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Continental J. Pharmaceutical Sciences 2: 1 - 14, 2008 ©Wilolud Online Journals, 2008.

GELLAN-BASED SYSTEMS FOR SUSTAINED OPHTHALMIC DELIVERY OF OFLOXACIN.

Farah Siddiqui1, M. Abul Kalam1, Nayyar Parvez2*, Suman Yadav3, Yasmin Sultana1, Asgar Ali1, M. Aqil1, A. K.

Mishra4 and K. Chuttani4

1Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi-110 062, India,

2Department of Pharmaceutics, Advanced Institute of Pharmacy, Palwal, Distt. Faridabad, Haryana, India, 3Department of Chemistry Advanced Institute of Technology, Palwal, Distt. Faridabad, Haryana, India. 4Department of Radiopharmaceuticals, Institute of Nuclear Medicine and Allied Sciences (INMAS), Brig. S.K. Majumdar Road,

Delhi-110054, India.

ABSTRACT It is common knowledge that the ocular bioavailability and therapeutic response of drugs applied topically as eye drops is very poor, this is due to rapid precorneal drainage of the instilled dose, which could be overcome by the use of in-situ gelling systems that undergo a sol-gel transition in the cul-de-sac. This work describes the ophthalmic formulation of an antibacterial agent ofloxacin, based on the concept of ion-activated in-situ gelation by using the polymer Gellan gum, a novel vehicle for sol-gel transition, which gels in the presence of mono/divalent cations present in the tear fluid. The formulations were characterized for in vitro drug release and rheological properties. The physical stability and interaction between the exipients of the selected formulations were determined following steam sterilization and storage at 25ºC and 40ºC. The optimized formulations were therapeutically efficacious and demonstrate to possess consistency that is favorable to prolong the drug release properties over an extended period of time (12 h). The physical stability, following steam sterilization and storage, the optimum viscosity combined with sustained release of the drug make the formulations “C” and “C-Beta” the promising formulations as an alternative to conventional eye drops.

KEYWORDS: Gelrite, ofloxacin, in-situ gelation, sustained release.

INTRODUCTION Topical application of antibacterial therapy to the conjunctival sac is usually an effective avenue for treating bacterial conjunctivitis. A very common disadvantage of using eye drops is a rapid elimination of the solutions containing drug and their poor bioavailability. The rapid elimination has different causes: the amount and the structure of the tear film present, the capacity of lower eyelid sac and the different defense mechanisms of the eye against foreign matter (Ooteghem, 1993). The poor bioavailability of eye drops is due to the short precorneal residence time (Cohen et al., 1997). The conventional therapy of bacterial conjunctivitis requires frequent administration of eye drops at an interval of 2-4 hours, which results in patient compliance problem. In addition to it the frequent administration of high concentration of drug in the eye leads to systemic side effects. To increase ocular bioavailability and duration of drug action, various ophthalmic vehicles such as viscous solutions, gels, ointments or polymeric inserts have been used (Mitra, 1993). Although the corneal contact time has been increased by these vehicles but they have not been widely accepted because of blurred vision (in the case of ointments) and lack of patient compliance (in the case of inserts). From the point of view of patient acceptability, a liquid dosage form that can sustained drug release and remain in contact with the cornea of eye for extended periods of time is ideal. Such delivery system consists of phase transition polymers that are instilled in the liquid forms and shifts to the gel phase once in the cul-de-sac of the eye. Three methods have been employed to cause phase transition on the eye surface: change in pH, change in temperature and ion-activation. The polymers mostly used in the in-situ gelling systems are viz. sodium alginate (Cohen et al., 1997), Carbopol and methyl cellulose (Kumar et al., 1994),

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Farah Siddiqui et al: Continental J. Pharmaceutical Sciences 2: 1 - 14, 2008

cellulose acetate phthalate latex (Gurney et al., 1985), poloxamer-407 (Miller and Donovan, 1982) and Gelrite (Sultana et al., 2004) etc. Gelrite is a gellan gum that is a high molecular mass, linear anionic heteropolysaccharide produced aerobically from the bacterium Auromonas (pseudomonas) elodea, renamed Sphingomonas paucimobilis. The polymer backbone is comprised of a tetrasaccharide repeat unit of glucose, glucuronic acid and rhamnose in the molar ratio 2:1:1. It has a characteristic property of temperature dependent and cation-induced gelation. The gelation involves the formation of an ordered state of gellan chains. X-ray diffraction studies have confirmed that a double helix of gellan chains is formed by complexation with cations and hydrogen bonding with water (Sanzgiri et al., 1993). Ofloxacin is a second-generation flouroquinolone derivative used to treat bacterial infections in the eye especially conjunctivitis caused by Gram negative bacteria. It affects bacterial DNA- gyrase without affecting mammalian DNA activity. The topical ophthalmic dose of ofloxacin is 2-3 drops of a 0.3% (w/v) solution in the affected eye(s) every 4 hour or at every hour in case of severe infection (Reynolds, 1989). The main aim of the present work was to develop an ion-activated in-situ gelling ocular formulation of ofloxacin. Gelrite was used as vehicle for the development of the formulation that could provide sustained release of the drug for 12 hours.

MATERIALS AND METHODS Materials Ofloxacin (Courtsey, Cadila, Ahmedabad, India), Gelrite (Courtsey, Kelco division of Merck, USA), Propyl paraben, Methyl paraben and β-Cyclodextrin (S.D. fine. Chem. Ltd, Mumbai, India), Tris maleate buffer (Scientific Delhi, India). Other formulation excipients were pharmaceutical grade and obtained from standard commercial suppliers.

Preparation of formulation (A) Preparation of in-situ gelling system containing free drug The polymer solutions were prepared by dispersing the polymer (0.3, 0.4, 0.5, 0.6, and 0.7 g) in Tris maleate buffer at pH 6.0 and stirring at 35°C for 24 hours. 0.27%, (w/v) of ofloxacin was added to it and mixed. The preservatives propyl paraben 0.01% and methyl paraben 0.05% were added to it and the formulations were made up to volume with Tris maleate buffer at pH 6.0 (Table 1).

(B) Preparation of in-situ gelling system containing complexed drug Preparation of drug and β-Cyclodextrin complex:

The solid inclusion complexes of ofloxacin and β-Cyclodextrin were prepared by freeze-drying. The drug and the polymer were dissolved separately in 25% aqueous ammonia solution. The molar mixture (1:1) was stirred at 30°C at room temperature for 24 hours and then freeze dried for 24 hours. The freeze-dried product was then sieved through 180 micromesh to give the final product. The inclusion complex containing drug, was added to the optimized formulation containing Gelrite 0.5% , propyl paraben 0.01%, methyl paraben 0.05% in Tris maleate buffer of pH 6.0 (The formulation was optimized on the basis of in-vitro release studies).The formulations were packaged in amber coloured glass vials, capped with rubber bungs and sealed with aluminum caps. The formulations in their final pack were subjected to terminal sterilization by autoclaving at 121°C and 15 psig for 20 min. Evaluation of Formulation The prepared formulations were evaluated for the in-vitro release studies and drug content was analyzed by UV spectrophtometry at 288 nm (Double beam UV spectrphotometer, Hitachi-110, Japan), viscosity studies (by Cone and Plate Viscometer, Physica Rheolab, Australia), clarity by visual observation against a black and white background in a well-lit cabinet, Osmolarity was determined by osmometer (Fiske Associates USA), refractive index was checked by Abbe’s Refractrometer (Scientific India), pH (Hanna Instruments), antimicrobial studies to

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Farah Siddiqui et al: Continental J. Pharmaceutical Sciences 2: 1 - 14, 2008 find out the sustained release of the drug from the prepared formulations, interaction studies and of course the stability study by HPTLC method.

In-Vitro Release Studies The in-vitro release of ofloxacin from the formulations was studied by flow through apparatus fabricated in laboratory (Ali and Sharma, 1992). The dissolution medium was Simulated Tear Fluid (STF) of pH 7.4. The in-situ gel forming system was converted to gel upon addition of STF to it. The gel containing 2.7 mg of the drug was added to the jacketed flow through cell. 125 ml of the STF of pH 7.4 was placed in the flask and 1ml sample was withdrawn at regular intervals and replaced with fresh buffer (STF). The buffer was allowed to flow through the artificial eye of the flow through apparatus by using the peristaltic pump. The flow was regulated with flow regulator to 10 drops per minute (~0.4 ml per min.). To compensate blinking of the eye, air bubbles were blown in the artificial eye through an aerator. The content of the flask was continuously stirred with the help of a magnetic stirrer. The whole assembly was maintained at 37±0.5°C by the circulation of warm water through the jacket. The water from the water bath maintained at 37±0.5°C was circulated through the flow through cell and then through flask and finally to the sink. The aliquots were diluted with STF and analyzed by UV spectrophotometer at λmax 288 nm.

Rheological Studies The viscosities of the solution and the gel formed were determined by cone and plate viscometer using MK-22 spindle. For viscosity determination, 1ml of the sample was placed on the plate and spindle was touched with the sample, temperature was adjusted to 25°C and system was started. Data were obtained and the graphs were plotted between shear stress and shear rate of the formulations. Antimicrobial Efficacy Studies The microbiological studies were carried out to ascertain the biological activity of ophthalmic sol-to-gel system against microorganisms. This was done by agar diffusion test method employing cup-plate technique. Marketed eye drops (Standard solutions) of the drug and the developed formulations containing free and complexed drug (Test solutions) were poured into cups made by sterile borer into sterile nutrient agar previously seeded with test organisms (S. aureus, P. aeruginosa, E. coli). After allowing diffusion of the solutions for 2 hours the agar plates were incubated at 37°C for 24 hours. The zone of inhibition (ZOI) in mm measured around each cup was compared with that of control. The entire operation except the incubation was carried out in a laminar flow unit. Each solution was tested in triplicate. Both positive and negative controls were maintained throughout the study.

Interaction Studies Interaction studies were done in order to investigate any interaction between drug and excipients and to study the effect of sterilization. The interaction studies were conducted by UV, FTIR and DSC methods. UV Scanning Ophthalmic gel before and after sterilization were dissolved in STF at pH 7.4. The solutions were filtered through Whatman filter paper no.42 and solutions were scanned. UV spectrophotographs recorded were taken as quantitative in order to assess the changes in peaks, pattern of curves etc.

FTIR Studies FTIR spectra of ophthalmic gel were compared with the spectra of pure drug. Spectra of drug and polymer were taken and analyzed for any major interaction. These were done qualitatively in order to assess the pattern of peaks and for comparison purpose. The FTIR spectra of the polymer and drug with polymer, β-Cyclodextrin and drug with β-Cyclodextrin were taken also.

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Farah Siddiqui et al: Continental J. Pharmaceutical Sciences 2: 1 - 14, 2008 DSC Studies DSC of the samples was performed using DUPONT model 910 (USA) systems. DSC of pure drug, polymer and polymer drug mixture were done for ophthalmic formulations. DSC of β-Cyclodextrin and mixture of β-Cyclodextrin with drug were taken for the ophthalmic gel. The samples were taken in solid state in the pan and were compressed with high-pressure press. All the samples (5 mg each) were treated at inert nitrogen atmosphere by oxidation method at the heat rate 10°C/min, flow of gas 35 cc/min and temperature range 50-350°C.

Stability Studies Stability studies were carried out as per ICH guidelines. The drug content in the package was analyzed by HPTLC (Srividya et al., 2003). Standard curve of ofloxacin was prepared using methanolic solution of ofloxacin in concentration range of 300-1000 ng/mL. Peak area versus drug concentration was treated by linear least square regression analysis.

Standard curve of ofloxacin by HPTLC The n-butanol: methanol: strong ammonia: water (4:1:0.9:1.3) system gave the most compact spots as well as separated the degraded products from the pure drug, this system was chosen as the mobile phase. The optimized formulation was then kept at condition of 75±5% relative humidity. Whole assembly was kept inside a hot air oven at temperature 40± 2°C for 90 days and samples were again analyzed after storage for any degradation. RESULTS AND DISCUSSION Selection of formulation ingredients Buffers play a pivotal role in formulating ophthalmic drops. They contribute significantly to chemical stability and clinical response and also influence the comfort and safety of the product hence the importance of selecting a suitable buffer, which ensures product stability and desired drug solubility. The studies in various buffer solution indicate that the drug is soluble in 0.01M Tris maleate buffer of pH 6.0 at the dosage level desired (0.27%, w/v). The solutions were stable at elevated temperature and autoclaving. The marketed eye drops were found a pH of 6.2. It has been reported that the ocular penetration of levofloxacin, the (-) isomer of ofloxacin is maximum at around pH 6.5.Tris maleate buffer; pH 6.0 was therefore selected as the vehicle for the formulated eye drops. Table 1: Formulae of the sol to gel systems containing free drug and complexed drug

S. No. Ingredients Amount (g) A B C D E C-Beta

1 Ofloxacin 0.270 0.270 0.270 0.270 0.270 ----- 2 Gelrite 0.30 0.40 0.50 0.60 0.70 0.50 3 Propyl paraben 0.01 0.01 0.01 0.01 0.01 0.01 4 Methyl paraben 0.05 0.05 0.05 0.05 0.05 0.05 5 Tris maleate

Buffer pH 6.0 (ml) 100 100 100 100 100 100

6 Complexed (Ofloxacin β-Cyclodextrin)

__ __ __ __ __ 1.243 g equivalent to 0.270 g of drug

Gelrite is a polysaccharide low acetyl gellan gum, which form clear gel in the presence of mono/divalent cations (Rozier et al., 1989). The pH of aqueous solution of Gelrite is neutral. Propyl paraben and methyl paraben were used as preservative in the concentrations 0.01 % (w/v) and 0.05 % (w/v) respectively.

