacobson, D. E. Nicolas and J. L. Mclaughin, 1982. Brine shrimp: a convenient general bioassay for active plant constituents. Planta Med., 45: 31- 34.
22. Murugan, A and M. Santhana Ramasamy, 2003. Biofouling deterrent natural product from the ascidian Distaplia nathensis. Indian J. Mar. Sci., 321: 62-164.
23. Nair, S. V., M. Burandt, A. R. L. Hutchinson, D. Raison and A. Raftos, 2001. A C - type from the tunicate, Styla plicata that modulates cellular activity. Comp. Biochem. Physio.129: 11- 24
. 24. Nanri, K., J. Ogawa and T. Nishikawa 1992. Tunic of pyurid ascidian Microcosmus hartmeyeri Oka is eaten locally in Japan. Nanki Seibutu., 34: 135
. 25. Pani Prasad and K. Venkateshwaran, 1997. Microhaemolytic assay, International Training Manual on Advance Techniques in Marine Biotoxinology; CIFE, India. 41pp
26. Raftos, D and S. Nair, 2004. Tunicate cytokine-like molecules and their involvement in host defense responses. Prog. Mol. Subcell. Biol., 34: 165-182.
27. Rajesh M and H. Abdul Jaffar Ali, 2008. Nutritional value and antimicrobial activity of marine ascidian species; Ascidian News. http://depts.washington.edu/ascidian/
28. Rinehart, K. L., J. B. Gloer, G. R. Wilson, R. G. Hughes, L. H. Li, H. E. Renis and J. P. McGovern 1983. Antiviral and antitumor compounds from tunicates. Federation Proceedings, 42: 87- 90.
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Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524128EnglishN-0001November30General SciencesGROWTH RESPONSE OF BROILER FINISHERS FED DIET WITH GRADED OF INDOMIE NOODLE WASTE MEAL AS REPLACEMENT FOR MAIZE
English4553AlabiEnglishO. M. AyoolaEnglishM.OEnglishThe replacement value of Indomie Waste (IW) in Maize-based Broiler finisher diets was investigated. One hundred and fifty (150) unsexed Hypecco strain of Broilers were randomly allotted into five isocaloric and isonitogenous dietary groups with graded levels of Indomie Waste in weight for weight replacement for maize.
Treatment 1(T1) contained 100% maize,T2(25%maize,75% IW),T3(50% maize,50% IW),T4(25% maize,75% IW) and T5(100% IW). The experiment lasted for five weeks(5 to 10 weeks of age) during which data was collected in respect of weekly body weight, daily feed intake while Feed Conversion Ratio(FCR), Feed
Conversion Efficiency(FCE) and cost of feed per kilogram body weight gained were calculated. Also, some internal organs were weighed in all the dietary group samples at the end of the experiment. Data analysis revealed that replacement of maize with IW in Broiler Finisher diets had significant (pEnglishBroiler finisher, Cost, Graded levels, Growth, Indomie Waste, Maize.Introduction
There has been a steady increase in the cost of conventional feed ingredients such as maize, groundnut cake, soyabean meal and fish meal in the past years and this has led to increase in the prices of animal protein sources [1] . While it is very easy to place ruminants on natural pastures, monogastrics must be provided with balanced diets with all nutrients both quantitatively and qualitatively. Over the years, the bulk of research works on livestock production has been on ways to reduce the cost of feeding the animals through the utilization of agro-industrial by-products (AIBs) and other materials that seem to be non important to the livestock. Various AIBs have been investigated to be useful for livestock feeding. Brewers Dry Grain (BDG), wheat offal, corn offal, rice bran, cassava sievate, cassava peels to mention few have been widely tested and incorporated into livestock feeding.[2,3,4,5,6] . These ingredients can be incorporated into the diet of monogastrics without any detrimental effect on the performance and health of the animals and thereby reducing the cost of feeding.
