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Pakistan Journal of Scientific and Industrial Research Vol. 51, No. 4 Golden Jubilee Issue July - August 2008

Messages

Shehryar Khan, Chairman PCSIR (iii)

Jamil Ahmed Chaudhry, Member Science PCSIR (v)

Editorial

Kaniz Fizza Azhar, Executive Editor (vii)

Special Paper

Fifty Years of Pakistan Journal of Scientific and Industrial Research-An OverviewWith Reference to PCSIR LaboratoriesShafiq Ahmad Khan and Muhammad Saleem 177

Physical Sciences

Phylogenetic and Computational Structure-Function Studies of ααααα-1, 4-Glucosidase(Maltase) From Baker's Yeast (Saccharomyces cerevisiae)Mushtaq Hussain, Javed A. Qureshi, Shagufta A. Shaikh and Liaquat Sultana 185

Study of Structure and Properties of Thermoplastic PolyurethanesA. Shokuhi Rad, M. Ardjmand and K. Mahmoodi 191

Preparation and Evaluation of Ciprofloxacin Hydrochloride Floating Oral Delivery SystemSujata Mohapatra, Sunit Kumar Sahoo, Subhakanta Dhal, Bhupen Chandra Behera, 201Bhakti Bhusan Barik and Bijan Kumar Gupta

Biological Sciences

Molecular and Biochemical Evaluation of Genetic Effects of Calotropis procera (Ait.)Latex on Aspergillus terreus ThomSameer H. Qari 206

Evaluation of Antiemetic Activities of Alcoholic Extract of Grewia asiatica inExperimental Model DogZahra Yaqeen, Tehmina Sohail, Atiq-ur-Rahman, Muhammad Saleem andZakir-ur-Rehman 212

Genetic Analysis of Fibre and Earliness Parameters in F2 Progenies ofIntra-hirsutum CrossesW. A. Jatoi, M. J. Baloch, N. F. Veesar, S.A. Panhwar, N. A. Panhwar and M. S. Majeedano 216

Occurrence and Severity of Arsenic in Urine, Hair and Nails Through ContaminatedDrinking Water in PakistanFarooq A. Khan, Ameera Javed, Javed Iqbal, Alia B. Munshi and Ishratullah Siddiqui 220

Review

Food and Drug Interaction - a Growing ConcernM. Humayoun Akhtar 225

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It is a privilege and honour to announce that Pakistan Journal of Scientific andIndustrial Research (PJSIR) completed fifty years of its publication (in the year 2007).This Journal was launched way back in 1958 by Prof. Dr. Salimuzzaman Siddiqui (Late),the renowned scientist and founder of PCSIR, to disseminate the research findings ofthe scientists and technologists, primarily those of PCSIR, for the benefit of the globalscientific community. Inspite of many technical and financial hurdles the Journal stuckto its commitment and continued its uninterrupted publication. I congratulate theEditorial Team of the Journal for the commendable work. It is also appreciated that theJournal gives exposure to the researches relating to the indigenous as well as third worldissues.

It is a matter of pride that PJSIR has kept pace with the times and continuouslyimproved its standard in keeping with the advances in knowledge. The world hasbecome a global village and it is not possible to work in isolation or keep the scientificdevelopments undeclared. The Journal conforms to the international standards and it ishoped that it will be enlisted with the International Scientific Institute (ISI) for the grantof Impact Factor. I wish and pray for continued success of the Journal in the times tocome.

Shehryar Khan Chairman PCSIR

(iii)

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It is a matter of great pleasure and pride in knowing that “Pakistan Journal ofScientific and Industrial Research (PJSIR)”, the bimonthly Journal of PCSIR has com-pleted fifty years of its uniterrupted publication.

Prosperity of industrialized countries is attributed to their advancement in the field ofscience and technology. Pakistan is blessed with abundant natural resources. With thepassage of time, more mineral deposits are being discovered and unexplored resourcesare being explored; what required is to make these valuable deposits utilizable throughresearch and developmental work. PCSIR has carried out tremendous work on theselines and its Journal (PJSIR) has performed the task assigned to it for disseminatingresearch findings in a standard format to scientific community globally.

In the light of current global challenges and threats posed by the internal and externalfactors, concerted R&D efforts are required to be undertaken by the scientificcommunity at national level to help in providing solutions for the critical problems beingfaced by industrial/S&T sector. Substantial amount has been allocated by the Govern-ment of Pakistan for human resource development and improvement of infrastructure atresearch institutes and technical training centres for the promotion of S&T basedresearch activities. It is hoped that, PCSIR in particular and the scientific community ingeneral, will put extra efforts towards this endeavour. I pray that Pakistan Journal ofScientific and Industrial Research (PJSIR) may continue its uninterrupted publicationas in the past and prosper further in future to achieve the highest standard of recognition.

Jamil Ahmed Chaudhry Member (Science) PCSIR

(v)

EditorialEditorialEditorialEditorialEditorial

Pakistan Journal of Scientific and Industrial Research (PJSIR), the prime Journal ofPCSIR, completed fifty years of its publication in the year 2007 and this year, its GoldenJubilee is being celebrated. The Journal disseminates the researches carried out by thescientists, primarily those of PCSIR, to the global scientific community. It conforms tothe international standards and the papers published in it are pre-reviewed by thesubject experts of international stature.

Two important projects, exclusively designed for the Golden Jubilee of the Journal, areon the anvil. One relates to the preparation of the Index to the Contents of the past fiftyvolumes of the Journal which is likely to be launched shortly. The second one is in theprocess of implementation with the collaboration of the Institute of Research Promo-tion (IRP); via the latter’s Pak Database (PDP), access to the abstracts of the paperspublished in the Journal during the last fifty years will be freely available.

A number of new features recently inducted emblazon the year of celebrations.Computerization of the overall activities has inducted a number of improvements in thesystem. The total time consumed in the processing of the papers has been reduced downto one month and the activation of the website of the Journal (www.pjsir.org) hasfacilitated on-line users, authors and referees. Lately introduced Manuscript TrackingSystem has made submission of papers and their book-keeping a lot easier.

The assistance of the reviewers of papers and the Higher Education Commission (HEC)is heartily acknowledged. Without scholarly contribution of the former and guidanceand financial assistance of the latter, it would have been difficult to implement manycorrective measures. It is hoped that valuable suggestions and guidance of the scientificcommunity to further improve the standard of PJSIR would remain available.

Dr. Kaniz Fizza Azhar Executive Editor

(vii)

IntroductionPakistan Journal of Scientific and Industrial Research (PJSIR)completed its fifty years of uninterrupted publication in theyear 2007; it started its publication in 1958 as a quarterly.Before going further into the subject matter underreview, it seems appropriate to reproduce here ‘The Foreword’that appeared in the first issue of the Journal. This Forewordwas written by Prof. Dr. Salimuzzaman Siddiqui, the founderChairman of the Pakistan Council of Scientific and IndustrialResearch (PCSIR); it describes not only the importance ofscientific research in the industrial development of thecountry but also provides a broad scenario that prevailed inthe country at that time and how steps were taken to build upthe infrastructure that is necessary for scientific andindustrial research. The need for bringing out the Journal ofScientific and Industrial Research is described in the said‘Foreword’ alongwith the desire to develop this Journal into amonthly publication ‘as early as possible’.

Foreword“Recognizing the importance of scientific research in theindustrial development and prosperity of a country, theGovernment of Pakistan took early steps to build upan organization for industrial research in the country andthe creation of the Department of Scientific and Indus-trial Research in 1949 under the Ministry of Industrieswas the first step in this direction. As a result of theactivities of this Department, the constitution of PakistanCouncil of Scientific and Industrial Research was drawnup with due reference to the structure of the organizationin the pre-independence period, and to the special needsand requirements of the greatly changed situation inPakistan and the Council was established as an autono-mous body in April, 1953.

Fifty Years of Pakistan Journal of Scientific and Industrial Research- An Overview With Reference to PCSIR Laboratories

Shafiq Ahmad Khana* and Muhammad Saleemb

a4-A, PCSIR Employees Cooperative Housing Society, Phase-I, Lahore, PakistanbDirector General, PCSIR Laboratories Complex, Ferozepur Road, Lahore-54600, Pakistan

(received February 15, 2008; revised March 4, 2008; accepted March 22, 2008)

The functions of the Council cover a wide range ofactivities generally directed towards the promotion andfostering of scientific research having a bearing on theindustrial development of the country and utilization ofits natural resources to the best economic advantage.While the scope of the activities of the Council has beenkept broad and comprehensive, due priority is given toproblems, which are of importance to the immediate needsof the country. In the pursuit of such a programme, how-ever, it is generally recognized that, if science is to get achance of effectively promoting the industrial develop-ment of the country, laboratory investigations in both pureand applied fields, followed by research developmentwork on a pilot plant scale, will all have to come underthe purview of the Council’s activities.

By far the most important among the aims and objects ofthe Council was the establishment of National Laborato-ries in the two wings of the country**. On the recom-mendations of a Planning Committee constituted by theCouncil, a plan was accordingly worked out for theestablishment of four such Laboratories, namely, theCentral Laboratories at Karachi, and three Regional Labo-ratories at Dacca**, Lahore and Peshawar, to take careof industrial problems specific to these areas. These Labo-ratories comprised 21 functional Research Divisions,allocated among them according to the requirements ofthe various regions. While the planning and constructionof these Laboratories are still proceeding, arrangementswere made early for setting up the nuclei of the Labora-tories in temporary buildings.

This was considered a matter of importance, because it isonly work done and results achieved that can lend mean-ing and reality to organizational activities and, moreover,the progress of the nucleus phase directed towards thesolution of urgent industrial problems of the country wouldensure a sound, organic growth of the research organiza-tion and its Laboratories. As an indication of the rapidpace at which the activities of the Council have

Pak. J. Sci. Ind. Res. 2008 51(4) 177-184

*Author for correspondence; E-mail: [email protected]**Editor’s note: At that time, there were two provinces of the coun-try, called East Pakistan (now Bangladesh) and West Pakistan; Daccais now included in Bangladesh.

177

Special Paper

progressed, it may be stated that, starting with comple-ment of only four research workers in 1953, the staff ofthe Central Laboratories, which are housed in temporarybuildings covering about 50,000 sq. ft. of floor area, hasgrown to over 280 members including scientific person-nel of various cadres and other technical and accessoryservices. The progress of the three Regional Laborato-ries, which started about two years later, is following thesame steeply ascending curve.

One of the aims of the Council relates to the collectionand dissemination of scientific and industrial informa-tion. To cope adequately with this sector of its activities,a scientific and technical documentation centre(PANSDOC)* has been recently set up under the Coun-cil with the assistance of UNESCO and the associationof three UNESCO expert advisers. With the phenomenalincrease in the volume of scientific research, keeping pacewith scientific literature even in a narrow, specialized fieldhas become a major problem in the advancement ofscientific knowledge, and a virtual despair of individualresearch worker. The establishment of PANSDOC will,it is hoped, considerably relieve the situation by supply-ing the needed publications to institutions and individualworkers in the form of microfilms and photocopies, andalso translations of research papers from other languagesinto English.

Alongside of these activities and the rapid progress madewith them, the need for bringing out a Journal ofScientific and Industrial Research under the Council hasbeen increasingly felt. At the same time, however, it wasrealized that it would be wiser to defer such an undertak-ing till research contributions from the various Laborato-ries of the Council and through research schemes financedby it at the Universities and other research institutions ofthe country had become available in sufficient volume torun the Journal successfully without adversely affectingthe publication of the existing Scientific Journals of thecountry. Happily, as a result of research work carried outunder the Council during the last four years over a widerange of problems relating to the development of the natu-ral resources of the country, a considerable volume ofpublishable material has accumulated, and it was felt thatthe publication of the Journal could not brook any furtherdelay. It will be initially published as a quarterly, but everyeffort will be made to develop it into a monthly Journalas early as possible. Such as it is, we are fully conscious

of the shortcomings of this first issue of the Journal. It isnonetheless hoped that it will be fulfilling an urgent needfor the dissemination of scientific and technologicalinformation on the one hand, and liaison with industry onthe other. The extent to which it may be able to fulfilthese dual functions, and further serve as an effectivemedium for the publication of results of originalresearches in both pure and applied fields, will be thetrue measure of its success”. (S.S)

Frequency changes and categorised publication. ThePakistan Journal of Scientific and Industrial Research (PJSIR)started its publication as a quarterly in the year 1958, thenwas converted to a bimonthly in the year 1971, which pro-vided greater coverage and also relieved pressure on theJournal as it continue to receive ever-increasing number ofresearch papers. The Publication Branch of PCSIR, estab-lished in 1956 at the PCSIR Head Office, launched the quar-terly Journal in 1958. The Branch, in 1984, was given thestatus of Division and, in 1989, after reorganization wasupgraded to the present Scientific Information Centre, at parwith other independent Centres of the Council. The Centrehandles all the matters relating to the Journal, from receipt ofmanuscripts submitted for publication to the Journal, to theirprocessing, to publishing/printing and distribution of theJournal. Among strict scrutiny on receipt, the papers undergopeer-review which is a sophisticated tool for sifting theoriginal work through review by the experts outside thein-house editional staff. The process exerts quality control overthe researches.

Since 1970, the Journal is being published regularly atbimonthly intervals and has been accommodating papers indiverse disciplines of science and research. A study of theJournal’s frequency reveals that since inception, it acceptedpapers in all disciplines of science. However, since 1970(vol. 13) the Journal did start assigning publications to thefollowing broad categories:

i) Physical Sciencesii) Biological Sciencesiii) Technology

This practice is continued currently as well. The major part ofthe published material in the earlier years was received fromthe Regional Laboratories of the Pakistan Council of Scientificand Industrial Research (PCSIR). The reason for this was thatthe country then had only a few Universities, which were notproperly staffed and equipped to undertake research activities.Also during that period there were virtually no other S&T andR&D Establishments and hence the work of PCSIR scientistsformed the bulk of contribution. With the passage of time, this

178

*Editor’s note: Now Pakistan Scientific and TechnologicalInformation Centre (PASTIC), a separate organization under theMinistry of Science and Technology.

Shafiq Ahmad Khan and Muhammad Saleem

scenario slowly changed and currently the Journal receivesresearch work from not only the R&D and S&T Organizationsand Universities (over 70) of the country but also from similarestablishments abroad.

All the PCSIR Laboratories were sub-divided into variousScientific Divisions in order to pursue activities related tothe assigned functions of these Divisions and to avoidduplication at the Laboratory level. As a policy the PCSIRre-organized itself during the decade of eighty and changedall its major Divisions to Centres with a view to achievingbetter efficiency and facilitating a smooth administrationof the expanded Laboratories. Presently three major multi-functional Laboratories of PCSIR are functioning at Lahore,Karachi and Peshawar. Recently, another such Laboratoryhas been established at Quetta, whereas, the scope of formerSolar Energy Research Centre at Hyderabad has beenexpanded to include other disciplines.

Centres of the Lahore Laboratories Complex• Applied Chemistry Research Centre.• Food and Biotechnology Research Centre.• Material Sciences Research Centre.• Glass and Ceramics Research Centre.• Applied Physics, Computers and Instrumentation

Centre.• Centre for Environmental Protection Studies.• Centre for Development of Laboratory Equipment.• Engineering Services Centre.

Centres of the Karachi Laboratories Complex• Pharmaceutical Research Centre.• Material Sciences Research Centre.• Food and Marine Resources Research Centre.• Applied Chemistry Research Centre.• Centre for Environmental Studies.• Centre for Development of Laboratory Equipment.• Engineering Services Centre.

Centres of the Peshawar Laboratories Complex• Medicinal Botanic Centre.• Food Technology Centre.• Material Sciences Centre.• Engineering Services Centre.

Divisions of the Quetta Laboratories• Food Technology Division• Mineral Technology Division• Chemical Engineering & Pilot Plant Division

Divisions of Hyderabad Laboratories• Analytical and Consultancy Division.• Food Preservation and Processes.

• Research Division.• Rural Development Research Division.• General Services Division.

Various mono-functional Centres are:-• Scientific Information Centre, Karachi• Environmental Analytical Laboratory, Islamabad.• Leather Research Centre, Karachi.• Fuel Research Centre, Karachi.• National Physical and Standard Laboratory, Islamabad.• Demonstration-Cum-Training Centre, Skardu.

Besides, a Training Centre, namely Pak-Swiss TrainingCentre was established at Karachi in 1965 to cater to thetechnical needs of the equipment used for research in thePCSIR Laboratories through training people in such diversefields as maintenance, repairs and operation of machines.Similar Centres are also operating at Karachi, Lahore,Peshawar and Quetta in addition to a degree awardingInstitute at Karachi.

The reorganization as well as the policy changes did notaffect the tempo of the assigned responsibilities of the researchscientists and technologists and consequently the Journal wasregularly provided with the research papers for publicationpurposes. It is of interest to state here that during the earlierphase, the research staff for the Laboratories, was recruited,and trained both inside on the job and abroad in variousUniversities, because of this the contributions to the Journalfrom the Laboratories were largely dependent on the numberof research workers available at any given time. Inspite ofthis, however, all Laboratories of the PCSIR continued to feedthe Journal for maintaining its regularity and continuity ofappearance.

The data collected for the past fifty years of the Journal’s pub-lications, in fifty volumes (1958-2007), is presented in Table 1and the number of publications from all the Laboratories (ofthe PCSIR) along with other contributing sources is tabulatedin it. Similarly the data regarding publications in the earlierstated three categories i.e., Physical Sciences, BiologicalSciences and Technology from 1970-2007, is provided inTable 2. It is to be mentioned that after establishment ofBangladesh (BD) in 1971, PCSIR Laboratories at Dacca andChittagong were taken over by BD. It is evident from the data(Table 1 and 2) that the Laboratories of the Council have beencontributing to the Journal not only regularly but also in all thethree categories under which the Journal has been publishingthe research papers since 1970 onwards.

The Journal published 4986 papers since 1958 to 2007, outof which 813 papers (16.3 %) were contributed by the Lahore,988 (19.8%) by Karachi, 357 (7.2%) by Peshawar and 113

179Fifty Years of Pak. J. Sci. Ind. Res. - An Overview

Table 1. Contributions from the PCSIR Laboratories and other sources to the Pakistan Journal of Scientific & IndustrialResearch (PJSIR)

Laboratory-wise Publications fromVolume Year Total Publications Lahore Karachi (a) Peshawar Dacca (b) Others (c) Foreign

1 1958 63 2 40 1 15 5 -2 1959 37 - 17 - 10 8 23 1960 57 4 24 4 12 12 14 1961 55 2 15 5 7 11 155 1962 67 6 18 9 20 10 46 1963 77 5 29 21 9 8 57 1964 76 9 33 12 6 12 48 1965 64 15 24 11 4 10 09 1966 104 20 37 20 9 17 110 1967 82 17 30 10 2 13 1011 1968 115 19 41 18 - 30 712 1969 115 18 37 18 1 33 813 1970 105 17 31 12 6 29 1014 1971 148 15 44 15 7 48 1915 1972 127 15 30 13 5 46 1816 1973 84 12 25 6 - 27 1417 1974 71 15 12 5 - 28 1118 1975 68 12 9 9 - 30 819 1976 78 12 5 8 - 30 2320 1977 99 30 7 5 - 32 2521 1978 65 9 5 3 - 28 2022 1979 94 31 6 3 - 35 1923 1980 70 17 8 3 - 24 1824 1981 54 13 3 4 - 24 1025 1982 63 10 11 2 - 31 926 1983 96 21 11 6 - 38 2027 1984 97 20 11 7 - 37 2428 1985 109 22 16 2 - 46 2329 1986 122 38 27 5 - 38 1430 1987 240 56 34 7 - 101 4231 1988 225 41 24 9 - 95 5632 1989 238 45 52 12 - 90 3933 1990 157 24 22 11 - 60 4034 1991 143 25 24 6 - 52 3635 1992 146 27 12 8 - 52 4736 1993 151 21 20 6 - 65 3937 1994 145 20 17 6 - 47 5538 1995 115 12 18 4 - 40 4139 1996 52 9 5 3 - 18 1740* 1997 30 4 4 3 - 8 1141 1998 70 7 14 5 - 18 2642 1999 91 14 13 4 - 25 3543 2000 82 7 27 3 - 21 2444 2001 88 4 23 4 - 32 2545 2002 99 11 16 2 - 34 3646 2003 101 10 13 5 - 30 4347 2004 96 10 14 9 - 27 3648 2005 89 10 8 1 - 17 5349 2006 85 20 11 7 - 14 3350 2007 81 10 11 3 113 20 37

Total 4986 813 988 357 113 1602 1113

(a) = inclusive of contribution from Head Office, Fuel Research Centre (FRC), Karachi; Leather Research Centre (LRC), Karachi; NationalPhysical and Standard Laboratory (NPSL), Islamabad and PCSIR Laboratories at Quetta and Skardu. (b) = inclusive of contributions fromother PCSIR Laboratories facilities in East Pakistan. (c) = contributions from Organizations (S&T and R&D), Universities and Faculties inPakistan other than PCSIR Laboratories. * = only two issues Jan-April and May-Dec.

180 Shafiq Ahmad Khan and Muhammad Saleem

Table 2. Categorized publications from the Lahore, Karachi and Peshawar Laboratories of the PCSIR to the Pakistan Journal ofScientific & Industrial Research (PJSIR).

Categorized Publications From Lahore, Karachi and Peshawar Laboratories

Lahore Labs. Karachi Labs. Peshawar Labs.Volume Year Phy. Sci. Biol. Sci. Tech. Phy. Sci. Biol. Sci. Tech. Phy. Sci. Biol. Sci. Tech.

13 1970 11 5 4 17 21 0 4 1 714 1971 5 8 2 20 30 1 10 2 315 1972 2 2 10 14 11 3 1 8 516 1973 4 3 6 7 10 5 2 1 417 1974 7 1 7 5 2 6 0 0 418 1975 2 2 10 7 2 0 2 1 519 1976 5 0 8 3 0 2 2 2 520 1977 2 0 29 1 2 4 1 1 321 1978 1 1 7 2 0 3 1 0 222 1979 0 0 30 0 2 4 0 0 223 1980 2 1 14 4 4 0 0 0 324 1981 4 0 9 2 2 0 0 0 325 1982 5 0 5 4 4 1 1 0 126 1983 6 4 12 2 3 5 4 0 227 1984 3 9 8 4 3 1 2 2 328 1985 4 8 8 5 7 3 1 1 129 1986 13 14 11 12 6 7 2 1 230 1987 20 19 18 8 14 9 3 4 031 1988 11 13 19 5 11 4 4 1 432 1989 4 18 20 10 24 11 4 2 733 1990 12 1 8 7 10 7 6 3 334 1991 5 12 8 9 6 6 3 1 235 1992 7 7 12 2 8 1 2 3 336 1993 4 5 11 4 11 1 2 2 237 1994 7 9 6 8 5 2 4 0 138 1995 1 6 5 5 4 5 1 1 239 1996 3 4 1 0 5 0 3 0 140 1997 2 0 1 2 2 0 2 0 141 1998 3 2 3 7 4 1 2 1 242 1999 1 11 2 9 3 0 1 0 343 2000 4 2 1 10 8 3 1 0 244 2001 4 6 2 4 6 2 1 1 045 2002 2 2 0 7 8 3 1 2 146 2003 4 7 0 9 3 1 3 2 247 2004 5 5 1 6 8 0 6 1 148 2005 7 1 2 3 1 1 0 1 049 2006 14 2 4 5 3 2 3 3 150 2007 6 - 4 5 4 1 1 - 1

Total 202 182 208 232 155 105 86 48 94


Table 3. Editorial Boards.

Volumes Name

1-10 Salimuzzaman Siddiqui (Chairman)(1958-1967) M.S.H. Siddiqui (Chairman)

M.K. AfridiAli AhmadNazeer AhmedTaskhir AhmadM. Innas AliM.O. GhaniM.H.KhundkarA.M. ChaudhuryM.A. KaziN.A. KhanM.I.R. KhanKamaluddin AhmedKhawaja SolahuddinMazhar-ul-HaqueS.HedayatullahQ.M.HussainKarimullahM. Qudrat-i- KhudaM. Raziuddin SiddiqiM.M. Qurashi (Editor, Secretary)Aftab Hassan (Co-Editor)M.A. Haleem (Associate Editor)M.Aslam (Associate Editor)Kamal Mohammad Habib (Associate Co-Editor)

11-20 M.S.H. Siddiqui (Chairman)(1968-1977) Abdul Ghani (Chairman)

Salimuzzaman SiddiquiA.F. AbbasiMuzaffer AhmedM. Zain-ul-AbedinM. Raziuddin SiddiqiM.H. HashmiM.O. GhaniM. AslamValentine G. DeSaS.Z. HaiderM.H. KhundkarA. Hameed KhanA.M. ChaudhuryM. AfzalMazhar-ul-HaqueKiam-ud-DinYousuf AhmedM.A. KaziM.K.BhattyA.H. ChotaniN.A. KhanH.A. KazmiS.M. QurashiM.I.R. KhanKamaluddin AhmedKhawaja SalahuddinS. Riaz Ali Shah

Afzal AhmadMirza Mansoor BegM. Inayat KhanM. Manzoor-i-KhudaM.H.KhanRiazuddinM.A. ShaukatM. ZafarullahAftab HasanM.M. Qurashi (Editor)M.A. Haleem (Chief Editor, Associate Editor)Kamal Mohammad Habib (Associate Co-Editor)

21-30 Abdul Ghani (Chairman)(1978-1987) M. Aslam (Chairman)

Naeem Ahmad Khan (Chairman, Chief Editor)Mirza Arshad Ali BegMuzaffer AhmadYusaf AhmadMirza Mansoor BegHeshamul HaqueM.K. BhattyN.M. ButtS. Fazal HussainM. IkramH.A. KazmiM.M. QurashiS.A. QureshiRiazuddin SiddiquiM. Ataur RehmanM. Shafi AhmadS. Riaz Ali ShahM.A. ShaukatM. ZafarullahM. Zain-ul-AbedinR.A. Khan TahirkheliA.R. AzmiS.H. Mujtaba NaqviM.H. QaziM. Ata-ur-RehmanMahmood Iqbal SheikhNasima M. TirmiziA.H. ChotaniA.T. KhanF.A. FarooqiS.A.QureshiS.M. QureshiMohammad Nazir RomaniM. A. Haleem (Chief Editor)A.H. Khan (Editor)Azmat Ali Khan (Executive Editor)

31-40 Naeem Ahmad Khan (Chairman, Chief Editor)(1988-1997) Qamar Iqbal (Chairman)

A.Q. Ansari (Chairman, Chief Editor)Syed Naeem Mahmood (Executive Editor)Anwar-ul-HaqAtta-ur-RehmanKhalid FarooqYasmin Badar


(2.3%) by the Dacca Laboratories; other R&D and S&Tinstitutions of the country contributed 1602 papers (32.1%),whereas, 1113 (22.3%) were contributed by the institutions offoreign countries. On an overall basis, the PCSIR’s share ofcontribution thus stands on an average, at about 45.55 over thepast 50 years. The data regarding the categorized contributionsfrom the Lahore, the Karachi and the Peshawar Laboratoriesof the PCSIR is given in Table 2. The basic purpose of thestatistics is to consolidate the data as well as to analyze andunderstand it with a view to knowing the significance of thecontributions from all the Laboratories of the PCSIR. It is hopedthis effort will provide authentic basis both for the policy plan-ners and the working scientists/technologists to better assessthe output. Consequently, the potential and S&T capabilities ofvarious groups will be better judged and the disadvantages, ifany, will be identified and removed for a better programme andefforts will be directed towards providing the needed facilitiesand opportunities to accomplish the assigned and planned tasks


Mohammad YusafShamshad Mehdi NaqviZafar Saied SaifyIsmail KhanS. Sadiq Ali RizviMirza Arshad Ali BegM.K. BhattyN.M. ButtS. Fazal HussainShafi Ahmad KhanAtta-ur-RehmanS.S.H. RizviEhsan AliS. Shahid HusainA. Q. AlviM. Ikram-ul-Haq DarA.H.K. YousufzaiKhushnood A. SiddiquiSaeed Iqbal ZafarR. B. QadriGhulam Abbas MianaM. Aslam ButtAbdul Rehman KhanMohammad Anwar WaqarSabir AliF.H. ShahMahmood Iqbal SheikhJamil Ahmad KhanNaseer SheikhIkram-ul-Haq DarM.A. QaziM.M. QureshiM. Shafi AhmadR.A. Khan TahirkheliA.R. AzmiS.H. Mujtaba NaqviM.H. QaziM. Ata-ur-RehmanMahmood Iqbal SheikhNasima M. TirmiziA.H. ChotaniA.T. KhanF.A. FarooqiS.A.QureshiS.M. QureshiMohammad Nazir RomaniS.M. Abdul Hai (Editor)Azmat Ali Khan (Executive Editor)J. N. Usmani (Executive Editor)Abdul Gaffar (Assistant Editor)

41-50 A.Q. Ansari (Chairman, Chief Editor)(1998-2007) Khalid Mahmood Khan (Chairman, Chief Editor)

Misbahul Ain Khan (Chairman)Anwar-ul-Haq (Chairman, Chief Editor)Javed Arshad Mirza, (Chairman, Editor-in-Chief)Saeed Iqbal Zafar (Editor-in-Chief)Syed Naeem Mahmood (Executive Editor)A. Rasheed Khan (Executive Editor)

Rabia Zuberi (Chief Editor)Khurshid Zaman (Executive Editor)Kaniz Fizza Azhar (Executive Editor)Atta-ur-RehmanKhalid FarooqM. Yasmin BadarMohammad YusafShamshad Mehdi NaqviZafar Saied SaifyIsmail KhanS.Sadiq Ali RizviH. AkhtarG. BouetW. LinertB.H. MehtaE. MiraldiM.J. QureshiF.M. SlaterM. A. WaqarMansoor Ahmad ButtS. Wazir Hussain ShahJ. OzgaM. AkhtarA.G. AttkinsM.A. KhanSuresh NarineJohn R. OgrenH.M. OrtnerNasima M. TirmiziAbul KalamAbdul Rehman MemonR.B. QadriM.A. KhanA. DiasproH. KhanM.W. Akhtar

for developmental purposes. The publications considered forthe present report have been gathered, thus, from 1970 to 2007(vol. 13-50) as they contain the publications under the abovestated categories.