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Preparation of Formulations

The two main prerequisites of an in-situ gelling system are viscosity and gelling capacity. The formulation should have an optimum viscosity that will allow easy instillation into the eye as a liquid (drops) which would undergo a rapid sol-to-gel transition triggered by the presence of cation in the tear fluid. Additionally the gel formed in-situ should preserve its integrity without dissolving or eroding for a prolonged period of time. Table1 shows the formulae of sol-to-gel system containing free drug and in situ gelling system containing complexed drug. The contribution of each ingredient to the osmotic pressure of the formulation was calculated in the concentration used in the terms equivalent to sodium chloride. Since the ingredients themselves contributed to the tonicity, no tonicity adjusting agents were added. Evaluation of Formulation The drug content was analyzed by UV spectrophtometry at 288 nm (Double beam UV spectrphotometer, Hitachi-110,

Japan), clarity by visual observation against a black and white background in a well-lit cabinet, viscosity studies (by Cone and Plate Viscometer, Physica Rheolab, Australia). Osmolarity by osmometer (Fiske Associates USA), refractive index with Abbe’s Refractrometer (Scientific India), and pH was determined by Hanna Instruments. And all of these physicochemical parameters of the formulations were found to be satisfactory as shown in Table 2. And both the optimized formulations were evaluated for the same parameters as mentioned above, after storage and the results were found to be satisfactory as shown in Table 3.

Table 2: Evaluation of Formulations

Formulations Clarity Drug content (%, w/v)

pH Ref. Index

Osmolarity (mosmol)

Surface Tension (mN/m)

Viscosity (cps) sol

“C” Clear solution

98 6.5 1.352 317 39 14800

“C-Beta” Clear solution

97 6.5 1.361 324 38 13700

Marketed preparation.

Clear solution

100 6.2 1.4 319 42 -------

The formulations were liquid at room temperature and it underwent rapid transition into the gel phase upon contact with the cations present in simulated tear fluid (STF) of pH 7.4. Terminal sterilization by autoclaving had no effect on the pH, gelling capacity and viscosity of the polymer Gelrite. Gelrite is only polymer, which is capable of standing high temperature of autoclaving without loss of its functional properties and transparency.

Rheological Studies The viscosity studies were done and the formulations “C’ and “C-Beta” were selected as optimized formulations for further studies, as they posses optimum viscosity. The formulations exhibited pseudoplastic rheology, as evidenced by shear thinning and an increase in the shear stress with increased angular velocity that can be observed in the Figures 1, 2 and 3. The viscosity was directly dependent on the polymeric content. No change in the viscosity of the formulations was observed after autoclaving. The desirable conversion of sol to gel was obtained on addition of STF (pH 7.4). It was supported by viscosity studies as shown in Figures 1, 2 and 3. The administration of ophthalmic preparations should influence as little as possible the pseudo-plastic character of the pre-corneal film (Ooteghem, 1993). Since the ocular shear rate is very high, ranging from 0.03 s–1 during inter-blinking periods to 4250–28500 s-1 during blinking (Bothner et al., 1990), viscoelastic fluids with a viscosity that is high under the low shear rate conditions and low under the high shear rate conditions are preferred for ophthalmic drug delivery.

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Farah Siddiqui et al: Continental J. Pharmaceutical Sciences 2: 1 - 14, 2008

(a) (b) Fig. 1 Rheogram of formulation “C” at 25 ºC (a) and with STF at 37 ºC (b)

(a) (b) Fig. 2 Rheogram of formulation “C-Beta” at 25 ºC (a) and with STF at 37 ºC (b)

Fig. 3 Effect of shear rate on viscosity of the formulation “C” and “C-Beta” with STF at 37 ºC

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Farah Siddiqui et al: Continental J. Pharmaceutical Sciences 2: 1 - 14, 2008 Antimicrobial Efficacy Study This study indicated that ofloxacin retained its antimicrobial efficacy in both the optimized formulations as shown in Fig. 4 and the formulations showed controlled release of the drug for 24 hrs.

Fig. 4 Zone of inhibition (mm) by the formulations “C” and “C-Beta”.

In vitro Drug Release Studies In-vitro drug release conditions were simulated to those, which are likely to be encountered in the eye.The cumulative percent of ofloxacin release from the free drug and complexed drug are shown in Fig. 5, complexation with β-Cyclodextrin alter the release profile of ofloxacin from in-situ gelling system. The results clearly showed that the gels have the ability to retain Ofloxacin in its matrix network and that the premature drug release will not occur.

Fig. 5 Release profiles of ofloxacin from sol to gels A, B, C, D, E and C-Beta each point represent the mean ± S.D, n = 3

To study the drug release mechanism, the release data were fitted to the general exponential function: Mt/M0 = ktn where M0 is the initial amount of drug (amount of drug released at time zero) and Mt is the amount of drug released at time t, n is a diffusion exponent characteristic of the release mechanism, and k denotes the properties of the polymer and the drug (Martin, 1993). According to Brazel and Peppas, 2000, this equation has been frequently used in the literature owing to its utility in describing the relative importance of Fickian (n = 0.5) and non-Fickian,

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Farah Siddiqui et al: Continental J. Pharmaceutical Sciences 2: 1 - 14, 2008 anomalous diffusion (n = 1.0). If the exponent n is 0.5, the drug release is represented by a square root equation; if n = 1, the fraction release is zero order. Values of n greater than 0.5 indicate anomalous diffusion, generally due to the swelling of the system in the solvent before the release takes place.

However, in vitro drug release conditions are completely different from those occurred in eyes, but the results showed that “C” and “C-Beta” had the ability to retain the drug for 12 hrs.

Stability Studies Stability studies by HPTLC showed that degradation product has Rf value of 0.43 and regressed value is found to be 0.98222 with Sdv is equal to 8.23. No interference was found from the excipient present in formulation. Thus, HPTLC method indicates stability of the packed product shown in Fig. 6.

(a) (b)

Fig. 6 Calibration curve (a) Stability Study curve by HPTLC (b)

Table3: Comparison of physical characteristics of ophthalmic gels “C” and “C –Beta” after storage

S.

No

PHYSICAL CHARACTERISTICS

Formulations Clarity pH Refractive index Surface tension (mN/m)

Osmolarity (mosmol)

Initial Final Initial Final Initial Final Initial Final Initial Final

1 C Clear solution 6.5 6.5 1.352 1.351 39 39 310 310 2 C-Beta Clear solution 6.5 6.5 1.361 1.362 38 38 317 317

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Farah Siddiqui et al: Continental J. Pharmaceutical Sciences 2: 1 - 14, 2008

Fig. 7 Release of drug in formulations “C”, “C-Beta” before and after storage Cumulative release of drug from both the optimized formulations before and after storage were performed as shown in Fig. 7 and it was observed that there were no change in the amount of drug released for 12 hrs.

Interaction Studies on ophthalmic gels

Interaction studies were done in order to investigate any interaction between drug and polymer as well as to study the effect of sterilization.

UV Scanning Ophthalmic gels before and after sterilization was dissolved in STF at pH 7.4. The solutions were filtered through Whatman filter paper no.42 and solutions were scanned. UV spectrophotographs recorded were taken as qualitative in order to assess the changes in peaks, pattern of curves etc and it was found that there were no changes in the peak patterns.

Fig. 8a FTIR of ofloxacin

IR Studies IR spectra of ophthalmic gel were compared with IR spectra of pure drug. IR spectra of drug and polymer is taken and analyzed for any major interaction. These were done qualitatively in order to assess the pattern of peaks and for comparison purpose. The IR spectra of the drug (Fig. 8a), polymer (Fig. 8b) and drug with polymer (Fig. 8c)

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Farah Siddiqui et al: Continental J. Pharmaceutical Sciences 2: 1 - 14, 2008 are taken. The IR spectra of the β-Cyclodextrin (Fig. 8d) and drug complexed with β-Cyclodextrin (Fig. 8e) are also taken.

Fig. 8b FTIR of polymer Gelrite

Fig. 8c FTIR of drug with polymer The pattern of peaks of drug (Fig. 8a) and drug with the polymer (Fig. 8c) are almost same and they differs from that of peaks obtained due to polymer alone (Fig. 8b), this showed that there were no any interactions between drug and polymer present in formulation “C”.

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Farah Siddiqui et al: Continental J. Pharmaceutical Sciences 2: 1 - 14, 2008

Fig. 8d FTIR of β-Cyclodextrin

Fig. 8e FTIR of drug complexed with β-Cyclodextrin

The pattern of peaks of drug (Fig. 8a) are same as those of drug complexed with β-Cyclodextrin (Fig. 8e) and they differs from that of peaks obtained by β-Cyclodextrin alone (Fig. 8d), this indicated that there were no any interactions between drug and β-Cyclodextrin present in formulation “C-Beta”.

DSC Studies DSC endotherms of pure drug (Fig. 9), polymer (Fig. 10a) and polymer with drug (Fig. 10b) were done for ophthalmic formulations. DSC endotherms of β-Cyclodextrin (Fig. 11a) and β-Cyclodextrin complexed with drug (Fig. 11b) was taken for the ophthalmic gel. The samples were taken in solid state in the pan and were compressed with high-pressure press. DSC of the samples was performed using DUPONT model 910 (USA) systems. All the samples (5 mg each) were treated at inert nitrogen atmosphere by oxidation method at the heat rate 10°C/min, flow of gas 35 cc/min and temperature range 50-350°C.

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Farah Siddiqui et al: Continental J. Pharmaceutical Sciences 2: 1 - 14, 2008

Fig. 9 DSC endotherm of drug (ofloxacin)

(a) (b) Fig. 10 DSC endotherm of polymer (a) and polymer with drug (b)

Comparison of DSC endotherms of drug, polymer and polymer with drug showed almost similar peaks which indicated that there was no any interaction between drug and polymer present in the formulation “C”.

Fig. 11 DSC endotherm of β-Cyclodextrin (a) and drug complexed with β-Cyclodextrin (b)

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Farah Siddiqui et al: Continental J. Pharmaceutical Sciences 2: 1 - 14, 2008

Comparison of DSC endotherms of β-Cyclodextrin showed some peaks as shown in (Fig. 11a) and when drug was complexed with β-Cyclodextrin as shown in (Fig. 11b), there were no any characteristic peaks of either one, indicated that the drug was completely entrapped within the β-Cyclodextrin molecules and there was no any interaction between drug and β-Cyclodextrin present in the formulation “C-Beta”. CONCLUSION The gellan gum (Gelrite) based in situ gelling systems containing ofloxacin, a broad spectrum antibacterial agent used in the treatment of ocular infections and various concentration of Gelrite for ocular administration have been prepared and evaluated successfully. The gel formed in situ afforded sustained drug release over an 12 hr period. The optimized formulations “C” and “C-Beta” showed satisfactory viscosity, release behavior and antimicrobial quality. Under the conditions 75±5% relative humidity and at 40± 2°C temperature, the dosage forms were stable for a period of 3 months. REFERENCES Ali, A., and Sharma, S. N., (1992) Fabrication of a flow through apparatus for in-vitro determination of drugs from ophthalmic preparations. Indian Drugs. 29, 157-160. Bothner, H., Waaler, T., Wik, O., (1990) Rheological characterization of tear substitutes. Drug Dev. Ind. Pharm. 16, 755–768. Brazel, C.S., Peppas, N.A., (2000) Modelling of drug release from swellable polymers. Eur. J. Pharm. Biopharm. 49,47–58. Cohen, S., Lobel, E., Trevgoda, A., Peled, Y., (1997) A novel in-situ forming ophthalmic drug delivery system from alginates undergoing gelation in the eye. J. Control. Rel. 44, 201-208. Gurney, R., Boye, T and Ibrahim H., (1985) Ocular therapy with nanoparticulate systems for controlled drug delivery. J. Control. Release, 2, 353-361. Kumar, S., Haglund, B.O., (1994) Himmelstein, K.J., In-situ forming gels for ophthalmic drug delivery. J. Ocul. Pharmacol.Ther. 10, 47-55. Martin, A., (1993) Coarse Dispersions, in Physical Pharmacy (Ed. G. H. Mundorff), Lea & Febiger, Philadelphia 496–497. M.M.M.Van ( 1993) Ooteghem. Formulation of ophthalmic solutions and suspensions. Problems and advantages. In: Edman, P (ed) Biopharmaceutics of ocular drug delivery. CRC press, Boca Raton Miller S.C. and Donovan M.D. (1982) Effect of Polaxamer-407 gel in the miotic activity of Pilocarpine nitrate in rabbits. Int.J.Pharm. 12,147-152. Mitra, A. K., (1993) Ophthalmic Drug Delivery Systems, Marcel Dekker, New York. Reynolds, J. E. F., (1989) Martindale, The Extra Pharmacopoeia, 29th Edition, The Pharmaceutical Press, London, 276-277. Rozier, A., Mazuel, C., Grove, J., Plazonnet, B., Gelrite, (1989) A novel, ion activated, in-situ gelling polymer for ophthalmic vehicles. Effects of bioavailability of timolol. Int. J. Pharm. 57, 163-168.

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Farah Siddiqui et al: Continental J. Pharmaceutical Sciences 2: 1 - 14, 2008 Sanzgiri, Y.D., Maschi, S., Crescenzi, V., Crescenzi, V., Callegaro, L., Toppa, E.M., Stella, V.J., (1993) Gellan based systems for ophthalmic sustained delivery of methylprednisolone. J. Cont. Rel. 26,195-201. Srividya, B., Cardoza, R. M., Amin, P. D., (2003) Stability indicating HPTLC method of analysis of ofloxacin. Indian Drugs. 40, 41-43. Sultana, Y., Jha, M.C., Ali, A., Aqil, M., ( 2004) A three-way comparative study on the efficacy of twin sol to gel systems and marketed eye drops of pefloxacin mesylate. J. Ocul. Pharmacol. Ther. 20 363-371.

Received for Publication: 27/03/2008 Accepted for Publication: 12/06/2008 Corresponding Author: Nayyar Parvez Department of Pharmaceutics, Advanced Institute of Pharmacy, Palwal, Distt. Faridabad, Haryana, India.

Email: nparvez1975@yahoo.co.in

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Continental J. Pharmaceutical Sciences 2: 15 - 26, 2008 ©Wilolud Online Journals, 2008.