However, seasonal variations in the availability of these AIBs have pushed researchers into the investigations of nonconventional feed ingredients with the intention of reducing the cost of production in order to make animal protein affordable to the populate [7]. Such non conventional feed ingredients include the seeds and leaves of some forest trees and forages. The extent to which some of these such as Acacia, albido pods [8] , Luffa cylindrica seeds meal [9]Solanum nigrum L.var, virginum leaves and seeds [10] , Stylosanthes guianensis [11] , Moringa olifera leaf meal [12],fluted pumpkin leaf [13] can be utilized by non-ruminants have been well investigated and documented. Meanwhile, many food factory waste products have been considered as ingredients that can be utilized by monogastrics. Examples are cassava meal, wheat and corn flour dusts, biscuit wastes, sorghum sprout, cocoa pod meal, poultry offal and Indomie noodle waste to mention few [14] . Indomie noodle waste (IW) is derivable from Indomie noodle which is very popular in Nigeria as a fast food for young and adults and is well accepted in the country. Apart from being well balanced with reasonable value of metabolizable energy, digestible protein with good aroma before, during and after cooking, the product is approved by the Nigerian Agency for Food, Drinks Administration and Control (NAFDAC). The major ingredients in Indomie noodle are wheat and palm oil which make it to be energy rich. The objective of this research work is to investigate the replacement values of Indomie waste for maize in broiler finishers diets in terms of growth response of the birds and the cost implications.
Materials and Methods The research was conducted at the Broilers section of the poultry unit of the Teaching and Research farm of Bowen University, Iwo, Nigeria. Two hundred (200) unsexed broilers of Hypecco strain were purchased at day old from a reputable hatchery in Ibadan, Nigeria. They were brooded for three weeks during which feed (commercial broiler starter mash) and water were provided ad libitum and other management practices in terms of medication and vaccination were strictly observed. Five isocaloric and isonitrogenous experimental diets were formulated at graded levels of maize and IW replacement on weight for weight basis At the end of the third week, 150 birds were weighed and randomly allotted to the five dietary groups with thirty chicks each. The groups were subdivided each into three replicates of ten birds each in a Completely Randomized Design (CRD). The dietary groups are: T1 (control with 100% maize with no IW); T2 (75% maize and 25% IW); T3 (50% maize and 50% IW); T4 (25% maize and 75% IW) and T5 (100%IW and no maize). The birds were allowed to adjust for one week while (4th week). Data collection started at the beginning of 5th week of age and lasted for five weeks. Parameters investigated were daily feed intake, weekly body weight while feed conversion ratio(FCR), feed conversion efficiency(FCE),unit cost of each diet, cost per weight gain were calculated.
Moreover, at the end of the 9th week, six birds were randomly selected per dietary group and were weighed, humanely bled, defeathered, eviscerated and the weight of some internal organs such as the gizzard ,proventriculus ,spleen heart, liver and abdominal fat were measured and expressed relatively as the percentage of the liveweight.
Statistical Analysis
Data generated were subjected to a oneway analysis of variance (ANOVA) using the Statistical Analysis Software [15] and significantly different means were separated with the Duncan?s option of the same software at 5% level.
Results
Table 1 shows the Gross Composition of the experimental diets with their respective cost as at the time of the research. The diets were isocaloric and isonitrogenous while the price per kilogram of maize was N50 and that of IW was N80. Table 2 shows the Performance Characteristics of the broiler finisher chickens fed with graded level of IW as replacement for maize. Parameters investigated on average basis were Initial Body Weight(IBW),Final Body Weight(FBW),Total Body Weight Gain(TBWG),Total Feed Consumed (TFC) per bird while Feed Conversion Ratio (FCR),Feed Conversion Efficiency(FCE) and Feed Cost Per Kilogram Weight Gained (CPW) were calculated.
No significant (p>0.05) difference was observed between the mean IBW of the broiler chicken. The value ranged from 0.65kg to 0.66kg. Other parameters were however influenced by the dietary treatments.
Birds on T5 (100%IW) had the highest average FBW of 2.50kg significantly (p0.05) difference was observed between the mean FBW of birds on T2 and T3 but they differed significantly (p0.05) difference was observed between the mean values for TFC of birds on T1, T2 but they differed significantly (pEnglishhttp://ijcrr.com/abstract.php?article_id=2230http://ijcrr.com/article_html.php?did=22301. Adejinmi.O.O,Hamzat,R.A,Fapohunda, J.B. Performance and Nutrient digestibility Rabbits fed fermented and unfermented cocoa pod..Nig.Jour.for Anim.Prod 2007;34(1): 63-68.