The data for the past 38 years ( i.e., 1970 to 2007, both yearsincluded, vol. 13-50 of Pakistan Journal of Scientific and In-dustrial Research)is presented in Table 2. It can be seen thatthe three Laboratories at Lahore, Karachi and Peshawarcontributed 202, 232 and 86 papers in the Physical Sciences,182, 155 and 48 in Biological Sciences and 208, 105 and 94 inthe Technology fields, respectively. Numerically speaking thecontributions, in the three specified categories add up to 592for the Lahore Laboratories, 492 for the Karachi Laboratoriesand 228 for the Peshawar Laboratories. Various Research Divisions/Centres of the three Laboratories, as already mentioned, carryout their research projects largely in relation to the scope of theassigned activities of the Division/Centre. However, there is nobar in the intra-Division/Centre or the intra-Laboratoriescollaboration and many a ublications are also the result ofsuch activities as it helps in using the expertise and facilitiesavailable to various research groups at different Laboratoriesfor the completion of the undertaken projects.

A closer look at the data in Table 2 indicates that all thethree research Laboratories undertake activities, in the speci-fied fields, according to their planned programmes thoughin varying degrees. The contributions from the Lahore Labo-ratories are less in the Physical Sciences and more in thefields of Biological Sciences and Technology, when com-pared with the Karachi Laboratories. The reason for thisvariation is only due to the pre-planned programmes of eachunit. On an overall basis, however, it can be said that all thethree contributing units have adhered well to a healthy com-petitive attitude inspite of variations in the availability ofresearch staff and facilities.

An improvement both in the quality and quantity of thecontributions to the Journal from the parent organization(PCSIR) can be achieved if intra-Laboratory task forces canbe created to undertake various technological projects whichare time targeted and their component bits are assigned togroups identified on the basis of technical expertise.

Such an approach, it is hoped, will encourage cooperationand provide access to facilities, if they are available at oneplace and not available at another, and help in better monitor-ing the progress of the undertaken project and bring it to auseful conclusion. A similar approach can also be practicedboth in the fields of Physical and Biological Sciences as theseprojects can also be divided and distributed to collaboratinggroups so that their assigned aspects are completed faster than

when the whole project is tackled at one place only. Thus, notonly collaboration but the output as well will increase and thecollaborating scientists/technologists will get more useful andwide exposure in their respective fields of expertise.

Editorial BoardsThe Editorial Boards of the Journal have been changing withthe appointments of the Chairmen of the Pakistan Council forScientific and Industrial Research (PCSIR). However, theboards always comprised of prominent scientists available notonly in the country but also from foreign lands. The majorshare of the board members has however, been occupied bythe senior scientists from the Laboratories and varioussubject specialists from the Universities. A list of the mem-bers of the Editorial Boards during the period under review ispresented in Table 3 for information only.

ConclusionThe Pakistan Journal of Scientific and Industrial Research(PJSIR), during the period under review (1958-2007), published4986 research papers in various disciplines out of which 45.55%papers were contributed by the PCSIR Laboratories Complexes,at Lahore, Karachi and Peshawar only. It is thus seen that theJournal during the period has continuously and regularly broughtout publications in all the disciplines in which its Laboratorieswere mandated to operate and accepted publications from otherS&T and R&D organizations of Pakistan (32.10%) and fromall over the world (22.30%).

AcknowledgementThanks are due to Dr. Kaniz Fizza Azhar, Executive Editorof the Pakistan Journal of Scientific and Industrial Research(PJSIR), and her other colleagues in the PCSIR ScientificInformation Centre, Karachi for their timely help andassistance and provision of information for this article.Acknowledgements are also due to Mr. Azmat Ali Khan,Dr. Jafar Nazir Usmani and Mr. S. Naeem Mahmood for notonly their cooperation and support but also for their keeninterest, useful criticism and encouragement in the prepara-tion of this manuscript.

ReferencesHames, I. 2007. Peer Review and Manuscript Management

in Scientific Journals: Guidelines for Good Practice,293 p, Blackwell Publishing, Massachusetts, Oxford,UK.

Pak. J. Sci. Ind. Res. 1958-2007. (all issues of the Journalfrom vol. 1 to vol. 50), PCSIR.

PCSIR Annual Report 2006-2007. PCSIR ScientificInformation Centre, Karachi.


progressed, it may be stated that, starting with comple-ment of only four research workers in 1953, the staff ofthe Central Laboratories, which are housed in temporarybuildings covering about 50,000 sq. ft. of floor area, hasgrown to over 280 members including scientific person-nel of various cadres and other technical and accessoryservices. The progress of the three Regional Laborato-ries, which started about two years later, is following thesame steeply ascending curve.

One of the aims of the Council relates to the collectionand dissemination of scientific and industrial informa-tion. To cope adequately with this sector of its activities,a scientific and technical documentation centre(PANSDOC)* has been recently set up under the Coun-cil with the assistance of UNESCO and the associationof three UNESCO expert advisers. With the phenomenalincrease in the volume of scientific research, keeping pacewith scientific literature even in a narrow, specialized fieldhas become a major problem in the advancement ofscientific knowledge, and a virtual despair of individualresearch worker. The establishment of PANSDOC will,it is hoped, considerably relieve the situation by supply-ing the needed publications to institutions and individualworkers in the form of microfilms and photocopies, andalso translations of research papers from other languagesinto English.

Alongside of these activities and the rapid progress madewith them, the need for bringing out a Journal ofScientific and Industrial Research under the Council hasbeen increasingly felt. At the same time, however, it wasrealized that it would be wiser to defer such an undertak-ing till research contributions from the various Laborato-ries of the Council and through research schemes financedby it at the Universities and other research institutions ofthe country had become available in sufficient volume torun the Journal successfully without adversely affectingthe publication of the existing Scientific Journals of thecountry. Happily, as a result of research work carried outunder the Council during the last four years over a widerange of problems relating to the development of the natu-ral resources of the country, a considerable volume ofpublishable material has accumulated, and it was felt thatthe publication of the Journal could not brook any furtherdelay. It will be initially published as a quarterly, but everyeffort will be made to develop it into a monthly Journalas early as possible. Such as it is, we are fully conscious

of the shortcomings of this first issue of the Journal. It isnonetheless hoped that it will be fulfilling an urgent needfor the dissemination of scientific and technologicalinformation on the one hand, and liaison with industry onthe other. The extent to which it may be able to fulfilthese dual functions, and further serve as an effectivemedium for the publication of results of originalresearches in both pure and applied fields, will be thetrue measure of its success”. (S.S)

Frequency changes and categorised publication. ThePakistan Journal of Scientific and Industrial Research (PJSIR)started its publication as a quarterly in the year 1958, thenwas converted to a bimonthly in the year 1971, which pro-vided greater coverage and also relieved pressure on theJournal as it continue to receive ever-increasing number ofresearch papers. The Publication Branch of PCSIR, estab-lished in 1956 at the PCSIR Head Office, launched the quar-terly Journal in 1958. The Branch, in 1984, was given thestatus of Division and, in 1989, after reorganization wasupgraded to the present Scientific Information Centre, at parwith other independent Centres of the Council. The Centrehandles all the matters relating to the Journal, from receipt ofmanuscripts submitted for publication to the Journal, to theirprocessing, to publishing/printing and distribution of theJournal. Among strict scrutiny on receipt, the papers undergopeer-review which is a sophisticated tool for sifting theoriginal work through review by the experts outside thein-house editional staff. The process exerts quality control overthe researches.

Since 1970, the Journal is being published regularly atbimonthly intervals and has been accommodating papers indiverse disciplines of science and research. A study of theJournal’s frequency reveals that since inception, it acceptedpapers in all disciplines of science. However, since 1970(vol. 13) the Journal did start assigning publications to thefollowing broad categories:

i) Physical Sciencesii) Biological Sciencesiii) Technology

This practice is continued currently as well. The major part ofthe published material in the earlier years was received fromthe Regional Laboratories of the Pakistan Council of Scientificand Industrial Research (PCSIR). The reason for this was thatthe country then had only a few Universities, which were notproperly staffed and equipped to undertake research activities.Also during that period there were virtually no other S&T andR&D Establishments and hence the work of PCSIR scientistsformed the bulk of contribution. With the passage of time, this

178

*Editor’s note: Now Pakistan Scientific and TechnologicalInformation Centre (PASTIC), a separate organization under theMinistry of Science and Technology.

Shafiq Ahmad Khan and Muhammad Saleem

scenario slowly changed and currently the Journal receivesresearch work from not only the R&D and S&T Organizationsand Universities (over 70) of the country but also from similarestablishments abroad.

All the PCSIR Laboratories were sub-divided into variousScientific Divisions in order to pursue activities related tothe assigned functions of these Divisions and to avoidduplication at the Laboratory level. As a policy the PCSIRre-organized itself during the decade of eighty and changedall its major Divisions to Centres with a view to achievingbetter efficiency and facilitating a smooth administrationof the expanded Laboratories. Presently three major multi-functional Laboratories of PCSIR are functioning at Lahore,Karachi and Peshawar. Recently, another such Laboratoryhas been established at Quetta, whereas, the scope of formerSolar Energy Research Centre at Hyderabad has beenexpanded to include other disciplines.

Centres of the Lahore Laboratories Complex• Applied Chemistry Research Centre.• Food and Biotechnology Research Centre.• Material Sciences Research Centre.• Glass and Ceramics Research Centre.• Applied Physics, Computers and Instrumentation

Centre.• Centre for Environmental Protection Studies.• Centre for Development of Laboratory Equipment.• Engineering Services Centre.

Centres of the Karachi Laboratories Complex• Pharmaceutical Research Centre.• Material Sciences Research Centre.• Food and Marine Resources Research Centre.• Applied Chemistry Research Centre.• Centre for Environmental Studies.• Centre for Development of Laboratory Equipment.• Engineering Services Centre.

Centres of the Peshawar Laboratories Complex• Medicinal Botanic Centre.• Food Technology Centre.• Material Sciences Centre.• Engineering Services Centre.

Divisions of the Quetta Laboratories• Food Technology Division• Mineral Technology Division• Chemical Engineering & Pilot Plant Division

Divisions of Hyderabad Laboratories• Analytical and Consultancy Division.• Food Preservation and Processes.

• Research Division.• Rural Development Research Division.• General Services Division.

Various mono-functional Centres are:-• Scientific Information Centre, Karachi• Environmental Analytical Laboratory, Islamabad.• Leather Research Centre, Karachi.• Fuel Research Centre, Karachi.• National Physical and Standard Laboratory, Islamabad.• Demonstration-Cum-Training Centre, Skardu.

Besides, a Training Centre, namely Pak-Swiss TrainingCentre was established at Karachi in 1965 to cater to thetechnical needs of the equipment used for research in thePCSIR Laboratories through training people in such diversefields as maintenance, repairs and operation of machines.Similar Centres are also operating at Karachi, Lahore,Peshawar and Quetta in addition to a degree awardingInstitute at Karachi.

The reorganization as well as the policy changes did notaffect the tempo of the assigned responsibilities of the researchscientists and technologists and consequently the Journal wasregularly provided with the research papers for publicationpurposes. It is of interest to state here that during the earlierphase, the research staff for the Laboratories, was recruited,and trained both inside on the job and abroad in variousUniversities, because of this the contributions to the Journalfrom the Laboratories were largely dependent on the numberof research workers available at any given time. Inspite ofthis, however, all Laboratories of the PCSIR continued to feedthe Journal for maintaining its regularity and continuity ofappearance.

The data collected for the past fifty years of the Journal’s pub-lications, in fifty volumes (1958-2007), is presented in Table 1and the number of publications from all the Laboratories (ofthe PCSIR) along with other contributing sources is tabulatedin it. Similarly the data regarding publications in the earlierstated three categories i.e., Physical Sciences, BiologicalSciences and Technology from 1970-2007, is provided inTable 2. It is to be mentioned that after establishment ofBangladesh (BD) in 1971, PCSIR Laboratories at Dacca andChittagong were taken over by BD. It is evident from the data(Table 1 and 2) that the Laboratories of the Council have beencontributing to the Journal not only regularly but also in all thethree categories under which the Journal has been publishingthe research papers since 1970 onwards.

The Journal published 4986 papers since 1958 to 2007, outof which 813 papers (16.3 %) were contributed by the Lahore,988 (19.8%) by Karachi, 357 (7.2%) by Peshawar and 113


Table 1. Contributions from the PCSIR Laboratories and other sources to the Pakistan Journal of Scientific & IndustrialResearch (PJSIR)

Laboratory-wise Publications fromVolume Year Total Publications Lahore Karachi (a) Peshawar Dacca (b) Others (c) Foreign

1 1958 63 2 40 1 15 5 -2 1959 37 - 17 - 10 8 23 1960 57 4 24 4 12 12 14 1961 55 2 15 5 7 11 155 1962 67 6 18 9 20 10 46 1963 77 5 29 21 9 8 57 1964 76 9 33 12 6 12 48 1965 64 15 24 11 4 10 09 1966 104 20 37 20 9 17 110 1967 82 17 30 10 2 13 1011 1968 115 19 41 18 - 30 712 1969 115 18 37 18 1 33 813 1970 105 17 31 12 6 29 1014 1971 148 15 44 15 7 48 1915 1972 127 15 30 13 5 46 1816 1973 84 12 25 6 - 27 1417 1974 71 15 12 5 - 28 1118 1975 68 12 9 9 - 30 819 1976 78 12 5 8 - 30 2320 1977 99 30 7 5 - 32 2521 1978 65 9 5 3 - 28 2022 1979 94 31 6 3 - 35 1923 1980 70 17 8 3 - 24 1824 1981 54 13 3 4 - 24 1025 1982 63 10 11 2 - 31 926 1983 96 21 11 6 - 38 2027 1984 97 20 11 7 - 37 2428 1985 109 22 16 2 - 46 2329 1986 122 38 27 5 - 38 1430 1987 240 56 34 7 - 101 4231 1988 225 41 24 9 - 95 5632 1989 238 45 52 12 - 90 3933 1990 157 24 22 11 - 60 4034 1991 143 25 24 6 - 52 3635 1992 146 27 12 8 - 52 4736 1993 151 21 20 6 - 65 3937 1994 145 20 17 6 - 47 5538 1995 115 12 18 4 - 40 4139 1996 52 9 5 3 - 18 1740* 1997 30 4 4 3 - 8 1141 1998 70 7 14 5 - 18 2642 1999 91 14 13 4 - 25 3543 2000 82 7 27 3 - 21 2444 2001 88 4 23 4 - 32 2545 2002 99 11 16 2 - 34 3646 2003 101 10 13 5 - 30 4347 2004 96 10 14 9 - 27 3648 2005 89 10 8 1 - 17 5349 2006 85 20 11 7 - 14 3350 2007 81 10 11 3 113 20 37

Total 4986 813 988 357 113 1602 1113

(a) = inclusive of contribution from Head Office, Fuel Research Centre (FRC), Karachi; Leather Research Centre (LRC), Karachi; NationalPhysical and Standard Laboratory (NPSL), Islamabad and PCSIR Laboratories at Quetta and Skardu. (b) = inclusive of contributions fromother PCSIR Laboratories facilities in East Pakistan. (c) = contributions from Organizations (S&T and R&D), Universities and Faculties inPakistan other than PCSIR Laboratories. * = only two issues Jan-April and May-Dec.


Table 2. Categorized publications from the Lahore, Karachi and Peshawar Laboratories of the PCSIR to the Pakistan Journal ofScientific & Industrial Research (PJSIR).

Categorized Publications From Lahore, Karachi and Peshawar Laboratories

Lahore Labs. Karachi Labs. Peshawar Labs.Volume Year Phy. Sci. Biol. Sci. Tech. Phy. Sci. Biol. Sci. Tech. Phy. Sci. Biol. Sci. Tech.

13 1970 11 5 4 17 21 0 4 1 714 1971 5 8 2 20 30 1 10 2 315 1972 2 2 10 14 11 3 1 8 516 1973 4 3 6 7 10 5 2 1 417 1974 7 1 7 5 2 6 0 0 418 1975 2 2 10 7 2 0 2 1 519 1976 5 0 8 3 0 2 2 2 520 1977 2 0 29 1 2 4 1 1 321 1978 1 1 7 2 0 3 1 0 222 1979 0 0 30 0 2 4 0 0 223 1980 2 1 14 4 4 0 0 0 324 1981 4 0 9 2 2 0 0 0 325 1982 5 0 5 4 4 1 1 0 126 1983 6 4 12 2 3 5 4 0 227 1984 3 9 8 4 3 1 2 2 328 1985 4 8 8 5 7 3 1 1 129 1986 13 14 11 12 6 7 2 1 230 1987 20 19 18 8 14 9 3 4 031 1988 11 13 19 5 11 4 4 1 432 1989 4 18 20 10 24 11 4 2 733 1990 12 1 8 7 10 7 6 3 334 1991 5 12 8 9 6 6 3 1 235 1992 7 7 12 2 8 1 2 3 336 1993 4 5 11 4 11 1 2 2 237 1994 7 9 6 8 5 2 4 0 138 1995 1 6 5 5 4 5 1 1 239 1996 3 4 1 0 5 0 3 0 140 1997 2 0 1 2 2 0 2 0 141 1998 3 2 3 7 4 1 2 1 242 1999 1 11 2 9 3 0 1 0 343 2000 4 2 1 10 8 3 1 0 244 2001 4 6 2 4 6 2 1 1 045 2002 2 2 0 7 8 3 1 2 146 2003 4 7 0 9 3 1 3 2 247 2004 5 5 1 6 8 0 6 1 148 2005 7 1 2 3 1 1 0 1 049 2006 14 2 4 5 3 2 3 3 150 2007 6 - 4 5 4 1 1 - 1

Total 202 182 208 232 155 105 86 48 94


Table 3. Editorial Boards.

Volumes Name

1-10 Salimuzzaman Siddiqui (Chairman)(1958-1967) M.S.H. Siddiqui (Chairman)

M.K. AfridiAli AhmadNazeer AhmedTaskhir AhmadM. Innas AliM.O. GhaniM.H.KhundkarA.M. ChaudhuryM.A. KaziN.A. KhanM.I.R. KhanKamaluddin AhmedKhawaja SolahuddinMazhar-ul-HaqueS.HedayatullahQ.M.HussainKarimullahM. Qudrat-i- KhudaM. Raziuddin SiddiqiM.M. Qurashi (Editor, Secretary)Aftab Hassan (Co-Editor)M.A. Haleem (Associate Editor)M.Aslam (Associate Editor)Kamal Mohammad Habib (Associate Co-Editor)

11-20 M.S.H. Siddiqui (Chairman)(1968-1977) Abdul Ghani (Chairman)

Salimuzzaman SiddiquiA.F. AbbasiMuzaffer AhmedM. Zain-ul-AbedinM. Raziuddin SiddiqiM.H. HashmiM.O. GhaniM. AslamValentine G. DeSaS.Z. HaiderM.H. KhundkarA. Hameed KhanA.M. ChaudhuryM. AfzalMazhar-ul-HaqueKiam-ud-DinYousuf AhmedM.A. KaziM.K.BhattyA.H. ChotaniN.A. KhanH.A. KazmiS.M. QurashiM.I.R. KhanKamaluddin AhmedKhawaja SalahuddinS. Riaz Ali Shah

Afzal AhmadMirza Mansoor BegM. Inayat KhanM. Manzoor-i-KhudaM.H.KhanRiazuddinM.A. ShaukatM. ZafarullahAftab HasanM.M. Qurashi (Editor)M.A. Haleem (Chief Editor, Associate Editor)Kamal Mohammad Habib (Associate Co-Editor)

21-30 Abdul Ghani (Chairman)(1978-1987) M. Aslam (Chairman)

Naeem Ahmad Khan (Chairman, Chief Editor)Mirza Arshad Ali BegMuzaffer AhmadYusaf AhmadMirza Mansoor BegHeshamul HaqueM.K. BhattyN.M. ButtS. Fazal HussainM. IkramH.A. KazmiM.M. QurashiS.A. QureshiRiazuddin SiddiquiM. Ataur RehmanM. Shafi AhmadS. Riaz Ali ShahM.A. ShaukatM. ZafarullahM. Zain-ul-AbedinR.A. Khan TahirkheliA.R. AzmiS.H. Mujtaba NaqviM.H. QaziM. Ata-ur-RehmanMahmood Iqbal SheikhNasima M. TirmiziA.H. ChotaniA.T. KhanF.A. FarooqiS.A.QureshiS.M. QureshiMohammad Nazir RomaniM. A. Haleem (Chief Editor)A.H. Khan (Editor)Azmat Ali Khan (Executive Editor)

31-40 Naeem Ahmad Khan (Chairman, Chief Editor)(1988-1997) Qamar Iqbal (Chairman)

A.Q. Ansari (Chairman, Chief Editor)Syed Naeem Mahmood (Executive Editor)Anwar-ul-HaqAtta-ur-RehmanKhalid FarooqYasmin Badar


(2.3%) by the Dacca Laboratories; other R&D and S&Tinstitutions of the country contributed 1602 papers (32.1%),whereas, 1113 (22.3%) were contributed by the institutions offoreign countries. On an overall basis, the PCSIR’s share ofcontribution thus stands on an average, at about 45.55 over thepast 50 years. The data regarding the categorized contributionsfrom the Lahore, the Karachi and the Peshawar Laboratoriesof the PCSIR is given in Table 2. The basic purpose of thestatistics is to consolidate the data as well as to analyze andunderstand it with a view to knowing the significance of thecontributions from all the Laboratories of the PCSIR. It is hopedthis effort will provide authentic basis both for the policy plan-ners and the working scientists/technologists to better assessthe output. Consequently, the potential and S&T capabilities ofvarious groups will be better judged and the disadvantages, ifany, will be identified and removed for a better programme andefforts will be directed towards providing the needed facilitiesand opportunities to accomplish the assigned and planned tasks


Mohammad YusafShamshad Mehdi NaqviZafar Saied SaifyIsmail KhanS. Sadiq Ali RizviMirza Arshad Ali BegM.K. BhattyN.M. ButtS. Fazal HussainShafi Ahmad KhanAtta-ur-RehmanS.S.H. RizviEhsan AliS. Shahid HusainA. Q. AlviM. Ikram-ul-Haq DarA.H.K. YousufzaiKhushnood A. SiddiquiSaeed Iqbal ZafarR. B. QadriGhulam Abbas MianaM. Aslam ButtAbdul Rehman KhanMohammad Anwar WaqarSabir AliF.H. ShahMahmood Iqbal SheikhJamil Ahmad KhanNaseer SheikhIkram-ul-Haq DarM.A. QaziM.M. QureshiM. Shafi AhmadR.A. Khan TahirkheliA.R. AzmiS.H. Mujtaba NaqviM.H. QaziM. Ata-ur-RehmanMahmood Iqbal SheikhNasima M. TirmiziA.H. ChotaniA.T. KhanF.A. FarooqiS.A.QureshiS.M. QureshiMohammad Nazir RomaniS.M. Abdul Hai (Editor)Azmat Ali Khan (Executive Editor)J. N. Usmani (Executive Editor)Abdul Gaffar (Assistant Editor)

41-50 A.Q. Ansari (Chairman, Chief Editor)(1998-2007) Khalid Mahmood Khan (Chairman, Chief Editor)

Misbahul Ain Khan (Chairman)Anwar-ul-Haq (Chairman, Chief Editor)Javed Arshad Mirza, (Chairman, Editor-in-Chief)Saeed Iqbal Zafar (Editor-in-Chief)Syed Naeem Mahmood (Executive Editor)A. Rasheed Khan (Executive Editor)

Rabia Zuberi (Chief Editor)Khurshid Zaman (Executive Editor)Kaniz Fizza Azhar (Executive Editor)Atta-ur-RehmanKhalid FarooqM. Yasmin BadarMohammad YusafShamshad Mehdi NaqviZafar Saied SaifyIsmail KhanS.Sadiq Ali RizviH. AkhtarG. BouetW. LinertB.H. MehtaE. MiraldiM.J. QureshiF.M. SlaterM. A. WaqarMansoor Ahmad ButtS. Wazir Hussain ShahJ. OzgaM. AkhtarA.G. AttkinsM.A. KhanSuresh NarineJohn R. OgrenH.M. OrtnerNasima M. TirmiziAbul KalamAbdul Rehman MemonR.B. QadriM.A. KhanA. DiasproH. KhanM.W. Akhtar

for developmental purposes. The publications considered forthe present report have been gathered, thus, from 1970 to 2007(vol. 13-50) as they contain the publications under the abovestated categories.

The data for the past 38 years ( i.e., 1970 to 2007, both yearsincluded, vol. 13-50 of Pakistan Journal of Scientific and In-dustrial Research)is presented in Table 2. It can be seen thatthe three Laboratories at Lahore, Karachi and Peshawarcontributed 202, 232 and 86 papers in the Physical Sciences,182, 155 and 48 in Biological Sciences and 208, 105 and 94 inthe Technology fields, respectively. Numerically speaking thecontributions, in the three specified categories add up to 592for the Lahore Laboratories, 492 for the Karachi Laboratoriesand 228 for the Peshawar Laboratories. Various Research Divisions/Centres of the three Laboratories, as already mentioned, carryout their research projects largely in relation to the scope of theassigned activities of the Division/Centre. However, there is nobar in the intra-Division/Centre or the intra-Laboratoriescollaboration and many a ublications are also the result ofsuch activities as it helps in using the expertise and facilitiesavailable to various research groups at different Laboratoriesfor the completion of the undertaken projects.

A closer look at the data in Table 2 indicates that all thethree research Laboratories undertake activities, in the speci-fied fields, according to their planned programmes thoughin varying degrees. The contributions from the Lahore Labo-ratories are less in the Physical Sciences and more in thefields of Biological Sciences and Technology, when com-pared with the Karachi Laboratories. The reason for thisvariation is only due to the pre-planned programmes of eachunit. On an overall basis, however, it can be said that all thethree contributing units have adhered well to a healthy com-petitive attitude inspite of variations in the availability ofresearch staff and facilities.

An improvement both in the quality and quantity of thecontributions to the Journal from the parent organization(PCSIR) can be achieved if intra-Laboratory task forces canbe created to undertake various technological projects whichare time targeted and their component bits are assigned togroups identified on the basis of technical expertise.

Such an approach, it is hoped, will encourage cooperationand provide access to facilities, if they are available at oneplace and not available at another, and help in better monitor-ing the progress of the undertaken project and bring it to auseful conclusion. A similar approach can also be practicedboth in the fields of Physical and Biological Sciences as theseprojects can also be divided and distributed to collaboratinggroups so that their assigned aspects are completed faster than

when the whole project is tackled at one place only. Thus, notonly collaboration but the output as well will increase and thecollaborating scientists/technologists will get more useful andwide exposure in their respective fields of expertise.

Editorial BoardsThe Editorial Boards of the Journal have been changing withthe appointments of the Chairmen of the Pakistan Council forScientific and Industrial Research (PCSIR). However, theboards always comprised of prominent scientists available notonly in the country but also from foreign lands. The majorshare of the board members has however, been occupied bythe senior scientists from the Laboratories and varioussubject specialists from the Universities. A list of the mem-bers of the Editorial Boards during the period under review ispresented in Table 3 for information only.

ConclusionThe Pakistan Journal of Scientific and Industrial Research(PJSIR), during the period under review (1958-2007), published4986 research papers in various disciplines out of which 45.55%papers were contributed by the PCSIR Laboratories Complexes,at Lahore, Karachi and Peshawar only. It is thus seen that theJournal during the period has continuously and regularly broughtout publications in all the disciplines in which its Laboratorieswere mandated to operate and accepted publications from otherS&T and R&D organizations of Pakistan (32.10%) and fromall over the world (22.30%).

AcknowledgementThanks are due to Dr. Kaniz Fizza Azhar, Executive Editorof the Pakistan Journal of Scientific and Industrial Research(PJSIR), and her other colleagues in the PCSIR ScientificInformation Centre, Karachi for their timely help andassistance and provision of information for this article.Acknowledgements are also due to Mr. Azmat Ali Khan,Dr. Jafar Nazir Usmani and Mr. S. Naeem Mahmood for notonly their cooperation and support but also for their keeninterest, useful criticism and encouragement in the prepara-tion of this manuscript.

ReferencesHames, I. 2007. Peer Review and Manuscript Management

in Scientific Journals: Guidelines for Good Practice,293 p, Blackwell Publishing, Massachusetts, Oxford,UK.

Pak. J. Sci. Ind. Res. 1958-2007. (all issues of the Journalfrom vol. 1 to vol. 50), PCSIR.

PCSIR Annual Report 2006-2007. PCSIR ScientificInformation Centre, Karachi.