A SURVEY OF ETHINOPHARMACOLOGY OF SINGLE HERBAL PREPARATIONS OF MEDICINAL

PLANTS IN ASENDABO DISTRICT, JIMMA

Nayyar Parvez 1 and Suman Yadav2 1. Department of Pharmaceutics, Advanced Institute of Pharmacy, Palwal, Distt. Faridabad, Haryana, India. Email:

nparvez1975@yahoo.co.in, 2. Department of Chemistry Advanced Institute of Technology, Palwal, Distt. Faridabad, Haryana, India.

ABSTRACT Medicinal plants have been used to prevent and treat various health problems. Several African and Asian nations are now encouraging traditional medicines as an internal component of their public health care programs. Indigenous medicines are relatively inexpensive and locally available and readily accepted by local population. Ethiopia has an enormous resource of plant species that are used in Traditional medicine. Among the 7000 higher plants species that are known to exist about 800 of them are employed in the traditional health care and 60% of pants are said to be indigenous with their healing potential. Majority of Ethiopians depend on medicinal plants as their only source of health care especially in rural areas where access to villages is lacking due to absence of vehicular roads. Vast knowledge on the traditional uses of these plants is not fully documented and most of the knowledge is conveyed from one generation to the text generation through words of mouth, especially in courtiers like Ethiopia. Thus the present survey is undertaken to explore the traditional systems of medicine in Asendabo region of Ethiopia. The survey was conducted from February 28 - May 5, 2006.

KEYWORDS: Ethiopia, Medicinal plants, WHO report, Traditional medicines, formulation

INTRODUCTION

Majority of the people on this world still rely on traditional medicines for their everyday health care needs. If is also a fact that one quarter of the medicinal prescriptions are formulations based on substances derived from plants or plant derived synthetic analogs and according to the WHO report 80% of the worlds population, primarily those of developing countries, rely on plant derived medicines for their health care. Peoples who use traditional remedies may not understand the scientific rationale behind their medicines, but they know from personal experience that some medicinal plants can be highly effective if used at therapeutic doses (1).

People in industrialized countries were seeking herbal medicine because of the fear of severe adverse effects of modern medicine. People believed that plant remedies used for mediation are less toxic than modern medicines, more over they seek plant remedies to satiety their high demand on secondary plant metabolites in drug discovery (2)

.In sub-Saharan Africa, thousands of kilograms of medicinal plants are collected and used by healers for treatment of different human and live stock diseases. (3) Ethiopian TM is composed of a number of skills such as use of plants, animal products and minerals as well as magic and suppression. It is also true in urban population, for instance, in Addis Ababa, where modern health service is relatively better; a significant percentage of the population has been using TM Social belief and various socio cultural reasons in Ethiopia made majority of the people continue to rely on indigenous remedies. The indigenous knowledge about many of medicinal plants has justified its existence by the biomedical benefits that have been established through observations of generations of people. This is demonstrated by the history of modern drug discovery from plants, which were employed in TM in other countries such as China and India (4).

Ethiopia has an enormous resource of plant species that are used in TM. Among the 7000 higher plants species that are known to exist about 800 of them are employed in the traditional health care and 60% of pants are said to be indigenous with their healing potential (4, 5).Majority of Ethiopians depend on medicinal plants as their only source of health care especially in rural areas where access to villages is lacking due to absence of vehicular roads.

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Nayyar Parvez and Suman Yadav: Continental J. Pharmaceutical Sciences 2: 15 - 26, 2008

Provision of modern health care through the construction of new hospitals, health centers and health posts, imported drug supplies and training of doctors and nurses are of little value at present time to the majority of the rural population (in excess of 40 million people). Medicinal plants and knowledge of their use provide a vital contribution to human and live stock health needs through out the country (6).

The increasing demand by the industrial courtiers for herbal remedies has put increasing pressure on the supply of raw materials available in developing countries. The enormous demand for medicinal plants is generally met by indiscriminate harvesting of the natural flora. As result many useful indigenous plant species are, therefore gradually disappearing due to deforestation and over consumption. Like many other countries cultivation of medicinal plants is not yet wildly practiced in Ethiopia. This scarcity of medicinal plant species causes traditional healers long distance to go for collection (7). Vast knowledge on the traditional uses of these plants is not fully documented and most of the knowledge is conveyed from one generation to the text generation through words of mouth, especially in courtiers like Ethiopia. The danger of losing valuable information is thus high considering the increasing acculturation, mobility and displacement of communities due to several factors, Moreover traditional healers have passed on their knowledge only to the members of their own family or apprentice considered to be “elect” under “oath” this practice of secretive transfer of information accompanied with the negligence of contemporary generation due to expansion of modern education and to some extent modern medicine has left traditional healers in a condition where they could hardly find successors(8). Like many other Ethiopians, the Asendabo people use medicinal plants for their primary health care. Ethinopharmacologically, these people have remained unexplored and there is no comprehensive accounting of their traditional medicinal practices. As it is happening else where in the country, both the traditional knowledge and plants utilized by these people are under threat due to the aforementioned reasons. Plants have formed the basis of sophisticated traditional medicinal systems that have been in existence for thousands of years and continue to provide mankind with new remedies. The modes of therapy of these herbal remedies are based on empirical of findings. Natural products and their derivatives represent more than 50% all the drugs clinically used in the world and higher plants contribute not less than 25% of the total natural products (9).A survey of medicinal plants in Indonesia was done in three tribes and 182 medicinal plants were collected for 45 health problems in Melayu tribe, 110 in Talang Mak for 58 cases and 101 medicinal plants for 54 cases in Anak Dalam tribe. The leaf part of the plant mostly used for preparation of remedies and delivers the active ingredients. Usually the leaves were boiled with water and taken orally, accounting for 199 traditional medicinal plants (10).80% of Mali population used traditional medicinal plants as their only source of medicine. Official medicinal attention is usually based on commercial drugs that have to be purchased with money while TM consultancy has a much lower cost. The majority of the remedies were prepared in the forms of decoction that account (65%), followed by infusion (13%) in which powders are used and maceration (11%) where roots are employed for preparation. Also, it was reported that most of the remedies were taken orally (11). Thus the present study was undertaken to study important medicinal plants used as single herbal prescription in Asendabo district of South West Ethiopia.The survey was carried out from Feb 28 May 05-2006. METHODS AND MATERIALS Study area and period. Asendabo District is one of the Districts in Jimma Zone. It is 50 km away from Jimma town and 291km south West of Addis Ababa. It has 9 kebeles with total area of 1589.4 km2, It has four climatic zones: wurch (0.03%.), dega (23.4%.), woyna dega (62.72%) and kola (13.8%), with the altitude ranging from 880-3344 meters above sea level; annual rainfall ranging from 900-6000mm and temperature ranging from 27—32oC .The topography consists of mountains (12%), hills (20%) and plaints (68%). Fifteen percent of area is covered by forest and 85% is used for agriculture (12). Study design Across-Sectional study was conducted to document indigenous Knowledge on medicinal plants. A structured questionnaire was used to collect ethinopharmcological information from healers.

17

Nayyar Parvez and Suman Yadav: Continental J. Pharmaceutical Sciences 2: 15 - 26, 2008

Population Source population People who live in Asendabo District of Jimma Zone.

Study population All healers in Asendabo District.

Sampling and sampling technique. All healers in the district were included in the survey. The healers were identified with help of community leaders and administrators of the kebeles.

Study variables Independent variables

- Age - Sex - Ethnicity - Religion - Education status - Economic status

Dependant variables

-Use of medicinal plants

- Plant parts used

- Formulation (method of preparation)

- Route of administration

- Disease treated.

Data Collection This Ethinopharmacological study was conducted among the healers about their knowledge on the medicinal plants and mode of practice by using a well-structured questionnaire, which was prepared in English and translated to Amharic at the time of interview. The questionnaire had the following components: Socio-demographic data, diseases treated by the healers, years of experience source of knowledge, information on medicinal plants such as parts of plant used and plant habitat methods of preparation. Specimen of the medicinal plants were collected and pressed for identification and documentation. Before data collection, the questionnaire was pre tested and necessary corrections were made. Quality control of Data The quality of data was assessed by checking the performances of data collectors in filling questionnaires with legible and easy words, completeness, and accuracy by the principal investigator. If there would be omission of questions and incompleteness, it was corrected during data clearance. Moreover the healers were interviewed twice in order to get more reliable information .In addition the principal investigator (PI) was visited some of the healers to be sure on the interviewers’ responsibility. RESULTS The study was carried out successfully.The socio-demographic characteristics of traditional healers is shown in Table 1 In the survey a total of 40 medicinal plants were collected. Majority of them were identified and few were not identified. The identified species were distributed in 42 families of which Solanaceae, Antiraceae, Euphobioniceae, labiatae, Ranunculaceae , Rutaceae, Verbenaceae, and Acanthaceae were the major families used for herbal preparation.The result is shown in table 2

18

Nayyar Parvez and Suman Yadav: Continental J. Pharmaceutical Sciences 2: 15 - 26, 2008 Table 1: Socio-demographic characteristics of traditional healers in Assendabo District.

Background characteristics Frequency Percent Sex

Male Female

7 4

63.6% 36.3%

Age 30-39 40-49 50-59 >60

2 5 3 2

16.6% 41.6% 25% 16.6%

Ethnicity Oromo Dawaro Amhara Kullo

6 2 4 1

42.8% 16.6% 28.5 7.14

Religion Muslim Christian

8 3

72.7% 27.2%

Educational status Illiterate Church education Literacy campaign Grade 1-6 Grade 7-8

7 5 3 2 1

38.8% 27.7% 16.6% 11.1% 5.5%

Occupational status Farmers Merchant Government employee

10 5 1

62.5% 31.25% 6.25%

Years of experience 5-10 11-15 16-20 24-25 >25

4 2 5 2 1

28.57 14.28 35.75 14.28 7.14

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Nayyar Parvez and Suman Yadav: Continental J. Pharmaceutical Sciences 2: 15 - 26, 2008 Table 2: Plant used as single herbal prescription in Asendabo district

Vernacular Scientific name Family name Indications & No

of satiations Part used Preparations and administration

Reje O

Veronica auriculifera Hein

Asteraceae

Chancroid (1) Wound (2)

W L

Pounded plant is macerated in water and the filtrate drunk. The leaves are squeezed and the juice applied on the wound.

Armagusa O Ajuga integrifolia Ham.Buch

Lamiaceae Diarrhea (3) Jaundice(ye’mariyam-mekent) (2)

W Whole plant is pounded: socked in hot water and the filtrate drunk

Dumuga0 Justilia schimperand (Hochst ex Nees) T. Alnder.

Acanthacea Goha-besheta (1) L The fresh leaves are squeezed and the juice taken: the residue is rubbed on the genitalia

Kelala 0 Stephania abyssinica (Dillonet A. Rich) walp

Menis permaceae T.Corporise (1) W The plant is pounded and applied to whole body.

Catto0 Albizia schimperiaha oliu . Fabaceae Facial fungus (chirete) (2) L The dried powdered leaves are mixed with better and applied on the affected area

Papaya A Cerica papaya Caricaceae Ameba (3), gardia (3), Malaria (6)

S L

The seeds are pounded and the powder mixed with honey and taken. The fresh leaves are socked in warm water and the filtrate taken.

Kobbo0 Ricins communis

Euphorbiaceae

Ameba (2)

R S

The fresh root is pulverized and mixed with garlic and honey and taken. The seeds are chewed and swallowed.

Ye’mariyam A mekent

Juandice(2) L The dried leaves are pounded and the powder dispersed in cup of tea and taken.

Baruda0 Tonsilitis (2) R The fresh roots chewed and the juice is for garglation.

(Note; L-leaf, R- root B-bark, s -seed, St-stem, La-latex, F –flower, Bb-bulb, O-Afan oromo, A-Amharic

20

Nayyar Parvez and Suman Yadav: Continental J. Pharmaceutical Sciences 2: 15 - 26, 2008 Table 2 continued Harbu O Ficussur foresee Moraceae Eczema (chiffea )(2) La The fresh latex of the leaves are applied on the affected

Damakese A Ocimum lamiifolium Labiatae Uv-skin reaction (mitchi)(4)

L The leaves are socked in warm water and the filtrate taken: the vapor for fumigation.

Ye’seythen kill A Lagenarin abyssinica (Hoof.f) c.Jeffery.

cucurbitaceae Epilpsy(ye’methele besheta (2)

L The leaves are squeezed and the juice applied to nose: the residue is subbed on the nose.

Lagiya O

Tuberculosis (ye’sameba-nekersa) (2)

R The dried roots are powdered and the powdered dispersed in to cup of tea and taken

Rabies (ye’wusha beshea (2)

R The dried roots powdered and the mixed with barley powder and water is poured in to the powder to make moist mast.

Botetesa Hemorriod (kintarote) (3) R The fresh roots are pounded and mixed with honey and taken.

Mareyata A Dodonea angusti folia L.f. Sapinaceae (madiate) (2) W The plant is pounded and applied on the face. Oumugulian Gonneria (chebete) (1) S The dried seed are pounded the powder is dispersed in

water and taken. Hiddao Clematis hirsute perr and

Gull Ranunlaceae Ameba (2) R The dried seeds are pounded and the powder is

dispersed in water and taken.

Table 2 continued zingibleA Zingier ofifcinal ross Zingiberaceae Abdominal cramp (Hode-

kurthet) (3) Rh The fresh rhizomes are pounded and macerated in

warm water and the filtrate Taken.

Shenfa o Lepidium satvuim. Cruciferceae Renal diseases (1)

The powdered seeds are mixed with honey and taken.

Oricha-ferengy o Gonnerria(1) S The dried seeds are powered, dispersed in water and taken.

Chkugn o Artemisa abyssinica Schtz Bipex Rich

Compositceae Evil eye(2) S The seeds are powered and dispersed in tea and taken.

Tufo o Ageratum conyzoides Asterceae Eipithaxis (ye’ne ser)(3) L The pounded leaves are applied on the nose.

Ludetta o

Goitor (1)

The fresh-pounded plant part is squeezed and the juice taken: the residue is rubbed on the neck.