2. Farinu,G.O.Effect of feeding a Compound Diet Based on NonConventional Feedstuffs on Growth and Carcass Characteristics of Rabbits. World Rabbit Science 1994;2(4):123- 126.
3. Ajayi, F.O.,Balogun, O.O., Ovuru, S.S. and Mgbere,O.O.(2005). Reproductive Performance of Rabbits fed MaizeMilling Waste Based Diets. African Journal of Biotechnology 2005;Vol.4(5):439-443.
4. Aderemi,F.A.,Alabi,O.M.and Lawal, T.E. (2006). Utilization of Whole cassava meal by Egg-type Chicken. In Raji AM,Oluokun JA, Odukoya SO.editors ASAN 06.Proceedings of the 11th Conference on Animal Science.2006.Sep 18-21.IARand T, Ibadan,Nigeria.p. 73-75.
5. Afolabi,K.D., Iyayi, E.A., Abu, O.A., Fakolade,P.O.and Adebiyi,O.A.Effects of solid Substrate fermentation by Aspergillus niger and Rhizopus spp.on the nutritional value Of cassava peel. In Raji AM, Oluokun JA, Odukoya SO.editors ASAN 06.Proceedings of the 11th Conference on Animal Science.2006.Sep 18- 21.IARand T,Ibadan,Nigeria.p.169-172.
6. Omole, A.J, Adejuyigbe, A,Ajayi, F.T, Fapohunda, J.B. Nutritive Value of Stylosanthes guianensis and Lablab purpureus as sole feed for Growing Rabbits. African Journal of Biotechnology 2007; Vol. 6 (18), pp.2171-2173
. 7. Olabanji, R.O., Ojebiyi, O.O., Tona, G,O.and Olaogun,O. Haematological and Serum Biochemical Response of Growing Rabbits fed Diets Containing Mango(Mangifera indica) Seed Kernel Meal. In Akinlade JA,Olayemi TB,Rafiu TA,Odunsi AA.editors ASAN 09. Proceedings of the 14th Conference on Animal Science, 2009. Sep 14-17. LAUTECH, Ogbomoso,Nigeria. p.270- 273.
8. Igwebuike, J.U., Anugwa, F.O.I., Raji, A.O., Ehiobu, N.G.and Ikurior, S.A. Nutrient Digestibility, Haematological and Serum Biochemical Indicies of Rabbits fed Graded Levels of Acacia albida Pods. ARPN Journal of Agricultural and Biological Sciences 2008;Vol.3,No.4:33-40.
9. Dairo, A. Performance and Haematological Evaluation of Weaner Rabbits Fed Loofah Gourd Seed Meal(Luffa cylindrical{M.J.ROEM}) . African Journal of Food,Agriculture Nutrition and Development 2008;8(4):451-463.
10. Akubugwo,I.E.,Obasi,A.N.and Ginika, S.C. Nutritional Potential of the Leaves and Seeds Of Black Nightshade-Solanum nigrum L.Var virginicum from Afikpo-Nigeria. Pakistan Journal of Nutrition 2007;6(4):323-326.
11. Omole, A.J., Adejuyigbe, A.,Ajayi, F.T. and Fapohunda,J.B. Nutritive Value of Stylosanthes guianensis and Lablab purpureus as sole feed for Growing Rabbits. African Journal of Biotechnology 2007; Vol.6 (18), pp.2171-2173.
12. Okeke, E.N., Usman, J.M., Osalusi, C.S., Akinola, O.O., Abiola, J.K. and Nwachi, A.C.Performance Characteristics and Haematological Profile of Adult Rabbits fed Morringa Oliefera Leaf Meal Supplements. In Akinlade JA,Olayemi TB, Rafiu TA, Odunsi AA.editors ASAN 09. Proceedings of the 14th Conference on Animal Science, 2009. Sep 14- 17.LAUTECH,Ogbomoso,Nigeria. p.400-401.