IntroductionThe functionality of baker’s yeast (Saccharomyces cerevisiae)is mediated by enzymes, amylase (α-1,4-glycosidase) (Tamaki,1978) and maltase (α-1,4-glucosidase) (Khan and Eaton, 1971).Genetically, the biosynthesis of maltase is regulated by theset of at least 5 unlinked genes ascribed as MAL loci. Theseinclude MAL1 through MAL4 and MAL6, each of whichhas different function (Charron et al., 1989; Michels andNeedleman, 1984). For instance MAL1 codes for a tranporterprotein, maltose permease, that causes internalization ofmaltose into the cell. MAL2 is a structural gene of maltasewhile MAL3 is the positive regulatory protein (Higgins et al.,1999). Functionally, maltase from S. cerevisiae belongs togroup of glucosyl hydrolases (EC 3.2.1.-) which is furthersegregated into 57 structural families (Henrissat and Bairoch,1996; Henrissat, 1991). More specifically, the enzyme (maltase)belongs to the family of 13 enzymes (α amylase family), whichtend to act on α-1,4- and/or α-1,6-glucosidic linkages presentin different carbohydrate moieties (Yamamoto et al., 2004;Henrissat, 1991). Primary structures of many of the mentionedproteins are known and have been rationally compared.Resultantly, four highly conserved regions (region I-IV) alongwith three acidic residues in the same have been reported inmost of the compared sequences, suggesting their role instructural and/or catalytic integrity of the molecules (Yamamotoet al., 2004; Brayer et al., 1995; Machius et al., 1995; Svensson,1988). However, the nature of amino acids responsible for theseparate recognition of α-1,4- and α-1,6-glycosidic linkages is

Pak. J. Sci. Ind. Res. 2008 51(4) 185-190

Phylogenetic and Computational Structure-Function Studies ofααααα-1,4-Glucosidase (Maltase) From Baker’s Yeast

(Saccharomyces cerevisiae)Mushtaq Hussaina, Javed A. Qureshib, Shagufta A. Shaikha and Liaquat Sultanaa*

aFood and Marine Resources Research Centre, PCSIR Laboratories Complex, Karachi - 75280, PakistanbHEJ Research Institute of Chemistry, International Centre for Chemical and Biological Sciences,

University of Karachi, Karachi - 75270, Pakistan

(received June 25, 2008; revised July 29, 2008; accepted July 30, 2008)

Abstract. Study, through blasting, cladogenesis, multiple sequence alignment and protein homology modeling, ofα,1,4 glucosidase from S. cerevisiae YJM789, showed the presence of different paralogues and orthologues of maltase indifferent genera of fungi and prokaryotes. The sequences of glucosidases contained 4 characteristic consensus regions. Inthe tertiary structure (modelled) of Baker’s yeast maltase, all the residues of consensus region were congregated in thecentral region of the folded protein, rendering the formation of catalytic groove. On the basis of the orientation and spatiallocation of residues in catalytic groove, Asp200 is proposed to be the second substrate-binding site.

Keywords: maltase, baker’s yeast, protein homology modelling

*Author for correspondence; E-mail: [email protected]

still debatable. Similarly, the cognizance of substrate bindingsite(s) of maltase from baker’s yeast S. cerevisiae is still basedon sequence comparison, site directed mutagenesis, chimericprotein and inhibition studies (Yamamoto et al., 2004; Yaoet al., 2003; Frandsen et al., 2002). Moreover, unavailabilityof any 3D structural data of α-1,4-glucosidase did not help indeciphering structural and functional aspects of the protein.

The present study has been designed to unravel the evolu-tionary course of the yeast maltase by generating multiplesequence alignment and consequently the phylogenetic tree.In addition to these tools, algorithms of protein homologymodeling and tertiary structure analysis have been exploitedto explore the structural and functional aspects of the enzymeat the molecular level. Fold recognition has been used to vali-date the findings in functional terms. At the best of our know-ledge this is the first attempt to study α-1,4-glucosidase ofS. cerevisiae at the tertiary scale level.

Materials and MethodsPrimary sequences, blasting and multiple sequence alignment.Amino acid sequence of α-1,4-glycosidase (GenBank acces-sion No. EDN 64908) from S. cerevisiae YJM789 was retrievedfrom NCBI data bank. The programmes FASTA and BLAST(Altschul et al., 1997) were subsequently used to retrieve theprimary sequences and structural homologues of the protein.Multiple sequence alignment was generated by defaultparameters of programme CLUSTAL X and manually adjustedwherever found necessary (Thompson et al., 1997). Thealignment file was analyzed using the programme GENE DOC

Physical Sciences

185

(Nicholas et al., 1997). Evolutionary lineage was calculatedusing the phylogeny inference programme, Phylip (Felsenstein,1993).

Homology modelling. The atomic coordinates of α-1,6-glucosi-dase of Bacillus cereus (PDBid;1UOK) was obtained fromBrookhaven Protein Data Bank (Bernstein et al., 1977). The 3Dmodels of yeast maltase were developed using the 1UOK astemplate (Watanabe et al., 1997). Briefly, the programmesSWISS-MODEL (Schwede et al., 2003) and 3D-JIGSAW(Bates et al., 2001) were used to construct the models withmanual input of PDBid.

Tertiary structure analysis. The structural coordinates ofyeast maltase were viewed using Swiss-Pdb viewer (Guex andPeitsch, 1997) and Weblab. Model. The structure were analyzedfor structural and thermodynamic stability using ANOLEA(Melo and Feytman, 1998); Verify 3D (Elsenberg et al., 1997);GROMOS (van Gunsteren et al., 1996) and Swiss-Pdb viewerand PROCHECK and Whatcheck (Laskowski and Kato, 1980).Fold recognition was performed by 3D-pssm algorithm (Kelleyet al., 2000).

Results and DiscussionSequence Comparison and Phylogeny. Holistically, thesequence identity among the sequences retrieved after non-redundant BLASTing of α-1,4-glycosidase from S. cerevisiaeYJM789 ranges from 58 to 99% with the target protein.However, multiple sequence alignment has shown non-homogenous distribution of sequence homology among theglucosidases of different organisms. Specifically, in compari-son to N-terminal, the C-terminal (middle) of the proteins hasbeen found with greater identities and/or similarities amongits different organismic versions. Importantly, despite thenonisofunctional nature of the proteins, the characteristicfour conserved regions I, II, III and IV (Yamamoto et al., 2004;Svensson, 1988) have been found in all homologues ofyeast α-1,4-glucosidase at least at partial scale. Precisely, inconserved region I (Asp97-Cys103) and II (Asp200-Gly208),the conservancy has been more pronounced, as discrepan-cies are only observed limited to substitution of residue withan iso-functional amino acid except for Cys103, which isconserved only in the homologues of yeast strains andreplaced by Thr in glucosidases of ascomycota andbasidomycota and Ser in some members of firmicutes at therespective position. The conserved region II (Asp200-Gly208) has shown minimum discrepancy in comparison toother three regions, suggesting its potential role in catalysis.In the region, only the last three residues showed somedegree of isofunctional variation among the glucosidases ofdifferent organisms. Additionally, at the first residue Asp200,

Ser has been found in multicellular members of ascomycotalike Aspergillus and Ustilago. It has been earlier stipulatedthat the acidic amino acids like Asp in the conserved (consen-sus) regions are crucial for enzymatic activity of maltase(Yamamoto et al., 2004; Brayer et al., 1995); in consideration ofthis, it is reasonable to assume that the yeast maltase may becatalytically more active than the maltase from multicellularfungi. Akin to conserved region II, the subsequent region IIIhas shown considerable similarities among glucosidases ofdifferent organisms. Absence of any acidic residue suggeststhat the region has structural importance rather than catalytic.In contrast to region II, region IV has completely conservedlast three residues among glucosidases of different organi-sms. However, the first three residues have exhibited somedegree of isofunctional substitutions (Fig. 1). As a whole theconsiderable degree of homologues of glucosidase suggeststhe indispensability of the enzymes in the survival of theorganisms at the transkingdom level. Moreover, patches ofstrong identity may also implicate gene duplication followedby the emergence of orthologs and paralogs (Cliften et al.,2006).

Phylogenetic tree clearly segregated the glucosidases homo-logues, present among different organisms, at the taxonomiclevel. Fundamentally, three nodal branches have been observed,two of them leading to ascomycota while the third bifurcatedinto basidomycota and prokaryotic bacterial group namelyfirmicutes. The branch leading to Saccharomyces also embarkson other unicellular members of ascomycota (Fig. 2). Theassembly of all Saccharomyces and other unicellular fungion the single nodal branch suggests their common ancestry(Cliften et al., 2006; Liti and Louis, 2005). BLASTing hasrevealed the presence of at least 9 homologues (orthologues)of α-1,4-glucosidase in the genome of S. cerevisiae YJM789;similarity among them ranges from 80% to 99%. The valuessuggest occurrence of extensive gene and/or genome duplica-tion events, which rendered the increased number of copies ofthe understudy gene. Indeed it has been earlier deduced thataround 72% of the yeast genome is the result of gene duplica-tion and subsequent substitution (Wong et al., 2002; Seoigheand Wolfe, 1999). According to Piskur (2001), genome duplica-tion has great influence on the fermentability of the yeast.The branch length of other unicellular fungi except forKluyveromyces excluded the possibility of lateral gene trans-fer among Saccharomyces and other single cell fungi (Liti andLouis, 2005). However, close proximity of Kluyveromyces andSaccharomyces homologue of α-1,4-glucosidase may implicatethe involvement of horizontal gene transfer. Earlier bifurcationof the nodal branch, leading to unicellular ascomycota into twosubbranches (one suggesting evolution of Saccharomycesand the second including other members of ascomycota),

186 Liaquat Sultana et al.

may be inferred in terms of gene duplication in the ancestralorganisms, followed by vertical gene transfer. However, it isworth mentioning here that almost all the homologues ofα-1,4-glucosidase are actually α-1,6-glucosidase in naturesuggesting the process of paralogous gene formation in thespecies distant to yeast (Cliften et al., 2006).

Three-dimensional structures. Overall structural featuresof modelled α-1,4-glucosidase from S. cerevisiae YJM789 aresimilar to those found in template α-1,6-glucosidase fromB. cereus. The fact is validated by the RMS deviation (1.4'Å)of Cα backbone between the modelled and template protein.The α-1,4-glucosidase molecule comprises of three domainsnamely N-terminal, followed respectively by subdomain andthe C-terminal domain as suggested by Watanabe et al. (1997).

The topology of N-terminal domain is similar to that found in a(β/α)8 barrel or TIM barrel containing proteins (Banner et al.,1975). Interestingly, the mentioned region is somewhat sand-wiched between the subdomain and C-terminal domain. Suchspatial location of N-terminal has also been observed in otherα-1,6-glucosidase for instance amylase (Matsuura et al., 1984),pig pancreatic amylase (Larson et al., 1994) etc. Both N-terminaland subdomain have been found to contain both α-helicesand β-pleated sheets with disparaging number with its tem-plate and other α-1,6-glucosidases. Intriguingly, the C-termi-nal of α-1,4-glucosidase is exteriorly localized and mainlycontains six antiparallel β-pleated sheets in contrast to 8such sheets, observed, in α-1,6-glucosidase from B. cereus(Watanabe et al., 1997). The helices and β-pleated sheets,particularly those present in N-terminal and subdomain

Fig. 1. Multiple Sequence Alignment: Selected conserved regions of different α-glucosidases are mentioned and annotatedaccording to their spatial order. Note the presence of two acidic residues (Asp; D) in conserved region II.

187Glucosidase from Baker’s Yeast

region are intervened by coils and loops of different span(Fig. 3). Despite the presence of 5 Cys residues, no disulphidebridges were noted in the modelled molecule; this is notsurprising in the light of intracellular nature of the enzyme.Absence of any transmembrane signature sequence in themolecule has further strengthened this notion. Rationally,the reducing environment inside the cell links the free end ofCys residues with hydrogen, thus halting the formation of any

cysteine bridge. Although the absence of cysteine bridgesmay suggest the vulnerability of the molecule, but contrarilythe total free energy (ΔG) of the molecule has been deducedas -18974 KJ/mole, indicating thermodynamic stability of theenzyme in the cytosol. Moreover, the spatial placement ofenzyme residues with reference to their Φ and ψ angles inthe Ramachandran plot (Ramachandran and Sasiskharan,1968) also validates the legitimacy of the protein on thearchitectural basis.

Catalytic Site. Constant conservancy of four regions dis-persed among different homologues of α-1,4-glucosidase sug-gests their importance in the structural and functional aspectsof the enzymes. In the modelled yeast maltase, it has beenobserved that despite the substantial difference along thedomain placement in the primary structure of the enzyme, onthermodynamically stable folding of the protein, these regionsof consensus has been found congregated at the specific cen-tral region. (Fig. 4). This conglomeration results in the forma-tion of catalytic groove through which an active residue couldpossibly bind with the substrate (Fig. 5). Electrostatic spacetopology suggests that the groove opening is sufficient forthe accessibility of the active residue to the disaccharide sub-strate molecule. Specifically, only the residues of consensusregions I, II and III were observed to be accessible to thesubstrate molecule. Indeed, as mentioned earlier, that acidicresidues (Asp) present in these regions are the key player inmediating the enzyme activity. Several site-directed mutage-nesis and enzyme inhibition studies have suggested thatAsp205 in the consensus region II is involved in the bindingto the substrate molecule (Yamamoto et al., 2004). Conversely,it has also been stipulated that single residue may not be

Fig. 3. Tertiary structure of α-1,4-glucosidase fromS. cerevisiae. All three domains are annotatedaccordingly.

Fig. 2. Phylogenetic tree as developed by neighbour join-ing method. The proximal right and left nodalbranches respectively belong to firmicutes andmembers of basidomycota. The distal right and leftnodal branches respectively embark unicellular andmulticellular members of ascomycota.

188 Mushtaq Hussain et al.

Acidobacteria

EAspergillusSchizosaccharomyces

Halothermothrix

Colprinopsis

Laccaria

Cryptococcus

JGibberella

Gibberella

PyrenophoraNeosartorya

AspergillusBAspergillus

CAspergillus

DAspergillus

PhaeosphaeriaChaetomium

Magnaporthe UstilagoNeurospora

LodderomycesPichia

APichiaDebaryomyces

Candida

EPichia

Kluyveromyces

Saccharomyces

RuminococcusHBacillus

CaldicellulosiruptorEXiguobacterium

FBacillusBacillus

StaphylococcusOenococcus

GBacillus

Fig. 4. Catalytic region of α-1,4-glucosidase from S. cerevisiae.Note the accumulation of cuddling of conservedregions residues at the central region of the molecule.Conserved region I, II, III and IV are respectivelycoloured as green, blue, orange and pink.

C-IIAsp200-Asp205

C-IAsp97-Cys103

C-IVTyr335-Asp340

C-IIIGly266-Asp269

N-terminal

C-terminal

Subdomain

dases. Importantly, all the conserved domain residues havebeen found congregated at the same site, forming a catalyticgroove in the tertiary structure of the compound. But it isinferred that only Asp200 and Asp205 are involved in bindingwith substrate. It is suggested here that experimental studieslike site-directed mutagenesis, enzyme inhibition and chimericprotein formation may explicate the details of the proposedsecond substrate binding site present in yeast α-1,4-glucosi-dase.

ReferencesAltschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang,

Z., Miller, W., Lipman, D.J. 1997. Gapped BLAST andPSI-BLAST: a new generation of protein database searchprograms. Nucleic Acids Res. 25: 3389-3402.

Banner, D.W., Bloomer, A.C., Petsko, G.A., Philips, D.C., Pogson,C.I., Wilson, I.A., Corran, P.H., Furth, A.J., Milman, J.D.,Offord, R.E., Priddle, J.D., Waley, S.G. 1975. Structure ofchicken muscle triose phosphate isomerase determinedcrystallographically at 2.5 angstrom resolution usingamino acid sequence data. Nature 255: 609- 614.

Bates, P.A., Kelley, L.A., MacCallum, R.M., Sternberg, M.J.2001. Enhancement of protein modelling by humanintervention in applying the automatic programs 3D-JIG-SAW and 3D-PSSM. Proteins, Suppl. 5: 39-46.

Bernstein, F.C., Koetzle, T.F., Williams, G.J., Meyer, E.F. Jr.,Brice, M.D., Rodgers J.R., Kennard, O., Shimanuchi, T.,Tasumi, M. 1977. The Protein data bank: a computer-basedarchival file for macromolecular structures. J. Mol. Biol.112: 535-542.

Brayer, G.D., Luo, Y., Withers, S.G. 1995. The structure ofhuman pancreatic alpha-amylase at 1.8Å resolution andcomparisons with related enzymes. Protein Sci. 4: 1730-1742.

Charron, M.J., Read, E., Haut, S.R., Michels, C.A. 1989.Molecular evolution of the telomere-associated MAL lociof Saccharomyces. Genetics 122: 307-316.

Cliften, P.F., Fulton, R.S., Wilson, R.K., Johnston, M. 2006.After the duplication: Gene loss and adaptation inSaccharomyces genomes. Genetics 172: 863-872.

Elsenberg, D., Luthy, R., Bowie, J.U. 1997. VERIFY3D: assess-ment of protein models with three-dimensional profiles.Methods Enzymol. 277: 396-404.

Felsenstein, J. 1993. PHYLIP (Phylogeny Inference Package)version 3.57C, distributed by the author. Department ofGenetics, University of Washington, Seattle, USA.

Frandsen, T.P., Palcic, M.M., Svensson, B. 2002. Substraterecognition by three family of 13 yeast α-glucosidases.Eur. J. Biochem. 269: 728-734.

Guex, N., Peitsch, M.C. 1997. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein

Fig. 5. Catalytic groove: Electrostatic surface establishedby the assemblage of conserved region residues.Importantly, a groove has an opening for the accessof substrate molecule to active residue. The activeresidues are labeled accordingly.

sufficient to bind with the two glucose moieties of the sub-strate, thereby suggesting possibility of the presence ofsecond substrate site (Yao et al., 2003). However, hitherto nosuch information has been extracted. In the current studies,it was observed that in addition to Asp205 of conservedregion II, Asp200 of the same region also exhibited orientationas that of Asp205 and was accessible to the substrate-bindingsite through catalytic groove. Here, it is plausible to inferthat Asp200 may act as a second binding site to the maltoseand subsequently cleaves it into 2 glucose moieties (Fig. 5).Furthermore, it is also anticipated that the immense negativecharge, developed by the presence of the two Asp residues atthe catalytic groove, may get counter balanced by the His101present in the consensus region I.

ConclusionSequence comparison and phylogenetic inference implicatesthat glucosidases are important and perhaps indispensableprotein for the normal metabolism in yeast and related organ-isms. Multiple sequence comparison has indicated that at leastfour consensus regions are always present with mostlyisofunctional discrepancies in all homologues of glucosidasesof yeast. It is likely that among unicellular fungi, the enzymeand its paralogues are evolved by the gene and/or genomeduplication followed by vertical gene transfer from the ances-tral organisms. However, the possibility of lateral gene trans-fer between Saccharomyces and Kluyveromyces could not beoverruled. Structurally, in contrast to similarities in the holisticcomparison of model and template proteins, the yeast maltaseappears less structurally defined than the other α-1,6-glucosi-

189Glucosidase from Baker’s Yeast

His101 Asp200

Asp205

modeling. Electrophoresis 18: 2714-2723.Henrissat, B., Bairoch, A. 1996. Updating the sequence based

classification of glucosyl hydrolases. Biochem. J. 316:695-696.

Henrissat, B. 1991. A classification of glycosyl hydrolasesbased on amino acid sequence similarities. Biochem. J.280: 309-316.

Higgins, V.J., Braidwood, M., Bell, P., Bissinger, P., Dawes, I.W.,Attfield, P.V. 1999. Genetic evidence that high non-inducedmaltase and maltose permease activities, governed byMALx3-encoded transcriptional regulators, determineefficiency of gas production by baker’s yeast in unsugareddough. Appl. Environ. Microbiol. 65: 680-685.

Kelley, L.A., MacCalluum, R.M., Sternberg, M.J. 2000.Enhanced genome annotation using structural profilesin the program 3D-PSSM. J. Mol. Biol. 299: 499-520.

Khan, N.A., Eaton, N.R. 1971. Genetic control of maltaseformation in yeast. I. Strains producing high and low basallevels of enzyme. Mol. Gen. Genet. 112: 317-322.

Larson, S.B., Greenwood, A., Cascio, D., Day, J., McPherson,A. 1994. Refined molecular structure of pig pancreaticα-amylase at 2.1Å resolution. J. Mol. Biol. 235: 1560-1584.

Laskowski, M.Jr., Kato, I. 1980. Protein inhibitors ofproteinases. Annu. Rev. Biochem. 49: 593-626.

Liti, G., Louis, E.J. 2005. Yeast evolution and comparativegenomics. Annu. Rev. Microbiol. 59: 135-153.

Machius, M., Wiegand, G., Huber, R. 1995. Crystal structure ofcalcium depleted Bacillus licheniformis α-amylase at 2.2Åresolution. J. Mol. Biol. 246: 545-559.

Matsuura, A.Y., Kusunoki, M., Harada, W., Kakudoi, M. 1984.Structure and possible catalytic residues of Taka-amylase A. J. Biochem. 95: 697-702.

Melo, F., Feytmans, E. 1998. Assessing protein structures witha non-local atomic interaction energy. J. Mol. Biol. 277:1141-1152.

Michels, C.A., Needleman, R.B. 1984. The dispersed, repeatedfamily of MAL loci in Saccharomyces spp. J. Bacteriol.157: 949-952.

Nicholas, K.B., Nicholas H.B. Jr., Deerfield II, D.W. 1997. GeneDoc: Analysis and visualization of genetic variation.EMBNEW 4: 14.

Piskur, J. 2001. Origin of the duplicated regions in the yeastgenomes. Trends Genet. 17: 302-303.

Ramchandran, G.N., Sasiskharan, V. 1968. Conformation ofpolypeptides and proteins. Advan. Prot. Chem. 23: 283-437.

Schwede, T., Kopp, J., Guex, N., Peitsch, M.C. 2003. SWISS-MODEL: An automated protein homology-modelingserver. Nucleic Acids Res. 31: 3381-3385.

Seoighe, C., Wolfe, K.H. 1999. Yeast genome evolution in thepost-genome era. Curr. Opin. Microbiol. 2: 548-554.

Svensson, B. 1988. Regional distant sequence homologybetween amylases, alpha-glucosidases and trans-glucano-sylases. FEBS Lett. 230: 72-76.

Tamaki, H. 1978. Genetic studies of ability to ferment starch inSaccharomyces: gene polymorphism. Mol. Gen. Genet.164: 205-209.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F.,Higgins, D.G. 1997. The CLUSTAL X windows interface:flexible strategies for multiple sequence alignment aidedby quality analysis tools. Nucleic Acids Res. 25: 4876-4882.

van Gunsteren, W.F., Billeter, S.R., Eising, A.A., Huenenberger,P.H., Krueger, P., Mark, A.E., Scott, W.R.P., Tironi, I.G. (eds.),1996. Biomolecular Simulations: The GROMOS96Manual and User Guide. VdF: Hochschulverlag AG ander ETH, Zürich, Switzerland.

Watanabe, K., Hata, Y., Kizaki, H., Katsube, Y., Suzuki, Y. 1997.The refined crystal structure of Bacillus cereus oligo-1,6-glucosidase at 2.0Å resolution: Structural characteriza-tion of proline substitution sites for protein thermo stabi-lization. J. Mol. Biol. 269: 142-153.

Wong, S., Butler, G., Wolfe, K.H. 2002. Gene order evolutionand paleopolyploidy in hemiascomycete yeast. Proc. Natl.Acad. Sci. USA. 99: 9272-9277.

Yao, X., Mauldin, R., Byers, L. 2003. Multiple sugar bindingsites in alpha-glucosidase. Biochim. Biophys. Acta.Proteins & Proteomics 1645: 22-29.

Yamamoto, K., Nakayama, A., Yamamoto, Y., Tabata, S. 2004.Val216 decides the substrate specificity of α-glucosidasein Saccharomyces cerevisiae. Eur. J. Biochem. 271: 3414-3420.

190 Liaquat Sultana et al.

IntroductionUrethanes were the first major elastomers that could be pro-cessed by thermoplastic methods. Thermoplastic polyure-thanes (TPUs) are derived from three basic compounds: longchain polyester or polyether diols, aromatic or aliphaticdiisocyanate, and short-chain diol as chain extender, whichin most cases is butanediol. They are characterized by asegmented structure consisting of flexible segments, thepolyester or polyether chains, and hard segments, the ure-thane groups. The hard segments are formed by the additionof a chain extender to the diisocyanate, and the soft segmentsinterconnects two hard segments. At temperatures lower thanthe order-disorder transition temperature (TODT), the lowmelting soft segments are incompatible with the polar andhigh-melting hard segments, which leads to a phase-separatedmicrostructure (Tien and Wei, 2001a).

TPUs come in a wide range of hardness grades. Amongelastomers, urethanes have outstanding abrasion resistance,good low temperature flexibility, excellent oil resistance andrank among the best for load-bearing capability. Additivescan improve dimensional stability and heat resistance, reducefriction and increase flame retardancy, fungus resistance andweatherability. The polyether types have better hydrolyticstability and low-temperature flexibility than the polyestertypes. However, mechanical properties of the polyestertypes are generally higher. The caprolactones offer a goodcompromise between the ether and ester types.

The morphology of PU determines its mechanical, thermal,and water absorption properties (Garrett et al., 2000; Market al., 1988; Miller, 1985; Wang and Cooper, 1983).

The physical properties of thermoplastic PUs are derived fromtheir molecular structure as well as the supramolecular struc-ture formed by interaction between the polymer chains. The

Pak. J. Sci. Ind. Res. 2008 51(4) 191-200

Study of Structure and Properties of Thermoplastic PolyurethanesA. Shokuhi Rada*, M. Ardjmanda and K. Mahmoodib

aDepartment of Chemical Engineering, South Tehran Branch, Graduate Faculty, Islamic Azad University, Tehran, IranbIran Khodro Co. 14 Km, Karaj Road, Tehran, Iran

(received May 12, 2008; revised July 14, 2008; accepted July 15, 2008)

Abstract. In the investigation on the effect of clay nano-composite on the structure and properties of thermoplasticpolyurethanes (TPUs), the mechanical properties of TPUs had maximum values with 8 wt % clay content. The wide angleX-ray diffraction patterns showed that the glycerol propoxylate had better compatibility with the organoclay thanpoly(propylene glycol).

Keywords: polyurethanes, thermoplastic polyurethanes, elastomers, clay nanocomposite, glycerol propoxylate


segmental flexibility, the chain entanglement, the interchainforces, and the crosslinking are all factors that influence theproperties and determine the use of the end products. Withwell designed combinations of monomeric materials, PUs canbe tailored to meet diversified demands of various applica-tions such as coatings, adhesives, fibers, thermoplastic elas-tomers and foams (Shokuhi and Ardjmand, 2008a). Hence, theyhave received wide attention in terms of their synthesis,morphology, chemical, and mechanical properties (Penczeket al., 1993; Szczepaniak et al., 1993). Increasing the strengthof PU elastomers without sacrificing its large elongation atbreak is an important object for elastomeric PU development.The mechanical properties of PU can be modified either byvarying PU chemical structure, resulting from step growthpolymerization of isocyanate resins with polyols or bydispersing inorganic or organic fillers into the pure PU.

Materials and MethodsPoly(propylene glycol) (PPG1, Mn 51000, and PPG2, Mn 52000),made by Bayer Chemical Co. Germany under the name ofDesmophen, poly- and di-isocyanate (based on TDI and HDIand MDI); made by Bayer Chemical Co. Germany under thename of Desmodur, glycerol propoxylate (GPO3, Mn≈ 3000,made by Dow Chemical Co.), Na-montmorillonite clay (thecation exchange capacity, CEC 5 90-100 mmol/100 g, with aparticle size of about 40-70 mm, made by Zhangjiakou QingheChemical Factory), sodium montmorillonite clay (Na-O-MMT),with cation exchange capacity (CEC) of 119 mEqu/100 g, wassupplied by Coniine Industry Co. (Japan) under trade nameKunipia-F. Styrene was supplied by Aldrich and used afterpurification by washing with 1M NaOH then drying overanhydrous sodium sulphate followed by distillation undervacuum. Maleic anhydride and ethylene glycol and 1,4-butanediol (from Merck-Schuchardt, Germany); 12-amino-dodecanoic acid, from Aldrich; tetrahydrofuran (THF) (from

191

Fluka) and ethanol (from Adwic, Egypt) were used, as obtainedwithout further purification.

In brief, NCO-terminated polymers of MDI and PEG or PPGwere prepared in the absence of any catalyst and with variousNCO:OH equivalent weight ratios. The reaction process wasfollowed by the removal of absorbed water by azeotropicmixture with toluene and then the water free PEG and TMPmixture was added to the kettle at 60-70 °C and the reactioncontinued at 70-80 °C for 6 h in N2 environment. To remove theoligomeric part and the moisture reacted part, MDI was heatedand filtered by a cloth before use. The synthesized isocya-nate-terminated polymers were reserved in plastic bottles. Apart of the polymer was taken in Teflon disc and kept in anoven at 80 °C for 6 h, and then the obtained polymer filmwas kept at room temperature and humidity for long untilcomplete disappearance of NCO band in FTIR spectrum wasobserved.

FTIR instrumentation. For the FTIR study, a small and diluteddrop of liquid polymers as well as nanocomposites (typically a1 wt % solution in ethyl acetate) were prepared and coated onKBr disks and stored at room temperature and humidity fora day, before spectral evaluation. To remove the residualsolvent, the disks were placed in a vacuum oven at 80 °C for6 h. The thickness of sample was controlled and kept at thesame uniform size. Fourier transform IR spectra (FTIR) wereobtained using a Thermo Nicolet Nexus 670 spectrometer. Aminimum of 32 scans was signal-averaged with a resolutionof 2/cm within the wavelength range of 400-4000/cm.

Synthesis of TPU/clay nanocomposites. The hard segment ofthe TPU was formed from 4,4 methylenebis (phenylisocyanate)(MDI) and 1,4-butanediol (BD). The soft segment was formedfrom PTMG. MDI and PTMG were dried in a vacuum ovenbefore use, and BD was stored on 4-A molecular sieve. Bothunmodified MMT (Na-MMT) and O-MMT were used afterheating at 130 °C for 24 h to remove molecular water in thegalleries. The TPU/clay nanocomposites were prepared by anin situ polymerization method; the molar ratio of MDI/BD/PTMG was 4 : 3 : 1 and was denoted as MBP431. The nano-composites were synthesized by a two-step reaction. In thefirst step, PTMG was reacted with an excess of MDI at 85 °Cfor 2 h to form a prepolymer end, capped with isocyanategroups. In the second step, clay dispersed in freshly distilleddimethyl acetamide was added to the prepolymer at roomtemperature; then, the chain extender (BD) mixed withdibutyltindilaurate was added. The chain extension reactionwas carried out at 85 °C for 2 h.