21

Nayyar Parvez and Suman Yadav: Continental J. Pharmaceutical Sciences 2: 15 - 26, 2008 Table 2 continued

Dechi Merecha o Evil eyes (2)

(ye’buamedanite) L The fresh leaves are squeezed and the juice is diluted

with water for drink.

Ye’ feres-zeng A Otostegia inter grifolia Benth

Lamiaceae Ascaris (1) R The powdered dried roots pounded and the juice is taken

Ye’ mider enboyi A Cucumis ficifolius Solanaceae Ya’ menmene besheta (1) R The powdered dried roots are mixed with cow milk

and taken. Tarigum o Epilepsy (1) R&L Fresh pounded leaves and roots are infused in water

for 7 days and taken. Ketetinna o

Verbascum siniaiticum Benth.

Scrophulariaceae. “Mister ye’meyasewota” (1)

F Fresh flower squeezed and the juice is applied on the forehead as a symbol of class.

Ye’shankila A medhanit Wound (2) L The fresh-pounded leaves squeezed and the juice is taken.

Attuch A Verbena officinalis L. Verbenaceeo Hode- kurtete(4) Ameba (3) Back pain (3)

R The fresh leaves are chewed with salt and the juice is taken.

Emboyi A Solanum incanum Solanaceae. Epitasis (3) L The powdered leaves are applied in to nose. Anchabi o Calpurna ourea

(Ait.) Benth Fabaceae Headache (1) L The pounded leaves are lited with fire and the smoke

inhaled. Bottoro o Snake bite (1) St The steams are ground, dispersed in water for drink.

22

Nayyar Parvez and Suman Yadav: Continental J. Pharmaceutical Sciences 2: 15 - 26, 2008 Table 2 continued

Kuttia o Gonorria (chebite)(1)

L The leaves are squeezed and the juice is taken.

BissanaA

Croton macrostachys

Euphorbionceae

T.corporise (3) Wound (2) Ameba (1) Blood coagulant (1)

La L L L

The fresh latex from leaves is applied on the affected area. The apexes of the plants are soaked in warm water and filtrate is taken

Kinin zafe A Hode kurthet(2) Toothache (2)

L The fresh pounded leaves juices poured in to tea and taken.

TosegnA

Thymus serrulatus

Labiatae

Renal diseases (5) Hypertension (1) T.capities (1)

L

The fresh leaves are soaked with warm water and the filtrate drunk. The fresh leaves are macerated in water for three days and the juice is applied on the head.

WanzaA Cordia faricanalam Boraginaceae T.capies (2) Lx The fresh apex of plant is squeezed and the juice is applied on the head.

Berbere A Capsicum annum L. Solanaceae Burn wound (2) L The fresh leaves are pounded and applied to the affected area.

Asaabella o Skin diseases (2) The fresh-pounded seed juice is taken: the residue is rubbed on the skin.

Edndod phytolacca dodecan dra L Herit

Phytolaccaeae Eczema (3) L&F The fresh pounded leaves and flower juice is applied on the affected area.

23

6.7%

49.4%

16.85%13.5%

2.25% 2.25% 2.25% 2.25% 2.25% 2.24%

0

10

20

30

40

50

60

leaf root seed wholeplant

stem bark flower latex apex other

Plant part

Per

cen

tag

e

Figure 1: Comparison of plant parts used as a source of medication In Asendabo District As indicated in figure 1 the most widely sought after plant part for preparation of remedies in the area were leaves followed by root and seed. Majority of the remedies were prepared in form of juice from freshly collected plant parts .The juice is usually prepared by pounding or crushing the plant parts by using traditional miller. Some remedies were prepared by squeezing the fresh plant and water was mostly used to dilute the juice. The majority of the preparations were of fresh plants and few remedies were prepared from dried and grounded plant parts. Regarding on the route of administration, most of the remedies were administered orally followed by topical application and inhalation/ fumigation.

Oral 63%Topical 28%In helation / fumigation 8.80%

63%

28%

9%

Oral

Topical

In helation /fumigation

Figure 2: Comparison of Route of administration of plant remedies in Asendabo in district

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Nayyar Parvez and Suman Yadav: Continental J. Pharmaceutical Sciences 2: 15 - 26, 2008

46.8%

12.76%10.6%

6.8% 6.3% 4.25% 4.25%2.2% 2.2% 2.1% 2.1%

05

101520253035404550

Wat

er

hone

y tea

salt

butte

reg

g

crop

pow

der

anim

al fa

tm

ilkm

ore

suga

r

Additives

Per

cen

tag

e

Figure 3: Additives used in the preparation of remedies in Asendabo district, Feb 28-May 5, 2005. As indicated in figure 3, water was the most commonly used additive followed by honey, tea, salt and butter in preparation of remedies. Most of the remedies had no measurement of dose where as some were measured by use of cups. Out of 40 types of herbal remedies some remedies had adverse effects; the most commonly occurring were nausea, vomiting gastrointestinal discomfort, diarrhea and fever. Some remedies were contraindicated for pregnant and nursling women. DISCUSSION This survey indicated that family members were the major source of the indigenous knowledge in this District, like other study conducted in Jimma zone. This is due to that the healers kept the knowledge as secrete with in the family members. Most of the remedies were derived from plants sources, but modern drugs and minerals were not yet used as a source of medicine (9). A study in Butajra (2003) indicated that 17.4% herbal medicines and 82.4% modern medicines were used for the treatment of different illness (2). Most of the medicinal plants utilized in this area were harvested from the wild source as in many parts of the country. A study done on Zay people also indicated that most plants were harvested from wild sources and only 6 types of specious are under cultivation (8). Harvesting from wild source decreases the supply of medicines and leads to destruction of potential medicinal plant species.

The most widely sought after the plant parts in the preparation of remedies in the area was leaf part. Colleting of leaves do not pose grate danger to the existence of an individual plant when compared with the collection of under ground parts, stem, bark, or whole plant, According to a study conducted in Shirka district roots were highly used for herbal preparation (7).

Most of the reported preparations in the area were prepared from freshly collected juice by crushing or pounding. Other study conducted in Jimma zone (2003) reported that 62% of the remedies were prepared by boiling. Preparing plant remedies by crushing or pounding is more advantageous than using decoction or concoction, since heat could affect the active constituents of the remedies. Majority of the reported preparations in the area

25

Nayyar Parvez and Suman Yadav: Continental J. Pharmaceutical Sciences 2: 15 - 26, 2008 were drawn from a singe plant and mixtures were used rarely. A study in Seka Chekorsa (2005) reported that 33 prescription remedies were polyherbal and 20 were prepared from single plant species. The use of mixture of plant species in treating particular ailment would be for synergic interaction or potentiation effect of one plant on the other (5). Most of the remedies in the area were administered though oral route. Other study conducted in Jimma zone reported that oral route was the major route of administration (8). In this survey water was the most commonly used additive in the preparation of remedies in many parts of the country. Water is safe and less toxic than other solvents and also it’s lest cost and easy availability may increase its demand for preparation of remedies. Other additives such as honey sugar, tea and salt were used as sweeteners to modify the taste of the remedies; butter and animal fats used as abase (carrier) in the formulation of topical preparations. Most of the remedies had no measurement of dose that is lack of precision in the determination of dose due to this different dose dependant adverse effects were seen. REFERENCES 1. Gurib Fakin A. (2006) Medicinal plants; traditions of yesterday and drugs of tomorrow. Molecular aspects

of medicine) 27: 5-13 2. Gedif T., Han HJ. (2003) Use of medicinal plants for self –care in Butagra central Ethiopia, J

Ethinopharmacol. 87: (155 –161) 3. World Bank (2001) Reconstruction and development Traditional medicine and the bridge to better health,

IK notes: No 35: 113 –115 www. World. Bank org/afr/Jk. Default. 4. Wolde B. and Gebre-Mariam T. (2002) Household herbal remedies for self-case in Addis Ababa. Ethiop.

Pharm.J. 20(1) : 61 –67. 5. Mesfin T., Hunde O., Getachew Y., Tadesse M. (2005) Survey of medicinal plants used for treatment of

human diseases in Seka Chekorsa, Jimma Zone Ethiopia. Ethiop. J Health Sci. 15 (2): 90 –95. 6. Agbovic T., Dennis F., Amponsah K. (2002) Conservation and Sustainale use of medicinal plants, in

Ghana; ethinopharmacology survey. Http// www. unep-wcwc. Org/species/ plants /Ghana. 7. Addis G., Abebe D., Urga K. (2001) Survey of traditional medicinal plants in Shirka District, Arisi Zone,

Ethiopia. Ethio. Pharm. J. 19: 30-34 8. Giday M., Asfaw z., woldu Z. (2002) An ethinobotanical study of medicinal plants used by the Zay people

in Ethiopia. J.Ethinopharmacol. 85:43-52. 9. Abera B. (2003) Medicinal plants used in traditional medicine Jimma Zone. Ethiop. J. Health Sci. 13(2): 86

–90 10. Pramon E. (2002) the commercial use of traditional knowledge and medicinal plants in Indonesia. PP.73-75

www. eisecier /locate/ jeb pharm.

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Nayyar Parvez and Suman Yadav: Continental J. Pharmaceutical Sciences 2: 15 - 26, 2008 11. Inngjerdingen K., Nergard C., Dillo D., mounkore PJ. (2004) An ethinopharmacological survery of plants

used for wound healing in Dogonland. Mali, West Africa .J. Ethinopharmacol. 92(2 –3): 233 –244. 12. Phase II Round I Team Training Students of Jimma University (2005) statistical and epidemiological

report in Asendabo health center (un published). Received for Publication: 27/05/2008 Accepted for Publication: 12/07/2008 Corresponding Author: Dr Nayyar Parvez Department of Pharmaceutics, Advanced Institute of Pharmacy, Palwal, Distt. Faridabad, Haryana. Email: nparvez1975@yahoo.co.in

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Continental J. Pharmaceutical Sciences 2: 27 - 31, 2008 ©Wilolud Online Journals, 2008. ETHNOMEDICINE AND PLANT FOOD IN KALABARI; THEIR PHARMACOGNOSY AND NUTRITIONAL

IMPLICATIONS.

Green Blessing O. Forestry and Environment Department, Rivers State University of Science and Technology, P. B. B 5080. Port Harcourt

ABSTRACT The socio-cultural impact of certain food and medicinal plants among the people of Kalabari (Rives State) was investigated. Twenty one plant taxa were implicated in this study. The finding revealed that plant nomenclature in Kalabari was based on their communal use and relationships among the plants popularly utilized within the community. The myth behind special plants and foods in Kalabari were also elucidated using current literature. KEYWORDS: Ethnomedicine, plant food, Kalabari, Pharmaceutical, Nutrition, Medicinal plants

INTRODUCTION In every culture, there are plants utilized for food and medicine which the orthodox taxonomist and physicians have not replaced (Olorode 1984). The earliest taxonomists were also naturalists who were physicians in their own right. Plants as autotrophs are useful in every sphere of life (Egunyemi 2002). Green, (1995) and Stace (19080) reported that the family Apocynaceace was useful as food (Genus Landolphia) and as medicine ( genus landolphia) and as medicine (genus Rauvolfia) Ogbonda (2002) reported numerous uses of plants including plants as single cell protein. Kalabari people also have those plants that are useful peculiar to them. There are also the exotic plants brought in as they interacted and transacted businesses with new neighbour. Kadans (1970) report that plant food and medicine are the most true and ancient food and medicine. In 1976, World Health organization recognized plant medicine as plants which when used will and generate health in the life of those that use them. Simpson and Ogorzaly (1995) proposed that plants used as medicine and food today were discovered by a perilous process of trial and error and That medicinal knowledge accumulated slowly as it was painstakingly passed on by word of mouth from generation to generation (Mann 1992). The history of medicinal plants started before written history. As early as 2500 BC; Sumerians hard already put up drawings of opium popy on their cave walls, suggesting a good knowledge of medicinal plants. However, a substantial record of medicinal plants came from the code of Hammurabi, caved under the directive of king of Babylon in 1770 BC (Black well, 1990) as Wrangham and Goodall (1989) reported, some medicinal plants were used by men who saw other primates using them. The doctrine of signature where plant structures are related to their function also aided the growth of medicinal plants (Ody 1993). Use of plants as food started with the early men. Zimmer (1991) reported the early use of plant as food and suggested that the establishment of man on earth must had been because he could eat the plants in his environment. Sketches of reports have been seen on useful plants of some Nigerian major tribe. The author seeks to present the folk taxonomy and useful plants of the less known Nigeria tribes; Kalabari being one of them. MATERIALS AND METHODS Plant taxa in this study were personal collection of the author form the various towns and villages that make up the Kalabari clan , Rivers State – Nigeria (Table 1) Eminent persons in culture and dialect of Kalabari were interviewed and the plants use generally accepted by all the people interviewed were reported as authentic.

28

Green Blessing O: Continental J. Pharmaceutical Sciences 2: 27 - 31, 2008 TABLE 1: PLANT TAXA. FOLK NAMES AND STATUS

BOTANICAL NAMES AND COMMON NAMES

FAMILIES

FOLK NAMES

STATUS

Avicennia-Africana managrove swamp taxon Avicenniceae Okopulo Medicine

NewbouIdia Laevis Ageratum conyzoides goat weed

Bignoniaceae Asteraceae

Odumdum Orowidiri

Medicine Sacred

Rryophyllum pinnatum Life plant Grassuloceae Ombusuwadiri Medicine

Solenostemon menostachyus Coctus afer inspid cane

Lamiaceae Costoceae

Mgbediri Okpete First aid Medicine

Acanthus montanus Acanthaceae Oguma Eardrop

Citrus aurantum insipid orange

Rutaceae Aguru elenda Medicine

C.-autantifoiia -lime Rutaceae Olomiri Medicine

Vernonia amygdalina bitter leaf

Asteraceae Pilama Vegetable, Medicine

Xylopia aethiopicum Annonaceae Enyi Spice

Piper guinensis- black Pepper Piperacaeae Ojija Spice

Gongronema latifolium Asclepidiaceae Otaji Spice

Ocimum gratussima- scent leaf Lamiaceae Ekiani Spice

Monodora myristica- Afr. Nutmeg Annonaceae Kukrakam Spice

Acrosticum aruem salt water fern aerum Adiantaceae Imingiye Sacred Plant

Musa paradisiaca- Plantain Musaceae Mbana Food

Colocasia esculentum cocoyam Areceae Iku Food

Dioscorea rotundata yam Dioscoreaceae Buru Food

Elaeis guinensis palm tree Arecaceae Eneme Oil

Baphia nitida chewing stick Papiliionaceae Duko Chewing stick

Fluerya ovalifolic-akubara African stinging nestle Urticaceae Akubara Medicine

RESULT AND DISCUSSION Spices and Ethnomedicines In Kalabari; Their Pharmacognosy And Nutritional Significance.

