80 | C a n Canadian Journal of Applied Sciences Effect of non-genetic and genetic factors on birth weight of mengali sheep of balochistan EDITOR IN CHIEF CO-EDITOR IN CHIEF PREVALENCE

80 | C a n J A p p S c i ; I s s u e 3 ; V o l u m e 1 ; O c t o b e r , 2 0 1 1

Volume 1 / Number 3/ 2011

Canadian Journal of Applied Sciences ISSN: 1925-7430. Committed to develop the scientific technologies of all disciplines;

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TABLE OF CONTENTS

Prevalence of disability in Oman: statistics and challenges. By; Taiseera Al-Balushi, ali h. Al-Badi, and Saqib Ali……………………………………………………………………………………………………………….…………………...81

Microbial assessment of herbal drugs available in local market of Lahore. By; Arifa Tahir and Madiha Aftab………………………………………………………………………………………………………….……….97

Bronchodilator, cardiotonic and spasmolytic activities of the stem barks of Terminalia arjuna. By; Janbaz, K. H., Hamid, I., Mahmood, M. H.2 and Gilani, A. H……………………………………………………….....…………..104

Effect of non-genetic and genetic factors on birth weight of mengali sheep of balochistan; Mohammad Masood Tariq, Masroor Ahmed Bajwa, Shakeel Babar, Abdul Waheed, Farhat Abbas Bukhari, Tahir Hameed, Illahi Bakhsh Marghazani, Yasir Javed………………………………………………………………………………………….....121

Influence of hyperglycemia on oxidative stress and dnaase-mediated genomic fragmentation coupled with apoptotic and necrotic cell deaths in kidney; Mohammed Saleem, Muhammad Shoaib Akhtar, Sidharta D. Ray, Bashir Ahmed……………………………………………………………………………………………………………….129

Depression and low serum levels of docosahexaenoic acid (dha) are associated with increasing number of pregnancies among postpartum omani women; Lamya Al Kharusi, Mostafa I. Waly, Hassan Mohammed Othman Nejib Guizani, Farhat Al Naabi, Amanat Ali……………………………………………………………………….144

EDITOR IN CHIEF CO-EDITOR IN CHIEF Prof. Dr. M. Shoaib Akhtar Dr.Taha Nazir

EDITORS Dr. Saswati Tripathy Dr. Amanat Ali Dr.Akin Peluola Jeffrey Purvis Sabahat Ahmad

MEMBERS Dr. Saswati Tripathy (Canada) Prof. Saringat Bin Bai @ Baie, (Malaysia) Dr. Taha Nazir (Canada) Dr. Akin Peluala, (Canada) Jeffrey Purvis (Canada) Sabahat Ahmed (UK) Dr. Syed Atif Abbas(Malaysia) Khalid Ahmad Sheikh(UK) Dr. Adul Haleem Khan (Pakistan) Dr. Nadeem Irfan Bukhari (Pakistan) M. Shamoon Chaudhary (Pakistan) Dr. M. Shoaib Akhtar(Pakistan) Dr. Riffat Ul Zaman (Pakistan) Muhammad Ilyas Insari (Pakistan) Naser Nimer Al-Khatib (Canada) ADDRESS; 115 V North Saskatoon Saskatchewan S7L3E4 Canada. Tel.: +1 306 261 9809, Email: [email protected], Web: www.canajas.com

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Canadian Journal of Applied Sciences; 2011; 1(3): 81-96, October, 2011 Intellectual Consortium of Drug Discovery & Technology Development Inc. ISSN 1925-7430 Available online http://www.canajas.com Original Research Article

PREVALENCE OF DISABILITY IN OMAN: STATISTICS AND CHALLENGES

Taiseera Al-Balushi, Ali H. Al-Badi, and Saqib Ali

Department of Information Systems, College of Commerce and Economics

Sultan Qaboos University, P.O. Box 20, P.C. 123, Al-Khoudh, Muscat, Sultanate of Oman

ABSTRACT

People with disabilities are among the most vulnerable groups all over the world.They have limited access to education, services, and employment. People with disabilities represent about 2.3% of the Omani population and the number is increasing. The research work was divided into three parts. The first part was a descriptive/interpretive analysis aiming to provide an outline of the current and relevant literature with regarddisability statistics and challenges worldwide. The second part aims to examine the state of disability and its demographics in Oman. The paper analyzes government legislations, major cause of disabilities, and the current initiatives of the Omani government to remedy the problems faced by disabled people in the country. Finally, the paper concludes with a set of recommendations suggesting ways to enhance the services provided for this group of population to integrate them in the society. The major findings of this study, despite the government effort in this regard, people with disabilities face a number of challenges as they lack basic services necessary to improve the quality of life and there is no updated statistics on the state of disability in Oman. This study will provide the foundations that might help the government and non-government organizations, disability researchers, and health professionals in devising a strategy for the integration of the disabled into Omani society.

Keywords:Disability, Impairment, Oman, Oman Census 2003, Disability Legislation, Oman Statistics

Corresponding Author:Dr. Saqib Ali, Room CCE~2011, Department of Information Systems, College of Commerce and Economics, Sultan Qaboos University, Al-Khoud, Muscat, P.O. Box 20, P.C. 123, Sultanate of Oman, Telephone: +968 2414 2928Email: [email protected]

1. INTRODUCTION

About 650 million people in the world live with disabilities of various types, and the number is growing due to a rise in chronic diseases, injuries, road accidents, violence,aging, and other such factors(World Health Organization, 2005). This number makes up approximately 10% of the world population. Of disabled population, nearly 80% live in low-income countriesand are poor


and have limited or no access to basic services including rehabilitation facilities. The number of persons with recognized disabilities in industrialized countries is between 8% and 20%, and on the other hand, developing countries often acknowledge much lower percentages officially(World Health Organization, 2003).

People with disabilities suffer from discrimination throughout the world, especially in the developing countries, and are frequentlyexcluded from social, economic, and political processes in their societies. Disability was long considered an individual problem that was treated from a medical and charitable viewpoint,but neglected in terms of equal rights for disabled persons (German Federal Ministry for Economic Cooperation and Development, 2006). Now disability has been recognized asa major social issue internationally,and the World Health Organization (WHO) is putting together great efforts to assist countries in developing policies for people with disabilities, ensuring early identification and treatment, providing equal opportunities, and promoting rights of the disabledpeople(World Health Organization, 2005).

1.1 Defining Disability

Disabilityhas different definitions depending on (national) social legislation and cultural standards. The definition of disability also varies according to the purpose for which it is being used, such as health care, rehabilitation, income protection, employment, and social assistance (ESCWA, 2010). Moreover, what is considered a disability in one country may not be perceived and labelled as such elsewhere (e.g., mental disability or female infertility). This variation makes it more difficult to establish reliable data on the number of the disabled people(German Federal Ministry for Economic Cooperation and Development, 2006).

World Health Organization’s definition formulated in 1976 iscurrently most widespread, which draws a three-fold distinction between impairment, disability and handicap, “An impairment is any loss or abnormality of psychological, physiological or anatomical structure or function; a disability is any restriction or lack (resulting from an impairment) of ability to perform an activity in the manner or within the range considered normal for a human being; a handicap is a disadvantage for a given individual, resulting from an impairment or a disability, that prevents the fulfilment of a role that is considered normal (depending on age, sex and social and cultural factors) for that individual” (World Health Organization, 1978). Mann in his book on Smart Technology for aging, disability, and independence defined disability as “a limitation in performing tasks, activities, and roles to levels expected within physical and social contexts. A person may have difficulty dressing (task), but this could be due to cognitive impairment, vision impairment, or motor impairment, each of which would require a different compensatory strategy”(Mann, 2005). According to Oman’s Ministry of National Economy a disabled person is “a person who is suffering from a shortage of some of its sensory or physical or mental moral, genetic factor, or disease, or accident, limiting his/her ability to perform his/her natural role in life compared to those in his/her age, and it constitutes all the needed care and rehabilitation until he/she can do their role in life” (Minisry of National Economy, 2008).