Results and DiscussionImprovement in TPUs properties. Montmorillonite claysinclude all clay minerals with an expanding structure, except

vermiculite, and also the high alumina end member of the group.Members of the montmorillonite group of clay minerals varygreatly in the mode of formation. Alkaline conditions and thepresence of magnesium particularly favour the formation ofthese minerals which occur in various modes in the soils,bentonites, mineral veins, marine shales, and as alterationproducts of other minerals. Recently formed sediments have afairly high content of montmorillonite.

TPUs have some disadvantages, such as low thermal stabilityand low mechanical strength, etc., (Table 1). To overcome thesedisadvantages, a great deal of effort has been devoted to thedevelopment of nanostructured PU (PU)/montmorillonite(MMT) composites in recent years (Tien and Wei, 2001b;Chen et al., 2000).

A wide variety of fillers, including talc, mica, and wollastonite,have been applied in TPU formulation to reduce cost and toincrease the rigidity (Varma et al., 1985). However, addition offiller frequently results in reduction in the elongation at break.In recent years, polymer/clay have attracted great attentionboth in industry and academia, after the Toyota groupdeveloped clay/nylon 6 nanocomposites with excellentmechanical properties (Solarski et al., 2005), creating interestin the use of clay as a reinforcement material for polymers(Rhoney et al., 2004). These composites exhibit remarkableimprovement over the conventional microcomposites invarious properties such as mechanical strength, heat resis-tance, gas permeability and flammability.

Table 1. Some advantages and limitation of TPUs in all formsAdvantages Limitations

Excellent abrasion resistance, Narrow hardness rangewear resistance, and tear strength compared to other TPEs

High tensile strength Low upper servicetemperature limit

High dynamic load-bearing Susceptible to hydrolyticcapacity attack by acid and base

solutions and by pure waterat elevated temperatures

Low coefficient of friction Limited resistance tohalogenated solvents

Good U V, weathering oxygen, Can be “tricky” to process;and fungus resistance TPUs are more sensitive to

temperature and shear thanother TPEs

Good resistance to Must be dried beforenon-polar fluids processing

192 A. Shokuhi Rad et al.

TPU clay/nanocomposites. Thermoplastic and thermosettingnanocomposites were reported to exhibit unusual propertiessuch as improved stiffness/strength/toughness balance,improved heat distortion temperature, strong barrier properties,and flame retardency (Ma et al., 2001; Pinnavaia and Beall,2000). Since the development of the nylon/MMT nanocom-posite (Usuki et al., 1989), a large number of new polymernanocomposites have been investigated (Shokuhi Rad andArdjmand, 2008a; Biasci et al., 1994). Most of the polymer/clay nanocomposites were based upon glassy polymers withhigh glass-transition temperatures, such as PMMA, PS, SANand epoxy, and studies of rubbery polymers have rarely beenreported.

Pinnavaia and Beall (2000) focussed on compatibility betweenorganoclay and polyols. They found that the montmorilloniteclay, exchanged with long chain onium ions (carbon number>12), had a good compatibility with several polyols commonlyused for synthesizing PU.

Tien and Wei (2001b) reported preparation of completelyexfoliated PU/clay nanocomposites by in situ polymerizationusing 12-aminolauric acid and benzidine. Pattanayak and Jana(2005) reported that exfoliated nanocomposites of TPUs canbe prepared by some unique method resulting in remarkableincrease in tensile strength and modulus along with opticalclarity when compared with pure PUs.

Zilg et al. (1999) reported that TPU nanocomposites contai-ning synthetic fluoromica could simultaneously increase theirtensile strength and elongation at break. Chen et al. (2000)synthesized a novel segmental PU/clay nanocomposite viapoly (e-caprolactone)/clay (PCL/clay) route. They reportedthat the mechanical properties were enhanced by introducinga small amount of PCL/clay in the composite.

However, the composite was transformed from an elastomer toa plastic as the amount of PCL/clay in the composite wasincreased (Jang et al., 2005).

The effect of used clay. The effects of clay type, clay content,and PU molecular structure on clay dispersion in thermo-plastic PU nanocomposites were studied by Cao et al. (2005).It was found that MMT clays exchanged with long chain oniumions (carbon number > 12), had good compatibility with polyol.The extent of gallery expansion of modified MMT is mainlydetermined by the chain length of the gallery onium ions (Wangand Pinnavaia, 1998). A good dispersion of layered silicate hasbeen found to improve the properties of PU elastomernanocomposites, such as mechanical properties, thermalstability and gas permeability (Chen et al., 2000). Good disper-sion of clays in the PU matrix has been achieved through themodification of MMT with active surfactants containing

more than two hydroxyl groups (Tien and Wei, 2001a). Thepresence of hydroxyl groups enhanced intragallery polyme-rization, which in turn led to better clay dispersion. However,the morphology of nanocomposites prepared by this approachwas still a combination of exfoliation/intercalation and themethod only worked at low clay content, i.e., less than 3 wt %,even for organoclay modified by surfactants with threehydroxyl groups. The exfoliated clay dispersion was onlyobserved at low reaction rates in solution polymerization(Tien and Wei, 2001b). PU foams account for the largestmarket among polymeric foams, estimated at nearly two billionkilograms in the US alone (Khemani, 1997). PU foams have aremarkably broad range of applications including thermalinsulation, cushioning, buoyancy, energy absorption (packa-ging), etc. Their low density also permits the design of lightand stiff components such as aircraft-interior panels, struc-tural shapes (transom cores, bulkhead core, stringers, motor-mounts, etc.), in fibre-reinforced plastic (FRP) for boat build-ing, impact-limiters and crash-pads, composite foam cores,mold-patterns and plugs, sports-equipment core material andcomposite tooling. Mechanical properties are importantconsiderations for such structural and semi-structuralapplications. Unlike thermoplastic foams, PU foams areformed by reactive processing, in which polymerization andfoam blowing occur simultaneously. Polymer structure mustbuild up rapidly to support the fragile foam, but not too fast tostop bubble growth. For thermoplastic foams, it was foundthat clay-filled nanocomposites could effectively reduce thecell size and increase the cell density (Han et al., 2003). Anexfoliated PS nanocomposite produced a foam with a cell sizefive times smaller than that of the pure PS foam. Severaladvantages can be expected in using a PU nanocomposite asthe matrix of PU foams. The presence of nanoparticles mayimprove the mechanical strength of the PU matrix and in turnthe strength of the PU foam. Nanoscale-dispersed clay mayact as nucleation agents during the foaming process to pro-duce finer cell structure and higher cell density. Furthermore,improved gas barrier properties provide another opportunityfor application of nanoclay in PU foams.

An alkylammonium modifying montmorillonite, a natural clay,was intercalated into the polyols to prepare the elastomericPU/clay nanocomposite by Ma et al. (2001). Their researchfocussed on effects of the clay content and route of claycompounding with polyols on the mechanical properties ofthe nanocomposites. Various types of polyol with differentmolecular weights and functionalities were used for preparingPU and PU/clay nanocomposites.

In the present work the ratio of the polyols was kept constantto ensure the same chemical structure of the PU and PU/MMT

193Structure and Properties of Polyurethanes

nanocomposites. It was found that the mechanical propertieshad maximum values with the clay content when it reached8 wt %. The maximum tensile stress and the maximum elonga-tion at break increased two and five times, respectively. TheWAXD patterns showed that the glycerol propoxylate hadbetter compatibility with the organoclay than poly(propyleneglycol). This corresponded to the formation of a nanocom-posite from the clay compounding with glycerol proxylatethat, in turn, resulted in the best mechanical properties. Themolecular weight of poly(propylene glycol) seemed to haveno obvious effect on the intercalative behavior as explainedby WAXD results. However, the nanocomposites from theclay compounding with the low molecular weight polyol routeshowed better mechanical properties than that from the claycompounding with the high molecular weight polyol route.The synthesis procedures, shown in Fig. 1, were used tosynthesize the PU/MMT1 and PU/MMT2 nanocomposites.The polyols of PPG1, PPG2 and GPO3 could be used inconstant ratio to ensure that all the nanocomposite sampleshad the same chemical structure.

meric characteristics. The hard segments (formed by MDI)form domains that are dispersed in a continuous matrix of thesoft, rubbery segments formed by PTMG segments.

Due to the dissolution of the hard domains in the soft matrixabove order-disorder transition temperature (TODT), it wasimportant to maintain the phase-separated structure duringvarious TPU processing operations. On transformation fromthe homogeneous state to the phase-separated state, mosthard segments aggregate and form hard domains in the softmatrix. Typically, PU is extensively hydrogen-bonded, withdonor being the NH group of the urethane linkage andacceptor being the carbonyl group of the urethane linkage ofthe neighbouring polymer chains. Since the hard segmentscontain urethane groups, they can form hydrogen bonds withother hard segments inside the hard domain, and the forma-tion of hydrogen bonding can be a strong driving force forphase separation. Therefore, one can monitor the extent ofphase separation by following the change in the degree ofhydrogen bonding. The order-disorder transition, sometimescalled the microphase separation transition of the TPU andits nanocomposites with O-MMT were studied by FTIR andDSC methods. Fig. 3 and 4 show C=O stretching peak regionin the FTIR spectra of pure TPU and TPU/clay nanocomposite.

Mechanical properties of TPU. The mechanical properties ofpure TPU, TPU/Na-MMT nanocomposites, and TPU/O-MMTnanocomposites are displayed in Table 2 and the stress-straincurves are shown in Fig. 5.

The tensile strength and ultimate elongation values of TPU/clay nanocomposites decreased with the addition of Na-MMTor O-MMT. However, a significant improvement in the Young’smodulus values was observed with increasing the levels ofboth Na-MMT and O-MMT. The same trend was observed

The reactions that occurred during the TPU/clay nanocom-posite syntheses by proposed methode are shown in Fig. 2.

Effect of clay on the order-disorder transition of TPU. TPU isa segmented block copolymer having thermoplastic elasto-

Fig. 1. A schematic diagram of clay compounding routefor synthesis of PU/MMT nanocomposites.

MMT

Organo-MMT

Route 3Route 2Route 1

GPO3M - 3000

prepolymer

+ TDI,80 C,2h

80-90 C,2h

PU/MMT3

PPG1M - 1000

PPG2M - 2000


Fig. 2. Synthesis route for the TPU/layered silicate claynanocomposites.

H O

O

O H

O -N N- C- C-N N=C=OO=C=N

Na MMT or O MMT, BD, Dibutyltindilaurate at 85 °C, for 120 min

Step 1

Step 2

Silicate layer

O H

O-C-N N-C-O

H O O H H O

N-C-OO-C-N

Hard segment Soft segment

Na MMT or O MMT nanocomposite

for TPU/layered silicate clay nanocomposites by Finniganet al. (2004). Interestingly, O-MMT caused a larger reductionin the tensile and elongation properties than Na-MMT.Meanwhile, the enhancement of Young’s modulus causedby O-MMT was higher than that caused by Na-MMT. Thisappeared to be due to enhanced interaction between TPU andO-MMT.

Effect of polyol molecular weight and functionality onintercalative behavior. In order to study the effect of mole-cular weight and functionality of the polyol on the intercalativebehaviour of the nanocomposites, a constant amount oforgano-MMT was intercalated with PPG1, PPG2 and GPO3, toobtain PU/MMT1, PU/MMT2 and PU/MMT3 nanocompo-sites, respectively.

Fig. 3. (Top): FTIR spectra of the C=O stretching peak ofthe pure TPU at various temperatures; (Bottom):relative fractions of two carbonyl peaks plottedas a function of temperature along with the DSCthermogram (solid line).

Free C=O

H.B.C=O

1800 1775 1750 1725 1700 1675 1650

40 C

Temp.

220 C

Wavenumber(cm )-1

70

60

50

40

3020 40 60 80 100 120 140 160 180 200 220 240

H.B.C=O

Free C=O

Temperature( C)

Fig. 4. (Top): FTIR spectra of the C=O stretching peak ofthe 1 wt % O-MMT/PU nanocomposite at varioustemperatures; (Bottom): relative fractions of twocarbonyl peaks plotted as a function of tempera-ture along with the DSC thermogram (solid line).

70

60

50

40

3020 40 60 80 100 120 140 160 180 200 220 240

Temperature( C)

H.B. C=O

Free C=O

Free C=O

H.B.C=O

1800 1775 1750 1725 1700 1675 1650

40 C

Temp.

220 C

Wavenumber(cm )-1

Table 2. Mechanical properties of the TPU/Clay nanocomposites

Sample Tensile Elongation Young’sstrength at break modulus(MPa) (%) (MPa)

MBP431(PU) 68.3 1420 24.1

1 wt % Na-MMT/PU 51.2 1180 24.4

3 wt % Na-MMt/PU 34.8 980 28.6

5 wt % Na-MMT/PU 37.1 998 49.1

1 wt % O-MMT/PU 28.6 990 26.2

3 wt % O-MMT/PU 27.9 660 46.3

5 wt % O-MMT/PU 31.0 575 53.3


Fig. 6 shows that the WAXD patterns of the PU/MMT1 andPU/MMT2 nanocomposites are more complex than that of thePU/MMT3.

Besides the characteristic peaks at 4.6 and 4.0 nm, PU/MMT1and PU/MMT2 have diffuse diffraction peaks at 2.2 and1.9 nm, respectively, which is close to the diffraction peak ofthe organo-MMT (Fig. 7). These results suggest that the GPO3has superior compatibility with organo-clay and is more easilyintercalated into the clay layers than PPG1 and PPG2. Fromthis observation, we expect that the PU/MMT3 nanocompositewould have the best mechanical properties. Since the WAXDpatterns of PU/MMT1 and PU/MMT2 nanocomposites showno obvious differences, it appears that the molecular weightof the polyols had no effect on the intercalative behavior.

Effect of clay content on the mechanical of the study of theeffect properties of the nanocomposites. The experimentalresults of the clay content on the tensile mechanical proper-

Fig. 7. Effect of clay content on WAXD of organo MMT,PU and PU/MMT3 nanocomposites.

1 2 3 4 5 6 7 8 9 1020/degree

4.5nm

PU/MMT(7.79%)1.9nm

PU/MMT(5.01%)

PU/MMT(2.36%)

PU(0%)

MMT

Fig. 5. Tensile stress-strain curves of TPU nanocompositescontaining (a) O-MMT and (b) Na-MMT.

(1) MBP431(PU)(2) 1wt% Na-MMT/PU(3) 3wt% Na-MMT/PU(4) 5wt% Na-MMT/PU

80

60

40

20

00 400 800 1200 1600

Strain (%)

(1)

(2)

(4)

(3)

(b)

(1) MBP431(PU)(2) 1wt% O-MMT/PU(3) 3wt% O-MMT/PU(4) 5wt% O-MMT/PU

80

60

40

20

00 400 800 1200 1600

Strain (%)

(1)

(2)(4)

(3)

(a)

ties of the nanocomposite are shown in Fig. 8. It was foundthat the clay content had a remarkably effect on the mecha-nical properties. Both the tensile strength and the elongationat break of the PU/MMT3 increased with increasing the claycontent in the range of 0-8 wt %. The maximum values of themechanical properties were reached when the clay contentwas 8 wt %. At higher clay content both the tensile strengthand elongation at break of PU/ MMT3 decreased. Theseresults could be explained in terms of the interaction betweenthe pure PU and the nanometer clay layers. When the claycontent was less than 8 wt %, the nanometer clay layers had agood compatibility and a strong interaction with the pure PU;when the clay content was higher than 8 wt %, the clay beganto aggregate, as suggested by the WAXD.

Effect of intercalative way on the mechanical properties ofthe nanocomposites. The route of the clay compoundingobviously affects the mechanical properties of the nanocom-posites. As compared to the pure PU, the tensile strength ofPU/MMT2 and PU/MMT1 nanocomposites increase by 25


Fig. 6. Effect of polyol molecular weight and functionalityon WAXD of PU/MMT nanocomposites with aconstant clay content of 5 wt %.

1 2 3 4 5 6 7 8 9 1020/degree

4.5nm

PU/MMT(7.79%)1.9nm

PU/MMT(5.01%)

PU/MMT(2.36%)

PU(0%)

MMT

physical and mechanical properties; the most publishedstudies have dealt with the processes responsible for degra-dation and ultimate failure (Hoyle and Kim, 1986).

Proposition for the formation of degradation layer. Noreticulation mechanism was found in the literature about thedegradation layer; however, by analogy with other polymers,the following schemes could be proposed. The photooxidationof poly(vinyl chloride) (PVC) leads to a radical, -CH2C• ClCH2-,which reacts according to the mechanism as presented inFig. 10. The alkoxy radical induces the formation of reticu-lation points. By analogy with PVC, it could be possible topropose the mechanism in Fig. 10 for TPU. The last radicalwould cause the reticulation points of the degradation layer.

The polyether photooxidation leads to the formation of analkoxy radical (Fig. 11), which could eventually inducereticulation points in TPU because the flexible segments ofTPU are based on polyether. In polyamides free radicals R•,derived from degradation, involve the abstraction of hydro-

and 55, respectively (Fig. 9a) and their elongation at breakincrease by 50 and 80% (Fig. 9b), respectively. The maximumtensile properties were found for the PU/MMT3 nanocom-posite, where its tensile strength is about twice that of purePU and its elongation at break is about 2.5 times that of purePU. These results can be interpreted by the intercalativebehavior of the polyols as shown in Fig. 3. There was acorrelation between the mechanical properties (Fig. 9) and theWAXD results of the nanocomposites (Fig. 6). As the charac-teristic diffraction peak shifted to a smaller angle, the mecha-nical properties were found to increase in the nanocomposites.

Degradation of TPUs. It is known that TPUs undergo signifi-cant structural changes on weather exposure, which have beenfound to depend on the structure and the morphology of TPUs(Wypych, 1995). These changes lead to a deterioration in


Fig. 8. Effect of the clay content on the tensile mechanicalproperties of PU/MMT3 nanocomposites.

1.6

1.4

1.2

1.0

1.8

1.6

1.42 0 2 4 6 8 10 12

Clay content (%)(a)

350

300

250

200

150

100

50

0 2 4 6 8 10Clay content (%)

(b)

Fig. 9. Effect of clay compounding route on mechanicalproperties of PU/MMT nanocomposites with a con-stant clay content of 5 wt %.

180160140120100906040200

(b)

PU PU/MMT2 PU/MMT1 PU/MMT3PU and PU/MMT

1.2

1.0

0.8

0.6

0.4

0.2

0.0PU PU/MMT2 PU/MMT1 PU/MMT3

PU and PU/MMT(a)

gen in the α-position from the amide bound, and leads to theformation of reticulation points (Fig. 12). By analogy withpolyamides, it could be possible to consider the hydrogenabstraction in the α-position from the urethane group. Itwould induce the reticulation mechanism given in Fig. 12.

Proposition for the development and stabilization of thedegradation layer. Directly initiated by UV with just suffi-cient intensity. Ultraviolet radiation, which goes through thealready formed layer, follows the Beer Lambert absorptionlaw i.e. the UV intensity decreases when the layer thicknessincreases. Consequently, UV could reach a boundary thatwould be the “nondegraded coating/degradation layer” inter-face where the UV intensity would be sufficient to causedegradation.

By the propagation of radical intermediate species initiatedby stronger UV in the layers close to the exposed surface.Initiation of intermediate species on surface and their propa-gation by reactions in the degradation layer thickness followsa radical mechanism. By means of termination reactions, theconcentration decreases when the layer thickness increases.The radical species could thus propagate themselves up to a“nondegraded coating/degradation layer” boundary where theconcentration is still sufficient to activate the layer formation.With these processes, the more the layer thickness increases,the more the layer formation rate decreases. The layer thick-ness is going to increase progressively up to a certain limit.This limit corresponds to the equality of the formation rate ofthe layer by the nondegraded part and of the degradation rateof the layer by the outside part. Thus a dynamic process is setup, involving a constant thickness of the degradation layer.

Thermoplastic PU coatings. TPU has been in use as a fabriccoating material in the textile industry for over 30 years. Thefirst application of TPUs was as a replacement for PVC wherethe “look and feel” of PVC was of value but the performance ofPVC was lacking in areas such as flex properties, plasticizermigration, abrasion resistance, low temperature properties andcleanability.

Thermoplastic PU may be described as the linear structuralblock copolymer of (SH)n type, where S is soft and H is hardsegment (HS). Due to the wide variety of properties between Sand H segment, phase separation may be observed in the finalmaterial. Phase separation occurs due to the intrinsic incom-patibility or thermodynamic immiscibility between the hard

Fig. 11. Reticulation mechanism of polyethers [97].

·

For polyethers

-(CH2) -O-(CH2) -4 4 + O2 -(CH2) -CHO-(CH2) -3 4

O

+ OHΔ

·


Fig. 12. Reticulation mechanism of polyamides [97] andpossible reticulation mechanism of TPU.

-CH2-NH-CO-CH2- + R -CH2-NH-CO-CH- + RH··

-CH2

2 -CH2

NH-CO-O-CH2-CH2

NH-CO-O-C H-CH2-

-CH2

-CH2

-CH2

NH-CO-O-C HC H2-

NH-CO-O-CH-CH2-

NH-CO-O-CH-CH2-

2 -CH2-NH-CO-CH- -CH2-NH-CO-CH-

-CH2-NH-CO-CH-

+ R

- RH

For TPU

·

· ·

·

Fig. 10. Reticulation mechanism of PVC [97] and possiblereticulation mechanism of TPU.

Cl Cl

ClCl

O2-CH2-C-CH2- -CH2-C-CH2- -CH2-C-CH2-

+RH

O-OH

+ R

Cl

O

-CH2-C-CH2- + OH-CH2-C-CH2-

O-OH

For TPU

C HO2

CH

O-O

CH

O-OH

+ RH

- RCH

O-O

CH

O-OH

CH

O

- OH

Δ

Δ

·

·

O-O·

·

·

··

·

··

·

For PVC For polyamides

For TPU

segments (HSs) and soft segments (SSs). The HSs, composedof polar materials, can form carbonyl to amino hydrogenbonds and thus tend to cluster or aggregate into ordered harddomains, whereas SSs form amorphous domains. The HS actsas filler particle as well as crosslinker to restrain the motion ofSS chains. Such a structure was first proposed by Cooper andTobolsky (1966). Their work and that of Schollenberger(1959), established that segmented PUs consist of high glasstransition temperature (Tg) or high melting temperature (Tm)HS microphase separated from relatively low Tg SS. Thedegree to which the hard and SSs phase separate, plays a vitalrole in determining the solid-state properties of these multi-block coatings. Properties of thermoplastic PU coatingsdepend upon several factors such as the composition of softand HSs, lengths of soft and HSs and the sequence of lengthdistribution, chemical nature of the units composing the poly-mer, anomalous linkages (branching, crosslinking), molecularweight and the morphology in the solid state. At room tem-perature, soft macroglycol segments are above their Tg andhave easy segmental rotations, which therefore impart thematerial its rubber-like behaviour or elastomeric properties.On the other hand, hard domains are below their glassy ormelt transition temperature and are thought to govern thehysteresis, permanent deformation, high modulus and tensilestrength and provide dimensional stability. Compositionalvariables and processing conditions such as structure of softand HSs, symmetry of diisocyanate, type of CE (diol or diamine)number of carbon in linear low molecular weight CE, the type(polyester or polyether) and chain lengths of SSs, crystal-lizability of either segment, thermal history of the PUs and themethod of synthesis are known to affect the degree of phasesegregation, phase mixing, HS domain organization, andsubsequent PU coating properties.

ConclusionIt has been found that MMT clays exchanged with long chainonium ions (carbon number>12) have good compatibility withpolyol. The extent of gallery expansion of modified MMT ismainly determined by the chain length of the gallery oniumions. By the study of a TPU/clay elastomeric nanocomposite,it was found that the nanocomposites have maximum mecha-nical property values with the clay content.

The presence of nanoparticles may improve the mechanicalstrength of the PU matrix and in turn, the strength of the PUfoam. Nanoscale-dispersed clay may act as nucleation agentsduring the foaming process to produce finer cell structure andhigher cell density.

It was found that the mechanical properties had maximumvalues with the clay content when it reached 8 wt %. The

maximum tensile stress and the maximum elongation atbreak increased two and five times, respectively. The WAXDpatterns showed that the glycerol propoxylate had bettercompatibility with the organoclay than poly(propyleneglycol); this corresponded to the formation of a nanocompositefrom the clay compounding with glycerol proxylate that, inturn, resulted in the best mechanical properties.

The tensile strength and ultimate elongation values decreasedwith the addition of Na-MMT or O-MMT. There was, however,a significant improvement in the Young’s modulus values withincreasing levels of both Na-MMT and O-MMT.

Besides the characteristic peaks at 4.6 and 4.0 nm, PU/MMT1and PU/MMT2 have diffuse diffraction peaks at 2.2 and1.9 nm, which is close to the diffraction peak of the organo-MMT. These results suggest that the GPO3 has superiorcompatibility with organo-clay and is more easily intercalatedinto the clay layers than PPG1 and PPG2. When the claycontent was less than 8 wt %, the nanometer clay layers had agood compatibility and a strong interaction with the pure PU;when the clay content was higher than 8 wt %, the clay beganto aggregate.

AcknowledgementThe authors are grateful to Iran Khodro Co. and Islamic AzadUniversity for financial support.

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Preparation and Evaluation of Ciprofloxacin HydrochlorideFloating Oral Delivery System

Sujata Mohapatraa*, Sunit Kumar Sahooa, Subhakanta Dhala, Bhupen Chandra Beheraa,Bhakti Bhusan Barika and Bijan Kumar Guptab

aUniversity Department of Pharmaceutical Sciences, Utkal University, VaniVihar, Bhubaneswar-751004, India bDepartment of Pharmaceutical Technology, Jadavpur University, Calcutta, India

(received April 4, 2008; revised August 10, 2008; accepted August 10, 2008)


Abstract . A sustained release system for ciprofloxacin hydrochloride designed to increase its residence time in thestomach was achieved through the preparation of floating microparticles by the solvent diffusion technique, usingEudragit S 100 and Eudragit RL 100 polymers. Eight different ratios of Eudragit mixture were used for the formula-tion, all of which showed good flow properties and packability. The drug retained in the microparticles decreased withincrease in Eudragit RL 100 content, whereas, the floating ability increased with increase in weight ratio of EudragitRL 100. There were differences between the formulations as to their appearance and size distribution. Fourier trans-form infrared (FTIR) spectrophotometeric study confirmed intactness of drug in formulations. The formulationcontaining ES: ERL in a ratio1.5:1 exhibited high percentage of floating particles with a controlled release of drug in0.1N HCl.

Keywords: ciprofloxacin hydrochloride; floating drug delivery, eudragit

IntroductionThe oral route is the most important means of delivery of drugsto the systemic circulation in a controlled manner. Generallyafter oral administration the bioavailability of these controlledrelease drug delivery system depends on the transit time ofthe dosage form through the absorbing area of the gastrointes-tinal tract (Groning et al., 2007). So this factor reduces theefficacy of drugs having an absorption window in thestomach and upper part of small intestine (Rouge et al., 1996)as dosage form escapes to lower regions of the gastrointesti-nal tract before the drug is completely released. A modifiedrelease drug delivery system with prolonged residence timein stomach is of particular interest for drugs, which arelocally active in stomach or with an absorption window in thestomach and upper part of small intestine (Deshpande et al.,1996). Various attempts have been made to prolong the reten-tion time of dosage forms in the stomach that includebioadhesive system, swelling type or plug type system, modi-fied shape systems and hydrodynamical balanced systems.(Baumgartner et al., 2000, Hwang et al., 1998; Moes, 1993).Floating drug delivery systems are expected to remain buo-yant and swim in gastric contents due to their lower densitythan gastric fluids (~ 1.004 g/cm3) for several hrs andrelease the drug in a controlled manner (Sato et al., 2003;Hilton and Deasy, 1992, Kawashima et al., 1991).

Ciprofloxacin HCl is a fluoroquinolone antibacterial agentwith wide spectrum of activity. It has a short half life of3-4 h.The absorption window of the drug lies in the stomachand upper part of small intestine. About 70% of drug isabsorbed from the above mentioned region (Tripathy et al.,2001). It possesses all the suitable characteristics for deve-lopment of a floating drug delivery system and hence it waschosen as the drug of choice in the above study. Eudragit S100 and Eudragit RL 100 which are generally biocompatibleand extensively used in the controlled release formulation wereused for designing of floating drug delivery system.

In the present investigation, attempts were made by using eightdifferent ratios of Eudragit mixture in order to develop asuitable delivery system which will not only control therelease of drug but also remain buoyant for several hrs in thegastric fluid so that the basic objectives behind the study couldbe achieved.

Materials and MethodsMaterials. Ciprofloxacin HCl (courtesy of Get-Rid Pharm.Pune, India), Eudragit S100 (ES) and RL100 (ERL) (Alkem,Mumbai), Ethanol and dichloromethane (Emerck Ltd., India)were used. All other chemicals were of analytical grade.

Methods. Floating microparticles containing ciprofloxacinHCl were prepared using the solvent-diffusion technique

Pak. J. Sci. Ind. Res. 2008 51(4) 201-205

201

(El-Kamel et al., 2001). The drug: polymer ratio used toprepare different formulations was 1:2. The polymer contentwas a mixture of ES: ERL, 4: 1 (F1), 3: 1 (F2), 2: 1 (F3), 1.5:1 (F4), 1: 1 (F5), 1: 1.5 (F6), 0: 1 (F7), and 1: 0 (F8). Thedrug-polymer mixture was dissolved in a solution of ethanoland dichloromethane (1:1), and dropped into 0.2% sodiumlauryl sulphate solution. The solution was stirred with a pro-peller type agitator at room temperature for 1 h at 150 rpm.The formed floating microparticles were filtered, washed withwater and dried at room temperature. The floatingmicroparticles were sieved and suitable fractions werecollected.

Percent yield. Microparticles dried at room temperature wereweighed and the yield of microparticles preparation wascalculated (Sahoo et al., 2007).

Measurement of micromeritic properties. The flowproperties of prepared microparticles were investigated bymeasuring the bulk density, tapped density and Carr’s index.The mean particle size of the beads was determined bysieving method (Sahoo et al., 2007).