29

Green Blessing O: Continental J. Pharmaceutical Sciences 2: 27 - 31, 2008 From pre-historic times, plants have contributed much in the ailment that troubled men. This is also the case with the Kalabari people. To treat any skin disease, the leaves of Avicinia sp is burnt and the ashes mixed with palm oil and warmed (oko-pulo) This is very popular in treating any skin disease in Kalabari likewise the extract of Bryophyllum (neonatic umbilical wounds), Acanthus sp (as first aid for fresh wounds), Costus sp (for measles) and Ageratum sp (for infection of reproductive tract especially female). It is of significance that without alkaloid analysis, the early Kalabari people chose those medicinal plants probably by trial error. Today many of these plants have proven medicinal values. For instance, Gill (1992) reported flavonoids (conyzongin, methoxybilentin) saponins, tannin and quiterpenoids which are effective against ulcers, inflammations, redness of the eyes and leprosy. In Ageratum conizoides. He also reported alkaloid trepanoid, eugenol and thymol in Ocimum gratussima (scent leaf) which is effective against cold and catearrh. Piper sp were also known to contain alkaloids pipline quinine and visine which are diuretic and effective against vomiting and tonsillitis (Delmarco 1994). Kalabari folks are known for their skill in culinary or cuisine culture. To prepare these items. Spices and vegetables are involved. Apart from peppers which they use a lot (due to freshfish dishes) there are other spices like Ocimum gratussima Monodora myristica XyIopiea ethiopicum, Piper guinensis and Vernonia amigdalina (which is the most relished vegetable. Table 1) these add vitamins, mineral, and essential medicinal alkaloids to their meals (George and Rogers l999, Mills 1991, Rubatzxy, 1997). The Rationale of Oral and Home Hygiene: among the people of Kalabari, species of Baphia were used as chewing sticks. The presence of tooth paste/brush had not reduced their cultural significance. It is also used for love expression and therefore usually given as a token especially to beloved ones. Pinnate leaflets of palm tree are peeled tied and used fro sweeping. The use of Baphia species as chewing stick must have come by trial and error but had great medicinal implication. Gill (1992) implicated curative saponins, tannin, iso-santalene and homo-pierocarpine in the stem and leaf of Baphia nitida. He also said that these made the plant efficacous in the eye, pains and spasm. Totem / Sacred Plants. Juvenile fronds of Elaesis guinensis were found tied around coffins during burials as well as on lands in dispute. It is also found in family memorial halls known as lkpu. To mark boundries, Newbouldia leavis is usually planted. It is believed that N. leavis wards off malevolent spirits. The leaves of Acrosticum species is used as exterior decor for shrines. Ageratum conyzoides is also significant as a cleansing plant and disinfectant t is an important plant as for as traditional mortuary practice is concerned. It was believed to have some mystic powers which warded off spirits for the mortuary attendance, corpse bearers or those women dressing the corpse. This myth was actually in the antibacterial effects of Agercitum sp (Gill, 1992, Green,2003) Typical Kalabari Meals: Many foods have been adapted from neighbours but none had replaced the most important traditional menu of kalabari. These were varieties of plantain meals. i ONUNU: Pastry made from ripe boiled plantain and boiled white yam (Dioscorea rotundata) while

mortaring the two, good quantity of red oil is added. The relish was not complete until it was complimented with local fresh fish pepper soup.

ii. UNRIPE PLANTAIN MEALS: Kalabari loved plantain especially in the unripe state. Consequent upon

this, Musa paradisiaca was divided into three on the basis of how it was prepared. Soote mbana (boiled and eaten with red oil and freshfish pepper soup), foite mbana (roasted and eaten with fish). Frying plantain is not cultural in Kalabari. Without being formally educated, the early Kalabari (since the settlement in their present location in the 18th century had made plantain especially the unripe one their most valued traditional meals. This wisdom has presently been explained by Gill (1992) who reported that unripe plantain contains high level of iron, protein, alkaloids, (inulin, noradrenaline and hydroxytryptamine) all of which are effective in the treatment of ulcer, diarrhea, hysteria and dysentery.

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Green Blessing O: Continental J. Pharmaceutical Sciences 2: 27 - 31, 2008 SUMMARY AND CONCLUSION: As a natural community, the Kalabari had unifying dialect and names for their indigenous plants. Plant nomenclature was based on their relationship and ethnobotany especially as food and medicine. Many ethnomedicinal plants and sacred plants in Kalabari were reputable pharmaceutically with documented alkaloids and glycosides. REFERENCE Blackwell W. (1990) Poisonous And Medicinal Plants. Engle Wood Cliffs, N.J Prentice-Hall. 101 pp. Delmarcor, C. (1994) Take Charge Of Your Body. Women’s Health Adviser. Luiulaw Well Woman Press 167pp. Egunyemi, E. (2002) Botanical Medicine Yesterday, Today And Tomorrow. Key Note Address Delieved At Annual Conference Of Botanical Society Of Nigeria March 10th - 13th. 2002. George D, And P. Rogers (1999) Encyclopaedia Of Medicinal Plants. Educational Health lib. Ed. Safelix publ. 690 pp. Gill, L. S. (1992) Ethnomedical Uses Of Plants In Nigeria. Benin Uniben Press. 240 pp. Green, B. O (1995) Taxonomic And Ethnobotaincal Studies On The Family Apocynaceae. A PhD Thesis University Of Port Harcourt Nigeria 280 pp. Green, B. O (2003) Folk Taxonomy In Kalabari And Its Systematic Implication For Ethnobotanical Studies In The Niger Delta Journ Of Pedagogy And Educational Dev. 9(1):205-212. Kadana J.M. (1970) Modern Encyclopaedia Of Herbs New York Parker Publishers 310 pp. Mann. (1992) Murder Maggic And Medicine Oxford, UK Oxford University Press. 320 pp. c b eW3,e,44h tex4 eapitat- p Mills, S. Y. (1991) The Essential Book of Herbal Medicine. London Cormorant Books 112.pp Ody P. (1993) The complete Medicinal Herbal London. Darling Kindersley Publishers. 260pp Ogbonda, K. H (2002) Biology In The Service Of Humanity Enugu. Odus Publ. 270 pp. Olorode, 0. (1984) Taxonomy Of West African Flowering Plants 1st Edition. London. Longman Inc. 228 pp.elli wi4 Simpson, B. B And Ogorzally. M. C. (1995) Economic Botany: Plants In Our World. 2nd Edition. New York MC Graw Hill Inc. 742 pp. Stace, A. C. (1980) Plant Taxonomy And Biosystematics. A Contemporary Biology. London Edward Arnold Publ. 283 pp. Rubatzky V.E (1997) World Vegetable, Principles, Production And Nutritive Valves 2nd Ed. USA Thomas PubI. 115 Pp. WHO (1976) African Traditional Medicine. Afro-Tech Rep Series World Health Organization. Brazaville. 1:2-4

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Green Blessing O: Continental J. Pharmaceutical Sciences 2: 27 - 31, 2008 Wrangham, R.W And Goodall J. (1989) Chimpanzee Use Of Medicinal Plant Leaves. Cambrige. Harvard University Press. 150 pp. Zimmer K,S (1991) The Regional Biogeography Of Native Potato cultivers In High Land Peru. Journal Of Biogeography. 18:165-178. Received for Publication: 10/07/2008 Accepted for Publication: 06/08/2008 Corresponding Author: Email: amayanabo7@yahoo

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Continental J. Pharmaceutical Sciences 2: 32 - 43, 2008 ©Wilolud Online Journals, 2008. PHYLLANTHUS AMARUS ATTENUATES ETHANOL-INDUCED OXIDATIVE STRESS IN RAT INTESTINE

1Toyin Yemisi Faremi, 2Stephen Monday Suru, , 1Udoka Eileen Obioha and 1Kikelomo Fausat Ola-Mudathir

1Nutritional Biochemistry Unit, 2Drug Metabolism and Toxicology Laboratories, Department of Biochemistry, Faculty of Basic Medical Sciences, College of Medicine, University of Ibadan, Ibadan, Nigeria.

ABSTRACT In recent years, there has been escalation in alcohol abuse with an attendant increase in alcohol-related gastrointestinal tract disorders. Alcohol is known to induce a dose-dependent increase in oxidative stress. This study evaluates the protective effect of Phyllanthus amarus on ethanol-induced oxidative stress in the intestine of rats. Animals were pre-treated with P. amarus (250 and 500mg/kg/day per oral) for one week and treatment continued for additional three weeks during which the animals were simultaneously challenged with ethanol (5g/kg/day, 20% w/v orally). Oxidative stress in the intestine tissue was estimated using lipid peroxidation (LPO) and glutathione (GSH) levels, as well as superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) activities. Ethanol administration caused a marked (p<0.05) reduction in body weight gain and organ weight albeit no significant difference in total protein content. LPO and GST levels were significantly (p<0.001) increased whereas GSH, SOD and CAT levels were significantly (p<0.001) decreased in the intestine of rats treated with ethanol alone. P. amarus administration successfully attenuated these effects, exerting a significant (p<0.001) increase in GSH, SOD and CAT levels, a marked (p<0.001) reduction in the levels of LPO and GST in intestine tissues. More so, treatment with 500mg/kg extract was more effective than 250mg/kg extract. In conclusion, P. amarus effectively protected the intestine against ethanol-induced oxidative stress by directly reducing the level of LPO and indirectly by enhancing the levels of endogenous antioxidants.

KEYWORDS: Ethanol; Oxidative Stress; Phyllanthus amarus; Intestine; Attenuates INTRODUCTION Alcohol beverages have been used and abused by a large number of individuals since the dawn of history. While light to moderate amounts of alcohol may have beneficial effects for cardiovascular health (Zakhari 1997), chronic consumption of alcohol can result in spectrum of abnormalities in many organ systems (Lindros 1995; Epstein 1997; Oscar-Berman et al 1997). Among the many organ systems that mediate alcohol’s effects on the human body and its health, the GIT and the liver play crucial roles (Bode and Bode 1997). The GIT is the primary site of alcohol absorption into the systemic circulation while the liver is the primary site of alcohol metabolism in the mammalian body (Lieber 2000). To a lesser extent, alcohol can also be oxidized (first pass metabolism) and produced in the GIT (Bode and Bode 1992). Although metabolism of alcohol in the GIT is quantitatively much lower compared to the liver, it is of importance because it affects the systemic availability of alcohol, local production of toxic acetaldehyde and ROS, which have been implicated in the pathogenesis of tissue injury (Bode and Bode 1997; Lieber 1997). Chronic alcohol consumption can exert deleterious effects on the structures and functions of all parts of the GIT (Bode and Bode 1997). The direct contact of alcohol with the GIT mucosa may elicit several metabolic changes among which is the induction of ethanol-inducible cytochrome P450 (CYP2E1) with an attendant generation of toxic acetaldehyde and reactive oxygen species (ROS) (Hakkak et al 1996). These metabolites are capable of depleting endogenous antioxidant status and may thus disturb the integrity of the mucosal epithelium (Zima et al 2001; Wu and Cederbaum 2003). This, may in concert with other mechanisms, contribute to the development of alcohol-related pathologies of the GIT such as gastrointestinal bleeding, inflammation, ulceration, diarrhoea and less well-recognized influences on other diseases especially cancer (Bode and Bode 1997; Zima et al 2001; Crabb et al 2004). More so, alcohol-induced mucosal injuries, especially in the vulnerable duodenum, have been reported to