13. Alabi.O.M., F.A.Aderemi., M.O. Ayoola, T.E. Lawal., A.A.A dewumi, A.O.Oguntunji,et al. Performance characteristics and gut Morphology of broiler chickens fed with oral supplementation of Telfaria occidentalis Leaf extract at finisher phase. In Akinlade JA,Olayemi TB,Rafiu TA,Odunsi AA.editors ASAN 09. Proceedings of the 14th Conference on Animal Science, 2009. Sep 14-17.LAUTECH, Ogbomoso, Nigeria. p. 354-356.
14. Eniolorunda, O.O., Taiwo, B.B.A., Oyewumi, O.O. and Adeyemi, O.A. Performance of Laying Hens Fed Graded Levels of Indomie Waste as Replacement for Maize in a Humid Tropical Environment. Research Journal of Animal Sciences 2008; 2(5):135-138.
15. SAS Statistical Analysis Systems. Institution Users Guide, SAS Institute, Cary, N.S;1999.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524128EnglishN-0001November30HealthcareNUTRACEUTICALS AND PHARMACEUTICALS:A COMPARATIVE ANALYSIS
English0306Ajay G. PiseEnglish Shilpa PiseEnglish D. SreedharEnglish Virendra LigadeEnglish Manthan JanodiaEnglish N. UdupaEnglishIntroduction:
Today healthcare market is flooded with the different new terminologies. Nutraceuticals, Cosmeceuticals, Biopharmaceuticals, Nanopharmaceuticals, Herbaceuticals, Dermaceuticals and Nutricosmeceuticals are new buzzwords in healthcare market. Different scholars have defined Nutraceuticals, but essence of all definition remains same i.e. “food as medicine”. Here in this study we have attempted to picture out the clear difference between Nutraceuticals and Pharmaceuticals.
EnglishConceptual Difference:
According to Thesaurus dictionary, meaning of pharmaceuticals is “A substance used in the treatment of disease” the term pharmaceuticals include its synonyms drug, medicament, medication, and medicine2 . Dictionary meaning of drugs says drug is a substance used in the diagnosis, treatment, or prevention of a disease or as a component of a medication. Drug is a substance as recognized or defined by the law of that country. A chemical substance, such as a narcotic or hallucinogen that affects the central nervous system, causing changes in behavior and often addiction also considered as drug2 . Every country has defined the term drug in their regulations for medicine, but essence of all the concepts of drugs remains same. The term "Nutraceutical" was coined in 1989 by Stephen De Felice, founder and chairman of the Foundation for Innovation in Medicine8,9. He fused the term Nutrition and Pharmaceuticals to derive the new term “Nutraceutical”. In his view “Food or parts of food that provide medical or health benefits, including the prevention and treatment of disease is called as Nutraceuticals”. As per this definition whole food or a part of food if taken for medical or health benefits is considered as nutraceuticals. This is the widely accepted definition of nutraceuticals. The definition adopted by Health Canada says, nutraceutical is “A product isolated or purified from foods, and generally sold in medicinal forms not usually associated with food and demonstrated to have a physiological benefit or provide protection against chronic disease”8 . As per this definition nutraceutical is a product isolated, derived or processed from natural sources and sold in the medicinal form (powder, tablet, capsules, etc. as OTC products) and its benefits can be explained scientifically in human being, such product referred as nutraceuticals. For example, if crude garlic is powdered or sold in the tablet form then it categorized as nutraceutical product. If the lemon oil is extracted from lemon grass and consumed for health benefits, it comes under the category of nutraceuticals. Many research scholars have different opinion on the nutraceuticals, functional foods, dietary supplements. Some group of scholars considers nutraceuticals, functional foods, dietary supplements are different terminologies, while others consider these all terminologies are parts of Nutraceuticals. USFDA defines functional food as “When food is being cooked or prepared using "scientific intelligence" with or without knowledge of how or why it is being used, the food is called functional food”5 . Dietary supplement is a product (other than tobacco) that is intended to supplement the diet that bears or contains one or more specified dietary ingredients. According to these definitions, our daily lunch and dinner items can be categorized as functional food. If we are supplementing our lunch or dinner with ghee or curd then it will considered as dietary supplements.