Encapsulation efficiency. About 50 mg of formulation wasdissolved in 50 ml of 0.1N HCl (with 2 ml ethanol). Theresulting mixture was agitated on mechanical shaker for 24 h.The solution was then filtered and the drug content wasestimated spectrophotometrically at 277 nm after suitabledilution (Sahoo et al., 2007).

Buoyancy test. In vitro evaluation of floating behaviorstudies were performed by placing 100 microparticles into500 ml 0.1 N HCl containing 0.02 % w/v tween 20 in a dissolu-tion vessel (USP dissolution tester) followed by paddlerotation at a speed of 100 rpm maintained at 37 °C (Choi etal., 2002). At predetermined time intervals (2, 4, 6, 8, 10, 12h) numbers of floating particles were counted by visualobservation. The percentage of floating microparticles wascalculated by formula:

number of floating microparticlesinitial number of microparticles

x 100

Scanning electron microscopy (SEM). Scanning electronmicroscope (JEOL, JSM-6360) was used to characterize thesurface topography of the microparticles after goldcoating.

X-ray powder diffractometric (XRD) study. X-ray powderdiffractometry was carried out to investigate the effect of microencapsulation process on crystallinility of drug. Powder XRDpatterns were recorded on Rigaku, Japan (Model-Meniflex)X-Ray diffractometer using Ni-filtered, Cuk α radiation, at30 kV and 25 mA. The scanning rate employed was 2°/min,

over 4° to 40° diffraction angle (2θ) range. The XRD patternsof drug powder and drug-loaded beads were recorded.

FTIR study. The IR spectra were recorded for pure drug anddrug-loaded microparticles using FTIR JASIO (Model No.410). Samples were prepared in KBr disks (2 mg sample in200 mg KBr). The scanning range was 400-4000/cm and theresolution was 2/cm.

Drug release study. The drug release rates from floatingmicroparticles were carried out in 500 ml 0.1N HCl main-tained at 37 ± 1 °C and stirred using USP basket type dissolu-tion apparatus (LABINDIA, DISSO-2000) at 100 rpm. 100mg microparticles were used in each test. Samples werewithdrawn at suitable time intervals and assayed at 277 nm.

Kinetics of drug release. In order to understand the mecha-nism of drug release, the first 60 % of the release drug wasfitted in Korsmeyer-peppas model (Sahoo et al., 2007; Ritgerand Peppas, 1987), wherein:

M t / M α = k t n,

where M t / Mα is the fraction of the drug released at time t,k is the rate constant and n is the release exponent. The nvalue is used to characterize different release mechanisms andis calculated from the slope of the plot of log of fraction ofdrug released vs. log of time.

Results and DiscussionThe percentage yield of floating microparticles determinedby weighing after drying to constant weight was found tolounge in the range 32.97-67.92 (Table 1). All formulations

Table 1. Micromeritic properties, yield and floating abilitiesof various formulations

Batch Bulk Tapped Carr’s index* Yield Floatingcode density density (%) particle at

(g/cm3) (g/cm3) the end of12 h (%)

F1 0.34 0.41 17.07±0.636 32.97 41F2 0.33 0.39 15.38±0.811 67.92 42F3 0.31 0.36 13.88±0.454 51.27 49F4 0.26 0.32 18.75±0.411 55.74 67F5 0.29 0.34 14.07±0.442 53.13 50F6 0.28 0.33 15.15±0.625 60.97 63F7 0.2 0.34 17.64±0.336 56.00 42F8 0.36 0.42 14.28±0.144 51.08 23Drug 0.25 0.36 30.55±0.112 ** **

* = each observation is the mean ± SD of three determinations;** = the test was not carried out for pure drug

202 Sujata Mohapatra et al.

showed excellent flow ability in terms of Carr’s Index (all < 18.75).The excellent flow properties suggest that the microparticlescan be easily handled during processing. The size distributionvaried among the formulations. Most of the floating micro-particles were collected above sieve range of 250 μm and thehighest amount was retained in the range 250-500 μm (Fig.1).The floating ability varied according to the polymer compo-sition of microparticles (Table 1). Compared to other formu-lations, the formulation without ERL content, F8, exhibitedlower floating ability. Combination of formulations F4 and F6

provided excellent results in terms of floating ability. Formu-lation F8 showed highest percentage of drug content andentrapment value and formulation F7 showed lowest percent-age of the retained drug (Table 2). The drug retention decreasedwith increase in ERL content of floating microparticles.This could be due to high permeation characteristic of ERLthat facilitated diffusion of a part of entrapped drug to thesurrounding medium during preparation of floating micro-

particles. Scanning election microscope photographs show themicroparticles to be spherical with irregular surface (Fig.2).Cross-sectional view exposed the characteristic open cell struc-ture of microparticles. From X-ray diffraction patterns (Fig.3)it is obvious that the pure drug exhibited crystalline charac-teristics while in formulations, drug peaks are still visiblebut with loss of its sharpness which is due to dilution withpolymer; hence it may be concluded that the drug retains thecrystalline structure in the formulation.

The FTIR report (Fig. 4) shows no significant difference inspectra of drug and formulations confirming absence of drugpolymer interaction. The dissolution study was carried out for6 h in 0.1 N HCl. It was found that drug release was faster incase of formulations containing higher percentage of ERL,whereas release rate was slow for formulations with higher %

Table 2. Entrapment efficiency and release kinetics offloating microparticles

Batch Drug encapsulated Korsmeyer-Peppas modelcode (%)* n r k

F1 52.56 ± 0.35 0.690 0.970 0.130F2 50.21±. 0.02 0.591 0.954 0.159F3 49.8± 0.045 0.600 0.976 0.169F4 49.48± 0.12 0.538 0.976 0.202F5 47.48± 0.866 0.574 0.981 0.224F6 45.63±.0.03 0.612 0.987 0.227F7 42.33±.0.03 0.563 0.992 0.258F8 62.15±.0.045 0.540 0.951 0.136

* = each observation is the mean SD ± of three determinations; dif-fusion exponent (n), correlation coefficients (r) and kineticconstants (k) are related to the mechanism of drug release, accord-ing to equation, Mt/Mα = Kt n

Fig. 2. SEM photographs of floating microspheres (A) withcross sectional view (B) of formulation F4.

(A)

(B)

203Ciprofloxacin HCl Oral Delivery System

Fig. 1. Particle size distribution of various formulations.

Particle size (microns)

80706050403020100 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

<250 250-500 500-1000 >1000

(Figures in order indicate formulations F1 to F8)

of ES (Fig.5). This may be attributed to the fact that ERL is aco-polymer of acrylic acid and methacrylic acid esters with ahigh content of quaternary ammonium groups which makesthem more permeable and gives an opportunity to the drug todiffuse out to bulk medium at a faster rate. But ES is of lowpermeability and insoluble in acidic medium. It is an anionicco-polymer of methacrylic acid and methyl methacrylatecontaining free carboxylic and ester group. Its very lowpermeability results from high intermolecular attraction.Hydrogen bonding between the hydroxyl group of carboxylicmoiety and the carboxyl oxygen of ester group increases thedegree of compactness of polymers and decreases the poros-ity and permeability, thereby decreasing the release rate. Themechanism of drug release was studied using Korsmeyer-Peppas model. The value of ‘n’ fell within the range of

Fig. 3. X-ray diffraction patterns (A): ciprofloxacin; (B): for-mulation F1; (C): formulation F4.

Fig. 4. FTIR spectra (A): ciprofloxacin (B) formulation F1;(C): formulation F4.

Fig. 5. Dissolution profile of ciprofloxacin from differentfloating microparticle formulations in 0.1 N HCl (n=3).

0.5-0.7 (Table 2), indicating anomalous or non-fickian typeof release, in which the rate of dissolution mediumuptake intothe polymer is largely determined by relaxation of polymerchain.

In conclusion, the floating microparticles of ciprofloxacincould be prepared successfully using suitable ratio of ES: ERLby solvent diffusion technique. Formulation F4 (ES: ERLratio = 1.5:1) provided better results regarding floatingbehavior with suitable controlled drug release trait among allother formulations.

AcknowledgementThe authors wish to thank M/s Get-Rid Pharm., Pune, Indiafor supply of ciprofloxacin HCl as a gift sample. The authorsare grateful to IIT Kharagpur for providing necessary facili-ties to carry out this research work.

ReferencesBaumgartner, S., Kristl, J., Vrecer, F., Vodopivec, P., Zorko,

B. 2000. Optimization of floating matrix tablets andevaluation of their gastric residence time. Int. J. Pharm.195: 125-135.

Choi, B.Y., Park,H.J., Hwang,S.J., Park, J.B. 2002.Prepara-tion of alginate beads for floating drug delivery system:Effects of CO2 gas-forming agents. Int. J. Pharm. 239:81-91.

Deshpande, A.A., Rhodes, C.T., Shah, N.H., Malick, A.W.1996. Controlled release drug delivery systems for pro-longed gastric residence: an overview. Drug Dev. Ind.Pharm. 22: 531-540.

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1000 2000 3000 4000(2 θ)

(A)

(B)

(C)

4000 3000 2000 1000 40020

Wavenumbers (Cm-1)

(C)

(B)

(A)

Sujata Mohapatra et al.

90

80

70

60

50

40

30

20

10

00 1 2 3 4 5 6 7

Time (h)

F1F2F5

F4F3F6F7F8

F1 ; F2 ; F3 ; F4 ; F5 ; F6 ; F7 ; F8

El-Kamel, A.H., Sokar, M.S., Al Gamal, S.S., Naggar, U.F.2001. Preparation and evaluation of ketoprofen floatingoral delivery system. Int. J. Pharm. 220: 13-21.

Groning, R., Cloer, C., Georgarakis, M., Muller, R. S. 2007.Compressed collagen sponges as gastroretentive dosageforms: In vitro and in vivo studies. Eur. J. Pharm. Sci.30: 1-6.

Hilton, A.K., Deasy, P.B. 1992. In vitro and in vivo evaluationof an oral sustained-release floating dosage form ofamoxicillin trihydrate. Int. J. Pharm. 86: 79-88.

Hwang, S.J., Park, H., Park, K. 1998. Gastric retentive drug-delivery systems. Crit. Rev. Ther. Drug Carrier Syst. 15:243-284.

Kawashima, Y., Niwa, T., Takeuchi, H., Hino, T., Ito, Y. 1991.Preparation of multiple unit hollow microspheres(microballoons) with acrylic resin containing tranilast andtheir drug release characteristics (in vitro) and floatingbehaviour (in vivo). J. Control Release 16: 279-289.

Moes, A.J., 1993. Floating delivery and other potentialgastric retaining systems, Current status on targeted drug

delivery to the gastrointestinal tract. Capsugel Library95-112.

Ritger, P.L., Peppas, N.A. 1987. A simple equation fordescription of solute release. II. Fickian and anomalousrelease from swellable devices. J. Control Release 5:37-42.

Rouge, N., Buri, P., Doelker, E. 1996. Drug absorption sitesin the gastrointestinal tract and dosage forms for site-spe-cific delivery. Int. J. Pharm. 136: 117-140.

Sahoo, S. K., Dhal, Mohapatro, S. P., Behera,B. Ch., Barik,B. B.2007. Effect of processing temperature on EudragitRS PO microsphere characteristics in the solvent evapo-ration process. Pharmazie 62: 638-639.

Sato, Y., Kawashima, Y., Takeuchi, H., Yamamoto, H. 2003.In vivo evaluation of riboflavin-containing microballoonsfor floating controlled drug delivery system in healthyhuman volunteers. J. Control Release 93: 39-47.

Tripathy, K.D. 2001. Essentials of Medical Pharmacol-ogy, 890 p., 5th edition, Jaypee Brothers MedicalPublishers Pvt. Ltd., New Delhi, India.

205Ciprofloxacin HCl Oral Delivery System

Pak. J. Sci. Ind. Res. 2008 51(4) 206-211

Molecular and Biochemical Evaluation of Genetic Effects ofCalotropis procera (Ait.) Latex on Aspergillus terreus Thom

Sameer H. QariDepartment of Biology, Teachers College, Umm Al-Qura University, P.O. Box 2203, Makkah, Saudi Arabia

(received January 1, 2008; revised July 2, 2008; accepted July 3, 2008)

IntroductionCalotropis procera (Ait.) of the Asclepiadaceae family,commonly known as Sodom apple, is widely distributed inSaudi Arabia and is one of giant milk weeds (Calvin, 1979).The plant is popularly known because it produces largeamount of latex, which is easily collected from its greenparts when the plant is wounded. The aspect of this naturalsecretion resembles that of rubber tree, Hevea brasiliensis.The latex of C. procera extract is prescribed in folkloricmedicine for the treatment of many diseases. Many recentstudies proved its potential activities as wound healing, anti-inflammatory, anti-diarrhoeal and anti-rheumatic drug(Kumar et al., 2001; Rasik et al., 1999; Sharma and Sharma,1999; Basu et al., 1997; Jain et al., 1996).

Some people use it successfully to combat some skin fungalinfections and malaria. The abundance of latex in the greenparts of the plant reinforces the idea that it is produced andaccumulated as a defence strategy against organisms such asvirus, fungi and insects, although wounded plants of Calotropisas well as insects visiting their leaves are also seen (Haqueet al., 2000; Larhsini et al., 1997). When injured, leaves or thenearest other green parts of the tree exude the latex which has aclingy effect, capable of immobilizing insects. Additionally, thepresence of plant defence-related proteins such as hevein,alpha-amylase inhibitor, among others, have been described tooccur in the latex secretion of other plants (Pereira et al., 1999;Wititsuwannakul et al., 1998) and this seems to be also the caseof Calotropis latex (Dubey and Jagannadham, 2003).

Scientific literature mentions other relevant activities ofthe latex of C. procera, such as among others antibacterial,analgesic or in vitro schizonticidal activity, (Alencar et al.,2004; Dewan et al., 2000; Sharma and Sharma, 2000; Jainet al., 1996). A brief communication pointed out the wholelatex of C. procera as a suitable source of active compoundsexhibiting larvicidal activity (Ramos et al., 2006). Thegenotoxicity of C. procera latex extract was demonstrated forthe first time by Baeshin and Al-Ahmadi (2004) employingroot tip meristems of Allium cepa and Vicia faba chromo-somal aberrations test. Cytotoxic and anti-tumour activitiesof the latex of C. procera were studied by Choedon et al.(2006). More investigations are required as a battery of teststo establish its genotoxic effect. The purpose of this study isto evaluate the genotoxic effects of the latex of C. procera bythe Aspergillus terreus auxotrophic mutant test and DNAfingerprinting by RAPD as well as protein profile by SDS-PAGE test.

Materials and MethodsLatex preparation. The latex of C. procera was collectedduring the last week of August, 2006 and January, 2007 fromplants growing in their natural habitats, located along the beltof C. procera in Al-Hajj Street of Makkah as described byMigahid (1988).

Fresh latex was collected from the healthy plants by makingsmall incisions near the youngest leaves in sterile 250 mlconical flask. The latex samples were gently handled in icebag under sterile conditions to maintain homogeneity andactivity during transport to the laboratory and transferred toE-mail: [email protected]

Abstract. On treating dense conidial suspensions of Aspergillus terreus Thom with different concentrations of Calotropisprocera latex, for investigating the genotoxicity of the latter, it was found that latex of Calotropis procera had potentlethal and mutagenic activities. Survival percentage decreased as concentration or time of exposure increased.Frequency of auxotrophic mutants increased with increase in concentration or exposure time. Most auxotrophicmutants were amino acid requiring mutants. DNA and total protein contents of each mutant was significantly lowerthan wild type of Aspergillus terreus. RAPD demonstrated polymorphic genetic bands of electrophoretic products ofPCR for all mutants compared with the wild type strain. SDS-PAGE results expressed a polymorphism of proteinbands as well. All these results indicated the mutagenicity of the latex of Calotropis procera.

Keywords: Calotropis procera, mutagenicity, Aspergillus terreus, genotoxicity

Biological Sciences

206

a refrigerator for 2-3 h; afterwards, they were centrifuged(3000-4000 rpm) at room temperature (25 °C) in a non-refrigerated bench top centrifuge for 15 min to remove intercoagulum (Atal and Sethi, 1962). The supernatant was thenfiltered through a membrane filter and the filtrate (the purelatex) was either used directly in the experiment or kept inthe refrigerator for no longer than 7 days for future use. Threeconcentrations of this stock extract were prepared withsterilized distilled water (1, 3, 9 and 27% of latex) for testingthe genotoxic activities.

Strains. Wild type strain.The haploid phase of the wild typestrain of Aspergillus terreus Thom was the test organism inthe present study. It was obtained from the Biology Depart-ment, Faculty of Sciences, King Abdul-Aziz University, Jeddahwhere it had been maintained for several years. It had beenisolated from Makkah Road by El-Sharkawi et al. (1981) andwas identified by the Common Wealth Mycological Institute,England.

Mutant strains.Induced mutant strains by C. procera latex inthis experiment were isolated and maintained on PotatoDextrose Agar (PDA) and used later in this study for deter-mining their nutritional requirements and to run RAPD andSDS-PAGE tests on them. Growth medium supplies werepurchased from Difco Laboratories.

Chemicals. Standard concentrations of hydrolysed casein(CAS), mixed vitamin solutions (VITS), hydrolysed yeastribonucleic acid (RNA), individual amino acids, individualvitamins and individual purines and pyrimidines were used assupplements to the minimal media (Table 1). All the chemi-cals used in the study were provided by Sigma Company,except DNA polymerase (from Perkin-Elmer Cetus) and thedNTPs (from Boehringer Mannheim). Difco Czapek Dox agar(DOX) was used as the minimal medium and Potato DextroseAgar (PDA) was employed as the complete medium.

Test of genotoxicity. Auxotrophic mutation. Latex ofC. procera was tested for biochemical genetic activitiesfollowing the method described by Sabir and Baeshin (1999)for the induction of auxotrophic mutants. A dense conidialsuspension was made and number of 1,000,000 conidia/mlwas estimated using a hemocytometer. Five ml of this suspen-sion was immediately added to 5 ml of the latex extract andone ml sample of this mixture was immediately diluted in9 ml sterile distilled water to serve as untreated (positive)control. Subsequent samples were taken at regular intervals(15 min) for a period of 1h after exposure and serially dilutedin sterile distilled water to halt the mutagenic treatment.Samples of the final dilutions containing about 100 conidia/ml were spreaded on PDA plates and incubated at 28 °C for

4 days. This was repeated for each of the four different con-centrations of Calotropis latex (1, 3, 9 and 27% of latex).

Mutants were isolated according to the method described byFincham et al. (1979). At each time interval, a monoconidialinoculum was inoculated in each of 26 loci/plate containingPDA and served as template. The template was in turn repli-cated on the minimal medium (DOX) to detect auxotrophicmutants. All replicates were incubated for 5 days at 28 °C.Auxotrophic mutants were those which failed to grow on DOXafter incubation for 5 days at 28 °C. All mutant colonies wereisolated on PDA templates and replicated on the followingsupplemented media for determination of their nutritionalrequirements:

1. DOX (minimal media).2. DOX (minimal media) + CAS ( add casein) + VITS (add

vitamins) – RNA (without ribonucleic acid)3. DOX (minimal media) + CAS ( add casein) - VITS (with-

out vitamins) + RNA (add ribonucleic acid)4. DOX (minimal media) – CAS (without casein) + VITS (add

vitamins) + RNA (add ribonucleic acid)5. DOX (minimal media) + CAS ( add casein) + VITS (add

vitamins) + RNA (add ribonucleic acid)

A colony which failed to grow on any of number 2, 3 or 4media requires the chemical, missing from that medium. Theauxanographic technique by Pontecorvo (1949) was used to

Table 1. Concentrations of medium supplements

Supplement Concentration Concentrationof stock solution used in media(mg/ml) (mg/ml)

Hydrolysed casein 60 3Individual amino acids 10 10-1

Hydrolysed yeast Ribonucleic acid 10 5 x 10-1

Individual purines andpyrimidines 4 2 x 10-2

Vitamins*Aneurin 5 5 x 10-4

Biotin 0.02 2 x 10-6

Choline chloride 20 2 x 10-3

Inositole 40 4 x 10-3

Nicotinic acid 10 10-3

Pyridoxine 5 5 x 10-4

P-aminobenzoic acid 1 10-4

Pantothenic acid 20 2 x 10-3

Riboflavine 10 10-3

* = mixed vitamins: 1 ml of each of the individual vitamin solutionswas mixed and 0.2 ml of this mixture was added to 200 ml of media(0.2 ml of 1/10 dilution/plate)

207Genetic Effects of Calotropis procera Latex

specify the particular nutritional requirement of each mutant.One ml of a dense conidial suspension of the mutant was mixedwith cooled molten DOX (45 °C) in dishes and left to solidify.A few crystals of the nutrients to be tested were placed atmarked positions around the periphery of the agar plate. Eachmutant grew after 5 days incubation in the immediate vicinityof the nutrient required.

RAPD profile. Auxotrophic mutants were incubated for5 days at 28 °C in broth PDA media, then, they were frozen inliquid nitrogen, ground with mortar and pestle and incubated in1.5 ml of extraction buffer (100 mM Tris-HCl pH 8, 100 mMNaCl, 50 mM EDTA pH 8, 2% SDS and 0.1 mg/ml proteinaseK) for 1.5 h at 37 °C. DNA was extracted according to Scott etal., (1991) with minor modifications.

PCR reactions were conducted using three arbitrary 10-merprimers (Operon Tech. Inc.). The names and sequences of theseoligoprimers are listed in Table 2. The reaction conditionswere optimized and mixtures (25 μl total volume) consistedof: P10X PCR buffer (pH 8.3), KCl (50 mM), MgCl2

(3 mM), dNTPs (2.5 mM), primer (20 μM), template DNA(30 ng/μl), Taq DNA polymerase (1 unit), all under a drop ofmineral oil. DNA amplification was carried out in a PerkinElemar G thermalcycler. Samples were preheated at 94 ºCfor 1 min. Amplification reactions were run for 40 cyclesconsisting of: 1 min at 94 ºC, 2 min at 36 ºC, 1 min at 72 ºCwith a final extraction time of 7 min at 72 ºC. Amplificationproducts were size-separated by electrophoresis in 2.5%agarose gel and visualized by ultraviolet illumination afterstaining with ethidium bromide.

Protein profile. Protein was extracted by crushing frozen fungiwhich were incubated for 5 days at 28 °C in broth PDA mediafrom each mutant and wild type in 500 μl sample buffer. Themixture was denatured for 5 min. SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) was performed on total proteinusing the slab gel apparatus (Hoffer SE 600 vertical slab unit)according to the method of Laemmli (1970). The proteincontents of both wild A. terreus and mutants were estimatedby the colorimetric method of Coomassie Brilliant Blueaccording to Bradford (1976).

Statistical Analysis. Data of survival percentage andfrequency of auxotrophic mutants were analyzed by

analysis of variance (ANOVA), with the calculations of theF-statistic and respective P values; P values were comparedwith the value of the significant difference for P=0.05% orP=0.01%.

Results and DiscussionAuxotrophic mutation. The survival percentage and recov-ery of auxotrophic mutants among survivors of A. terreuscultured cells, as a function of treatment with differentconcentrations of C. procera latex, are presented in Table 3and 4.

Table 2. Primers and their sequences

Primer Sequence G+C (%)

OPB-16 5‘ TTT GCC CGG A′3 60OPC-20 5‘ ACT TCG CCA C′3 60OPE-03 5‘ CCA GAT GCA C′3 60

Table 3. Effect of concentrations of C. procera latex andexposure time on survival percentage (SP) of conidia ofAspergillus terreus

Extract Exposure time (min) Mean ofconc. SP (%)(%) within

conc.

0 15 30 45 60

1 100 96.74 87.91 78.37 76.74 87.95*3 100 94.96 87.65 82.62 83.62 89.77*9 100 79.48 70.51 65.64 64.87 76.10**27 100 60.87 15.11 46.17 45.18 60.66**

* = significant from wild type (control at 0 time) at P>0.05;** = significant from wild type (control at 0 time) at P>0.01

208 Sameer H. Qari

Table 4. Effect of concentrations of C. procera latex andexposure time on auxotrophic mutation percentage (AMP) for208 isolated colonies of A. terreus

Extract Exposure time (min) Mean ofconc. AMP (%)(%) within

conc.

0 15 30 45 60

1 0.00 0.00 0.00 0.48 0.48 0.192*

3 0.00 0.00 0.48 0.00 0.96 0.288**

9 0.00 0.00 0.48 0.48 0.96 0.384**

27 0.00 0.00 0.00 0.48 0.00 0.096**

* = significant from wild type (control at 0 time) at P>0.05;** = significant from wild type (control at 0 time) at P>0.01

The highest possible percentage of mutation (1.92%) isachieved with the dose of 9% at exposure time of 60 minwhich is the optimal dose for the induction of auxotrophic

Fig. 1. RAPD-DNA genetic bands of auxotrophic mu-tants in Aspergillus terreus caused by C. proceralatex (OPB-16).

Table 5. List of auxotrophic mutants recovered fromC. procera latex treated conidia of A. terreus

Mutants code no. Latex conc. Exposure Requirement(%) time (min)

AMC1T45 1 45 glutamineAMC1T60 1 60 lysineAMC3T30 3 30 proline (or)

tyrosineAMC3T45-a 3 45 tyrosineAMC3T45-b 3 45 glutamineAMC9T30 9 30 lysineAMC9T45 9 45 tyrosine (or)

prolineAMC9T60-a 9 60 glutamineAMC9T60-b 9 60 lysineAMC27T45 27 45 lysine

Table 6. Quantities of DNA and total protein isolated fromwild type and auxotrophic mutants of A. terreus

Aspergillus Exposure Treatment DNA Total %terreus time conc. (%) quantity protein of decreasestrains (min) (μg/ml) quantity total protein

(μg/ml)

Wild Type - - 600 3190 0

AMC1T45 45 1 543* 2820 11.59**

AMC1T60 60 1 445** 2930 8.15**

AMC3T30 30 3 340** 3009 5.67**

AMC3T45-a 45 3 310** 2706 15.17**

AMC3T45-b 45 3 315** 2742 14.04**

AMC9T30 30 9 300** 2685 15.83**

AMC9T45 45 9 221** 2689 15.70**

AMC9T60-a 60 9 260** 2627 17.64**

AMC9T60-b 60 9 247** 2349 26.36**

AMC27T45 45 27 117** 2476 22.38**

* = significant from wild type of A. terreus at P>0.05; ** = signifi-cant from wild type of A. terreus at P>0.01

209

RAPD profile. The RAPD results illustrated polymorphic num-bers of the genetic bands (Fig. 1), which were the electrophoreticproducts of PCR for all mutants compared with the wild type.Table 7 illustrates that the highest number of polymorphic bandsamong mutants was generated in reactions with the primersOPB-16 which was 78 genetic bands and the lowest number ofpolymorphic bands obtained with primer OPE-03 which was46, whereas the number of polymorphic bands of primersOPC-20 was 58. The percentage of polymorphic bands to sourceof polymorphism are 42.86, 25.27 and 31.86 for OPB-16,OPE-03 and OPC-20, respectively.

Genetic Effects of Calotropis procera Latex

mutants with C. procera latex. All of the auxotrophic mutantsrecovered from C. procera latex-extract-treated conidia ofA. terreus were amino acid-requiring mutants (Table 5). Someof the mutants restored growth with any one of different alter-native nutritional requirements (e.g., tyrosine or proline).

DNA and total protein quantity of mutants (Table 6) decreasedsignificantly (P> 0.01) as compared to the wild type which isa consequence of molecular changes in the genetic materialof A. terreus.

Protein profile. The results of total protein profile andapplied SDS-PAGE technique are expressed as a decreasein the total protein in all mutants compared to wild type(Table 7); a polymorphism of protein bands are clear inFig. 2. All these results strongly point out the mutagenicityof the latex extract of C. procera.

It has been clearly shown that the increase in concentrationand exposure time to C. procera latex lead to a decrease insurvival percentage and an increase in auxotrophic mutationpercentage of A. terreus. These results are generally in agree-ment with the rule mentioned by Fincham et al. (1979) whostated that by using chemical mutagens, there was a constantrelation between the dose and the mutation percentage whichincreases to a certain limit with the increase in the dose. Thisprovides additional evidence for the genotoxicity of this la-tex extract earlier demonstrated by Baeshin and Al-Ahmadi(2004) for the first time.

The present study confirms that C. procera latex extractexhibits strong mutagenic activities as compared to the po-tent chemical mutagenic agent N.T.G. It was found by Baeshinand Sabir (1987) that N.T.G. yielded 3.8% of auxotrophs inA. terreus with the optimal dose of 0.0075 g/10 ml on 70min. exposure, whereas C. procera latex extract resulted in

Table 7. Polymorphic bands of each genetic primer andpercentage of polymorphism in auxotrophic mutants ofA. terreus

Primers Total Polymorphic Polymorphic Polymorphic Polymorphicbands bands to bands to bands to bands to

control total bands source of total bandsbands (no.) of primers polymor- (%)

(%) phism (%)

OPB-16 161 78 48.45 42.86 19.84

OPE-03 109 46 42.20 25.27 11.70

OPC-20 123 58 47.15 31.86 14.75

Total 393 182 - - -

1.92% auxotrophic mutants with a dose of 9% at 60 min ex-posure, in the present study.

All of the auxotrophic mutants recovered from C. proceralatex extract treated-conidia of A. terreus were amino acid-requiring mutants. Some of the mutants restored growth withany one of different alternative nutritional requirements. Theseare probably leaky auxotrophic mutants i.e., they fail to com-pletely prevent the action of a gene and permit some residualfunctions, specifically of glutamine, proline, tyrosine andlysine. According to the selected target, locus of this extractat the molecular level is suggested. Similar results wereobtained by Sabir (2005), Baeshin and Sabir (1987) and Tayl(1975). This observation led to a molecular study of DNAand protein of the auxotrophic mutants with the wild type ascontrol.