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Toyin Yemisi Faremi et al: Continental J. Pharmaceutical Sciences 2: 32 - 43, 2008 render the gut more permeable to bacterial toxins. These toxic substances have been implicated in the pathogenesis of alcohol-induced damage to the liver and other organs (Bode and Bode 1997). In addition, alcohol-induced functional alterations and mucosal damage have been reported to cause disturbances in the digestion, absorption and assimilation of other nutrients into the body, thereby contributing to the malnutrition and weight loss often observed in alcoholics (Thomson and Pratt 1992; Bode and Bode 1997; Lieber 2000). Phyllanthus amarus Schum and Thonn, family Euphorbiaceae, is a small erect, annual herb that grows 30 to 40cm in height (Khanna et al 2002; Jaleel et al 2007). It is indigenous to the rainforest of the Amazon and other tropical areas throughout the world. P. amarus is locally referred to as Eyin-olubisowo or dobiosowo (Yoruba), Geeron-tsuntsaye (Hausa), Buchi oro (Ibo), Oyomokeso (Efik). It is widely used in the folk medicine of different countries (Calixto et al 1998). It is a well known hepatoprotective (Sane et al 1995) and antiviral (Thyagarajan et al 1990) agent. Some other studies also reported that P. amarus possesses antioxidant, anti-diabetic (Rapheal et al 2002), radioprotective (Kumar and Kuttan 2004) and antimutagenic (Rapheal et al 2002) properties. Recent study in our laboratory (Odetola and Akojenu 2000) showed that P. amarus is a formidable source of protection against castor oil-induced diarrhoea in experimental model. On the basis of these considerations, the aim of the present study was to evaluate the protective effect of P. amarus on ethanol-induced oxidative stress in the intestine. In this study, P. amarus was administered prior and during ethanol intoxication in order to determine preventive and protective effects on ethanol-induced oxidative injury. Our hypothesis is that the use of herbal medicine may make it possible to prevent and alleviate the incidence of alcohol-induced gastrointestinal disorders via inhibition of oxidative stress. MATERIALS AND METHODS Preparation of P. amarus Extract Aerial parts (stem and leaves) of P. amarus were collected in April 2005 from Ado-Ekiti town, Nigeria. It was authenticated at the Forestry Research Institute Nigeria (FRIN), Ibadan and a voucher specimen (no FHI 107623) has been deposited at the Forestry Herbarium Ibadan (FHI). The leaves were air-dried at room temperature, powdered and 100g were extracted with 500 ml of 80% methanol by stirring overnight at room temperature (25oC). The mixture was then centrifuged at 2,500 rpm to separate the supernatant, the supernatant was filtered (Whatman No 3) and evaporated to dryness at 45oC with a rotary evaporator. The extract was dissolved in water at a concentration of 3g/100ml to form P. amarus solution. Phytochemical studies carried out with P. amarus aerial parts have demonstrated the presence of many classes of constituents, such as tannins (phyllanthusin D, repandusinic acid, 1,6-digalloyl glucopyranose, geraniin, amariin, furosin, geraniinic acid B, amariinic acid, amarulone, corilagin, furosin and elaeocarpusin) (Foo and Wong 1992; Foo 1993; Foo 1995), alkaloids (securinine, norsecurinine, epibubbialine and isobubbialine) (Houghton et al 1996) phenolics (gallic acid and 4-O-galloylquimic acid) (Foo 1993) and flavonoids (catechins, gallocatechin, quercetin, rutin and quercetin-3-O-glucopyranoside) (Morton 1981; Foo 1993), polyphenols (ellagic acid, phenazine and phenazine derivatives) (Foo 1993) Animals and Treatments A total of 30 male albino-Wistar rats weighing 180 ± 5g were used for the experiment. The animals were purchased from the Institute of Medical Research and Training (IMRAT), University College Hospital, Ibadan. They were kept in specially designed, well ventilated plastic cages under standard conditions and were maintained on normal laboratory chow (Bendel Feed and Flour Mill Ltd, Edo-State) and water ad libitum. All animal experiments were conducted without anaesthesia and according to the Ethical Norms on Animal Care and Use approved by IMRAT and Faculty of Basic Medical Sciences, College of Medicine, University of Ibadan. After two weeks of acclimatization, the rats were randomly divided into five treatment groups of six animals each (Table 1). The PA, ETPA and ETPAPA groups were pre-treated with P. amarus extract for one week and treatment continued for additional three weeks during which ET, ETPA and ETPAPA groups were treated with ethanol solution while the normal and PA groups were treated with isocaloric glucose solution. P. amarus extract and ethanol were administered by gavages respectively, two hours apart and once daily. At the end of the experimental

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Toyin Yemisi Faremi et al: Continental J. Pharmaceutical Sciences 2: 32 - 43, 2008 period of four weeks, the final body weight of the rats was determined and they were fasted 12 hours prior to when they were sacrificed by cervical dislocation. Collection and Preparation of Tissue All rats were carefully dissected and the proximal portion (15cm) of the small intestine was excised, washed and flushed thoroughly with ice-cold physiological saline, blotted on filter paper, weighed, and homogenized in ice-cold 1.15% KCl (1g/3ml) in a Potter-Elvehjem type homogenizer. The homogenates were centrifuged at 5000 rpm for 15min at 4oC. Aliquots of the supernatant were used for the following biochemical assays. Biochemical analysis The total protein content of the aliquot was determined by the method of Lowry et al (1951), using bovine serum albumin as standard. Lipid peroxidation: Lipid peroxidation was determined by estimating malondialdehyde (MDA) content using the thiobarbituric acid test (Beuge and Aust (1978). Briefly, the stock solution contained equal volumes of trichloroacetic acid 15% (w/v) in 0.25 N hydrochloric acid and 2-thiobarbituric acid 0.37% (w/v) in 0.25 N hydrochloric acid. One volume of the test sample and two volumes of stock reagent were mixed in a screw-capped centrifuge tube, vortexed and heated for 15min on a boiling water bath. After cooling on ice the precipitate was removed by centrifugation at 4000rpm for 15min and absorbance of the supernatant was measured at 532 nm against blank containing all the reagents except test sample4. TBARS were calculated using an extinction coefficient of 1.56 × 10-5 M-1cm-1 and expressed as µm MDA (g tissue)-1. Reduced glutathione: Reduced glutathione level was assayed by the method of Jollow et al (1974) based on the reaction with 5,5’-dinitro bis (2-nitrobenzoic acid) (DTNB). Briefly, 1.0ml of sample (10%) was deproteinized by adding an equal volume of 4% sulfosalicyclic acid. The sample was kept at 4oC for at least 1 hour and then centrifuged at 4200 rpm for 15min at 4oC. 0.5ml of the resulting supernatant was added to 4.5ml of DTNB in phosphate buffer (0.1 M, pH 7.4). The yellow colour developed was read immediately at 412 nm on a spectrophotometer. A blank was prepared by adding 0.5ml of 4% sulfosalicyclic acid to 4.5ml DTNB. Antioxidant enzyme activity: The activity of SOD (EC 1.15.1.1) was determined spectrophotometrically at 480 nm by the epinephrine method (Misra and Fridovich 1972) and expressed in units of enzyme activity per milligram protein. CAT (EC 1.11.1.6) activity was estimated spectrophotometrically at 570 nm by the method of Sinha (1971) by measuring the rate of decomposition of hydrogen peroxide (H2O2). The method of Habig et al (1974) was followed to assay the activity of GST (EC 2.5.1.18) by monitoring the appearance of the conjugated complex of GSH and 1-chloro-2,4-dinitrobenzene (CDNB) at 340 nm. Statistical analysis All results are presented as mean ± SD, and differences were analyzed by one-way analysis of variance (ANOVA) followed by Fischer’s LSD post hoc test using SPSS 11 software (SPSS Inc, Chicago). When appropriate, Independent sample t test was done to evaluate the significance of the differences between means. Statistical significance was considered at p<0.05. RESULTS During the experiment, the body weight increased in all experimental rats (Table 2). However, the increase was significant in the normal, PA, ETPA and ETPAPA groups (p<0.01, p<0.001, p<0.01 and p<0.05, respectively). Treatment with ethanol alone significantly (p<0.05) reduced the weight of the intestine although no significant decrease in the total protein content. Co-treatment of varied doses of P. amarus with ethanol had no significant effect on intestine weight and total protein content albeit a general improvement in these parameters relative to ET-treated rats (Table 2). The level of lipid peroxidation (LPO) and reduced glutathione (GSH) are presented in Table 3. The level of lipid peroxidation, as assessed by malondialdehyde (MDA), in the intestine was significantly (p<0.001) increased in ET-treated group relative to normal, ETPA and ETPAPA groups (Figure 1). A significant (p<0.001) decrease in LPO

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Toyin Yemisi Faremi et al: Continental J. Pharmaceutical Sciences 2: 32 - 43, 2008 level was observed in ETPAPA-treated group as compared with ETPA-treated group. There was a marked (p<0.001) decrease in the intestine GSH level of ET-treated group compared to normal, ETPA- and ETPAPA-treated groups as well as a marked (p<0.001) increase in ETPAPA-treated group relative to ETPA group. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione-S transferase (GST) are presented in Table 4. The activity of SOD in the intestine as shown in Table 4 followed a similar pattern as in reduced GSH level (Table 3). The activity of CAT in the intestine was significantly (p<0.001) reduced in ET-treated group relative to normal and ETPAPA-treated groups but not significant relative to ETPA-treated group. More so, CAT activity was significantly (p<0.01) enhanced in ETPAPA-treated group compared to ETPA-treated group. The activity of GST in the intestine increased significantly (p<0.001) in ET-treated group relative to normal, ETPA and ETPAPA groups. A marked (p<0.05) decrease in GST activity was observed in ETPAPA-treated group relative to ETPA-treated group. DISCUSSION There is increasing evidence that chronic alcohol consumption can exert deleterious effects on the structures and functions of all parts of the GIT via oxidative stress (Bode and Bode 1997). The direct contact of alcohol with the GIT mucosa has been reported to disturb the integrity of the mucosal epithelium leading to mucosal injuries particularly in the duodenum (Bode and Bode 1997). This may interfere with digestion and absorption of essential nutrients in the GIT (Lieber 2003). Poor nutritional status, as a consequence of alcohol-induced primary and secondary malnutrition, has been reported to contribute to loss in body weight and the development of other alcohol-related pathologies (Lieber 2003; Poschl et al 2004). Hence, in our study, there was a significant increase in body weight gain in all animals except the ET-treated group. More so, a marked reduction in organ weight was observed, albeit no significant difference in total tissue protein in ET-treated group relative to the normal and other groups. Treatment with varied doses of P. amarus markedly restored the ethanol-induced alterations in body and organ weights. These positive effects may be attributed to the protective effect of P. amarus extract on the GIT. A similar observation has been reported by Odetola and Akojenu (2000). It is a well established fact that alcoholic patients and experimental animals exposed to chronic ethanol display biochemical signs of oxidative damage (Wu and Cederbaum 2003; Rukkumani et al 2004; Uzun et al 2005). GIT metabolism of alcohol is quantitatively much lower compared to the liver but it is of importance because it plays a major role in the local accumulation of toxic acetaldehyde and ROS, implicated in mucosal injury (Lieber 1997; Seitz and Oneta 1998). Excessive generation of ROS, as a result of CYP2E1 induction in the GIT after chronic ethanol consumption, has been reported to play a major role in peroxidative damage of epithelial membrane lipids (Albano and Clot 1996; Hakkak et al 1996). In our present study, there was a marked increase in the level of LPO by 284% in the intestine of ET-treated animals relative to control animals. Other authors have reported similar observations (Bode and Bode 1992; French et al 1993). The antioxidant defence system protects the aerobic organism from the deleterious effects of ROS (Halliwell 1999). Glutathione (GSH), an important endogenous non-enzymatic antioxidant is involved in the protection against free radicals and other toxic compounds such as acetaldehyde (Rukkumani et al 2004). Glutathione-S transferase (GST) is a key component of cellular antioxidant defenses, offering protection against cell damage (Dinkova-Kostova and Talalay 1999). Catalase (CAT), which acts as preventative antioxidant and SOD, a chain-breaking antioxidant, play a vital role in protection against the deleterious effects of LPO (Rukkumani et al 2004; Uzun et al 2005). In our study, chronic ingestion of ethanol challenges the antioxidant defence system by causing a remarkable decrease in the level of GSH, SOD and CAT by 73.29%, 53.43% and 63.04% respectively and a marked increase in GST activity by 85.58%. These findings are indicative of an obvious change in the oxidant-antioxidant balance in the intestine of rats, which may culminate in gastric and duodenal ulcer (Poschl et al 2004). Some investigators have reported similar findings in the level of GSH, SOD and CAT (Pronko et al 2002; Ozaras et al 2003; Rukkumani et al 2004; Uzun et al 2005). The observed depletion in GSH level may be attributed, in part, to increase utilization to counteract ROS and lipid peroxidative products and toxic acetaldehyde. In addition, alcohol itself has been reported to inhibit the biosynthesis of new GSH (Lieber 2003). The inverse correlation between ethanol-induced LPO and the activities of SOD and CAT is indicative of an overwhelming oxidative stress which is further corroborated by heightened GST activity.

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Toyin Yemisi Faremi et al: Continental J. Pharmaceutical Sciences 2: 32 - 43, 2008 The fact that ethanol intoxication can induce a state of oxidative stress implies that administration of antioxidants before or after ingestion of alcohol could possibly prevent or diminish the processes of alcohol-induced oxidative damage in the tissues (Molina et al 2003; Ozaras et al 2003; Rukkumani et al 2004). The results obtained in this study agree with this hypothesis. Treatment of ethanol-intoxicated rats with varied doses of P. amarus extract (250 and 500 mg/kg) significantly reduced the level of LPO by 28.22% and 61.81% respectively. The antioxidant defence system was positively modulated. The ethanol-induced decrease in the levels of GSH, SOD and CAT were significantly restored in the ETPA (250mg)-treated group by 91.07%, 51.37% and 12.52% respectively, and in the ETPAPA (500mg)-treated group by 160.71%, 103.29% and 39.66% respectively relative to the ET-treated group. In addition, the ethanol-induced elevation of GST activity was significantly reduced in the ETPA- and ETPAPA-treated animals by 29.57% and 44.36% respectively relative to the ET-treated animals. Phytochemical analysis of the extract show that P. amarus contains several bioactive ingredients such as benzenoids, tannins and flavonoids of proven antioxidant activity in-vitro (Joy and Kuttan 1995; Calixto et al 1998). Thus, the observed protection against LPO may be attributed to the antioxidant phytochemicals present in P. amarus (Sane et al 1995; Raphael et al 2002). More so, the restoration of the antioxidant defence system in this study may be due, in part, to the antioxidant-sparing actions of the phytochemicals, particularly, the polyphenolic compounds (Sane et al 1995; Calixto 1998; Raphael et al 2002). Apart from antioxidant sparing action, polyphenolic compounds have been reported to enhance the biosynthesis of reduced GSH by up-regulating the expression of γ-glutamylcysteine synthetase, the rate-limiting enzyme in the biosynthesis of reduced GSH (Moskaug et al 2005). CONCLUSIONS In conclusion, our study shows that ethanol intoxication resulted in an increased oxidative stress in rat intestine. It also demonstrated that administration of P. amarus extract prior and during ethanol intoxication may exert a marked protective effect on ethanol-induced oxidative damage. This finding is plausible as it can be seen from the positive effects of the extract on the parameters considered above in the PA-treated group compared to the normal group. Our study, therefore, suggests a role for P. amarus as a safe, cheap and effective phytotherapy in the prevention and management of ethanol-induced oxidative damage, a causative factor in duodenal ulcer and other GIT disorders. ACKNOWLEDGEMENTS The authors gratefully acknowledged the Department of Biochemistry, University of Ibadan, Ibadan, Nigeria for providing the basic facilities used in this study. REFERENCES Albano, E., Clot, P. (1996). Free radicals and ethanol toxicity. In: Preedy, V.R., Watson, R.R. (eds) Alcohol and the

Gastrointestinal Tract. Boca Raton, FL: CRC Press, pp. 57-68.