1.2 Importance of Disability studies:

Disability studies are imperative and in many developed countries, disability rights activists grabbed the attention of the society to the numerous social, cultural, economic, and political barriers faced by many people with impairments. Increasingly, such movements have gathered support for disabled people around the world and, many campaigns have influenced


governments’ disability policy (Colin & Geof, 2010). On the other hand, people with disabilities in some of the developing countries are still deprived of their basic rights of education, health, and housing. Therefore, disability research on various issues (e.g., economic, social, education, employment, and health) will educate and inform policy makers to initiate health programs and take measures that can provide remedy to reduce the burden of disability(Sangram, 2009).

In Oman,according to the 2003 Ministry of National Economy Census there were about 41,303 people with various types of disabilities(Ministry of National Economy, 2010b). However, there is a lack of reliable information on the number of people with disabilities. Thus, this article aims at analysing the state of disability in Oman, which can help policy makers, government and non-government agencies, and disability researchers in understanding the impact of disability on the economical and social development in the country. The paper offers a deeper understanding of the disability issue in Oman by analyzing disability legislation, causes of disability, and the government’s initiatives against disability. Recommendations for coping with the issue effectively are also made.

2. LITERATURE REVIEW

The 2003 WHO report estimatedthat about 80% of people with disability are from developing countries (World Health Organization, 2003). The day-to-day activities of 25% of the world’s population are affected by disability, which affects entire families, not just the individual.It is argued that between 14% and 34% of children in developing countries havesome sort of disability according to the United Nations Children’s Fund (UNICEF) and 8% of the world’s disabled population live in low-income countries; a majority of them are poor and cannot access basic services including rehabilitation facilities(The United Nations Children’s Fund, 2009). According to the United Nations Educational, Scientific and Cultural Organization (UNESCO), the death rates among children with disabilities are 80% and that98% of kids with disabilities in developing countries are not enrolled in schools. This limitation considerably reduces opportunities for their training and employment (UNESCO, 2009).

Disability is caused by numerous factors, such as disease, malnutrition, incorrect treatmentor non-treatment, violence and war, accidents due toinadequate protection at the workplace and in road accidents, andincreasingly age-related diseases. People with disabilities suffer from discrimination throughout the world andare frequently excluded from social, economic, and political processes in theirsocieties(German Federal Ministry for Economic Cooperation and Development, 2006).A report by Economic and Social Commission For Western Asia (ESCWA) claimed that countries of the ESCWA region have very low disability rates in contrast to other regions (ESCWA, 2010). For example, this region shows a disability rate of 0.5% in Saudi Arabia, 0.6% in Egypt, 1.9% in Yemen, and 4.8% in Sudan; which is very low considering the population in these countries; causing doubt about the truth of these statistics.However, this might beunderstandable since these communities considerdisability as a source of shame or disgrace. Also, this situation arises due to the narrow description for impairments in surveys and censuses, which mainly focus on the most severe types of disability like blindness and deafness rather than activity-limiting disabilities like learning difficulties and psychiatric disorders. It is argued by people with disabilities that if culture, physical environment, and attitudes towards them change then the effect of their disabilities would be reduced. As a result, there will be equality in the society. This can be achieved by providing them easy access and better facilities in doorways, corridors, toiletfacilities, parking spaces, and other such benefits. Also, support


such as flexitimework schedules will help this segment of the society to overcoming strict rules, protocols, and procedures in the workplace. In addition, attitudes toward disabled people must be changed because people may adopt a discriminatory attitude by considering them not as talented as normal persons (Disabled Living Foundation, 2006).

Thus, in developed countries such as the USA, a number of disabled rights legislations have been introduced by the federal government such as Section 508 of public law 99-506 (Federal Discrimination Law), the Americans with Disabilities Act of 1990,and Public Law 101-336(U.S. Department of Justice). These laws establish guidelines for access to electronic equipment in the federal workplace. These laws also helpthe disabled by providing funds to the States in their efforts to improve access for people with disabilities, to support individual research projects, and to provide graduate fellowships for people with disabilities(Glinert & York, 1992).

In Oman, according to the Ministry of National Economy, there are nearly 41,303 persons reported disabled in 2003 Census (Ministry of National Economy, 2010a),and only about 23% of the total number of people with disabilities have been admitted to different rehabilitation centers. It is important to note that there are no statistics since 2003 about the number of disabled people in Oman while it is estimated that the number is increasing dramatically. There are about 37rehabilitation centres across the country (see Table 1). However, it is important to note that these centres/associationshave twofold objectives. Some of them provide help to disabled people to improve their quality of life and enable them to live independently. They also provide basic educational and vocational training to help them integrate in the society. While the main concern of the other associations, as indicated in Table 1 (Oman Disabled Welfare Association and Al-Noor Association for Blind), is to call for the rights of people with disability and promote awareness abouttheir rights.

Table 1. Centers/Associations for the disabled and the beneficiaries, 2008

Center/Association Number Beneficiaries Al-Wafa Social Voluntary Centre 22 2,167 Disabled Welfare and Rehabilitation Centre

1 125

Disabled Children Welfare Association

9 431

Association of Early Intervention 1 128 Oman Disabled Welfare Association 1 130 Al-Noor Association for Blind 3 74 Total 37 3,055

Source: *(Ministry of National Economy, 2010b)

There are a number of barriers concerning the studies about people with disabilities in Oman that areincluding a lack of:

• Clear and specific statistics on the number of people with disabilities, types of their special needs, and causes of such disabilities.

• Vocational training program for the youth with disabilities in order to help them to join the workforce and work independently. As indicated in Table 1, a big number of centers are


male

56%

female

44%

Distribution of disability by Gender in

Oman

Community Based Training (CBR) and the personnel who work in these centers are volunteers who lack the necessary skills in dealing with people with special needs.

• Human and financial resources in these centers as most of the people working there are volunteers with little or no experience about how to deal with people with special needs. These centers lack financial resources, which is crucial to provide better facilities for people with disabilities (e.g., Information Technology).

• Awareness and acceptance of the disabled.

• Empowerment for disabled people by giving them equal access to opportunities and the development of technologies to compensate for physical impairment.

Disability demographics in Oman

The Sultanate of Oman is administratively divided into 5 regions and four governorates with 61 Wilayats (or districts). The regions are: Ad-Dakhliyah, Ash Sharqiyah, Al-Batinah, Adh-Dhahirah, and Al-Wusta, and the governorates are: Muscat, Dhofar, Musandam and Al-Buraymi(Ministry of Health, 2009). According to mid-year census for 2009, the Omani population is about 3.17 millionof which 2.01 million were Omanis and 1.15 millionwere expatriates. (Ministry of Health, 2009). The proportion of population below 15 years of age is 34.5%.Femalesaged 15 to 49 years represent 58.3% of the total female population and the fertilityrate is 3.9. According to (Nour, 2005), the population projections for countries in the MENA region indicates that the number of childrenwill increase by about 30% and this expansion will be faster in countries such as Yemen, Saudi Arabia, Palestine, and Oman.

6324

13700

750

308369187561

386

2581

0100000200000300000400000500000600000700000

Distribution of disability by region in Oman

Disabled Population

Total Population

Figure 1.Disability by region

Figure 2. Disability by gender


26.48

8.58

9.37

4.713.076.45

10.67

6.23

1.051.56

21.82

Distribution of disability by type in Oman

Visual Hearing Speech

Hearing & Speech Learning Behavioral disorders

Figure3. Types of disabilities in Oman

Moreover, according to genderdistribution 56% were males and 44% were females(Ministry of National Economy, 2010a). Among the disabled in Oman, visually disabledindividuals were the largest (26.5%) followed by people with movement impairments(10.7%) and speech disability (9.37%).

As mentioned in section 2.2, it was estimated that the total number of disabled people in Oman is about 41,303 according to the 2003 census. Since there is no data consistency with regards to the number of people with disability from the years 2003, 2006, and 2010, the authors used the total population data for 2003, 2006, and 2009 from the Ministry of National Economy (2010) and then calculated the population growth rate for these three years. Based on the pollution growth rate, the total number of disabled people was estimated. In addition, the growth rate for the years 2010-2015 was predicted from the United Nations (2008) figures by calculating the population and the number of disabledin Oman for the year 2012,as depicted in Table 2.By looking at the predicted data, we can assume that the government must give special attention to this segment of the society.