The DNA and protein study revealed a significant decrease inall auxotrophic mutants as compared with the wild type,suggesting a molecular change causing a deletion at one ormore loci; this probably affected gene expression andconsequent interruption in biochemical pathways of DNA andprotein synthesis; alkaloids, glycosides and functional enzymes

Fig. 2. Protein bands of auxotrophic mutants in A. terreuscaused by C. procera latex.

in C. procera latex extract often exhibit similar activity. Theseresults are consistent with the results obtained by Choedonet al. (2006).

The auxotrophic mutation resulted due to a change (defectin the metabolism pathway) on genomic DNA; the resultsof RAPD genetic bands have given strong evidence thatauxotrophic mutants induced genomic DNA changes. Sothe observation of the RAPD results have provided goodevidence of the ability of C. procera latex to induce pointmutation as a result of deletion of at least one nucleotide;this is revealed by the disappearance of many genetic bandsas compared with the wild type. Some of the componentsof C. procera may act as intercalation agent or generatefree radicals which interact with DNA to account for theobserved deletions; similar results were obtained by Ansahet al. (2005) in their study of Cryptolepis sangvinolehta.Also a polymorphism of protein bands, obtained as a resultof PAGE protein profile, confirms the ability of C. procerato cause frame shift mutation in A. terreus. All theseresults strongly point out the mutagenicity of the latexextract of C. procera in A. terreus.

Generally, latex of C. procera showed mutagenic effect onthe conidian cells of A. terreus in increasing manner alongwith concentration and treatment time. Further studies needto be made to separate the active ingredient of that latex whichproduces the mutagenic effect and to explore the toxicity andother pharmacological assay in order to make herbal safetyproducts as mentioned in our local folkloric medicine.

ReferencesAlencar, N.M., Figueiredo, I.S., Vale, M.R., Bitencourt,

F.S., Oliveira, J.S., Ribeiro, R.A., Ramos, M.V. 2004.Anti-inflammatory effect of the latex from Calotropisprocera in three different experimental models: peritoni-tis, paw edema and hemorrhagic cystitis. Planta Med. 70:1144-1149.

Ansah, C., Khan, A., Gooderham, N. 2005. In vitro genotoxi-city of the West African anti-malaria herbal Cryptolepissanguinolenta and its major alkaloid crytolepine. Toxicol.208: 141-147.

Atal, C.K., Sethi, P.D. 1962. Proteolytic activities of someIndian plants. II. Isolation, properties and kinetic studiesof Calotropis. Planta Med. 10: 77-84.

Baeshin, N.A., Al-Ahmadi, M.S. 2004. Assaying single andcombined genotoxicity of Calotropis procera (Ait.)latex and chlorcyrin in root tip meristems of Allium cepaand Vicia faba. In: Proc. 3rd Int. Cont. Biol. Sci., 3: 184-199, Fac. Sci.,Tanta Univ., Egypt.

210 Sameer H. Qari

Baeshin, N.A., Sabir, J.S. 1987. Some genetic studies on As-pergillus terreus Thom. Res. Sci., K.A.U. 215-225.

Basu, A., Sen, T., Pal, S., Mascolo, N., Capasso, F., Chaudhuri,A.K.N., Nagchaudhri, A.K. 1997. Studies on the anti-ulcer activity of the chloroform fraction of Calotropisprocera root extract. Phytotherapy Res. 11: 163-165.

Bradford, M.M. 1976. A rapid and sensitive method for thequantification of microgram quantities of proteinsutilizing the principle of protein-dye binding. Anal.Biochem. 72: 248-254.

Calvin, M. 1979. Petroleum plantations for fuel and materi-als. BioScience 29: 533-538.

Choedon, T., Mathan, G., Arya, S., Kumar, V.L., Kumar, V.K.2006. Anticancer and cytotoxic properties of the latex ofCalotropis procera in a transgenic mouse model ofhepatocellular carcinoma. World J. Gastroenterol. 12:2517-2522.

Dewan, S., Sangraula, H., Kumar, V.L. 2000. Preliminarystudies on the analgesic activity of the latex of Calotropisprocera. J. Ethnopharmacol. 73: 307-311.

Dubey, V.K., Jagannadham, M.V. 2003. Procerain, a stablecysteine protease from the latex of Calotropis procera.Phytochemistry 62: 1057-1071.

El-Sharkawi, H., Malibari, A., El-Shaieb, M., Tawfik, K. 1981.Some edapho-ecological factors controlling the distribu-tion of soil fungi in the western region of Saudi Arabia.Bull. Fac. Sci. K.A.U. Jeddah 5: 103-115.

Fincham, J.R.S., Day, P.R., Radford, E. 1979. Fungal Genet-ics, University of California Press, Berkeley, California,USA.

Haque, M.A., Nakakita, H., Ikenaga, H., Sota, N. 2000. De-velopment-inhibiting activity of some tropical plantsagainst Sitophilus zeamais Motschulsky (Coleoptera:Curculionidae). J. Stored Prod. Res. 36: 281-287.

Jain, S.C., Sharma, R., Jain, R., Sharma, R.A. 1996. Antimi-crobial activity of Calotropis procera. Fitoterapia 67:275-277.

Kumar, S., Dewan, S., Sangraula, H., Kumar, V. L. 2001.Antidiarrhoeal activity of the latex of Calotropis procera.J. Ethnopharmacol. 76: 115-118.

Laemmli, U.K. 1970. Cleavage of structural proteins duringthe assembly of the head of bacteriophage T4. Nature227: 680-685.

Larhsini, M., Bousaid, M., Lazrek, H.B., Jana, M., Amarouch,H. 1997. Evaluation of antifungal and molluscicidal

properties of extracts of Calotropis procera. Fitoterapia68: 371-373.

Migahid, A.M. 1988. Flora of Saudi Arabia, 3rd edition, KingSaud University Press, Riyadh, Saudi Arabia.

Pereira, L.S., Gomes. V.M., Fernandes, K.V.S., Sales, M.P.,Xavier-Filho, J. 1999. Insecticidal and antifungal proteinsof the latex from Manihot glaziovii Muell. Arg. Revta.Bras. Bot. 22: 27-30.

Pontecorvo, G. 1949. Auxanographic techniques in biochemi-cal genetics. J. Gen. Microbiol. 3: 122-126.

Ramos, M.V., Bandeira, G.D.P., Freitas, C.D.T., Nogueira,N.A.P., Alencar, N.M.N., de Sousa, P.A.S., Carvalho,A.F.U. 2006. Latex constituents from Calotropis procera(R. Br.) display toxicity upon egg hatching and larvae ofAedes aegypti (Linn.). Mem. Inst. Oswaldo Cruz. 101:503-510.

Rasik, A.M., Raghubir, R., Gupta, A., Shukla, A., Dubey, M.P.,Sirvastava, S., Jain, H.K., Kulshrestha, D.K. 1999. Heal-ing potential of Calotropis procera on dermal wounds inGuinea pigs. J. Ethnopharmacol. 68: 261-266.

Sabir, J., Baeshin, N.A. 1999. Single and combined mutageniceffects of the insecticide Furadan and cadmium chloridein Aspergillus terreus. In: Proc. Third Int. Symp.NewGenetical Approaches to Crop Improvement, Tando Jam,Pakistan.

Sabir, J.S.M. 2005. Assaying the single and combinedmutagenicity of Calotropis procera Ait. latex andChlorcyrin in Aspergillus terreus. J. Agric. Res. TantaUniversity 31: 820-839.

Scott, D., Galloway, S.M., Marshall, R.R., Ishidate, Jr. M.,Brusick, D., Ashby, J., Myhr, B. C. 1991. Genotoxicityunder extreme culture conditions. A report from ICPEMCTask Group 9. Mutat. Res. 257: 147-205.

Sharma, P., Sharma, J.D. 2000. In-vitro schizonticidal screen-ing of Calotropis procera. Fitoterapia 71: 77-79.

Sharma, P., Sharma, J.D. 1999. Evaluation of in vitro sch-izonticidal activity of plant parts of Calotropis procera -an ethnobotanical approach. J. Ethnopharmacol. 68:83-95.

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211Genetic Effects of Calotropis procera Latex

Evaluation of Antiemetic Activities of Alcoholic Extract ofGrewia asiatica in Experimental Model Dog

Zahra Yaqeen*, Tehmina Sohail, Atiq-ur-Rahman, Muhammad Saleem and Zakir-ur RehmanPCSIR Laboratories Complex, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280, Pakistan

(received April 25, 2007; revised July 4, 2008; accepted July 10, 2008)

IntroductionEmesis or vomiting is a common problem especially infemales during pregnancy (morning sickness); allopathicdrugs used to control the condition are costly and likely topresent adverse side effects. Herbal medicines have beenused as substitutes of allopathic drugs to stop vomiting andto prevent adverse side effects of these drugs. Earlier Prunusdomestica (Brown, 2001), Emblica officinale (Yaqeen et al.,1998) Nelumbium speciosum (Yaqeen et al., 1990) andZingibar officinale (Qureshi et al., 1988) have been reportedfor their antiemetic action. The present study is in continua-tion of this programme, aimed at developing safe herbal drugsas substitutes of allopathic medicines, for stopping vomit-ing especially in cases of pregnancy. Keeping this objectivein mind Grewia asiatica, a popular folklore/medicinal plant,was selected to evaluate the antiemetic activity of its fruitson scientific lines.

Grewia asiatica belongs to the family Tiliaceae. It is a nativeof Pakistan, India and Southeast Asia (Chundawat and Singh,1980; Hays, 1953) but on a commercial scale, it is cultivatedmainly in the northern and the western states of India (Sastri,1956; Hays, 1953). The fruit of G. asiatica is slightly sour intaste and mostly used in preparation of beverages andpickles as well as in several health related problems includinginflammation, respiratory, cardiac and blood disorders and infever (Yadav and Li, 1998; Morton, 1987).The bark of theplant contains a mucilaginous juice and its infusion is used asa demulcent in rheumatism. Leaves and the buds areapplied to pustules and eruptions (Prajapati and Kumar, 2003;Nadkarni and Nadkarni, 1954). According to the nutritional

study of G. asiatica fruit by Morton (1987) in Philippines, itcontains 725 calories/kg of edible fruit, 81.13% moisture,1.58% protein, 1.82% fat, 1.77% crude fibre and 10.27% sugar.

Materials and MethodsCollection of plant material. Grewia asiatica wild fruits(about 5 kg) were purchased from local market and identifiedby taxonomist. Individual fruit measured 1-2.0 cm in diam-eter, 0.8-1.7 cm in height and 0.4-2.2 g in weight.

Extraction. Crude alcoholic extract of the fruits was preparedby the method of Alad and Irobi (1993) with minor modifica-tions. Briefly, fruits of the G. asiatica were washed thoroughlywith water and dried in shade at room temperature. The driedand milled fruits (5 kg) were soaked in 5 lit of 95% ethanol for96 h at room temperature. The mixture was stirred every 24 h,using a sterile glass rod and then filtered and pooled in a con-tainer. The process was repeated thrice for obtaining maximumquantity of extract. Pooled solvent was completely removedunder vacuum at 60 ±1 °C and the crude dark brown, viscousalcoholic extract (300 g) was stored at 4 °C in a well stopperedglass container for further study.

Selection of animals. Healthy albino mice and albino ratsweighing 25-30 g and 150-200 g, respectively, were selectedfor acute oral toxicity test. Healthy dogs (mongrel) of eithersex weighing 16-18 kg were selected for anti-emetic study. Theywere housed in standard animal cages of the animal house ofPakistan Council of Scientific and Industrial Research (PCSIR)Laboratories Complex Karachi, at room temperature (24±2 °C),relative humidity (60% -70%) and exposed to 12:12 h light:dark cycle. The animals were fed standard diet and water. Theywere grouped into test and control groups.

Abstract. The fruits of Grewia asiatica were evaluated for the antiemetic activity in the experimental model dogs,whereas, acute oral toxicity test was carried out in mice and rats. Maximum oral dose of 200 mg/kg and 600 mg/kg ofcrude alcoholic extract was found non toxic in mice and rats. Oral dose of crude alcoholic extract (120 mg/kg bodyweight) caused antiemetic effect in dogs in 3 h and controlled emesis centrally induced by Apomorphine (0.044 mg/kgbody weight). This activity of G. asiatica was comparable with standard commercial anti-emetic drugs like Maxolon(Metoclopramide) and Largactil tablets 10 mg (Chlorpromazine) of M/s. Aventis Pharma., Pakistan.

Keywords: antiemetic activity, Grewia asiatica fruits, alcoholic extract

Pak. J. Sci. Ind. Res. 2008 51(4) 212-215

*Author for correspondence

212

Drugs and chemicals. Standard drugs, Maxolon tablets(Metaclopramide) 10 mg, M/s Glaxo Smith Kline, Pakistan;Largactil tablets, 10 mg, (Chlorpromazine), M/s. AventisPharma., Pakistan and Apomorphine of analytical gradeof Aldrich were used for this study.

Acute oral toxicity test. Acute oral toxicity of alcoholicextract of G.asiatica was determined by oral administeringthe extract to albino mice and rats, weighing between 25-30 gand 150-200 g, respectively (Loomis, 1978; Clarke andClarke,1975). Animals were divided into four groups; threeof them were test groups and received extract while the fourthwas treated as control group which received bread soakedin distilled water. Each group comprised of six animals(3 male and 3 female).

The crude alcoholic extract of G.asiatica at a dose of 50 mg,100 mg and 200 mg/kg body weight was partially dissolvedin distilled water and administered orally after mixing withbread to each test group of animals. However, in case ofrats, test groups received doses of 150 mg, 300 mg and 600mg/kg body weight mixed with bread. (Table 2). The controlgroups of both mice and rats received bread soaked indistilled water only (as negative control). All animals werekept under observation for 15 days for gross physiologicaland behavioural changes and mortality.

Antiemetic studies. Antiemetic activity of alcoholic extractof G. asiatica was studied on dogs. Vomiting was induced byApomorphine (Piala et al., 1959), with slight modification(Qureshi et al., 1988). Dogs were divided into five groups;each group comprised of four animals. Four groups were con-sidered as test groups and one as control group. Each animalin test group was fed, six slices (135 g) of bread mixed withbeef (150 g) and extract, 3 h before subcutaneous injection ofapomorphine.(0.044 mg/kg body weight); the dose ofApomorphine 0.044 mg/kg body weight that induced emesisin each dog was determined experimentally before conduct-ing the study (Qureshi et al., 1990).

For the comparative study of alcoholic extract of G. asiatica,with two synthetic drugs i.e., Largactil and Maxolone,animals were divided into four groups. Group-I for study ofalcoholic extract, group-II of Largactil, group-III of Maxoloneand group-IV of distilled water. All animals were injected Apo-morphine subcutaneously in the dose of 0.044 mg/kg bodyweight, 3 h prior to the comparative study.

Results and DiscussionOral administration of alcoholic extract of G. asiatica to threegroups of albino mice and albino rats, each in doses of 50,100 and 200 mg/kg and 150, 300 and 600 mg/kg body weight,

respectively, did not show any toxic effect in 24 h (Table 1).The animals were kept under observation for 15 days and nophysical and behavioural abnormalities were observed. Thusfor the control of emesis, oral administration of fruit extractof G. asiatica, even in high doses, was safe.

Alcoholic extract of G. asiatica at dose of 20 mg/kg bodyweight did not show any antiemetic effect in the test dogs(Table 2). All animals of this test group showed emesis imme-diately after subcutaneous injection of Apomorphine.(0.044mg/kg body weight) and frequency of vomiting was 4-6 eachdog. In test group II, all the animals also showed vomitingimmediately after intramuscular injection of Apomorphine butthe frequency of vomiting was reduced to 3-4 in each dog.In group III animals, the frequency of vomiting decreased to1-2. Whereas in group IV, at dose of 120 mg/kg, emesis wascompletely controlled and no animal showed any indicationof vomiting after injecting Apomorphine. These resultsreveal that with increasing doses of crude alcoholic extract ofG. asiatica, the frequency of vomiting decreased and com-plete control was achieved at the dose of 120 mg/kg bodyweight .This maximum effective dose is within safe dose rangeof toxicity study in mice (200 mg/kg) and rats (600 mg/kg).Comparative effects of this effective dose (120 mg/kg)ofcrude alcoholic extract with synthetic drugs are presented inTable 3. The results indicate that Maxolon tablet (10 mgmetaclopramide) and Largactil tablet (10 mg chlorpromazine)in dose of 0.142 mg/kg body weight antagonized the emeticstimulus induced by Apomorphine (0.044 mg/kg body weight)while G. asiatica exhibited the same action after adminis-trationof a comparatively larger dose of 120 mg/kg bodyweight in crude form. Furthermore animals receiving Largactilshowed many side effects like drowsiness, lethargy and dry-ness of mouth and the animals receiving Maxolon showedweakness and hunger but the animals receiving alcoholicextract of G. asiatica were calm and quiet, lying comfortablyon ground.

Table 1. Acute oral toxicity test in albino mice and rats

Albino mice Albino ratsGroup no. Mean Alcoholic Mean Alcoholic Observation

body wt. extract body wt. extract(g) mg/kg (g) mg/kg

body wt. body wt.

1 28.5 ± 1.24 50 180.00 ± 4.8 150 normal2 28.3 ± 1.08 100 180.00 ± 5.9 300 normal3 28.2 ± 0.77 200 180.3 ± 5.31 600 normalControl 28.5 ± 1.04 bread 180.1 ± 3.5 bread normalgroup. soaked soaked

with dist. with dist.water. water

213Antiemetic Activities of Grewia asiatica

Apomorphine used in the present study stimulates theChemorveceptor Trigger Zone (CTZ) which causes vomiting(Guyton and Hall, 1996; Goodman and Gillman 1985;Gosh,1981). The extract of G. asiatica antagonized the actionof Apomorphine thus confirming the ability of G. asiatica toinhibit/prevent centrally induced emesis by suppressing thevomiting centres.

Continuous emesis (as in pregnancy, morning sickness andmotion sickness) results in loss of salts and water which causesmetabolic alkalosis and dehydration. As fruits of G. asiaticacontain 17 nutritional constituents (Table 4) (Yadav, 1999) intheir intake can not only make up the losses but also providenutrition to body. During the experiment, it was observed thatafter administration of extracts, dogs became more active andenergetic as compared to the normal animals. From the abovestudy it is concluded that G.asiatica is safe in use for control-ling the nausea and vomiting without producing any untowardside effects and can be used as an alternative medicine.

Table 2. Antiemetic effect of alcoholic extract of G. asiatica in test dogs

Test No. of Average Alcoholic Time interval Dose of No. of animals Frequencygroups animals body wt. (kg) extract b/w extracts Apomorphine showing emesis of vomiting

in each (mg/kg and drumg (mg/kggroup body wt.) administration body wt.)

with bread (in hours)

I 04 17.5 ± 0.47 20 3 0.044 4 4-6 ± 1.4II 04 17.2 ± 0.64 40 3 0.044 4 3-4 ± 0.7III 04 17.7 ± 0.28 80 3 0.044 4 1-2 ± 0.70IV 04 17.5 ± 0.43 120 3 0.044 0 0

Control 04 17.2 ± 0.68 bread soaked with 3 0.044 4 4-6 ± 0.5group distilled water

ReferencesAlade, P.I., Irobi, O.N. 1993. Antimicrobial activities of

crude leaf extracts of Acalypha wilkesiana. J.Ethnopharmacol. 39: 171-174.

Brown, D.J. 2001.Ginger alleviates nausea and vomiting ofpregnancy. HerbalGram 53: 21-22.

Chundawat, B.S., Singh, R.1980. Effect of growth regulatorson phalsa (Grewia asiatica). I: Growth and fruiting.Indian J. Hort. 37: 124-131.

Clarke, E.G.C., Clarke, M.L. 1975. Garner’s VeterinaryToxicology, Ist edition, Bailliere, Tindall and Cassel,London, UK.

Ghosh, R. 1981. Pharmacology, Materica Medica and Thera-peutics, 25th edition, Hilton and Co., Calcutta, India.

Goodman, Gilman 1985. The Pharmacological Basis ofTherapeutics, 7th edition, The Macmillan Co. Inc., NewYork, USA.

Guyton, C.A., Hall, J.E. 1996. Text Book of Medical

Table 3. Comparative assessment of antiemetic activity ofalcoholic extract of G. asiatica with Maxolone and Largactil

No. of Extracts/ Minimum Dose of Observations Emesisgroups drugs dose at which Apomor- present/absent

vomiting stop phine to(mg/kg) induce

vomiting

Group-1 alcohlic 120 0.044 calm and absentextract quite

Group-2 largactil 0.142 0.044 drowsiness, absentlethargy,

Group-3 maxolone 0.142 0.044 weakness,hunger

Control bread - 0.044 vomiting, presentgroup soaked weakness

withdist.water

Table 4. Nutrient values of G. asiatica

Nutrient analyzed Nutrient values/ Nutrient analyzed Nutrient values/in 1998 100 g fruit in 1998 100 g fruit

Calories (Kcal) 90.5 phosphorus (mg) 24.2Caloriesfrom fat (Kcal) 0.0 iron (mg) 1.08Moisture (%) 76.3 potassium (mg) 372Fat (g) < 0.1 sodium (mg) 17.3Protein (g) 1.57 vitamin A (μg) 16.11Carbohydrate (g) 21.1 vitamin B1, 0.02

thiamin (μg)Dietary Fiber (g) 5.53 vitamin B2, 0.264

riboflavin (mg)Ash (g) 1.1 vitamin B3, 0.825

niacin (mg)Calcium (mg) 1.36 vitamin C (mg) 4.385

214 Zahra Yaqeen et al.

Physiology, 9th edition, W.B. Saunders Co., Philadelphia,USA.

Hays, W.B. 1953. Fruit Growing in India, 2nd revisededition, Kitabistan, Allahabad, India.

Loomis,T.A. (ed.) 1978. Essentials of Toxicology, 3rd edition,Lea and Febiger, Philadelphia, USA.

Morton, J.F. 1987. Phalsa. In: Fruits of Warm Climates,pp. 276-277, Julia F. Morton, Miami, Florida, USA.

Nadkarni, A.K., Nadkarni, K.M. 1954. India Materia Medica,vol. 1, pp 10-15 and 844-845, 3rd edition, Popular BookDepot, Bombay, India.

Piala, J.J., Hogh, J.P., Hassert, G.L. Jr., Bruke, J.C., Craver, B.N.1959. Pharmacological and acute toxicological compari-son of triflupromazine and chlorpromazine. J. Pharmacol.Exp. Therap., 127: 55-65.

Prajapati, N.D., Kumar, U. 2003. Agro’s Dictionary ofMedicinal Plants, pp. 223, Agrbios, Jodhpur, India.

Qureshi, I.H., Yaqeenuddin., Yaqeen, Z., Mirza, M. 1990.Determination of critical dose of Apomorphine for inductionof emesis in dogs. Pak. J. Sci. Ind. Res. 33: 62-63.

Qureshi, I.H., Yaqeenuddin., Yaqeen, Z., Mirza, M.,Qureshi, S. 1988. Evaluatuion of the antiemetic actionof Prunus domestica, Linn. Pak. J. Sci. Ind. Res. 31:774-776.

Sastri, B.N. 1956. Grewia Linn. Tiliaceae. In: The Wealth ofIndia: Raw Material, pp. 260-266. Council of Scientificand Industrial Research, New Delhi, India.

Yadav, A.K. 1999. Phalsa: A potential new small fruit forGeorgia. In: Perspectives on the New Crops and NewUses, J. Janick (ed.), pp.348-352, ASHS Press, Alexan-dria, Verginia, USA.

Yadav, A.K., Li, X.Y. 1998. Investigating feasibility of growingphalsa (Grewia asiatica) in temperate climate of middleGeorgia. p. 4, Fort Valley State University, AgriculturalExperiment Station, Fort Valley, Georgia, USA.

Yaqeen, Z., Mirza, M., Yaqeenuddin., Ahmed, K.F., Badar, Y.1998. Pharmacological evaluation of the antiemeticaction of the Nelumbium speciosum-wild seeds. Pak. J.Sci. Ind. Res. 41: 203-207.

215Antiemetic Activities of Grewia asiatica

IntroductionPhenotypic variations in plant populations are expressed bythe joint action of both genetic and environmental factors;the proportion of genetic variation can be estimated from theavailable total phenotypic variations. For proper selection,the information regarding the amount of genetic variability,transferable to the progeny or heritability is important inimproving any quantitative trait. The additive variance andadditive genes, which pass from one generation to the next,play a significant role in selection and improving polygenictraits. A lot of work on genetic variability and heritabilityestimates has already been carried out, yet differences intheir magnitude always exist due to difference in eithermaterial and methodology used or environment in which thematerial is tested (Baloch, 2004; Baloch and Bhutto, 2003;Moser and Percy, 1999; Efe and Gencer, 1998; Khadi et al.,1998; Meredith, 1984).

Customarily, two types of heritability in broad and narrowsense, help plant breeders in phenotypic selection. However,when heritability for a trait is high, phenotypic selection ismore effective (Baloch, 2004). The volume of genetic variationspresent in plant populations may also differ for quantitativetraits. Hence to improve polygenic traits, segregating popula-tions should retain more genetic variability in a smaller lowsample size (Reed and Frankham, 2001).

The main objective of the present study, therefore, was todetermine heritability and genetic variances so as to predictgenetic responses to selection for fibre and earliness para-meters in intra-hirsutum F2 progenies.

Pak. J. Sci. Ind. Res. 2008 51(4) 216-219

Genetic Analysis of Fibre and Earliness Parametersin F2 Progenies of Intra-hirsutum Crosses

W. A. Jatoi, M. J. Baloch*, N. F. Veesar, S. A. Panhwar, N. A. Panhwar and M. S. MajeedanoDepartment of Plant Breeding and Genetics, Sindh Agriculture University, Tandojam, Sindh, Pakistan

(received April 12, 2007; revised July 8, 2008; accepted July 12, 2008)

Abstract. Study of ten F2 progenies of cotton along with their ten parental lines showed that the mean performance ofgenotypes differed significantly for the traits fibre length, seed index, lint index, micronaire value and earliness. Among F2hybrids, progeny CIM-499 x NB-111/S exhibited maximum heritability percentage for lint index and seed index, whereasprogeny VH-142 x CRIS-134 expressed high genetic advances for fibre length and micronaire value. Progeny BH-147 xCIM-511 exhibited fair amount of genetic variance for earliness. The studied breeding material hence may reliably be usedas a potential segregating population to improve fibre and earliness attributes in cotton.

Keywords: genetic analysis, fibre and earliness characters, intra-hirsutum crosses, Gossypium hirsutum


Materials and MethodsTen parents of upland cotton were randomly crossed and tenintra-hirsutum F2 hybrids were developed so as to determinegenetic parameters such as genetic variance (σ2g), heritabilitypercentage in broad sense (h2%) and genetic advance (GA)for fibre and earliness parameters in intra-hirsutum F2 popula-tions. The experiment was conducted in the Botanical Gardenof the Department of Plant Breeding and Genetics at SindhAgriculture University Tandojam, during 2005. The trial waslaid-out in a Randomized Complete Block Design (RCBD) withfour replications. Seeds of ten intra-hirsutum F2 hybrids alongwith their ten parents was sown in the field through handdibbling. Three seeds per hill were dibbled; however, after15 days of planting, number of seedlings were thinned to oneper hill. Distance between plant to plant and row to row waskept at 30 and 75 cm, respectively. The inputs like fertilizer,irrigation and insecticides were applied as and when required.For recording the data, ten plants per genotype were taggedat random from each replication and treated as index plants.The data were collected on fibre length, lint index, seed index,micronaire value and earliness. The analysis of variance wascarried out according to the statistical procedures adoptedby Gomez and Gomez (1984), whereas genetic parameters werecalculated from variance components, according to formulae/procedures developed by Falconer (1989).

Results and DiscussionFor developing new varieties with the desirable traits, geneticvariability in segregating populations and high heritabilityestimates for various characters are the reliable parameters.Present research was designed to estimate heritability

216

percentage and genetic advance at 10% selection intensity inintra-hirsutum F2 populations. The traits studied were fibrelength, lint index, seed index, micronaire value and earliness.The analysis of variance (Table 1) revealed significant diffe-rences among the genotypes for all the characters suggestingthe existance of genetic variations in the mean performance ofF2 progenies and their parents. The mean performance of F2

hybrids and their parental lines (Table 2) indicated thatparent CIM-707 exhibited maximum fibre length (31.81 mm)while the next scoring was CIM-499 (29.68 mm). Among theF2 progenies, BH-147 x CIM-707 and CIM-499 x CIM-511recorded maximum fibre length with mean values of 31.7 and31.11 mm, respectively. Genetic parameters (Table 3) indicatedthat progenies from cross VH-142 x CRIS-134 were among thetop scorers for genetic variance (σ2g=1.12) and genetic advance(1.600), yet ranked second scorer in heritability estimates(73.68%). Though, majority of the progenies expressedmoderate to high heritability estimates, yet CIM-499 x CIM-511exhibited maximum heritability (86.27%) suggesting presenceof more additive variance and additive genes; thus it couldbe promising breeding material for selection of plants fromsegregating populations with longer fibres.

El-Adly et al. (2006), Ulloa (2006) and Baloch (2004), recordedhigh heritability percentage of 81.8% while other researcherslike Shaheen et al. (2001) and Chen et al. (1999) reportedmoderate heritability sense of 38.00% for staple length.

For lint index (Table 2) mean performance demonstrated thatamong the parents CIM-511 and CRIS-470 produced maximumlint index of 4.67 and 4.53 g, respectively. While among the F2

hybrids, progenies VH-142 x CIM-511 (5.1 g), CIM-499 xCIM-511 (5.07 g) and BH-147 x CIM-511 (5.04 g) were the first,second and third top rankers, respectively, in producing lintindex. Genetic parameters (Table 3) indicated that progeniesfrom CIM-499 x NB-111/S with high genetic variance (0.1300)also manifested high heritability percentage (86.25%) that wasassociated with high genetic advance (0.60) for lint index.While some other progenies like FH-925 x NB-111/S (h2=81.48),BH-147 x NB-111/S (h2=76.00) and VH-142 x CRIS-134 (h2=76.00)also recorded high heritability estimates. Genetic parametersthus suggested that progenies from cross CIM-499 x NB-111/S, FH-925 x NB-111/S, BH-147 x NB-111/S and VH-142 xCRIS-134 may be the choice material to be exploited for selec-tion in subsequent selfing generations so as to improve thelint index in cotton. Present results are in consonance withthose of Shaheen et al. (2001) and Jagtap and Mehetre (1998)who also reported that high heritability was associated withhigh genetic gains for lint index.