Beuge, J.A., Aust, S.D. (1978). Microsomal lipid peroxidation. Methods Enzymol. 52: 302-310.

Bode, C., Bode, J.C. (1997). Alcohol’s role in gastrointestinal tract. Alcohol Health and Research World 21:76-83. Bode, J.C., Bode, C. (1992). Alcohol malnutrition and gastrointestinal tract. In: Watson, R.R., Watzl, B. (eds)

Nutrition and Alcohol. Boca Raton, FL: CRC Press, pp. 403-428. Calixto, J.B., Santos, A.R., Filho, V. (1998). A review of the plants of the genus Phyllanthus, their chemistry,

pharmacology and therapeutic potential. Med. Res. Rev. 18:225-258. Crabb, D.W., Matsumoto, M., Chang, D., You, M. (2004). Overview of the role of alcohol dehydrogenase and

aldehyde dehydrogenase and their variants in the genesis of alcohol-related pathology. Proc. Nutr. Soc. 63:49-63.

Dinkova-Kostova, H., Talalay, P. (1999). Relation of structure of curcumin analogs to their potencies as inducers of

phase-2 detoxification enzymes. Carcinogenesis 20:911-914.

37

Toyin Yemisi Faremi et al: Continental J. Pharmaceutical Sciences 2: 32 - 43, 2008 Epstein, M. (1997). Alcohol’s impact on kidney function. Alcohol Health and Research World 21:84-93. French, S.W., Nash, J., Shitabata, P., Kachi, K., Hara, C., Chedid, A., Mendenhall, C.L. (1993). Effect on ethanol on

cytochrome P450 2E1 (CYP2E1), lipid peroxidation, and serum protein adduct formation in relation to liver pathology pathogenesis. Seminars in Liver Disease 13:154-169.

Foo, L.Y. (1993) Amarulone, a novel cyclic hydrolysable tannin from Phyllanthus amarus. Natural Product Letters

3: 45-52. Foo, L.Y. (1995) Amarinic acid and related ellangitannins from Phyllanthus amarus. Phytochemistry 39: 217-224. Foo, L.Y., Wong H. (1992) Phyllantusin D, an unusual hydrolysable tannin from Phyllanthus amarus.

Phytochemistry 31: 711-713. Habig, W.H., Pabst, M.J., Jakoby, W.B. (1974). The first enzymatic step in mercapturic acid formation. J. Biol.

Chem. 249: 7130-7139. Hakkak, R., Karourian, S., Ronis, M.J., Ingelman-Sundberg, M., Badger, T.M. (1996). Effect of diet and ethanol on

the expression and localization of cytochromes P450 2E1 and P480 2C7 in the colon of male rats. Biochem. Pharmacol. 51:61-69.

Halliwell, B. (1999). Antioxidant defense mechanisms: From the beginning to the end. Free Radic. Res. 31:261-272. Houghton, P.J., Woldemariam, T.Z., O’Shea, S., Thyagarajan, S.P. (1996). Two securinega-type alkaloids from

Phyllanthus amarus. Phytochemistry 43: 715-717. Jaleel , C.A. , Manivannan, P., Lakshmanam, G.M., Sridharan, R., Panneerselvam, R. (2007). NaCl as a

physiological modulator of praline metabolism and antioxidant potential in Phyllanthus amarus. C. R. Biol. 330: 806-813.

Jollow, D.J., Michell, J.R., Zampaglione, N., Gillete, J. (1974). Bromobenzene-induced liver necrosis: protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacology 11: 151-169.

Joy, K.L., Kuttan, R. (1995). Antioxidant activity of selected plant extracts. Amala Res. Bull. 15:68-71. Khanna, A.K., Rizvi, F., Chander, R. (2002). Lipid lowering activity of Phyllanthus niruri in hyperlipemic animals.

J. Ethnopharmacol. 82: 19-22. Kumar, K.B.H., Kuttan, R. (2004). Protective effect of an extract of Phyllanthus amarus against radiation-induced

damage in mice. J. Radiat. Res. 45:133-139. Lieber, C.S. (1997). Cytochrome P450 2E1: Its physiological and pathological role. Physiol. Rev. 77:517-544. Lieber, C.S. (2000). Alcohol: Its metabolism and interaction with nutrients. Ann. Rev. Nutr. 20:395-430 Lieber, C.S. (2003). Relationships between nutrition, alcohol use, and liver disease. Alcohol Research and Health

27:220-231. Lindros, K.O. (1995). Alcoholic liver disease: pathobiological aspects. J. Hepatol. 23:7-15.

38

Toyin Yemisi Faremi et al: Continental J. Pharmaceutical Sciences 2: 32 - 43, 2008 Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951). Protein measurement with the Folin phenol

reagent. J. Biol. Chem. 193: 265-275. Misra, H.P., Fridovich, I. (1972). The role of superoxide anion in the autooxidation of epinephrine and a simple

assay for superoxide dismutase. J. Biol. Chem. 247: 3170-3175. Molina, M.F., Sanchez-Reus, I., Iglesias, I., Benedi, J. (2003). Quercetin, a flavonoid antioxidant, prevents and

protects against ethanol-induced oxidative stress in mouse liver. Biol. Pharm. Bull. 26:1398-1402. Moskaug, J.O., Carlsen, H., Myhrstad, M.C.W., Blomhoff, R. (2005). Polyphenols and glutathione synthesis

regulation. Am. J. Clin. Nutr. 81: 277S-283S. Morton, J.F. (1981). Atlas of medicinal plants of Middle America. Library of Congress Cataloging in Publication

Data, Thomas Books. p 1420 Odetola, A.A., Akojenu, S.M. (2000). Anti-diarrhoeal and gastro-intestinal potentials of aqueous extract of

Phyllanthus amarus (Euphorbiaceae). Afr. J. Med. Sci. 29:119-122. Oscar-Berman, M., Shagrin, B., Evert, D.L., Epstein, C. (1997). Impairments of brain and behaviour: The

neurological effects of alcohol. Alcohol Health and Research World 21:65-75. Ozaras, R., Tahan, V., Aydin, S., Uzun, H., Kaya, S., Senturk, H. (2003). N-acetylcysteine attenuates alcohol-

induced oxidative stress in rats. World J. Gastroenterol. 9: 791-794. Poschl, G., Stickel, F., Wang, X.D., Seitz, H.K. (2004). Alcohol and cancer: genetic and nutritional aspects. Proc.

Nutr. Soc. 63:65-71. Pronko, P., Bardina, L., Satanovskaya, V., Kuzmich, A., Zaimatkin, S. (2002). Effect of chronic alcohol

consumption on the ethanol- and acetylaldehyde-metabolizing systems in the rat gastrointestinal tract. Alcohol and Alcoholism 37:229-235.

Raphael, R.K., Sabu, M.C., Kuttan, R. (2002). Hypoglycemic effect of methanolic extract Phyllanthus amarus

Schum and Thonn on alloxan-induced diabetes mellitus in animals and its relation with antioxidant potential. Indian. J. Exp. Biol. 40:905-909.

Rukkumani, R., Aruna, K., Suresh, V.P., Menon, V.P. (2004). Influence of ferrulic acid on circulatory prooxidant-

antioxidant status during alcohol and PUFA induced toxicity. J. Physiol. Pharmacol. 55:551-561. Sane, R.T., Kuber, V.V., Menon, S. (1995). Hepatoprotection by Phyllanthus amarus and Phyllanthus debilis in

CCl4-induced liver dysfunction. Curr. Sci. 20:1243-1246. Seitz, H.K., Oneta, C.M. (1998). Gastrointestinal alcohol dehydrogenase. Rev. Nutr. 56:52-60. Sinha, K.A. (1971). Colorimetric assay of catalase. Anal. Biochem. 47: 389-394. Thomson, A.D., Pratt, O.E. (1992). Interaction of nutrients and alcohol: absorption, transport, utilization, and

metabolism. In: Watson, R.R., Watzl, B. (eds) Nutrition and Alcohol. Boca Raton, FL: CRC Press, pp. 75-99. Thyagarajan, S.P., Jayaram, S., Vallimmai, T. (1990). Phyllanthus amarus and hepatitis B. Lancet 336:949-950. Uzun, H., Simsek, G., Aydin, S., Unal, E., Karter, Y., Yelmen, K.N., Vehid, S., Curgunlu, A., Kaya, S. (2005).

Potential effects of L-NAME on alcohol-induced oxidative stress. World J. Gastroenterol. 11:600-604.

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Toyin Yemisi Faremi et al: Continental J. Pharmaceutical Sciences 2: 32 - 43, 2008 Wu, D., Cederbaum, A.I. (2003). Alcohol, oxidative stress, and free radical damage Alcohol Research and Health

27:277-284. Zakhari, S. (1997). Alcohol and the cardiovascular system: Molecular mechanisms of beneficial and harmful action.

Alcohol Health and Research World 21:21-29. Zima, T., Fialova, L., Mestek, O., Janebova, M., Crkovska, J., Malbohan, I., Stipek, S., Mikulikova, L., Popov, P.

(2001). Oxidative stress, metabolism of ethanol and alcohol-related diseases. J. Biomed. Sci. 8:59-70. Received for Publication: 24/07/2008 Accepted for Publication: 12/12/2008 Corresponding Author: Stephen Monday Suru, Drug Metabolism and Toxicology Laboratories, Department of Biochemistry, Faculty of Basic Medical Sciences, College of Medicine, University of Ibadan, Ibadan, Nigeria.

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Toyin Yemisi Faremi et al: Continental J. Pharmaceutical Sciences 2: 32 - 43, 2008

Table 1 Experimental design

Notes: n = 6 for each treatment group.

Group Treatment

Control Isocaloric glucose solution

ET Ethanol solution (5g/kg/day)

PA P. amarus extract (250mg/kg/day) and Isocaloric glucose solution

ETPA P. amarus extract (250mg/kg/day) and ethanol solution (5g/kg/day)

ETPAPA P. amarus extract (500mg/kg/day) and ethanol solution (5g/kg/day)

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Toyin Yemisi Faremi et al: Continental J. Pharmaceutical Sciences 2: 32 - 43, 2008

Table 2 Effects of ethanol and Phyllanthus amarus treatments on the body weight, intestine weight and total protein content of

rats.

Notes: Values are means ± SD. n = 6 for each treatment group. Body weight data were analyzed by Independent sample t test and comparison was done between initial weight and final weight. Intestine weight and total protein content were analyzed by one-way analysis of variance (ANOVA) followed by Fischer’s LSD post hoc test. a = Control vs. other groups; b = ET vs. ETPA and ETPAPA; c = ETPA vs. ETPAPA; * = P<0.05; # = P<0.01; ‡ = P<0.001.

Experimental

Group

Body Weight Intestine/body weight

ratio (×10−2 g)

Total Protein Content

(mg/ml) Initial (g) Final (g) % Change

Control 180.00 ± 0.00 216.67 ± 16.23 20.37 # 3.01 ± 0.82 40.14 ± 9.46

ET 180.00 ± 8.16 183.33 ± 21.91 1.89 2.27 ± 0.20 *a 38.68 ± 11.12

PA 180.00 ± 0.00 223.33 ± 8.16 24.07 ‡ 2.75 ± 0.30 44.32 ± 5.88

ETPA 180.00 ± 0.00 196.67 ± 8.16 9.26 # 2.60 ± 0.54 45.12 ± 7.94

ETPAPA 180.00 ± 0.00 206.67 ± 24.22 14.82 * 2.39 ± 0.37 41.91 ± 6.10

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Toyin Yemisi Faremi et al: Continental J. Pharmaceutical Sciences 2: 32 - 43, 2008 Table 3 Effects of Phyllanthus amarus extract on the levels of lipid peroxidation and reduced glutathione in the intestine of ethanol-exposed rats. Notes: Values are means ± SD. n = 6 for each treatment group. Data were analysed by one-way analysis of variance (ANOVA) followed by Fischer’s LSD post hoc test. a = Control vs. other groups; b = ET vs. ETPA and ETPAPA; c = ETPA vs. ETPAPA; * = P<0.05; # = P<0.01, ‡ = P<0.001.

Experimental group

MDA content (µg/g tissue)

Glutathione content

(µg/g tissue)

Control 14.07 ± 1.93 6.29 ± 0.43

ET 54.04 ± 4.15 ‡a 1.68 ± 0.32 ‡a

PA 10.13 ± 1.23 *a 6.80 ± 0.36 *a

ETPA 38.79 ± 3.92 ‡b 3.21 ± 0.26 ‡b

ETPAPA 20.64 ± 1.81 ‡bc 4.38 ± 0.25 ‡bc

Experimental group

MDA content (µg/g tissue)

Glutathione content

(µg/g tissue)

Control 14.07 ± 1.93 6.29 ± 0.43

ET 54.04 ± 4.15 ‡a 1.68 ± 0.32 ‡a

PA 10.13 ± 1.23 *a 6.80 ± 0.36 *a

ETPA 38.79 ± 3.92 ‡b 3.21 ± 0.26 ‡b

ETPAPA 20.64 ± 1.81 ‡bc 4.38 ± 0.25 ‡bc

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Toyin Yemisi Faremi et al: Continental J. Pharmaceutical Sciences 2: 32 - 43, 2008 Table 4 Effects of Phyllanthus amarus extract on the activities of superoxide dismutase, catalase and glutathione-S transferase in the intestine of ethanol-exposed rats.

Notes: Values are means ± SD. n = 6 for each treatment group. Data were analysed by one-way analysis of variance (ANOVA) followed by Fischer’s LSD post hoc test. a = Control vs. other groups; b = ET vs. ETPA and ETPAPA; c = ETPA vs. ETPAPA; * = P<0.05; # = P<0.01, ‡ = P<0.001. Catalase feiahigkeit or “Kat f” is equivalent to µmole H2O2 consumed/min/mg protein.