Table2. Population to Disability ratio

Source: *(Ministry of National Economy, 2010b), **United Nations, 2008

2003 2006 2009 2012

Population *2,341,000 *2,577,000 *3,174,000 3,357,000 Growth Rate NA 10.08 % 23.17% NA Annual Growth Rate NA 3.36% 7.72% **1.92% No. of Disabled People 41,303 45,467 56,002 59,228


3. DISABILITY LEGISLATIONS

3.1 Disability legislations Worldwide

In Australia, the Equal Opportunity Act of 1984 was enacted by the Australian government to protect its citizens against certain discriminations, such as sexual, racial, age-related, anddisability discrimination. The Act also appointed a Commissioner for Equal Opportunity who was charged with overseeing the implementation of this law(Equal Opportunities Act, 1984). In 1992, the Australian Disability Discrimination Act (DDA) was passed as a broad piece of legislation that attempts to label and outlaw any form of discrimination against any kind of disability(Disability Discrimination Act, 1992). The act enforces fair and equal treatment for those with disabilities in areas such as education, employment, and housing, and access to premises, clubs, sports or other gatherings. The Australian Human Rights Commission enforces the Disability Discrimination Act and it covers those with physical, intellectual, psychiatric, sensory, neurological, and learning disabilities, as well as individuals with physical disfigurements or disease-causing organisms. The act also covers family members or others connected to the disabled.

In the UK, the Disability Discrimination Act (DDA) was passed in 1995 by the Parliament. DDA is an act that makes it unlawful to discriminate against disabled persons in connection with employment, the provision of goods, facilities and services or the disposal or management of premises; to make provision about the employment of disabled persons; and to establish a National Disability Council (Disability Discrimination Act, 1995).In the United States, Americans with Disabilities Act (U.S. Department of Justice) of 1990 was introduced as a wide-ranging legislation intended to make American society more accessible to the people with disabilities. The Act is divided into five titles: employment, public service, public accommodation, telecommunication, and miscellaneous.

In 1993, the UN General Assembly adopted “Standard Rules” for establishing equal opportunities for disabled people, which were developed along the lines of the World Program of Action. The Standard Rules provide a universal framework for activities to integrate the rights of persons with disabilities into national legislation. However, the “Standard Rules” are not binding beyond their significance as a political guideline, which means that the needs and rights of disabled persons are still not sufficiently reflected in many national and international development strategies (United Nations Enable, 1993). The Convention on the Rights of People with Disabilities and its Optional Protocol was adopted on 13 December 2006 at the United Nations Headquarters in New York, and was opened for signature on 30 March 2007. The purpose of the present Convention was to promote, protect and ensure the full and equal enjoyment of all human rights and fundamental freedoms by all people with disabilities, and to promote respect for their inherent dignity. Persons with disabilities include those who have long-term physical, mental, intellectual or sensory impairments which in interaction with various barriers may hinder their full and effective participation in society on an equal basis with others. There are currently 147 signatories and 99 ratifications of this convention (United Nations Enable, 2011).

3.2 Disability legislations in Oman

Oman has made headway inpassing laws and regulations for the social welfare of disabled people. In 2008, a royal decree was introduced to publicize the law on the care and rehabilitation


of disabled people where the Ministry of Social Development supervises and regulates the needed actions. Also, the decree announced the establishment of the National Committee for the Welfare of Disabled People. This committee is responsible to study and prepare a general plan for the care and rehabilitation of people with disabilities in Oman. The committee develops special programs for rehabilitation, care, employment, and development of the disabled, as well as promotes plans and programs for the awareness of all types of disability and its possible prevention. In addition, hardware and compensatory rehabilitation tools imported by rehabilitation centres and disabled individuals are exempted from customs duties Also, the activity requirements of the rehabilitation centres are exempted from taxes and fees. Moreover, the government is working on the preparation and training of disability specialists for early detection of disabilities and provision of appropriate assistance and services for handicapped people(Ministry of Social Development, 2008).Some benefits/services are assured by law to disabled people,which will be discussed in details in the coming sections, such as medical care, rehabilitation,counselling, employment, training, financial security, and others.

The government provides health care and treatment to the disabled including rehabilitation and assistive devices that help them in movement, education, training, and other daily life activities. Also, the government helps families having disabled persons who cannot afford to buy assistive equipmentson their own. With regards to educational services for the disabled, Omani government is obligated to provide education appropriate to the physical and mental state of disabled people. Also, they are eligible for appropriate professional qualifications and certificatesawarded to them from the rehabilitation centres support them in the labourmarket. Any public or private organization thatholds fifty workers or more are obliged by law to hire qualified disabled persons nominated by the Ministry of Manpower. For the benefit of public services, the government ensures smooth access to public places, roads, buildings, religion places, markets, car parking,and areasthat might be visited by the disabled. Moreover, the government improved the public transportation to facilitate their movement in the society. Finally, the government is committed to facilitate disabled people in social activities, sports and cultural work as well as to ensure their participation in local, regional, and international camps/sports competitions(Ministry of Social Development, 2011).

4. AN OVERVIEW ON CAUSES OF DISABILITIES IN OMAN

There are numerous studies available worldwide (in developed as well as developing countries) regarding the causes of death, but very few such studies are available on the causes of disability in developing countries. The causes of disabilities vary according to various age group(working age and older age) and gender(Sangram, 2009). According tointernational agencies, governmental departments, and non-governmental sources the major causes of disability include heredity, birth defects, lack of care during pregnancy and child birth, insalubrious housing, natural disasters and accidents, illiteracy and the resulting lack of information available on health services, , congenital diseases, malnutrition, marriage between close relatives, respiratory diseases, metabolic diseases (diabetes, kidney failure etc.), drugs and alcohol, old age, poliomyelitis, and measles (Sangram, 2009)

According to the 2003 census, one-third of the causes of disability in Oman were congenital reasons and about 15% of the disabilities were caused by age. Statistics also revealed that one-fifth of congenital disabilities were speech difficulty, 18.4% were categorized under other types


of difficulties, and 12% were vision difficulties. Furthermore, of the disabilities caused by illnesses, one-third was vision difficulty, 27.5% wereother difficulties, and 11% were mobility difficulties. Car accidents on the other hand accounted for3% of disabilities in 2003(Ministry of National Economy, 2010a). Children of parentswith familial relationshipshave higher risk of inheriting from each of their parents a matching faulty gene that could result in some form of disability. It was estimated by the Centre for Genetics Education (2007) that 2 to 3% of blood-related parents had a risk of having a kid with a disability or a genetic birth defect. If parents are first cousins, the threat is almost two-times, about 5% to 6%. In Oman, about 36% of marriages were between first and second cousins, and additional 20.4% of the marriages were within members of the same tribe, which makes a total of 56.5% of the population (Rajab, 2000). This justifies the huge percentage of 34.3% of congenital disability rate in Oman (Ministry of National Economy, 2010a) .

Road accidents are considered to be another main cause of disabilities in Oman. Every year, according to the statistics, 1.2 million people are known to die in road accidentsworldwide. Millions of others sustain injuries with some suffering permanent disabilities. No country isfree of thisdeath toll, especially among the youth. Thus, enormous human resources isbeing destroyed, which leads to grave social and economic consequences(World Health Organization, 2004).According to statistics by the Ministry of Health (2009), about 20% of total deaths of children in Oman were caused by road accidents. It is predictable that road accidents every year cause around 500 deaths and more than 6000 injuries. Almost 7,570 road accidents occurred in Oman in the year 2010 (see Table 3), where 2,896 accident were only in Muscat governorate (Al-Rajhi, 2011).