Market Segments:
Pharmaceutical products are targeted in different market segments which includes segments as per different diseases, age, and lifestyle. Nutraceutical products are mainly promoted and targeted for prevention of disease or avoid future consequences of diseases. For example, garlic tablets are mainly promoted to avoid risk of heart related problems. Nutraceutical products are targeted in different segments in healthcare market; it includes products for men?s health, woman?s health, energy products, weight management products, and disease preventive products. These segments are viewed as new and highly potential for nutraceutical products.
Regulatory status:
Very few countries have accepted the term “Nutraceuticals”. India government has not yet adopted this terminology and product category relevant to nutraceuticals, functional food and dietary supplements are brought under “Prevention of Food Adulteration Act”. No separate guidelines are available for nutraceuticals, functional food and dietary supplements in India. United States has mentioned the terminologies in Federal Food Drug and Cosmetic Act and given guidelines for it. Canada government has adopted the term Nutraceuticals and framed its own definition. Health Canada has given guidelines for nutraceuticals under the Food and Drugs Act. In India several companies are in fray, Lupin, Wockhart, Morepan, Dabur, Parry Nutraceuticals and Himalaya are doing well in nutraceuticals market. Annual growth of nutraceuticals market (25%) is higher than the annual growth of pharmaceuticals. The global market potential for nutraceuticals is estimated $80 by 2008
Nutraceuticals Vs. Pharmaceuticals:
In healthcare, a substance used in the treatment of disease is called as drug and generally it would be from synthetic source. Nutraceutical is a substance used to prevent disorders and generally referred as health products. Nutraceuticals can be from plant, animal and mineral source. License is needed from regulatory body for selling the pharmaceutical product (other than OTC products). No license is needed to sale the nutraceutical product. Pharmaceutical products can be purchased only by prescriptions (other than OTC products). No prescription is required for purchasing nutraceutical products. Stringent guidelines have to be followed for manufacturing the pharmaceutical product. Conclusion: It is observed that very few countries have accepted the terminology of Nutraceuticals and designed regulatory guidelines. Absence of regulatory guidelines for nutraceuticals may flood the healthcare market with spurious and inferior quality products. This is high time for India government to accept and define these new terms of Nutraceuticals, Functional foods and Dietary supplements. Government also has to prepare proper regulatory guidelines to control the healthcare market and to restrict the entry of spurious and inferior quality nutraceutical products. As these terms are very new in healthcare market, enough data on research is not yet available. Nutraceutical companies can focus more on their research and development activities and develop more data to support the claims of their products. There are tremendous opportunities and potential for nutraceutical products in healthcare market, nutraceutical companies should explore these opportunities.
Englishhttp://ijcrr.com/abstract.php?article_id=2231http://ijcrr.com/article_html.php?did=2231References:
1. “Global Regulatory information” cited at http://www.globalregulatory.com /labclass/nutraceutical_consultants.cf m
2. “Definition of Pharmaceuticals” cited at http://en.wikipedia.org/ wiki/Pharmaceuticals
3. Information on nutraceuticals cited at http://www.aapspharmsci.org/ view.asp? Art=ps050325
4. Editorial, “OvaRex(r) clinical trial for primary analysis prompts steps toward approval” cited at http://www.pharmabiz.com/article/d etnews.asp?articleid=1203§ioni d=47
5. Presentation for FDA public hearing, June 8, 1999- Washington, DC by Gary L.Huber (American Nutraceuticals Association).
6. Information cited at http://www.bccresearch.com/food/G A085R.html
7. Information on nutraceuticals cited at http://www.teknoscienze.com/ agro/pdf/nov_ dec03/bioceuticals.pdf
8. “Nutraceuticals: dietary supplements a legal dilemma” by V. D. Deshmukh IDMA 30th June 2005 no. 24, pg 38-39
9. “Nutraceuticals: dietary supplements a legal dilemma” by V. D. Deshmukh, IDMA 30th June 2005 no. 24, pg 38-39
10. “Nutraceuticals to gain ground globally” by Raja Prasanna published in „The Pharma Review? vol 4 no. 20 (Feb. 06)
11. “Nutraceuticals: Borderline Between Food and Drugs” by R. K. Rishi Published in „The Pharma Review? vol 4 no. 20 (Feb. 06)