Experimental group

Superoxide dismutase Activity

(U/mg protein)

Catalase Activity (Kat f)

Glutathione-S transferase Activity

(µg/min/mg protein)

Control 3.93 ± 0.16 28.52 ± 0.64 2.15 ± 0.42

ET 1.83 ± 0.22 ‡a 10.54 ± 1.92 ‡a 3.99 ± 0.81 ‡a

PA 4.28 ± 0.28 *a 30.05 ± 1.72 1.99 ± 0.26

ETPA 2.77 ± 0.28 ‡b 11.86 ± 1.57 2.81 ± 0.40 ‡b

ETPAPA 3.72 ± 0.17 ‡bc 14.72 ± 1.48 ‡b #c 2.22 ± 0.40 ‡b * c

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Continental J. Pharmaceutical Sciences 2: 44 - 48, 2008 ©Wilolud Online Journals, 2008.

SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF SOME BENZIMIDAZOLE DERIVATIVES

D.N. Patil1, S.C.Chaturvedi1, D.L. Kale1, R.B. Kakde1 and S.B. Dahikar2

School of Pharmacy, Devi Ahilya Vishwavidyalaya, Indore (M.P.), INDIA-452017 1Department of Pharmaceutical Sciences, R.T.M. Nagpur University, Nagpur (M.S.), INDIA-440033

2Sanjivani Institute of Pharmacy and Research, Kopargaon (M.S.), INDIA-423603

ABSTRACT A series of substituted benzimidazole compounds were synthesized by phase transfer catalyst (PTC) method using quaternary ammonium salt. Reaction has been carried out by conventional method in inert gas. Synthesized compounds were confirmed by IR, NMR, mass spectral and elemental analysis. The synthesized compounds were screened for their antibacterial and antifungal activity using paper disc diffusion method against some microorganism such as Escherichia coli, Bacillus pumilis, Staphylococcus aureus, Shigella sonnei, Proteus vulgaris, Pseudomonas aeruginosa, Aspergillus niger, Candida albicans. Almost all compounds shows potent antibacterial and antifungal activity. KEYWORDS: Benzimidazole derivatives, synthesis, antibacterial activity, antifungal activity

INTRODUCTION Synthesis of benzimidazole compound is emerged as essential need for development of new pharmaceutical entity. It may provide scaffolds on which pharmacophores can be arranged to yield potent and selective drug (Cramer et al,1988) . Every type of biological action detected, irrespective of the compounds involved in its induction, presents a potential lead (Cohen et al, 1990). The compounds responsible for the action have to be identified(Ozden et al, (2004), Rajasree et al, 2005). The range of the biological actions of potential interest is wide. Detection of biological action and identification of the chemical compounds involved to constitute the main and nearly unlimited source of leads for drug design(Natesh et al, (2003), Jarrahpour et al, (2004). The literature survey shows that in past recent years large number of compounds with different structures has been reported which exhibited antimicrobial activity (Han and Water (1998), Dunn et al, 1976). However, their clinical usefulness is still restricted because of their side effects. The use of antimicrobial is limited mainly due to development of resistance power. The aim of our paper is to synthesize benzimidazole derivatives and evaluation of their antimicrobial activity which can be used for antimicrobial therapy. MATERIALS AND METHODS Synthesis of benzimidazole derivative (Vogel, (2006), Seshaiah et al, (2001), Murat et al, (2005), Yingjie et al, (2005). All chemicals used in the synthesis were of synthetic grade. In a round bottom flask provided with efficient double surface condenser, 0.043 moles of aniline, 0.066 mol of carbon disulphide and 6.35 ml of ethanol were placed. The apparatus was set up in the fuming cup-board and heated on an electrically heated water bath for 8 hr or till content solidifies. Condenser was arranged for downward distillation, to remove the excess of carbon disulphide and alcohol. Residue in the flask was shaken with excess of dil. HCl (1:10) to remove any aniline present, It was filtered then washed with water and dried. The crude drug was recrystallized with rectified spirit. STEP II: Synthesis of benzimidazole derivatives: Synthesis was carried out by phase transfer catalysis method by using quaternary ammonium salt. Six milliliter solution of potassium hydroxide (50% in water) and 0.25 mg of tetra butyl ammonium bromide (Q+X-) were added in necked round bottom flask filled with inert gas and fitted with septum. The solution was then continuously stirred for 30 min with magnetic stirrer. First step product was added drop wise in above solvent and stirred for 4 hr. It was then filtered and recrystallized with solvent and dried. Synthesized benzimidazole derivatives with their respective yields are given in Table-1.

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D.N. Patil et al: Continental J. Pharmaceutical Sciences 2: 44 - 48, 2008 General chemical scheme:

NH 2

NH

C ONH

R

R

R

C 2H 5 OHC S 2

NNH

O

R

R

Q +X -PTC

Step I

Step II

Diphenyl Urea

Substituted Benzimidazole Derivatives STEP I: Synthesis Thiocarbanilide (Diphenyl Thiourea): C2H5OH 2 C6H5NH2 + CS2 S = C (NH.C6H5) 2 +H2S STEP III: Structure Confirmation: Synthesized compounds were confirmed by IR, NMR, mass spectra and elemental analysis. Table 1: Synthesized benzimidazole derivatives.

Comp. Code Substituent-R M.P. (°C) Yield (gm)

DP-1 H 258-260 2.3

DP-2 p-CH3 257-259 1.8

DP-3 m-CH3 281-283 1.5

DP-4 m-NO2 296-297 2.5

DP-5 o-NO2 284-286 2.1

DP-6 p-OCH3 274-276 1.2

DP-7 o-Br 282-284 1.8

DP-8 p-Cl 285-287 2.3

DP-9 m-Cl 285-287 1.1

DP-10 COOH 291-293 1.4

Where DP-Benzimidazole Derivatives with substituent R

STEP-IV: Antimicrobial screening: The standard pathogenic microorganism was procured from School of Life Sciences, Devi Ahilya Vishwavidyalaya, Indore, India and used in the study is given in table-2. 0.1ml of broth was inoculated in 10ml sterile nutrient broth and incubated at 37°C for 3hr. Turbidity of culture was measured with the help of Nephelo-turbidimeter. Viable count (106) was measured by standard plate and used in the study.

46

D.N. Patil et al: Continental J. Pharmaceutical Sciences 2: 44 - 48, 2008 Disc diffusion method as described by the National Committee of Clinical Laboratory Standards (2002) was used to determine the antibacterial activity of the various synthesized compounds. For antibacterial properties, 0.1ml bacterial suspension of 105CFU/ml was uniformly spread on Muller Hinton agar and Potato Dextrose Agar plate to form lawn cultures. The solutions of synthesized compounds were prepared 100µg/ml in tetrahydrofuran (THF). The blotting paper discs (6 mm diameter) were soaked in prepared solutions, and tested for their antibacterial and antifungal activity by disc diffusion technique (NCCS 2000). After incubation of 24 hr at 37°C, zone of inhibition of growth was measured in mm. The antibacterial activity was classified as highly active (>21mm), mild active (15-21mm) and slightly active (12-15mm) and less than 12mm was taken as inactive. Gentamycin 10mcg/disc (Hi-Media disc) for antibacterial and Griseofulvin for antifungal was used as positive control, while discs soaked in THF were placed on lawns as negative control. Experiment was performed in triplet to obtain persistent result.

Table 2: Standard pathogenic microorganism with ATCC No. (American type culture collection)

Microorganism ATCC No.

Escherichia coli 2109

Bacillus pumilis 2327

Staphylococcus aureus 2079

Shigella sonnei --

Proteus vulgaris 2813

Pseudomonas aeruginosa 2036

Aspergillus niger 545

Candida albicans --

RESULTS AND DISCUSSION In this work, series of compounds were synthesized and evaluated for biological activity against micro organism. The anti-bacterial activity of the ten compounds was evaluated by paper disc diffusion method using Gentamycin as a standard. The minimum inhibitory concentration of the compound was determined. Compounds DP-1, DP-3 and DP-10 were good activity against E. coli, while DP-2 and DP-6 were moderately active. Compounds DP-1, DP-2, DP-4 and DP-10 have greatest activity against B. pumilus, while DP-3 and DP-5 were moderately active where as DP-6, DP-7 and DP-8 shows zero activity. All Compounds have moderate activity against S. aureus, except DP-6 and DP-7 which are poorly active and DP-8 with zero activity. DP-10 was active, while DP-5, DP-6 and DP-7 show zero activity and rest were poorly active against S. sonnei. Compounds DP-1, DP-2, DP-3, DP-7, DP-8 and DP-9 have moderate activity against P. vulgaris, while DP-4 and DP-6 were moderately active where as DP-5 and DP-10 shows zero activity. Compounds DP-3, DP-9, and DP-10 have moderate activity against P. aeruginosa, where DP-5 has zero activity and rest shows poor activity. The anti-fungal activity of the ten compounds was evaluated by paper disc diffusion method using Griseofulvin as a standard. All compounds except DP-8, DP-9 and DP-10, which has zero activity shows moderate activity against A. niger as well as C. albicans. Detail results are depicted in Table-3. Synthesized Compounds DP-1 to DP-10, were confirmed by IR, NMR, mass spectral and elemental analysis. CONCLUSION Our finding suggests that the compounds DP2 and DP8 with methyl and Cl substitute at para position are active molecule comparing to the previously synthesized derivatives. One can also synthesize the different substitute on methyl moiety to increase antimicrobial activity with minimal side effect. More potent benzimidazole derivative with substitute on methyl group at para position may find the active drug candidate in antimicrobial activity. The area of research on this molecule is still open to develop very potent agent in antimicrobial therapy.

47

D.N. Patil et al: Continental J. Pharmaceutical Sciences 2: 44 - 48, 2008 ACKNOWLEDGEMENT The authors are thankful to the Panacea Biotech India Ltd. for providing NMR, Mass structural analysis report and Head of Department, School of Life Sciences, Devi Ahilya Vishwavidyalaya, Indore, India for providing pathogenic microorganisms. Authors are also thankful to Head of Department, School of Pharmacy, Devi Ahilya Vishwavidyalaya, Indore, India for providing necessary facilities to carry out experimental work. Table 3: Antimicrobial activity of benzimidazole Derivatives against microorganism with zone of inhibition (mm)

Compounds code E. coli B. pumilis S. aureus S. sonnei P. vulgaris P. aeruginosa A. niger C.albicans

DP-1 18 17 16 15 14 11 18 17

DP-2 17 18 17 14 14 12 17 16

DP-3 18 15 14 16 15 14 16 14

DP-4 15 16 16 13 12 10 15 16

DP-5 - 14 15 - - - 15 14

DP-6 17 - 11 - 11 12 16 11

DP-7 14 - 09 - 14 09 16 13

DP-8 15 - - 14 13 10 - -

DP-9 14 13 14 15 14 13 - -

DP-10 18 16 15 18 - 14 - -

Gentamycin 19 16 18 19 15 16 - -

Griseofulvin - - - - - - 22 21

Negative control - - - - - - - -

REFERENCES 1. Cramer R.D., Patterson D.E. and Bunce J.D. (1988): Recent advances in comparative molecular field

analysis (CoMFA), J.Am.Chem.Soc. 170:5959. 2. Cohen N.C., Blaney J.M., Humblet D.E., Gund P., Barry D.C. (1990): Molecular modeling software and

methods for medicinal chemistry, J.Med.Chem., 33:883. 3. Ozden S., Atabey D., Yildiz S. and Goker H., (2004): Synthesis and potent antimicrobial activity of some

novel 4-(5,6-Dichloro-1h-benzimidazol-2-yl)-N-substituted derivatives, Arch. Pharm. Pharm. Med., 337,556-562.

4. Rajasree R., Nair G.V., Ekambaram H.S., Seshaiah K.S., Perumal P., (2005): Synthesis of schiff bases of 4-(4-aminophenyl)-morpholine as potential antimicrobial agents, Euro. Jour. of Med. Chem.,40,225-229.

5. Natesh R., Mohan A., Ekambaram H.S., Raju I., Seshaiah K.S., (2003): Synthesis of 6-fluoro-1,4-dihydro-4-oxo-quinoline-3-carboxylic acid derivatives as potential antimicrobial agents, Eur. J. Med. Chem., 38,1001-1004.

6. Jarrahpour A.A., Motamedifar M., Pakshir K., Hadi N. and Zarei (2004): Synthesis of novel azo schiff base and their antibacterial and antifungal activity, Molecule, 9,815-824.

7. Han V., Water B., (1998): Glossary of terms used in computational drug design, Annual Reports in Med. Chem., 33,404.

8. Dunn, W.J., Greenbag, W.J. and Callejas, S.S., (1976): Use of cluster analysis in the development of structure-activity relations for antitumor triazenes. J. Med. Chem., 19,1299.

9. Vogel’s introduction to chemical synthesis, (2006), 10th edition, Avs pub 994. 10. Seshaiah K.S., Muniyandy S., Atmakuru R., (2001): Synthesis and antibacterial screening of hydrazones,

schiff and mannich bases of isatin derivatives, Eur. J. Med. Chem., 36,615–625.

48

D.N. Patil et al: Continental J. Pharmaceutical Sciences 2: 44 - 48, 2008

11. Murat K., Misir A., Alaaddin C., and Cavit K., (2005): Studies on the synthesis and reactivity of novel benzofuran-2-yl-[3-methyl-3-phenylcyclobutyl] methanones and their antimicrobial activity, Molecules, 10,747-754.

12. Yingjie C., Yaxian D., Yushe Y., Shuhua Z. and Ruyun J., (2005): Synthesis and antibactral activity of a series of 3- (pyridine -3-yl)-2-oxazolidinone, Euro. J. of Med. Chem., 40,209-214.

Received for Publication: 04/11/2008 Accepted for Publication: 20/12/2008 Corresponding Author: Deepak N. Patil Sanjivani Institute of Pharmacy and Research, Kopargaon (M.S.), INDIA-423603 E-Mail: dnp_pharma@yahoo.co.in