Table 3.Road accidents for the year 2010

Road Accident Total Percentage

Collision between vehicles 3,441 45.5

Run over 1,091 14.4 Loss of control 1,160 15.3 Hitting into solid objects 1,879 24.8 Total accidents 7,571 100%

5. CHALLENGES FOR DISABLED PEOPLE IN OMAN

5.1 Education

Despite the continuous effort to raise the level of education in Oman and the government’s strategy to minimize the illiteracy at all ages and gender, yet a huge percentage of illiteracy is prevails among people with disability (ages 15 years and above). According to the 2003 census (Ministry of National Economy, 2010a),75% of disabled people were illiterate and almost 55.6% of them had already reached the age of 60 or above. However, only 24.4% were in the age group of 15-24 years. Another important point is that females represent a higher percentage in illiteracy and the majority of them come from Al-Batinah region as shown in Figure 1.


Figure 4 below presents the distribution of people with special needs by education level. It is clear from the figure that a huge percentage of disabled people are illiterate and those who were fortunate to get some education beyond high school are only 1.7% male and 0.9% female. In fact, higher education is very crucial to have a decent life and be part of the labour force in most of the scenarios(Ministry of National Economy, 2010a).

It is important to note that major attemptswere made to integrate children with special needs in public schools since 2000-2001 academicyear. According to theMinistry of Education, two educational programsare offered by the ministry: Learning Difficulties Program and Special Needs Integration Programs. Learning Difficulties Program offers special educational courses for students with disabilities in government schools rather than sending them to specialized schools. The program was launched in two schools in 2000,by the academic year 2006-2007 it was successfully integrated in 178 schools all over the Sultanate. Special Needs Integration Programs was launched in 2005-2006 as separate classes in public schools, and these classes werereserved for students with hearing and intellectual disabilities. The program started in two schools in Al-Batina and Al-Sharqyia regions, and by year 2006-2007, it had covered other four regions (Ministry of Education Portal, 2011).

Figure 4: Disabled people distribution by education level

However, there were only three specialized schools for children with special needs, and all of them are located in Muscat governorate. As shown in Figure 5below, people with visual, speech, or both disabilities are most fortunate when it comes to education,because the current specialized schools are only equipped to support these types of disabilities. It is important to note that in order to provide education to people with other types of disabilities; specialized schools, equipment, and human resources are required. The schools should also be set up in other regions in Oman, not only Muscat, so that lessadvantaged childrencould be educated.Proper education and training will increase their chances of integration in the community and will help them seek their rights. These steps will lead the country to stability and social development.

0

5000

10000

15000

20000

25000

Illiterate reads and

writes

Below

high

school

High

school

above

high

school

not stated

Distribution of people with special needs Aged (5-29)

years by education level

Female

Male


Figure 5. Education and types of disability

5.2 Employment People with disability represent approximately one-sixth of the working age population globally (Bruyère & Ruiz-Quintanilla, 2000). In Oman, according to the 2003census (Ministry of National Economy, 2010a),disability has a major impact on the economy of the Sultanate. It was evident that 15.7% of people with disability were active economically (i.e., they can have jobs and be independent) and about 65% were unable to do any type of job. Once again female contribute less, only 5% compared to 24% males who can actively involve themselves in labour force. The public sector is playing a great role in employing people with disability as compared to other types of work sectors. Figure 6 illustrates the distribution of the disabled in various sectors. Section17 of Oman’s Labour Law states that “people with disability should be employed in both public and private sectors,” and it clearly necessitates the employer, who employs 50 or more employees, to offer job opportunities according to skills and abilities of a certain number of disabled persons. As a follow-up, the Ministry of Manpower remains in contact with the private sectors to enforce the law(Ministry of Social Development, 2011).

0

5

10

15

20

25

30

35

40

Percentage of education status and type of diability

Enrolled now

Enrolled befoe

Never enrolled

Not stated


Figure 6. Distribution of people with disability and work sectors

6. GOVERNMENT INITIATIVES TO REMEDY DISABILITY PROBLEMS IN OM AN

The government of Oman is making great efforts to fulfil the needs of people with disabilities. The government has adopted and implemented a number of laws to ensure equal opportunities for disabled people. Provision of special education and rehabilitation services in Oman is shared by the Ministry of Education with a special education division that was established in 1974, the Ministry of Social Development and Manpower, the Ministry of Health, and non-governmental organizations. In the following sub-section, each government entity’s role with regards to catering the needs of people with disabilities is discussed in details. 6.1 Ministry of Education The Ministry of Education is providing educational services for children with special needs, both in special schools and in integrated educational facilities. The Ministry established a Special Education Division in 1974. There are only three specialized education schools in Oman: one is for children with hearing impairments which was established in 1978 and another for children with cognitive impairments that wereset up in 1985. The third specialized education,named Omar bin Al-Khattabfor the blind, was established in 2000 (Ministry of Education Portal, 2011). This institute is accompanied by a technology center to train studentsin basic IT skills. The ministry is also running other educational programs for students with special needs,such as Learning Difficulty Programs and Integration Program with normal schools. With regards to higher education, students with mobility-related disabilities can be enrolled at any institution of higher education in the Sultanate. However, the blind get very limited options at Sultan Qaboos University since the College of Arts is the only College that caters for the needs of blind students. In some other cases, students are sent tothe neighboring countries, such as Kuwait, Bahrain, and Egypt for further education and rehabilitation.

6.2 Ministry of Social Development The Ministry of Social Development established a Directorate for Special Care in 1980 with the primary purpose of improving services for persons with disabilities, elderly people, and juvenile rehabilitation. The main responsibilities of this directorate include: setting up vocational rehabilitation centres,offering rehabilitation personnel preparation programs, adopting policies and regulations in relation to employment of persons with disabilities, and supervising and

49%

30%

20%

0%1%0%

Distibution of working poeple with special needs (15

years and above) and work sectors

Public

Private

Family

National

Other

Not stated


coordinating national efforts for vocational training and integration of people with disabilities into community life(Ministry of Social Development, 2011). The directorate has amended labour, civil service, government pension, social security, and social insurance laws to protect the rights of the disabled. In this regards, major amendments made so far are as follows: (a) promoting accessibility to public buildings, (b) providing assistance for purchasing individual means of transportation, (c) supporting the participation of persons with disabilities in cultural activities and sports, (d) providing financial support for adapting homes for persons with disabilities, (e) offering social security benefits and pensions for unemployed individuals and their families, and (f) promoting the employability of the disabled in the public and private sectors(Hadidi, 1998; Ministry of Social Development, 2011). The Sultanate of Oman is gradually realizing the importance of community-based rehabilitation (CBR), and thus, it was adopted as a strategy to address the increasing number of people with disability with various demographic characteristics and geographical location in order to reach a larger number of people with disabilities (Ministry of Social Development, 2011). Therefore, a number of CBR centres were founded in 1980s inseveral governorates. Al-Wafacentres one of the key CBR initiatives and it has 22branches all over the Sultanate, which accommodates over 2,000 children of age group 3-14 years and has 234 volunteer workers. These CBR centresfunction toimprove the quality of life for disabled children so that they can integrate easily with the society(Ministry of Social Development, 2011). Some recent significant achievements for the social and economic development of this segment of society is the outcome of collaboration between Information Technology Authority (ITA), Oman International Bank, and Oman Arab Training Institute. A major achievement was training hearing and speech impaired persons to be certified trainers on the program International Cambridge Diploma for information Technology Skills(AlRoya, 2011). 6.3 Ministry of Health The Ministry of Health has been involved in prevention of disability and provision of health services to people with disabling conditions. The Ministry collaborates with some agencies of the UN in developing programs for disease prevention and control, comprehensive health care, environmental health, and development of women (Hadidi, 1998). With regards to road accidents issue that resulted in a significant number of people with disabilities, Oman with other countries has brought up the issue of road safety to the UN General Assembly and played a major role in raising global awareness to the growing impact of deadly road traffic injuries, especially in the developing world.The magnitude of the problem encouraged the UN General Assembly to adopt a special resolution (No. 58/9) on this issue and the WHO declared the year 2004 as the year of road safety(World Health Organization, 2004). 1. DISCUSSION In the last 40 years, Oman has made remarkable development in various fields, such as social, economic, health, education as well as technology. Although there have been a significant investment in human resource development in general,the Sultan of Oman has recently granted 100 million Omani Riyals for human resource development programs for Omanis enabling them to pursue higher education in specialized areas (e.g., Medicine, Engineering, Information Technology, and Economics) or those needs in the society(Oman News Agency, 2010). Yet the people with special needs are underrepresented in all these plans. Oman lacks


qualifiedprofessionals whocan deal with issuesfaced by people with special needs.Technologies are now helping the disabled to participate in society more fully and exert great control over their own future. Technical advances and the continuing convergence of computing and telecommunication are reducing barriers and many people have benefited from them, but for people with disabilities the impact is more profound. The increasing range of assistive devices, from low-technology self-care mobility devices through the high technology “smart homes” are all contributing to minimize and well manage a “disabling condition”(Mann, 2005) .