The mean performance in respect to seed index (Table 2)indicated that parents CRIS-470 (7.75 g) and BH-147 (7.34 g)

gave maximum seed index values. Whereas among the F2

hybrids, crosses VH-142 x CIM-511 (7.73 g) and CIM-499 xCIM-511 (7.61g) exhibited maximum mean performance. Thegenetic parameters (Table 3) revealed that progenies fromcross VH-142 x CRIS-134 though expressed high heritabilitypercentage (h2=64.28%), yet were associated with relativelylow genetic variability (σ2g=0.018) and low genetic advance(GA=0.18). Though CIM-499 x NB-111/S expressed next maxi-mum heritability (h2=60.86) and genetic variance (σ2g=0.140)but was associated with high genetic advance (GA=0.50).Moderate heritability estimates were also observed in otherprogenies like BH-147 x CIM-511 (h2=62.74%) and NB-999 xCRIS-470 (h2=50.00); these progenies thus also expressedfair amount of genetic advance. Results generally suggestedthat progenies CIM-499 x NB-111/S, VH-142 x CRIS-134 andBH-147 x CIM-511 may be prospective breeding material for

Table 1. Mean squares from analysis of variance for fibre andearliness parameters in F2 progenies of intra-hirsutum crosses

Source of Degree Staple Lint Seed Micronaire Earlinessvariation of freedom length index index value

Replication 3 0.008 0.022 0.001 0.002 0.056Genotypes 19 7.793** 0.365** 0.101** 0.092** 54.562**Error 57 0.011 0.063 0.003 0.001 00.223

** = significant at 1% probability level

217

Table 2. Mean performance of parents and F2 progenies forfibre and earliness traits in upland cottonParents and Fibre Lint Seed Micronaire EarlinessF2 progenies length index index value (%)

(mm) (g) (g) (μg/inch)

ParentsCIM-499 29.68 4.38 6.43 4.44 10.00NB-111/S 29.53 4.20 6.45 4.40 8.40CIM-511 27.99 4.67 7.22 4.75 9.56NB-999 27.93 3.33 5.87 4.45 10.08VH-142 27.25 3.97 6.38 4.72 9.55CRIS-134 28.31 4.31 7.24 4.70 8.49BH-147 27.25 4.27 7.34 4.70 8.18CRIS-470 27.34 4.53 7.75 4.66 8.63FH-925 28.37 4.35 6.90 4.36 7.94CIM-707 31.81 4.05 6.70 4.65 6.76

F2 ProgeniesCIM-499 x NB-111/S 29.85 4.49 6.63 4.49 13.43CIM-511 x NB-999 28.27 4.25 7.17 4.80 10.45VH-142 x CRIS-134 28.75 4.05 6.94 4.75 13.79BH-147 x CIM- 707 31.70 4.31 7.09 4.80 18.16BH-147 x CIM-511 28.70 5.04 7.55 4.80 19.44BH-147 x NB-111/S 30.96 4.68 7.53 4.76 15.21NB-999 x CRIS-470 29.65 4.56 7.52 4.81 16.26FH-925 x NB- 111/S 29.68 4.82 7.52 4.58 15.49CIM-499 x CIM-511 31.11 5.07 7.61 4.70 11.40VH-142 x CIM-511 28.49 5.10 7.73 4.90 13.37LSD (5%) 0.149 0.269 0.072 0.026 0.668

Genetic Analysis of Cotton Progenies

selection to improve seed index in cotton. Mahmoud et al.(2004), Basbag and Gencer (2004) and Patel et al. (2005) alsoobserved moderate to high heritability percentage (h2=30 to93%) and genetic advance (GA=10-51) for seed index in cotton.Fibre fineness of cotton has a unique importance in textileindustry for the manufacture of fine quality fabrics. Themean performance (Table 2) indicated that parent CIM-511

(4.75 K μg/inch), followed by VH-142 (4.72 μg/inch), CRIS-134and BH-147(4.70 μg/inch) gave higher micronaire values whileamong the F2 hybrid progenies, VH-142 x CIM-511 (4.9 μg/inch) followed by NB-999 x CRIS-470 (4.8 μg/inch) andBH-147 x CIM-707 (4.81 μg/inch) registered maximum meanvalues. Genetic parameters (Table 3) indicated that progeniesBH-147 x CIM-511 with high heritability percentage (h2=68.42)was associated with fair amount of genetic advance (GA=0.150);it implied that this particular cross retains more additive genesand hence can be promising material for further improvementin micronaire value of cotton. Present results are in line withthose of Yuan et al. (2002), Shaheen et al. (2001) and El-Adlyet al. (2006) who also observed moderate to high heritabilityestimates. Larik et al. (1997) also observed high heritabilitypercentage (h2=89.75-99.74%) for micronaire value. Earlymaturing varieties in cotton are very desirable in that theyrequire less fertilizer, irrigation and labour. They are alsoexposed for shorter period to biotic stresses, especially toinsects as compared to the late maturing cotton. The meanperformance presented in Table 2 indicated that among theparents, NB-999 and CIM-499 opened maximum number ofbolls (10.08% and 10.00%, respectively). The genetic para-meters (Table 3) revealed that progenies from cross BH-147 xCIM-511 which recorded high genetic variance (σ2g=11.24)and maximum heritability estimates (h2=70.03) were associatedwith maximum genetic advance (GA=4.93). Earliness resultsby and large suggested hybrids BH-147 x CIM-511, FH-925 xNB-111/S and NB-999 x CRIS-470 may prove potential bree-ding material for selection of plants for early maturity in cottonin subsequent generations of selfing. Other researchers, Godoyand Palomo (1999), Ji and Zhou (1994) and Baloch (2004) alsorecorded moderate heritability estimates while characterizingearliness in cotton (Gossypium hirsutum L.).

ConclusionFrom the present research, it may be concluded that amongthe parental lines, CIM-707 by producing longer fibres, CIM-511 by giving maximum lint index and micronaire value andNB-999 by showing more earliness in maturity could provebest to be utilized in hybridization programme for improvingrespective characters. Heritability estimates being indicator ofthe degree of transmission of characters from one generationto the next suggested that among the F2 hybrid progenies,cross CIM-499 x NB-111/S, which expressed maximum heri-tability estimates and more genetic advance for seed indexand lint index, may best be exploited in subsequent genera-tions of selfing to improve both the characters. However forfibre length, progeny CIM-499 x CIM-511 and for earlinessand micronaire value, progeny BH-147 x CIM-511 could servepotential segregating populations for these characters.

Table 3. Genetic parameters for fibre and earliness traits in F2progenies of intra-hirsutum crossesF2 progenies Fibre Lint Seed Miconaire Earlinessand genetic length index index value (%)parameters (mm) (g) (g) (μg/inch)

1. CIM499 x NB-111/SGenetic variance (σ2g) 0.17 0.130 0.140 0.0068 1.44Heritability (h2) 38.58 86.25 60.86 52.30 14.32Genetic advance (GA) 0.440 0.60 0.50 0.100 0.80

2. CIM-511 x NB-999Genetic variance (σ2g) 0.06 0.035 0.026 0.0022 1.19Heritability (h2) 36.66 62.50 45.61 44.00 16.78Genetic advance (GA) 0.240 0.25 0.18 0.054 0.78

3. VH-142 x CRIS -134Genetic variance (σ2g) 1.12 0.019 0.018 0.0210 0.44Heritability (h2) 73.68 76.00 46.18 67.74 6.17Genetic advance (GA) 1.600 0.20 0.18 0.200 0.28

4. BH-147 x CIM-707Genetic variance (σ2g) 0.02 0.001 0.011 0.0045 4.43Heritability (h2) 25.00 5.00 34.77 37.50 63.46Genetic advance (GA) 0.097 0.12 0.10 0.066 2.94

5. BH-147 x CIM-511Genetic variance (σ2g) 0.04 0.022 0.032 0.0130 11.24Heritability (h2) 41.57 62.85 62.74 68.42 70.03Genetic advance (GA) 0.210 0.20 0.24 0.150 4.93

6. BH-147 x NB-111/SGenetic variance (σ2g) 0.13 0.038 0.065 0.150 5.91Heritability (h2) 43.33 76.00 54.16 65.21 56.50Genetic advance (GA) 0.410 0.29 0.33 0.170 3.21

7. NB-999 x CRIS-470Genetic variance (σ2g) 0.38 0.001 0.028 0.0063 5.00Heritability (h2) 79.16 5.55 50.0 65.62 59.55Genetic advance (GA) 0.960 0.01 0.20 0.110 3.03

8. FH-925 x NB-111/SGenetic variance (σ2g) 0.03 0.044 0.057 0.0043 8.00Heritability (h2) 17.64 81.48 51.80 48.71 60.46Genetic advance (GA) 0.120 0.33 0.30 0.780 3.85

9. CIM-499 x CIM-511Genetic variance (σ2g) 0.88 0.019 0.004 0.0088 2.66Heritability (h2) 86.27 45.23 42.55 46.31 35.36Genetic advance (GA) 1.530 0.16 0.22 0.090 1.70

10. VH-142 x CIM-511Genetic variance (σ2g) 0.05 0.0007 0.004 0.0110 5.79Heritability (h2) 60.20 7.52 16.66 64.70 54.46Genetic advance (GA) 0.30 0.01 0.043 0.140 3.12

218 M. J. Baloch et al.

ReferencesBaloch, M.J., Bhutto, H.U. 2003. Design-II analysis for

estimating general and specific combining ability effectsof cotton leaf curl virus resistant inbred parents. Zagazig.J. Agric. Res. 30: 635-649.

Baloch, M.J. 2004. Genetic variability and heritability estimatesof some polygenic traits in upland cotton. Pak. J. Sci.Ind. Res. 47: 451-454.

Basbag, S., Gencer, O. 2004. Investigations on the heritabilityof seed cotton yield, yield components and technologicalcharacters in cotton. Pak. J. Biol. Sci. 7: 1390-1393.

Chen, A., Hu, B., Wang, P., Abulaiti, Weijun, Shi. 1999. Ananalysis of the combining ability and heritability ofquantitative characters of upland cotton. China Cottons26: 9-10.

Efe, L., Gencer, O. 1998. Inheritance of important properties inhalf-diallel hybrids of some glandless cotton (Gossypiumhirsutum L.) cultivars. In: New Frontiers of Cotton Re-search, Proc. of World Cotton Research Conference-2,pp. 239-243, Athens, Greece.

El-Adly, H.H., Mohmed, S.A.S., Hemaida, G.M. 2006. Geneticdiversity of some cotton genotypes (Gossypiumbarbadense L.). Egypt J. Agric. Res., Abstracts 84: 1549-1560.

Falconer, D.S. 1989. Introduction to Quantitative Genetics,pp. 187-192, 3rd edition, Longman Scientific and Technical,New York, USA.

Godoy, A.S., Palomo, G.A. 1999. Genetic analysis of earlinessin upland cotton (Gossypium hirsutum L.) morphologicaland phonological variables. Euphytica 105: 155-160.

Gomez, K.A., Gomez, A.A. 1984. Statistical Procedures forAgricultural Research. 2nd edition, John Wiley & SonsInc., New York, USA.

Jagtap, D.R., Mehetre, S.S. 1998. Genetic variability in inter-varietal crosses of upland cotton. (Gossypium hirsutumL.). Ann. Agric. Res. 19: 130-132.

Ji, W.C., Zhou, Y.Y. 1994. Genetic analysis of earliness and

fibre properties in upland cotton. Acta Agric. Universi-tatis Pekinensis 20: 389-396.

Khadi, B.M., Kulkarni, V.N., Narajji, S.S. 1998. Achievingmultiple pest tolerance through manipulation of morpho-logical features in cotton. In: New Frontiers of CottonResearch, Proc. of World Cotton Research Conference-2, pp. 201-203, Athens, Greece.

Larik, A.S., Ansari, S.R., Kumbar, M.B. 1997. Heritabilityanalysis of yield and quality components in Gossypiumhirsutum L. Pak. J. Bot. 29: 97-101.

Mahmoud, A.M., El-Ameen, T.M., Mohamed, A.A, Ali, M.A.2004. Inheritance of some agro-economic traits in interspecific cotton crosses. Asian. J. Agric. Sci. 35: 95-106.

Meredith, W.R.J. 1984. Quantitative Genetics, R. J. Kohel andC. F. Lewis (eds.), pp. 131-150, Cotton. ASA Inc., CSSA.Inc. Pub., Madison, USA.

Moser, H.S., Percy, R.J. 1999. Genetic improvement of yield,yield components and agronomic characters of Pimacotton 1944-1991. In: Proc. of Beltwide Cotton Conf.pp. 488-489, Natl. Cotton Council, Am., Memphis, TN,USA.

Patel, K.V., Varghese, S., Patel, M.L., Patel, U.G., Patel, J.S.,Darji, V.B. 2005. Genetic variability and heritability ofsome characters of cotton seed. Res. on Crops 6: 100-103.

Reed, D.H., Frankham, R. 2001. How closely correlated aremolecular and quantitative measures of genetic variation?A meta-analysis. Evolution 55: 1095-1103.

Shaheen, A.M.A., Gomaa, M.A.M., Esmail, R.M. 2001.Response to selection for yield, yield components andfibre properties in three Egyptian cotton crosses. Ann.Agric. Sci. 45: 491-506.

Ulloa, Mauricio. 2006. Heritability and correlations ofagronomic and fibre traits in an okra leaf upland cottonpopulation. Crop Sci. 46: 1508-1514.

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219Genetic Analysis of Cotton Progenies

IntroductionArsenic has long been classified as a poison. This odourlessand tasteless substance, when ingested at high concentra-tions, may cause death, whereas lower levels of exposure toarsenic can yield detrimental results such as cancer and anumber of other diseases. The most common route of arsenicinto human body is through ingestion of contaminateddrinking water (NRC, 1999).

Arsenic in groundwater results from water-rock interaction,leaching of mine tailings and from arsenic rich fumes infil-trating through the soil (Armienta et al., 1997). Arsenic istypically released to the environment in an inorganic form,and it tends to adsorb strongly to soil which may either run offinto surface water bodies or leach into shallow groundwater.

If exposure of an individual to arsenic is suspected, examina-tion of biomarkers can provide useful information The mostcommonly used biomarkers are hair, nails, blood and urine.The use of these indicators, however, is not without problems,with hair and nails being particularly prone to externalcontamination. In humans, the gastrointestinal tract absorbs60-90% of ingested soluble arsenic. Acute arsenic poisoningresults in vomiting, oesophageal and abdominal pain, bloodyurine, diarrhoea, anuria, shock, convulsions, coma and death(Hughes, 2002). Chronic exposure to arsenic can lead to detri-mental effects involving skin, lung, liver and bladder cancer,diabetes and vascular diseases, as well as pregnancy irregu-larities including stillborns and spontaneous abortions(Armienta et al., 1997; Gorby, 1994).

Humans eliminate most of the ingested arsenic (e.g., 40-80%of ingested arsenite or arsenate) through urine. Generally, 40-60% of the daily intake of inorganic arsenic is excreted eachday in urine (Farmer and Johnson, 1990; Buchet et al., 1981).

Pak. J. Sci. Ind. Res. 2008 51(4) 220-224

Occurrence and Severity of Arsenic in Urine, Hair and NailsThrough Contaminated Drinking Water in Pakistan

Farooq A. Khan, Ameera Javed*, Javed Iqbal, Alia B. Munshi and Ishratullah SiddiquiCenter of Environmental Studies, PCSIR Laboratories Complex, Karachi - 75280, Pakistan

(received October 2, 2007; revised June 26, 2008; accepted July 7, 2008)

Abstract. The study on contamination of well waters of Hyderabad city with arsenic and its effect on urine, hair and nailsof people consuming this water revealed the concentration of arsenic in well waters to be 25.413 to 1286.47 ppb and thatin urine of the people to be 2.032-33.906 ppb, in hair 105.7-427.96 ppb and in nails 8.579-71.033 ppb.

Keywords: Pakistan, groundwater, arsenic contamination, Hyderabad city


Normal arsenic values in urine range from 0.005 to 0.04 ppm(Takagi et al., 1988; Narang et al., 1987). The concentration oftotal arsenic in urine is generally used and accepted as a goodand recent indicator of the absorbed amount of arsenicals(Vahter and Lind, 1986). However, contradictory findings havebeen recorded regarding the correlation of arsenic intake andits concentration in human urine.

Arsenic concentrations are normally higher in hair and nailsthan in other parts of the body because of the high content ofkeratin, which might bind trivalent inorganic arsenic (Hostýneket al., 1993; Curry and Pounds, 1977; Hopps, 1977). The concen-tration of arsenic in the root of the hair is in equilibrium withthe concentration in the blood. Hair might be considered anexcretory pathway; once incorporated in the hair, arsenic isnot biologically available. Arsenic poisoning may have hairconcentrations varying, from 10 ppm to 100 ppm. In peoplewith no known exposure to arsenic, the concentration ofarsenic in hair is generally 0.02-0.2 mg/kg (Kurttio et al., 1998;Rogers et al., 1997; Paulsen et al., 1996; Vienna et al., 1995;Wolfsperger et al., 1994). Hair arsenic levels can provideuseful information in chronic arsenic poisoning. However, theingested arsenic and that derived from external contaminationare both bound to the outer surface of the hair and these twosources cannot be differentiated by any known technique.

Normal arsenic values in nails appear to range from 0.02 to 0.5ppm (Takagi et al., 1988; Narang et al., 1987). A single dose ofarsenic can be detected at the distal tip of the nails about 100days after exposure (Pirl et al., 1983). Presumably, arsenic isdeposited in the nail roots from the blood and then migratesdistally as the nails grow (at about 0.12 mm a day).

Arsenic concentration in hair and nails might increase as aresult of surface contamination. The source of arsenic on theouter surface of hair is claimed to be both ingestion and

220

external contamination, such as via air, water, soaps/shampoosetc. which may produce arsenic concentrations of severalthousand ppm and therefore can mislead investigatorsattempting to diagnose chronic arsenic poisoning (Hindmarshand McCurdy, 1986).

The present study is a cross sectional research on the sus-pected contamination of the ground water (well water) ofHyderabad city of Pakistan with arsenic and arsenic concen-tration in hair, nails and urine of the residents of the city whohad been consuming such water from wells since decades;these people had manifested typical symptoms of acutearsenic poisoning.

Materials and MethodsSamples of nails, hair and urine of the people suffering fromthe typical symptoms of arsenic poisoning as well as the wellwater used by them for drinking purpose were tested intriplicate for arsenic concentration. Samples of water wereanalyzed with standard reference material. Recovery test forarsenic in 3 samples of drinking water and 2 samples of urine,hair and nails was also performed.

Hair and nail samples were washed twice with deionized water(5 ml) and then methanol (5 ml) to reduce any external materialwithout leaching arsenic out of the hair and nails. Sampleswere dried and digested by boiling in a mixture of nitric/per-chloric/sulphuric acids for 3 h until the acid evaporated. Theremaining volume was reconstituted to 25 ml with deionizedwater and an aliquot was analyzed. Samples of urine and waterwere analyzed by wet digestion method with concentratednitric acid. Total arsenic was detected by continuous-flowhydride generation atomic absorption spectrometry (AAS)using Atomic Absorption Spectrophotometer (Hitachi ModelZ-5000), equipped with Zeeman background corrector anddata processor, ZAA and Hydride Formation Unit (AOAC,1990). Working conditions adopted during analysis of arsenicare given in Table 2.

Results and DiscussionArsenic has long been known to be toxic to humans and hasbeen classified as a human carcinogen (IARC, 1987). The grea-test health concern with regard to regular exposure to arsenicderives from consumption of drinking water containingelevated concentrations of arsenic which is a major pathwayinto the human body. The WHO guideline for arsenic in drin-king water is 10 ppb (WHO, 1999) which is exceeded in manyareas of the world. However, the US Environmental ProtectionAgency (2000) has suggested that the concentration ofarsenic in drinking water should be no more than 5 or even

2 ppb. Other existing standards for arsenic in drinking waterare given in Table 1.

Working conditions of the procedure used in the study aredescribed in Table 2, whereas accuracy of the procedure wasdetermined through comparison with standard referencematerial (Table 3). For further rectification of any discrepancy,arsenic was added to the samples and percentage recoverywas determined (Table 4 and 5).

Concentration of arsenic in ten samples of well water used asdrinking water in Hyderabad city revealed that arsenic concen-tration in all the samples were above the permissible standard(Fig. 1); statistics are given in Table 6. Concentration ofarsenic was low in some samples of well water and too high inothers. This might be due to solubility of arsenic in water, timeof digging of well or geology of underground water of wells.Such high values are dangerous for the health of the peopleconsuming such water as well as occupational risk related totheir use in agriculture (Hinwood et al., 2003).

Table 2. Working conditions adopted for determination ofarsenic

Element Wavelength Slit Flame Reductant Acid(nm) (nm) composition channel channel

Arsenic 193.7 0.5 Air-acetylene 1% NaBH4, 1.2N HCl20% KI

Table 3. Accuracy and precision of analytical procedure(standard reference material: trace elements in natural waterSRM-1640)Element Certified conc. Observed conc. Number of RSD

(ppb) (ppb) determinations(Mean value ± SD) (Mean value ± SD) (%)

Arsenic 26.67 ± 0.41 26.384 ± 0.205012 5 0.77

Table 1. Existing standards of arsenic in drinking water

International Standards Maximum permissiblelimit

World Health Organization (WHO) 10 ppb

US Environmental Protection 10 ppb Agency (US EPA)

Bangladesh Standards (BSTI) 50 ppb

Pakistan Standard Quality Control 10 ppb Authority (PSQCA)

Indian Standards (IS) 50 ppb

221Arsenic in Urine, Hair and Nails Through Water

proportion of this could be relatively non-toxic organic forms.Though high arsenic concentration in the urine is indicativeof continued exposure to arsenic, it is not always diagnosticof chronic arsenic toxicity. (Chowdhury et al. 1997). Therehave been contradictory findings in respect of the correlationof the intake of arsenic-contaminated water and correspon-ding concentration of arsenic in urine. Our findings also donot show a good correlation between arsenic in water to thatin urine (Fig. 3).

Conc. of arsenic (ppb)

Matrix

Fig. 2.Arsenic found in patients.

Different biomarkers reflect different timescales of exposure toarsenic; hair and nails reflect past or long-term exposure, whileblood and urine levels reflect recent exposure. Concentrationof arsenic in 10 samples of urine, hair and nails are shown inFig. 2 and statistics is given in Table 6. Measurement of totalarsenic in urine needs to be treated with caution as a large

Fig. 3.Comparision of arsenic in urine, hair and nails withthat in well water.

222Ameera Javed et al.

Fig. 1.Comparative overview of arsenic in well water withexisting standards.

Table 4. Recovery of arsenic added in 2 samples of urine, hairand nails eachMatrixSpikingMean detectedRecovery± SD

level (ppb)conc. (ppb)(%)

Urine5.04.89597.900.0148495.04.91698.32

Hair2.01.94597.250.0311132.01.98999.45

Nail0.50.48797.40.0028280.50.49198.2

Table 5. Recovery of arsenic added in 3 samples of well water

Actual conc.Detected conc.Recovery± SD(ppb)(ppb)(%)

0.50.47494.80.0080.50.48296.40.50.49098.0

Table 6. Statistical analysis of arsenic in urine, hair, nails and well waterMatrix (ppb)No. of samplesRangeMeanMedian±SDRSD%

Urine102.032-33.90616.60618.05050.0080.061Hair10105.7-427.96286.52329.3950.0030.033Nails108.579-71.03332.60530.5700.0030.010Water0825.42-1286.68496.683232.2150.0500.014

0

50

100

150

200

250

300

350

400

450

12345678910

urinehairnail

Conc. of arsenic (ppb)

1 2 3 4 5 6 7 8WHO

US EPA

BSTI

PSQCAIS

Standards Well water samples

0

200

400

600

800

1000

1200

1400

Mean concentation (ppb)

MatrixUrine/Well water Hair/Well water Nails/Well water

0

100

200

300

400

500

600

exposed to arsenic in drinking water in southest Hun-gary. Arch. Toxicol. 66: 77-78.

Buchet, J.P., Lauwerys, R., Roels, H. 1981. Comparison of theurinary excretion of arsenic metabolites after a singledose of sodium arsenite, monomethylarsonate ofdimethylarsinate in man. Int. Arch. Occup. Environ. Health48: 71-79.

Chowdhury, T.R., Mandal, B.K., Samanta, G.K., Basu, G.K.,Chowdhury, P.P., Chanda, C.R., Karan, N.K., Lodh, D.,Dhar, R.K., Das, D., Saha, K.C., Chakraborti, D. 1997.Arsenic in groundwater in six districts of West Bengal,India: the biggest arsenic calamity in the world: the statusreport up to August 1995. In: Arsenic Exposure andHealth Effects, C. O. Abernathy, R. L. Calderon andW. R. Chappell (eds.), pp. 93-111, Chapman and Hall,London, UK.

Curry, A.S., Pounds, C.A. 1977. Arsenic in hair. J. For. Sci.Soc. 17: 37-44.

Das, D., Chatterjee, A., Mandal, B.K., Samanta, G., Chakraborti,D., Chanda, B. 1995. Arsenic in groundwater in six dis-tricts of West Bengal, India: The biggest arsenic calamityin the world. Part 2. Arsenic concentration in drinkingwater, hair, nails, urine, skin-scale and liver tissue (biopsy)of the affected people. Analyst 120: 917-924.

Farmer, J.G. , Johnson, L.R. 1990. Assessment of occupationalexposure to inorganic arsenic based on urinary concen-trations and speciation of arsenic. Br. J. Ind. Med. 47:342-348.

Gonzales, T.A., Vance, M., Helpern, J., Umberger, C. 1954.Legal Medicine Pathology and Toxicology, 2nd edition,Appleton-Century-Crafts, New York, USA.

Gorby, M.S. 1994. Arsenic in human medicine. In: Arsenic inthe Environment, Part-II: Human Health and EcosystemEffects, J. O. Nriagu (ed.), pp. 1-16, John Wiley & SonsInc., New York, USA.

Grantham, D.A., Jones, J.F. 1977. Arsenic contamination ofwater wells in Nova Scotia. J. Am. Water Works Assoc. 69:653-657.

Harrington, J.M., Middaugh, J.P., Morse, D.L., Housworth, J.1978. A survey of a population exposed to high concen-trations of arsenic in well water in Fairbanks, Alaska. Am.J. Epidemiol. 108: 377-385.

Hindmarsh, J.T., McCurdy, R.F. 1986. Clinical and environ-mental aspects of arsenic toxicity. Crit. Rev. Clin. Lab.Sci. 23: 315-347.

Hinwood, A.L., Sim, M.R., Jolley, D., deKlerk, N., Bastone,E.B., Gerostamoulos, J., Drummer,O.H. 2003. Hair and toe-nail arsenic concentrations of residents living in areaswith high environmental arsenic concentrations-Environ-mental Medicine. Environ. Health Perspect. 111: 187-194.

Hair and nails suffer from contamination problems and it isdifficult to ascribe what is likely to be due to external exposure.Some workers have suggested that human hair and nails cangive estimates of arsenic body load (Chowdhury et al., 1997).In some other cases, there was no accordance between hairarsenic content and total life intake. The most probableexplanation of this finding is that of external contaminationdue to the use of contaminated water for washing purposes.However, most of the studies favour a correspondencebetween the amount of arsenic in drinking water and that inhair. (Das et al., 1995; Borzsonyi et al., 1992; Hindmarsh andMcCurdy, 1986; Valentine et al., 1979; Harrington et al., 1978;Grantham and Jones, 1977). According to the results of abovestudies, arsenic concentration of hair can be used as a goodmeasure of past exposure to arsenic via drinking water. In ourstudy, most of the patients had arsenic concentrations in hairfrom 50 to 100 ppb, a few persons showed concentrationsover 150 ppb and some had near 500 ppb.Thus a good rela-tionship between arsenic concentrations in drinking waterand in hair of patients was observed (Fig. 3). Patients who takedrinking water with average arsenic concentration of 496.683ppb had average arsenic concentration of 286.52 ppb in hair.Our findings showed that hair arsenic concentration can beused as a good proxy for chronic arsenic ingestion via drink-ing water.

Data on arsenic in nails are sparse. There appears to be nofixed maximum limit for the amount of arsenic that can beabsorbed in body organs so it is not necessary that arsenicaccumulates equally in hair and nails. (Agahain et al., 1990;Gonzales et al., 1954). Large amounts of arsenic have beenreported in cases of chronic arsenic poisoning (Das et al.,1995). The average concentration of arsenic for our referencegroup was 8.579-71.033 ppb (Table 4); in our findings, nosignificant correlation between arsenic in nails and waterwas seen.

ReferencesAgahain, B., Lee, J.S., Nelson, J.H., Johns, R.E. 1990. Arsenic

levels in fingernails as a biological indicator of exposureto arsenic. Am. Ind. Hyg. Assoc. 51: 646-651.

AOAC. 1990. Official Method of Analysis, 15th edition, Asso-ciation of Official Analytical Chemists, Washington DC.,USA.

Armienta, M.A., Rodriguez, R., Cruz, O. 1997. Arsenic contentin hair of people exposed to natural arsenic pollutedground-water at Zimapan, Mexico. Bull. Environ. Contam.Toxicol. 59: 583-589.

Borzsonyi, M., Berecsky, A., Rudnai, P., Csanady, M., Horvath,A. 1992. Epidemiological studies on human subjects

223Arsenic in Urine, Hair and Nails Through Water

Hopps, H.C. 1977. The biological bases for using hair and nailfor analyses of trace elements. Sci. Total Environ. 7: 71-89.