2. CONCLUSION Despite thegovernment efforts to enforce law and develop awareness about disabilities as well as to improve the quality of life ofthe disabledpeople, a significant number of them are underrepresented in education, employment, health, and other facilities. Those who are fortunate get a chance to receive basic education (only for those withlearning difficulties, visual impairment or hearing impairment), but the majority of them do not get a chance to pursue higher education. The fortunate ones (who receive basic education) are very few compared to the total number of people with disabilities in Oman. A major challenge in this study that the authors faced was the lack of up-to-date statistics on people with disability in Oman as the most recent resource is the 2003 census(Ministry of National Economy, 2010a). In addition, there is no information on what services/facilities are available for disabled persons and where these services are located. This paper, however, provided some insight about the state of disabilities in Oman and offered and overview of a number of issues, such as the demographics,legislation, and day-to-day problems regarding disabilities in the Omani society. The paper explored some of the causes of disabilities in Oman and highlightedthe issue of traffic accidents, which has become a prime cause of disabilities recently.The paperalso discussed the government initiatives to overcome these problems throughcoordinated effortsof various ministries.It is hoped that specificrecommendations put forward in this paper will facilitate the policy formulation process for the welfare of the disabled. RECOMMENDATIONS In order to guarantee a quality life and equal opportunity for people with special needs in Oman, the following issues should be taken into consideration: • Nationwide statistics about the number and types (and sub-types) of disabilities should be

available by gender, age, and region, because theavailable list is not comprehensive and there are additional types of disabilities.

• Arrangements for capacity building of Omani professionals who are trained in counseling, training, and teaching people with special needs should be made.

• the awareness should be enhanced that with the current technologies anything can be achieved and the disabled can join the workforce and become effective workers as long as resources and proper training is provided

• Infrastructure necessary to cater for people with special needs in Oman should be developed by raising awareness about the opportunities that ICT can provide to empower the disabled.

• Involve and encourage the local businesses to invest in CBR due to the limited number of these centers, currently 22, which are managing a big number of children with disabilities with limited resources(Minisry of National Economy, 2008).


• Local businesses should utilize technology in order to expand their services to people with special needs.

• Utilize computing technology for tasks such as reading and writing documents, communicating with others, and searching for information on the Internet.People with disabilities are capable of handling a wide range of activities independently.

• By accessing government’s e-services from home, it makes lifeof the disabled easy, especially if they find it difficult to go outof their homes. It also makes pursuing higher education via e-learning possible, if attending university/college is physically difficult.

ACKNOWLEDGMENTS The Research leading to these results has received Research Project Grant Funding from the Research Council of the Sultanate of Oman Research Grant Agreement No [ORG SQU ICT 10 004]. 3. REFERENCES

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12. Glinert, E., & York, B. (1992). Computers and People with Disabilities. Communications of the ACM, 35(5).

13. Hadidi, M. (1998). Education of Children with Vision Impairments in the Sultanate of Oman. International Journal of Disability, Development and Education, 45( 4), 423 - 429

14. Mann, W. C. (Ed.). (2005). Smart Technology for Aging, Disability, and Independence: John Wiley & Sons, Inc.,.

15. Minisry of National Economy. (2008). "Population" Series: Disabled Statuses in Sultanate of Oman. Ministry of National Economy.

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National Economy website: http://www.mone.gov.om/book/SYB2010/2-population.pdf 20. Ministry of Social Development. (2008). Laws and regulations for the care and rehabilitation of

disabled people. Retrieved from Ministry of Social Development website: http://www.mosd.gov.om/rules_disable.asp

21. Ministry of Social Development. (2011) Retrieved April 26th 2011, from http://www.mosd.gov.om 22. Nour, O. (2005). Child Disability in some countries of the MENA region: Magnitude, Characteristics,

Problems and Attempts to Alleviate Consequences of impairments. Paper presented at the the XXVth IUSSP International Population Conference, Tours, France. http://iussp2005.princeton.edu/download.aspx?submissionId=50279

23. Oman News Agency. (2010). HM Grant for Human Resources Development. Oman Newspaper. Retrieved from http://www.omannews.gov.om/ona/newsDetails.jsp?newsID=40873

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25. Rajab, A., and Patton, M. A. (2000). A study of consanguinity in the Sultanate of Oman (Vol. 27, pp. 321-326.): Annals of Human Biology.

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28. U.S. Department of Justice. Americans with Disabilities Act. Retrieved from Americans with Disabilities Act. website: http://www.ada.gov/

29. UNESCO. (2009). Policy Brief on Early Childhood – Inclusion of Children with Disabilities: The early childhood imperative. 46. Retrieved from The United Nations Educational, Scientific and Cultural Organization website: http://unesdoc.unesco.org/images/0018/001831/183156e.pdf

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Canadian Journal of Applied Sciences; 2011; 1(3): 97-103, October, 2011 Intellectual Consortium of Drug Discovery & Technology Development Inc. ISSN 1925-7430 Available online http://www.canajas.com

Original Research Article

MICROBIAL ASSESSMENT OF HERBAL DRUGS AVAILABLE IN LOCAL MARKET OF LAHORE

Arifa Tahir 1* and Madiha Aftab 1

1Environmental Science Department, Lahore College for Women University, Lahore, Pakistan.

ABSTRACT

The present work was carried out considering the increased use of herbal products as alternative medicines in Pakistan. It is necessary to set appropriate standards for microorganisms in herbal drugs in order to reduce the risks for consumers' health. The presence of fecal coliform and moulds represents a potential risk of contamination. The microbial contamination of herbal drugs both branded (Qarshi ghutti, Khamira gaozban, Surficol, Sadooori, Carmina, Sauline and Toot Siah syrup etc.) and unbranded was determined. Forty eight samples of herbal drugs were purchased from local market and were analyzed for total viable count, spore former (aerobic and anaerobic), mold, yeast and coliforms. Total viable count ranged from 1.1x 105 - 9.3 x 105/g. The aerobic spore former varied from 1-59/g. Moisture content varied from 4-20%. Non fecal coliform were present in most of the samples but fecal coliforms were also present in few samples. Aspergillus, Mucor and Penicillium species were predominant in most herbal drugs.

Key words: Herbal drugs, microbial assessment, moisture content

Corresponding author: Dr. Arifa Tahir, Associate Professor, 1Environmental Science Department, Lahore College for Women University, Lahore, Pakistan. Tel.:92-42-9203091, Fax: 92-42-9203077, Email: [email protected]

INTRODUCTION

About 30% of the pharmaceutical drugs are still extracted from different plant materials (Gloria and Asuncion, 1994). These are source of very affordable and effective drugs. At present, most of the medicinal plants have been processed into dosage forms that are more convenient to use and more readily available than raw materials. W.H.O estimates that 80% of the world population use herbal medicines for some aspects of primary healthcare (Chan, 2003) . The increasing popularity of natural drugs made their use a Public Health problem due to the lack of effective surveillance of the use, efficacy, toxicity and quality of these natural products (Bugno et al., 2006). The adverse effects of long-term herbal use, adulteration with toxic compounds and contamination by pathogenic microbials or natural toxins like mycotoxins have been reported for herbal products and medicinal plants (Mandeel et al., 2005, Rizzo et al., 2004, Tassaneeyakul et


al., 2004). The concern over quality of these products is mainly due to their potential contamination, considering their natural origin. Natural drugs should meet the requirements of quality, safety and efficacy (Calixto, 2000). Practices used in harvesting, handling, storage, production and distribution make medicinal plants subject to contamination by various fungi, which may be responsible for spoilage and production of mycotoxins (Mandeel et al., 2005, Tassaneeyakul et al., 2004).

The major sources of microbial contamination is from raw materials, handling during processing, storage and transportation. This may pose a serious problem to the consumers health as microbial contaminants may cause intoxication or other forms of illness (Nahrstedt et al., 1996). The object of microbial examination of any food or food product is to look their freedom from fecal pathogens, toxic bacteria and high viable count and spore formers. These types of studies have been reported by workers (Ramaday and Elnaby 1962). Coliform organisms are members of Enterobacteriaceae which ferment lactose with production of gas within 48h at 35°C. Coliform were instituted as indicators of fecal pollution of water in the United States by the Public Health Service (Sneath et al., 1986). The coliform group of bacteria includes all the aerobic and facultatively anaerobic, gram negative, non sporulating bacilli. The classical species of this group are Escherichia coli and Aerobacterearogens.

MATERIALS AND METHODS

Collection of samples

20 herbal drugs for oral use in human were selected based on microbial caused diseases. These drugs were obtained from local market. A total of approximately 100g of the sealed samples were placed in the sterilized plastic bags and stored at 4°C until needed. The samples and their prescribed uses along with their manufacturers are given in table 1.

Microbial Assesment

25g of herbal drug was shaked in 250ml distilled water. 1ml of sample was added to maconkey bottles containing 9ml of sterilized saline water. Ten fold serial dilutions were performed up to 10-5, in buffered peptone water (MERCK).

Enumeration of microorganisms was performed by pour plating method using Nutrient agar medium for bacteria and potato dextrose agar medium for moulds. Plates were incubated upside down at 37°C and 26 ± 1ºC for bacteria and fungi respectively. After incubation, the microbial colonies were counted, recorded and the number of colony-forming units (c.f.u) per gram were calculated. Results were compared with US Pharmacopoeia, 2005.

Thermophilic spore formers

The nutrient agar medium was used for aerobic spore former and Brain Heart Infusion Agar for anaerobic spore former. Spore formers were isolated by heat shock method. The plates were incubated at 37°C for 24-48h. The numbers of bacterial colonies were counted after 24-48h.

Detection of Coliform

The coliform organisms were determined by standard multiple tube fermentation technique (Alcala, 1990).


Table 1: Herbal drugs and their prescribed uses

Samples Prescribed uses Qarshi Ghutti Medicines to evacuate the bowels of newborn babies and infants,

constipation in children. Surficol Effective in cough, cold, bronchitis and catarrh. Khamira Gaoziban

As a remedy for perplexity and restlessness, sore throat, cold and flu.

Johar joshanda As a remedy for flu, cough, cold, catarrh, irritation of throat and fever. Sharbat Toot Siah Effective in swelling and pain of throat, hoarseness of voice and helps

relieve swelling of throat in diphtheria. Zanjabeen Effective in hyperacidity, nausea and vomiting, digestive system and

migraine. Musaffine Medicine used in blood purification, natural glow to skin and face,

pimples, prickles and boils associated with blood disorders. Sauline As a remedy for irritation of throat. Carmina As a remedy to correct indigestion, liver disorders, dyspepsia and

constipation. Sadoori Effective in cough and congestion of lungs, chronic and acute bronchitis,

cold, catarrh and esthma. Dawa Al-shifa Medicine used in hysteria, restlessness and tension. Safi Medicine used to purify blood, spurs the system on to start elimination

of the accumulated morbid matter, the root cause of all ailments, through the intestines, kidneys, and skin. Corrects digestive system, constipation and prevents pimples.

Maseehi (Ajmali) Effective in tuberculosis. Hab-e-Banafsha Effective in pain of throat, flu and influenza. Unbranded As remedy for common cold and flu. Unbranded Medicine used in fever, sore throat and irritation of throat.

RESULTS AND DISCUSSION

The data of (table 2) shows moisture content of different herbal drugs. Moisture content varied from 6% (Qarshi ghutti) to 20% (unbranded samples). It was found that branded samples had low moisture content as compared to unbranded sample. High amount of moisture allows the growth of microorganisms at a higher rate. As the water activity (aw) decreases, the growth of microbes is slowed or prevented (Scott, 1957 and Troller, 1973). The growth of mold is dependent on temperature, water activity, atmosphere and substrate i.e., pH, nutrients and inhibitors. At a given water activity, the growth response will change if any of the factors is optimal (Christian,1963).The herbal drugs were found contaminated with both pathogenic and non-pathogenic microorganisms.


Table 2: Microbial examination of herbal drugs

No Product Manufacturer

Sporeformer (c.f.u /gm)

coliform

Fungi Moisturcontent

Total viable count

Aerobic Anaerobic Fecal Non-fecal

Mold

Yeast

1 Qarshi ghutti

Qarshi industries (Pvt.)Ltd. Pakistan

19 7 - + 4 - 6 5.2x105

2. Surficol Qarshi industries (Pvt.)Ltd. Pakistan

13 7 - - 9 - 7 5.4x105

3. Khamira Gaozban


58 32 - + - - 9 6.2x105

4. Johar joshanda


17

3 - + 9 - 6 4.2x105

5. Sharbat Toot Siah


9 6 - - - - 6 2.1x105

6. Zanjabeen Qarshi industries (Pvt.)Ltd. Pakistan

22 15 - - 6 - 6 6.2x105

7. Musaffine Qarshi industries (Pvt.)Ltd. Pakistan

40 28 -

- 6 - 9 3.5x105

8. Sualine Hamdard Laboratries (waqf) Pakistan.

30 22 - - 12 - 6 6.6x105

9. Carmina Hamdard Laboratries (waqf) Pakistan.

45 32 - + - - 5 2.0x105

10. Sadoori Hamdard Laboratries (waqf) Pakistan

6 3 - + 3 - 5 2.9x105

11. Dawa Al-Shifa

Dawa khana Hakeem ajmal khan (Pvt) Ltd. Pak.

9 5 - - 12 - 4 1.5x105

12. Safi Hamdard Laboratries (waqf) Pakistan

15 8 - - 19 - 5 3.2x105

13. Maseehi Ajmali

Dawa khana Hakeem ajmal khan (Pvt) Ltd. Pak.

40 32 - + - - 6 4.3x105

14. Hab-e-Banafsha

Dawa khana Hakeem ajmal khan (Pvt) Ltd. Pak

59 42 + + 9 2 6 5.4x105

15. Khamira Unbranded 42 32 + + 9 - 20 9.3x105

16. Cough syrup

Unbranded 70 52 + + 30 9 20 9.2x105

The number of microorganisms were maximum (9.3 x 105 c.f.u/g) in unbranded sample. This contamination may be due to unhygienic conditions during processing. The presence of contaminants and the lack of proper labelling and appropriate packaging in samples were clear indications that the manufacturers did not comply with existing regulations on herbal drug production.The presence of mold and yeast indicate the potential for spoilage and mycotoxigenesis and causes the spoilage of herbal drugs (Mandeel, 2005). They secrete enzymes such as lipases, amylases and proteases, which can degrade lipids, starch and proteins. The maximum fungal load (30 c.f.u/g) was observed in unbranded cough syrup. The genus Aspergillus was the most dominant genus followed by Penicillium (Figure 1). Our results are in


agreement with other workers (Bugno et al.,2006). These moulds have been reported to have ability to produce mycotoxins.

Fig 1: Mycoflora detected in herbal drugs

The non fecal coliform bacteria were present in most of the samples under study but the fecal coliform bacteria were present in all unbranded samples and one branded sample (Hab-e-banafsha). The presence of Enterobacter and Escherichia coli in drug samples not only indicated improper hygienic conditions in processing but it was also definitely contradictory to the claims of the manufacturer. The sample which is supposed to remedy and cure many health problems might be dangerous to the consumer’s health. In most of the studies reported on the microbial contamination of food items, coliform bacteria have been common isolates (Robinson). Enteric disease caused by coliform bacteria result most likely from ingestion of material contaminated with these organisms (Stainer et al.,1986). The presence of these potentially hazardous contaminant microoraganism in seven of the sixteen drug samples was clearly hazardous to health since contaminant microorganisms alter the physical and chemical properties of materials they attack and may produce secondary metabolites like toxin. Moreover, these microorganisms are potential hazards themselves when ingested. There is therefore a high health risk in consuming herbal drugs unless regulatory agencies strictly require and monitor Good manufacturing Practices (GMP) among herbal drug manufacturers.