Hostynek, J.J., Hinz, R.S., Lorence, C.R., Price, M., Guy, R.H.1993. Metals and the skin. Crit. Rev. Toxicol. 23: 171-235.

Hughes, M.F. 2002. Arsenic toxicity and potential mechanismsof action. Toxicol. Lett. 331: 1-16.

IARC 1987. Overall evaluations of carcinogenicity. An upda-ting of IARC monographs 1-42. In: IARC Monographson the Evaluation of Carcinogenic Risks to Humans,Suppl. 7, International Agency for Research on Cancer,Lyon, France.

Kurttio, P., Komulainen, H., Hakala, E., Kahelin, H., Pekkanen,J. 1998. Urinary excretion of arsenic species after expo-sure to arsenic present in drinking water. Arch. Environ.Contam. Toxicol. 34: 297-305.

Narang, A.P.S., Chawla, L.S., Khurana, S.B. 1987. Levels ofarsenic in Indian opium eaters. Drug Alcohol Depend.20: 149-153.

National Research Council (NRC) 1999. Arsenic in DrinkingWater, Sub-committee on Arsenic in Drinking Water,Committee on Toxicology Board on EnvironmentalStudies and Toxicology Commission on Life Sciences,National Academy Press, Washington DC., USA.

Paulsen, F., Mai, S., Zellmer, U., Alsen-Hinrichs, C. 1996. Bloodand hair arsenic, lead and cadmium analysis of adults andcorrelation analysis with special referene to eating habitsand other behavioral influences. Gesundheitswesen 58:459-464.

Pirl, J.N., Townsend, G.F., Valaitis, A.K., Grohlich, D., Spikes,J.J. 1983. Death by arsenic: A comparative evaluation of

exhumed body tissues in the presence of external con-tamination. J. Anal. Toxicol. 7: 216-219.

Rogers, C.E., Tomita, A.V., Trowbridge, P.R., Gone, J.K., Chen,J., Zeeb, P., Hemond, H.F., Thilly, W.G., Olmez, I., Durant,J.L. 1997. Hair analysis does not support hypothesizedarsenic and chromium exposure from drinking water inWoburn, Massachusetts. Environ. Health Perspect. 105:1090-1097.

Takagi, Y., Matsuda, S., Imai, S., Ohmori, Y., Masuda, T., Vinson,J.A., Mehra, M.C., Puri, B.K., Kaniewski, A. 1988. Surveyof trace elements in human nails: An international com-parison. Bull. Environ. Contam. Toxicol. 41: 690-695.

US Environmental Protection Agency (2000). Drinking waterpriority rulemaking - Arsenic; accessed on 4/5/00 at URLhttp://www.epa.gov/OGWDW/ars/arsenic.html

Vahter, M., Lind, B. 1986. Concentrations of arsenic in urine ofthe general population in Sweden. Sci. Total Environ. 54:1-12.

Valentine, J.L., Kang, H.K., Spivey, G. 1979. Arsenic levels inhuman blood, urine and hair in response to exposure viadrinking water. Environ. Res. 20: 24-32.

Vienna, A., Capucci, E., Wolfsperger, M., Hauser, G. 1995. Heavymetals in hair of students in Rome. Anthrop. Anz. 53: 27-32.

Wolfsperger, M., Hauser, G., Gossler, W., Schlagenhaufen, C.1994. Heavy metals in human hair samples from Austriaand Italy: Influence of sex and smoking habits. Sci. TotalEnviron. 156: 235-242.

World Health Organization. 1999. Arsenic in drinking water:Fact Sheet 210, accessed on 4/5/00 at URL http://www.who.int/inf-fs/en/fact210.ht

224 Ameera Javed et al.

IntroductionBoth food and drugs (medicine) are essential for a good,healthy and productive life. Food consists of nutrients(macro: carbohydrates, fats, proteins, minerals such ascalcium, potassium, sodium; micro: vitamins, trace mineralssuch as (copper, iodine, iron, selenium, zinc...) that are thesource of energy and other materials necessary for sustenance,whereas drugs prevent and cure illnesses. Food and drugsare taken mainly orally (by mouth) and follow processesbefore imparting desired benefits. On consumption of food,nutrients are digested and absorbed from the intestine.

When a drug is taken orally, it is absorbed from the intestine,distributed as is or after metabolism and finally excreted(pharmacokinetic step). The effect of a drug to the body isreferred to as pharmacodynamic. Another often used term indrug efficacy is its therapeutic index. A drug with highertherapeutic index provides a greater degree of safety com-pared to the one with low value i.e., toxic dose and effectivedose are very close. In order to be effective, a drug must bebioavailable at the site i.e., it is absorbed from the intestine(before and/or after enzymatic reactions mainly by P450)and transported by P-glycoprotein (P-GP). Both P450 andP-glycoprotein are in high concentration in the human liverand small intestine.

Since both food and drug are integral part of human healthand a productive lifestyle, they should be compatible witheach other for wellness as well. Unfortunately, this is not

Pak. J. Sci. Ind. Res. 2008 51(4) 225-234

Food and Drug Interaction−−−−−a Growing ConcernM. Humayoun Akhtar

Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, Ontario, N1G 5C9, Canada

(received March 12, 2008; revised August 22, 2008; accepted August 24, 2008)

Abstract. Food and drug are taken for the survival and well-being of human race. Food-drug interaction occurs when foodincluding beverages taken orally affects the properties of intestinal enzymes (P450), which alters pharmacokinetics,bioavailability and pharmacodynamics of the drug. The bioavailability of a drug determines its therapeutic value. Anincrease in the amount of a drug would produce undesired toxic effects; whereas a reduced amount would result in seriouscomplications that could be termed as therapeutic failure. Such health hazards are more pronounced in elderly persons andthose who are taking potent drugs to control/treat chronic diseases. Also, the severity of adverse effect depends on thetherapeutic index of a drug, more pronounced for a drug with low therapeutic index. The food (nutrient)-drug interaction hasbeen recognized as a major health issue in western countries. Popular health magazines, newspapers etc. often summarizeresearches published in scientific journals, proceedings of conferences in simple to understand language to educate and warnreaders on the consequences of food (nutrient)- drug interaction etc. Several national health agencies are also involved inconducting surveys and devising policies to minimize food-drug interactions.

Keywords: food-drug interaction, foods, drugs

E-mail: [email protected]

always the case. The individual’s age, weight, body-mass index(BMI) and general health as well as the food composition andtime of intake may have significant impact on the absorption,distribution and bioavailability of pharmaceuticals (drugs).Such impact is often referred to as food-drug interaction.After World War II, and since the 1980s, there have beenconsiderable changes in travel, migration, eating, cookinghabits and tastes. This is more common among the moreaffluent middle to older generations because of financialindependence. With advancing age, the adult population isalso prone to several health related issues including chronicdiseases. This group also uses more herbal supplements inaddition to prescription drugs, often without the knowledgeof medical practitioners, mainly to manage aches and pains.Thus, this group is the most affected by food-drug interac-tions and has raised serious health concerns.

A recent survey done in Canada reported that 47% of thosewho use prescription drugs and natural health productssimultaneously encountered adverse effects, which couldrange from mild to severe rashes to headaches and seriouseffects for those using prescription drugs such as bloodthinner, insulin. These authors concluded that the use ofnatural drugs with prescription drugs should be treated withrespect (Charrois et al., 2007). Natural products commonlyreferred to are vitamins, herbal supplements (garlic, carrot andothers), trace minerals etc. The aim of this article is to reviewcurrent popular health magazines and scientific literature onthe interaction of food and drug to enlighten public, healthcarepractitioners, scientific communities and those interested in

Review

225

self-health management programme on the hazards of somefoods including supplements taken routinely, most impor-tantly with life saving drugs.

What are the issues? Modern day consumers worldwide wantfood that not only provides basic energy, but contributes toenjoyment, productivity, fun and a lifestyle that is free fromdiseases (infirmity) i.e., food should contribute to health andwellness and support:

“Let food be your medicine and medicine be your food”(Hippocrates)

Health and wellness (in some circles it is also referred to asfitness and wellness) is routinely referred in the developedcountries to describe a productive, healthy and happy life-style. We all understand health means fit, but wellness hasevolved and continues to evolve to describe a lifestyle withgood physique (good and attractive body), stable mentalcapacity, longevity, free from disability (infirmity). Wellnessis a holistic approach for taking control and making a commit-ment where body, soul and mind are in harmony for an enjoy-able lifestyle. This requires managing proper food intake aspart of a regular diet that not only provides energy but alsocontributes to improving other physiological benefits. Hencefood in this overview refers to substances that provide notonly the basics for sustained living, but also beverages andother natural products with healthy benefits, commonlyreferred to as natural health products (NHP). The Ayurvedic,Chinese Traditional medicine, Unani-Tibbi (also referred to asIslamic medicine that has been practiced for over 800 yearsand promotes nutrition management for a healthy lifestyle)and native medicines in the Americas, Australia and othercountries are still the first line of health care. Even in the 21st

century, about 70% of the world population rely on alterna-tives to conventional medicine (western medicine). In thedeveloped countries, they are called by a variety of namessuch as folk medicine, complementary and alternativemedicine/therapy (CAM/T).

How do we achieve health and wellness? Health and wellnessare a pre-occupation with modern day consumers, especiallyin the developed countries. Recent studies have clearlyestablished a link between health and diet. Consumers wantto take control and manage their own health. They are influ-enced by constant bombardment through the media on thebenefits of green tea, garlic, tomato and spices e.g., turmeric(Daniells, 2006). This has resulted in marked increase in theuse of herbal products as supplements and complementaryalternative medicine (CAM), mainly as the booster ofimmune system (health benefits) (Ness et al., 1999). There

are serious demands for new products with physiologicalbenefits along with better information. Literature and anec-dotal reports for concomitant oral intake of natural productsand therapeutic drugs affect drug metabolism and havesignificantly increased risks with serious clinical adverseevents (safety issue). However, the effect of concomitantadministration of the 42,000 or so herbal products on thesafety and efficacy of conventional drugs has not beenunequivocally established.

Examples of food-drug interaction. Many patients take a widerange of NHP in addition to their western medicine (Eisenberget al., 1998). The effect of concomitant administration of herband/or herbal medicine on the safety and efficacy of conven-tional drugs has not been fully determined (Anastasio et al.,1997; Kozryskyj, 1997; De Smet, 1995). Literature and anec-dotal report suggests that concomitant oral adminis-tration ofnatural products and therapeutic drugs may affect human drugmetabolism and significantly increase the risk of seriousclinical adverse events (Foster et al., 2002, 1999; Ameer andWeintraub, 1997). In recent years several excellent reviewsand analysis reports have appeared on the interaction ofnatural health products with therapeutic drugs includingantiretroviral agents (Lee 2006; Foster et al., 2005; Haddadet al., 2005; Guo and Yamazoe, 2004; Bailey and Dresser, 2004;Raucy, 2003). One of the most popular over-the-counterherbal products to treat depression and other mental disor-ders is St. John’s Wort. It has been implicated in several druginteractions (Boullata, 2005; Markowitz et al., 2003; Izzo andErnst, 2001). It is recommended that St. John’s Wort shouldnot be taken concurrently with most of the drugs, especiallydigoxin (digitalis), immunosuppressive medications, proteaseinhibitors (drugs for HIV/AID treatment) loperamide, oral con-traceptives, reserprine (used in high blood treatment) andWarfarin (anticoagulant) (National Centre for Complementaryand Alternative Medicines (NCCAM): http://nccam.nih. gov/health/stjohnswort/#cautions). Similarly, garlic is used in cook-ing as herb, and as supplement to control cholesterol level,high blood pressure, to reduce blood sugar levels and as anti-fungal agent among others. On the other hand, there is con-cern that use of garlic would be detrimental when taking bloodclotting drugs such as Warfarin, Plavix and Aspirin andantiretroviral drugs (Mills et al., 2005). An interaction betweengarlic and chlorpropamide caused hypoglycemia in a diabeticPakistani woman, who consumed a curry dish that consistedof karela (Momordica charantia, also known as bitter gourd)and garlic (Allium sativum) (Aslam and Stockley, 1979). Fosteret al. (1998) reported that garlic extracts interfered withCYP3A4-mediated metabolism, which may have been respon-sible for observed interaction of garlic in diets.

226 M. Humayoun Akhtar

During the In last 20 years or so, numerous articles haveappeared in medical journals as well in the popular magazinesincluding lay press that describe incidences of adverse drugreaction. Interactions between foods and drugs can haveprofound influence on the success and failure (side effects)of many drugs (Medical University of South Carolina HealthTopic Library, 2008; McLachlan and Ramzan , 2006; Ayo et al.,2005; Bland, 1998). A careful review of literature showed thatincidences are more frequent among senior citizens becauseof multiple medication usage and several age-related physio-logical changes that affect drug absorption, distribution,metabolism and excretion. Frequent and serious incidencesof food drug interactions among seniors often lead tohospitalization, significant morbidity and even death. Recentstudies predict the population of seniors (over 60 years ofage) to reach about 22% of the total globle populationincluding the North America by 2050. But two Asian coun-tries, South Korea (37.3%) and Japan (36.5%), are expected tolead the pack, followed closely by Italy (34.4%) and otherEuropean countries (>27%) (United Nations, 2007; SCIN,2005). Increase in health care costs in most of the majordeveloped countries is already out-pacing the revenuegrowth, and with the projected increase in number of seniors,the burden on treasury would increase substantially. Food/supplements (herbs)-drug interactions are becomingimportant and major public health issues due to their impacton fitness, productive and enjoyable lifestyle and of courseon health care cost (Cheng, 2006; Health Canada, 2006;Boullata and Armaenti, 2004; Leibovitch et al., 2004; McCabeet al., 2003; University of Florida, 2003; Thomas and Burns,1998).

How does food-drug interaction occur (mechanism of action)?The “food-drug interaction” initially suggests a chemicalinteraction between food and a drug that produces toxicsubstance(s). It is far from the truth. It refers to the effect ofnatural products including some foods that enhances orinhibits the activities of major metabolizing enzymes locatedin the human intestine. The efficiencies of these enzymes canalter pharmacokinetics and of course the pharmacodynamicsof a drug by either increasing or decreasing the amount of adrug in the blood stream.

In order for a drug to be effective, it must be absorbed (mostoften unchanged), distributed and localized to the site,metabolized and excreted from the body. Any deviation in theprocess has the potential for side effects, the degree of whichis highly dependent on the potency of the drug (therapeuticindex) i.e., a drug with lower therapeutic index has greaterpossibility of toxic effects.

A drug is administered orally, subcutaneously or intravenously,but it is the orally administrated drug that interacts with foodsincluding beverages that often cause side effects. Orally adrug can be taken with water (highly recommended), with foodor in empty stomach. In all cases the purpose is to deliver thedrug at the site for maximum action. An orally taken drug mustfollow the following steps: absorption, distribution, metabo-lism and excretion in timely fashion for both benefits andavoidance of toxic side effects. In simple terms a drug must bebioavailable in right quantity at the right cell in timely fashion.The absorption and the bioavailability of a drug could easilybe altered by the type, quantity and composition of food andbeverages, which is highly dependent on several factors suchas (i) dosages of the drug (medication); (ii) patient’s age, size(BMI), state of health; (iii) the relationship between drug andfood intake. A drug is of clinical significance only if it isbioavailable and reaches the target, most often in theunchanged state.

The effectiveness of a drug is measured by its desired thera-peutic outcome. Natural products are known to interfere withdrugs used for some chronic diseases such as neuroendro-chrine (e.g., sedative, MAO inhibitors); cardiovascular e.g.,hypotensive and hypertensive; hematological (high vitaminK containing products); metabolic (hyper/hypoglycemic);immune (immune modifier, salicylate containing compounds)(Dharmananda, 2003). Hence the choice of carrier (mode ofadministration) for drug is very important and advice shouldbe based on the nature and chemistry of drug under consi-deration. Food can either retard or accelerate absorption of adrug depending on its chemistry and its effect on CYP 450.For example, Coumadin, Warfarin (anticoagulant) when takenconcomitantly with foods with high vitamin K content (foundmainly in kale, spinach, turnip greens, collards, Swiss chard,parsley, mustard greens, the cabbage or cruciferous typevegetables such as broccoli, cauliflowers and lettuce) can beless effective due to low bioavailability. It is a cause ofconcern for therapeutic failure and in certain instances mayresult in death. Similarly, high-protein, low carbohydratediets can accelerate hepatic metabolism of a number ofmajor drugs resulting in therapeutic failure with seriousconsequences (Leibovitch et al., 2004; Hansten and Horn,2003; Singh, 1999; Winstanely and Orme, 1989).

Food/beverages. The extent of absorption of a drug is highlydependent on its chemistry, timing and composition of thefoods/beverages. A physician when prescribing a drug and itsmode of administration takes into consideration the healthand age of the patient and the timing and type of food intake.The food factors that influence absorption as well as thebioavailability include (i) chelation, (ii) poor acid stability,

227Food and Drug Interaction

(iii) acid dependency; (iv) bile acid or fat enhanced drugsdissolution, (v) physical binding/adsorption (vi) reducedgastric emptying (McLachlan and Ramzan, 2006):

Aging causes following significant changes: (Thomas andBurns, 1998):

a) Absorption: Gastrointestinal (GI) system (i.e., increasedgastric pH; decreased GI tract blood flow; increasedgastric emptying system);

b) Distribution: Body composition (BMI factor (i.e., decreasedlean body mass; increased fatty tissue; decreased totalbody water, decreased plasma protein i.e., albumin);

c) Metabolism: Hepatic system influences metabolism(decreased liver size, decreased microsomal enzymeactivities, decreased hepatic blood flow; increasedincidences of drug-induced hepatitis);

d) Excretion: Renal changes (i.e., decreased creatinine level;decreased renal size; decreased renal blood flow; reducedkidney function e.g., decreased glomerular filtration rate(GFR) and number of functioning glomerulli).

Effect of foods/beverages on human Cytochrome P450(CYP450). Aging causes change in hepatic systems, whereasxenobiotics including foods/beverages affect the enzymaticactivities: neutral, enhance or retard. For food and beverages,it is the changes in the intestinal enzymatic activities thatinfluence the absorption/ bioavailability of a drug. A thera-peutic failure would result if the drug is degraded by enhancedmetabolizing activity of the enzymes, where a toxic effect isexpected when retardation of enzymatic activities allows moredrugs in the system. In both cases there are health issues.Food and drugs when consumed affect mainly the P450enzymes located in the human intestine. Human P450 consistsof two major classes of enzymes, but only isozymes CYP1,CYP2, CYP3 and CYP4 are involved in the metabolism ofpharmaceuticals (medicine).CYP3A is responsible for hepaticmetabolism (breakdown) of approximately 60% of currentlyavailable human medicines, whereas CYP2, CYP1 are estimatedfor 20 and 15%, respectively (Chen and Raymond, 2006;Christians, 2004; Dahan and Altman, 2004; Kivisto et al., 2004),but specifically CYP3A4 and P-glycoprotein (PGP) in theintestine. PGP transports many drugs from the intestine intothe blood (Sharom et al., 2005).

Beverages. Juices are highly recommended as functionalfood because of their physiologically active components e.g.vitamins, fibre, bioavailability of certain drugs etc. The mostoften recommended is apple, grapefruit or orange juice. Baileyet al. (1991) reported a pharmacokinetic interaction of grape-fruit juice with the calcium-channel blockers felodipine andnifedipine. These researchers gave 5 mg of felodipine to six

men with borderline hypertension with water, grapefruitjuice or orange juice. The mean bioavailability of the drugwas 284% (range 164-469%) with grapefruit compared to thatwhen taken with water. Similarly the availability of 10 mg ofnifedipine to male adult was 134% (range 108-169%) of thatof water when taken with orange juice. Further, it generatedconsiderable interest in this area globally. This group latershowed that grapefruit juice mediated inhibition of CYP3A4in the intestine was responsible for increased bioavailabilityof felodipine- an increased felodipine concentration in plasma(Bailey et al., 1988). These are landmark studies on the food/beverage interaction with therapeutic drugs, which wereresponsible for considerable research in food and drugresearch globally. A group of researchers (Paine et al., 2006)fifteen years later reported that felodipine plasma concen-tration did not increase when adults were given furanocou-marin-free grapefruit juice (but contained other flavonoids).They also observed that the maximum concentration offelodipine in plasma was similar to that of orange juice, butconsiderably lower than regular grapefruit juice that contai-ned furanocoumarins, most abundant are 6',7'-dihydroxybergamottin and bergamottins, derived from psoralenfollowing metabolism (structures in Fig. 1). Thus, theseauthors concluded that furanocoumarins are the active ingre-dients for observed increased bioavailability of felodipine.It should be mentioned here that there has been no reportedincidence of interaction of apple juice with a drug. However,most recently, Bailey (2008) reviewed his pioneering workwith grapefruit juice at the American Chemical SocietyMeeting. He also reported that patients who took an antihis-tamine with grapefruit juice absorbed only half the drug com-pound compared to those who took the pill with water. He alsosuggested inhibitory effects of other juices including apple,orange, and the whole fruits.

Collectively, the vast numbers of published studies withgrapefruit juice have resulted in the most drug regulatoryauthorities advising consumers and patients to avoid orgreatly limit their intake of grapefruit products. A recent articlein Ladies Home Journal, compiled by Wagle (2008) reproducedfrom Karch (2004) a complete list of drugs that includedseveral anthelmintic, antileptic, calcium-channel blockers,hormone replacements, which interact with grapefruit juice.The article advocated “to avoid grapefruit juice within a fewhours of taking medication, or perhaps even at all”. Recently,an interesting article titled “The grapefruit challenge: the juiceinhibits a crucial enzyme, with possibility fatal consequences”was posted at the Mayo clinic site (Jan 23, 2007).

Such advisories will have serious negative impact on grape-fruit growers and processing industries. Psoralen derivatives


are also present in many foods including, but not limited to,peas, beans, figs, jackfruit, carrot, celery, dill, fennel, parsnip,Baizhi (Angelica dahurica root), Hamaudo (Angelicajaponica), Qianghuo (Notopterygium incisum), and Fang-feng (Saposhnikovia divaricata) (Guo and Yamazoe, 2004).Many but not all of the psoralen derivatives can causemechanism-based inactivation; hence foods with lower levelsof the active derivatives would be useful and provide a marketadvantage. A recent article reviews the literature on mecha-nism, extent and relevance of drug interactions with grape-fruit juice and concludes that the predominant mechanismfor this interaction is the inhibition of cytochrome P450 3A4in the small intestine resulting in a significant reduction ofdrug presystemic metabolism (Dahan and Altman, 2004).

Reported incidences of food, including beverages, and druginteractions have directed researchers to look into thebehaviour of other commonly used beverages that arepromoted for health benefits. The unexpected observationof grapefruit juice resulted in investigation of other juices.A study by Malhotra et al. (2001) also identified Sevilleorange juice to interact with felodipine by a commonmechanism-based inactivation of intestinal CYP3A4.

Pomegranate juice, derived from a fruit that has been prescribedas medicine (complementary medicine) in Eastern and Medi-terranean cultures from pre-historic times, is a new addition asbeverage in the west, and is promoted in lay magazines andnewspapers for its health benefits (Bowden, 2008; Underwood,2005). Most of the health benefits in pomegranate have beenassociated to its rich antioxidant polyphenol compoundsthat may have more activity than highly touted red wineand green teas (Malik et al., 2005). However, some studies(Summer, 2006; Hidaka et al., 2005) have also reportedinhibition of CYP450 by pomegranate juice similar to that ofgrapefruit juice in rats.

Cranberry juice is a popular addition to beverages for itsantioxidant properties. However, concerns have been raisedfor its interaction with Warfarin, a most often prescribeddrug for anticoagulation therapy (Suvarna et al., 2003).But a review of published research could not support thishypothesis because the two clinical studies with patientstaking Warfarin and cranberry juice did not conclusivelyshow cranberry as the sole source for observed increase inthe international normalized ratio (Pham and Pham, 2007).

Star fruit (Averrhoa carambola) another delicious fruit found,grown and eaten in many tropical countries – has now beenintroduced in the west. It has also shown to inhibit humancytochrome P450 3A (Hidaka et al., 2004).

To date there has been no report of verifiable actual incidenceof interaction of any other juice and star fruit with any lifesaving drug. There is no information in the published litera-ture on the nature of bioactives substances in pomegranateand star fruit.

As a general rule, drugs such as Lipitor, Toprol XL, VytorinLexapro, Synthroid, Prevacid, Norvasc taken to control highcholesterol level, high blood pressure, depression should notbe taken with food and/drug to avoid and/or minimize thera-peutic failure or side effects. Most often it is better to takethese drugs with water.

Herbs in food. A large population of senior citizens often alsouse herbal supplements in combination with prescription medi-cations; most often they do not inform their physicians. A fewof these herbs and supplements are also used regularly indiets as flavouring agents or as vegetables. Not unexpectedly,there are many reported incidences of herb-drug interactionswhich require separate detailed studies. Garlic and carrot havebeen used both as food and herbal supplements for theirantioxidative properties. Garlic is used for flavor in many Asianand Mediterranean diets, especially after frying in oil untilgolden brown for aroma. It is also used as supplement tocontrol cholesterol level, high blood pressure and as disinfec-


Fig. 1. Chemical structures of major furanocoumarins(Psoralen based) in foods/beverages.

Psoralen

Bergapten Bergaptol

Bergamottin

6',7'-Dihydroxybergamottin

Fig. 1. Chemical structures of major furanocoumarins(Psoralen based) in foods/beverages.

Psoralen

Bergapten Bergaptol

Bergamottin

6',7'-Dihydroxybergamottin

tant among others. However, there is also some concern thatthe use of garlic would be detrimental when taken in conjunc-tion with blood clotting drugs such as Warfarin, Plavix andaspirin; or antiretroviral drugs (to treat HIV/AIDS patients).A survey revealed that the use of herbal drinks/supplementsis considerably higher in persons with AIDS/HIV who alsotake antiretroviral and other prescribed medicines (Fairfieldet al., 1998). It has been reported that HIV-infected patientshave carotene deficiency most likely caused by general mal-absorption, fat malabsorption or altered metabolism_ a causeof concern for increased oxidative stress (Keating et al., 1995;Pace and Leaf, 1995). For this reason HIV/AID patients oftentake beta-carotene supplements in combination with pres-cription drugs such as nelfinavir (NLF, a protease inhibitorrecommended for use by HIV/AID patients). Several studieswere undertaken to assess the interaction of beta-carotenewith CYP 3A4 and P-glycoprotein in vitro (Chauhan et al.,2005; Foster et al., 2002). This was extended to an in vitro-study on the effect of beta carotene on the pharmacokineticsof NLF or its major metabolite, commonly referred to as M8,which indicated no potential for significant interaction(van Heeswijk et al., 2004). In order to assess the benefit ofbeta-carotene supplements on the absorption of NLF, theteam conducted clinical trials on 24 persons with AIDS. Datasuggested that beta-carotene given twice daily at a dose of25000 IU with regular dose of NLF for more than 2-week didnot significantly alter the steady-state pharmacokinetic ofNLF and M8, though it delayed NLP absorption, and is safe(Sheehan et al., 2005; 2008).

Foster et al. (2003) studied in vitro inhibition of human P450-mediated metabolism of marker substrates by natural pro-ducts. The products studied included several routinely usedspices, herbal and black teas and soybeans and investigatedtheir capacity to inhibit metabolism of drug marker substratesby human cytochrome P450 (CYP) isoforms (CYP3A4).Products were purchased from local markets: 6 spices (clover,ginger, oregano, sage, thyme leaves, turmeric), 20 teas: single-entity herbal teas (Cat’s claw bark, Chamomile herb, Feverfewleaf, Goldseal Herb, Gotu Kola herb; Kava kava, Siberianginseng, St. John’s Wort; Black tea: (Darjeeling tea, Earl Grey,English Breakfast, Irish Breakfast, Orange Pekoe) and Herbalmixture (Echinacea plus; Echinacea Special, Echinacea andGoldenseal, Ginger mix, Ginko biloba Special, Green tea withKombucha and Chinese herbs; and Green tea with TripleEchinacea and Kombucha), 7 soybean varieties and 4 puresoy isoflavones. Data showed that all products includingspices had significant varied inhibition of P450 metabolismof the substrates. Further, the conducted screening clearlydemonstrated the potential of these products to interfere

with human CYP mediated drug metabolism. Surprisingly, thesingle-entity herbal teas exhibited lower inhibition than othercategories. Also, soybean extracts also inhibited CYP, whichis in agreement with previously reported inhibition of puremajor isoflavones.

ConclusionFood-drug interaction occurs regularly, but is poorly reportedto the appropriate authorities. Recent activities includingresearch reports, articles in lay magazines and surveys ofvarious stakeholders highlight the growing health concernamong health care professionals. Now more and more people,especially the senior citizens _who have the financial resour-ces to travel to many exotic places and try a variety of foodsincluding exotic foods and fruits and juices _without havingknowledge of ingredients that may interact with prescriptiondrugs, fall victims to such reactions. In addition, a lot of exoticfruits, beverages are now found at local groceries in manyparts of the developed countries. It is highly advisable thathigh risk patients taking regular prescription drugs consulttheir physicians before they venture out in tasty, deliciousfruits and foods that may not be good for health, and maybecome fatal. Although, the food-drug interactions mostoften are seen in negative context, a recent presentation byReddy et al. (2007) reported the bioavailability of very expen-sive breast cancer drug lapatinib (Tykerb, approved by theUS Food and Drug Administration on March 13, 2007 to betaken in empty stomach- fasting) is greatly increased byfood, especially a high-fat meal. In a commentary Ratain andCohen (2007) estimated a cost saving of 60% or $1740 permonth if lapatinib is taken with food instead of in emptystomach. They also recommend marketing of a 500 mg formu-lation of the drug to be taken with food instead of currentlyrecommended 1250 mg formulation. The estimates andrecommendation may appear very simple, but one should bemindful of various competing factors that come into play fordelivery and efficacy of a drug. This suggests comprehensivestudies of the use of food for delivery of expensive drugs atreduced dosages for the maximum benefits.

AcknowledgmentThe author acknowledges valuable assistance of MichaelBryan and Francesco Lai.

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