The data of (Table 2) shows aerobic spore former (in branded and unbranded samples). Maximum aerobic (70/g) and anaerobic sporeformer (52/g) load was present in unbranded cough syrup (sample 16). This might be due to unhygienic conditions during processing. The presence of sporeformer might be a result of contamination of raw materials due to highly resistant spores, the bacteria are able to withstand adverse environmental conditions. It was found that population varied quite markedly in each of the samples.

CONCLUSION

0

10

20

30

40

50

60

Aspergillussp.

penicilliumsp.

mucor sp. alternariasp.

No

. o

f is

ola

tes


Our results demonstrated that all of the samples exceeded the limit determined by the US Pharmacopoeia. This will open the way to investigate the raw materials, the processing procedure and general hygienic conditions under which drugs packed. Broadly speaking such marked change in bacterial counts among different samples may be due to unclean utensils and containers, particularly place and atmosphere where herbal drugs were packed.

REFERENCES

ALCALA, J.C., 1990. Bureau of food and drugs (BFAD), Alabang, Muntinlupa, Metro manila Philippines.

BUGNO, A., ALMODOVAR, A.A.B., PEREIRA, T.C., PINTO, T.J.A., SABINO, M., 2006. Occurrence of toxigenic fungi in herbal drugs. Braz. J. microbiol. 37: 47-51.

CALIXTO, J.B., 2000. Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (phytotherapeutic agents). Braz. J. Med. Biol., 33: 179-189.

CHAN, K., 2003. Some aspects of toxic contaminants in herbal medicines.Chemosphere. 52(9):1361-1371.

CHRISTIAN, J.H.B., Water activity and growth of microorganisms. In Recent advances in food sciences. Ed. Leitch, J.M., Phodes, D.N., 1963. 3: 248. Butterworth and co., London

GLORIA, D., ASUNCION, K.R., 1994. Microbial analysis of herbal drugs. 2(1): 1-16.

MANDEEL, Q. A., 2005. Fungal contamination of some imported spices. Mycopathologia. 159: 291-298.

NAHRSTEDTA, A., 1996. Quality of herbal medicines. Phytomedicines of Europe: their chemistry and biological activity. Orland FI, August. 27-28.

RAMADAY, ABED. E., 1962. Microbial examination of food and food products. Journal of food science. 45.

RIZZO, I., VEDOYA, G., MAURUTTO, S., HAIDUKOWSKI, M., VARSAVSKY, E. 2004. Assessment of toxigenic fungi on Argentinean medicinal herbs. Microbiol. Res.159 (2): 113-120.

ROBINSON, R.K., The microbial of milk 2nd ed. Elseiver. Applied science London and New York. 341-345.

STAINER, R.Y., INGRAHAM, J.L., WHEELS, M.L., PRINTER, P.R., 1986. The microbial world. 5th ed. New jersey: Prentice- hall. 496-50

SCOTT, W.J., 1957. Water relations of food spoilage microorganisms. Adv. Food research. 7: 84.

SNEATH, P.H.A., MAIR, N.S., SHARPE, M.E., HOLT, J.G. Bergey's manual of systematic bacteriology. Baltimore: William and wilkins, 999-1509.

TASSANEEYAKUL, W., RAZZAZI-FAZELI, E., PORASUPHATANA, S., BOHM, J. 2004. Contamination of aflatoxins in herbal medicinal products in Thailand. Mycopathologia. 158: 239-244.

The United States Pharmacopeia. 2005. 28. ed. Rockville: United States Pharmacopeial Convention.


TROLLER, J.A., 1973. Water relations of food born bacterial pathogens: A review. J. Milk food technol. 36:276.




BRONCHODILATOR, CARDIOTONIC AND SPASMOLYTIC ACTIVITIES OF THE STEM BARKS OF TERMINALIA ARJUNA

Janbaz, K. H.1, Hamid, I.1, Mahmood, M. H.2 and Gilani, A. H.2

Faculty of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan1. Department of Biological and Biomedical Sciences, The Aga Khan Medical College, Stadium Road, Karachi,

Pakistan.2

ABSTRACT

Terminalia arjuna, Roxb barks has a folkloric repute to possess spasmolytic, bronchodilator and cardiotonic, properties. The present study was undertaken to evaluate possible effects of methanolic extract of barks of Terminalia arjuna (Ta.Cr) in gastrointestinal, respiratory and cardiovascular systems via in vitro experiments to rationalize its use in traditional system of medicine. The phytochemical analysis of crude methanolic extract of Terminalia arjuna barks revealed the presence of tannins and saponins as methanol soluble extractable constituents. The spontaneous contractions in isolated rabbit jejunum were relaxed at tissue bath concentrations of 0.01-5.0 mg/ml, however, addition of propranolol to the tissue bath reduced the extent of relaxation and same level of effect was achieved at increased concentrations, i.e., 0.1-10.0 mg/ml. The Ta.Cr showed a weak relaxant effect on high K+(80 mM) low K+ (25 mM) and carbachol (1 µM)-induced contractions. Similarly, it also caused partial relaxation of high K+ (80 mM) and low K+ (25 mM)-induced contractions in isolated rabbit tracheal preparations. However, it relaxed completely the carbachol (1 µM)-induced contractions, but addition of propranolol (1 µM) to the tissue bath minimized the relaxant effect. Moreover, the Ta.Cr caused partial relaxation of the phenylephrine (1 µM)-induced contractions in isolated rabbit aorta preparations. Furthermore, the Ta.Cr showed a positive inotropic and positive chronotropic effects at lower tissue bath concentrations in isolated rabbit paired atria preparations but these effects were found to be antagonized by the presence of propranolol in the tissue bath. Further addition of Ta.Cr to the tissue bath caused a decrease in the magnitude of observed inotropic and chronotropic effects in isolated rabbit paired atria; which was found to be abolished on addition of atropine. Thus; in conclusion, the crude methanolic extract of barks of Terminalia arjuna, Roxb exhibited spasmolytic, bronchodilator and cardiotonic properties which are likely to be mediated through presence of dominant β-adrenergic agonistic activity along with presence of some weak muscarininc agonistic activity.


Key words: Terminalia arjuna, bronchodilator, cardiotonic, spasmolytic,

Corresponding author: Janbaz, K. H. Faculty of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan

INTRODUCTION

Terminalia arjuna, Roxb. (Combretaceae) is commonly known as Arjun (Khare, 2007) and is found throughout India and Pakistan (Kiritikar and Basu, 1987). It is a large evergreen tree with buttressed trunk and drooping branches, having smooth grayish bark. Leaves are simple, oblong and unequal sided, green from above but pale brown beneath. Flowers are white in panicles of spikes with linear bracteoles. Fruits are ovoid or oblong with 5-7 short, hard angles or wings (Kumar and Prabhaka, 1987).

Phytochemical investigations on different plant parts revealed the presence of constituents like triterpenoid (arjunolic acid) (Ramesh et al., 2011), triterpenes diglucoside terminolitin (23-deoxyarjunolitin) (Singh et al., 1995), oleanane-type triterpene (terminoside A), from stem bark (Ali et al., 2003a), triterpene glycoside like arjunetoside, oleanolic acid, arjunic acids from root barks (Upadhyay et al., 2003), oleanane type triterpene glucosyl esters like arjun glucosides IV and arjun glucosides V from bark (Wang et al., 2010a), 18,19-secooleanane-type triterpene glycosyl esters, namely arjunasides A, arjunasides B, arjunasides C, arjunasides D and arjunasides E from the bark (Wang et al., 2010b). Cardenolides like 14, 16 dianhydrogitoxigenin-3-ß-D-xylopyranosyl (1→2)-O-ß-D-galactopyranoside from seeds (Yadav and Rathore, 2000) and 16,17-dihydroneridienone 3-O-ß-D-glucopyranosyl-(1→6)-O-ß-D-galactopyranoside from roots (Yadav and Rathore, 2001). Similarly, naphthanol glycoside, i.e., arjunaphthanoloside from the stem barks (Ali et al., 2003b) and ursane type triterpene glucosyl esters including 2α,3ß-dihydroxyurs-12,18-dien-28-oic acid 28-O-ß-D-glucopyranosyl ester from the bark (Wang et al., 2010c). Moreover, ellagitannins like arjunin (Kandil et al.., 1998) and ellagic acid (Bajpai et al., 2005), gallitanins, (gallic acid and ethyl gallate) (Petit et al., 1996), hydrolyzable tannin like casuarinin (Kuo et al., 2005ab), flavones (luteolin) (Petit et al., 1996) and flavonoids (Bhuyan and Saikia, 2005).

In indigenous systems of medicine, the barks of Terminalia arjuna, Roxb are found to be useful in asthma (Gruenwald et al., 2000);, blood diseases (Kirtikar and Basu, 1975; Gruenwald et al., 2000), dysentery (Kirtikar and Basu, 1975; Gruenwald et al.,., 2000; Jain and deFillips, 1991), ulcers, tumors (Hartwell, 1982), and relieves fatigue (Kiritikar and Basu, 1987). Moreover, it helps to manage anaemia (Kirtikar and Basu, 1975; Gruenwald et al., 2000), diabetes (Hansel et al., 1998; Gruenwald et al., 2000), cardiopathy (Burkill, 1966; Kirtikar and Basu, 1975; Bone, 1996), cirrhosis of the liver (Bone, 1996; Gruenwald et al., 2000), hypertension (List and Hohammer, 1969-1979; Kapoor, 1990; Bone, 1996; Gruenwald et al., 2000), leucorrhoea (Gruenwald et al., 2000), otalgia (Kirtikar and Basu, 1975; Jain and deFillips, 1991), inflammations (Bone, 1996), internal and external blood loss (Kumar and Prabhaka, 1987)

Scientific investigations revealed inhibition of platelet function (Malik at el., 2009), improve gastric ulcer healing (Devi et al., 2007ab), protective effect against CCl4- induced oxidative stress to heart (Manna et al., 2006; Manna et al., 2007) and cardio-protective effect (Karthikeyan, et al., 2003).


Aims and objectives

Terminalia arjuna, Roxb has traditionally been used for the relief of asthma, bronchitis, and dysentery, but it has not been thoroughly investigated to rationalize its use in gastrointestinal and respiratory tract ailments. The present study was undertaken to explore its therapeutic potential in the management of ailments pertaining to gastrointestinal and respiratory systems to validate its folkloric use in native systems of medicines.

MATERIAL AND METHODS

Plant Material

The plant material pertaining to Terminalia arjuna, Roxb. was collected from the residential area of the Bahauddin Zakariya University Multan, Pakistan. The plants was authenticated by the kind cooperation of an expert taxonomist (Prof. Altaf Hussain Dasti), at the Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan and a voucher specimen was deposited at the herbarium of the Institute. The plant material was rendered free from adulterated materials and vegetative debris through manual picking and grinded to coarse powder with the help of a special herbal grinder.

Crude extract

The powdered plant material (1 kg) was subjected to maceration in 70% aqueous-methanol in amber coloured bottle at room temperature for 8 days with occasional shaking. (Harborne, 1973). The soaked material was passed through muslin cloth to remove the vegetative material and the fluid obtained was filtered through Whattman-1 filter paper. The filtrate was evaporated on a rotary evaporator (Rotavapor, BUCHI labrotechnik AG, Model 9230, Switzerland) at 37ºC under reduced pressure to thick paste like consistency (approximate yield was 12%) and the extract obtained was stored at -4°C in air tight jars. The plant extract (0.3 g) was dissolved in 1ml of distilled water to produce stock solution. From this stock solution further dilutions were made. Solutions were freshly prepared on the day of experiment.

Preliminary Phytochemical Screening:

Ta.Cr was subjected to phytochemical screening for the detection of saponins, tannins, phenols, coumarins, alkaloids and anthraquinones as possible constituent of the plant (Harborne, 1984; Harborne, 1998). The saponin presence was detected by the froth formation on vigorous shaking of the aqueous extract solution. Development of blue green or dark green coloration on mixing of aqueous FeCl3 with extract solution indicated presence of phenols and tannins. The coumarins as plant constituents were detected on emission of fluorescence in UV light from pieces of filter paper which were exposed to the vapors emerging from boiling aqueous solution of plant extract, subsequent to treatment with NaOH. The alkaloid presence was noted by the appearance of yellowish brown coloration on mixing of Dragendorff’s reagent with HCl treated aqueous plant extract solution. The appearance of pink, violet or red coloration on exposure to NH4OH of the mixture of benzene with aqueous solution of plant extract already acidified with 1% HCl was taken as presence of anthraquinones among the plant constituents.


2.4. Chemicals:

The chemicals, solvents, drugs and reagents used in these experiments were of highest purity and of reagent analytical grade. Acetylcholine chloride, carbachol (carbamylcholine), isoprenaline, propranolol, potassium chloride (KCl) and verapamil hydrochloride were purchased from Sigma Chemical Company, St. Louis, MO, USA, while calcium chloride (CaCl2) was purchased from Merck (Merck, Darmstadt, Germany). All the solutions were freshly prepared in distilled water on the day of experiment.

Animals and housing conditions

Rabbits, guinea-pigs and mice of either sex of local breed were used for the experiments and kept at the animal house of The Aga Khan University, Karachi, maintained at 23-25°C and were given standard diet and tap water. Food was withdrawn 24 hr prior to the experiments from animals but had free access to water. Guinea-pigs were sacrificed by cervical dislocation and rabbits sacrificed by a blow on back of the head. Mice were used for the in vivo anti-diarrhoeal study.

Isolated Rabbit Jejunum Preparation

The anti-spasmodic activity of plant material was studied using isolated jejunum as described by Gilani et al. (2005a; 2006).

Rabbit was killed through cervical dislocation, abdomen was incised; jejunum segment of 2 cm length was dissected out and was rendered free from adhering mesenteries. Each segment was mounted between two stainless steel hooks in l0 ml tissue bath containing normal Tyrode’s solution (pH 7.4), maintained at 37°C and aerated with carbogen (5% CO2 and 95% O2). A pre-load of 1 g was applied and tissue was allowed to equilibrate for a period of 30 minutes, during which the tissue was washed with fresh fluid at an interval of every 10 minutes prior to the addition of any drug. The spontaneous contractions were recorded isotonically through Powerlab Data Acquisition system (AD instruments, Sydney, Australia).

Subsequent to equilibration, the tissue preparation was repeatedly treated with acetylcholine (0.3 µM) at 3 min intervals to stabilize the preparation. When three successive responses to acetylcholine were found to be identical in magnitude, the preparation was considered stable and exposed to test materials for the evaluation of antispasmodic action in a cumulative fashion. The test material-induced relaxant effect was measured as percent change in spontaneous contractions of rabbit jejunum immediately before the addition of test materials.

Determination of Calcium Channel Blocking Activity

The possible mechanism of the relaxant action on the part of test material was determined by its effects on high K+(80 mM)-induced contractions in isolated rabbit jejunum preparations as described by Faree et al. (1999). The isolated rabbit jejunum preparations on exposure to high tissue bath concentration of K+(80 mM), exhibits a sustained contractile response. Test materials were added to the tissue bath in a cumulative fashion to achieve concentration-dependant relaxant responses in the isolated rabbit jejunum preparations (van-Rossum, 1963). The extent of relaxation of pre-contracted intestinal preparation was expressed as percent of the control responses mediated by K+(80 mM). The smooth muscle contraction on exposure to high K+(80 mM) is mediated through the influx of Ca+2 from extra-cellular fluid and the test material capable


to inhibit this contraction is likely to exert its effect through Ca+2 channel blockade (Bolton, 1979).

The proposed calcium channel blocking mechanism of the test materials was confirmed further by the methods described by Gilani et al (2005a). The isolated rabbit jejunum preparation was allowed to stabilize in normal Tyrode’s solution, followed by 45 min exposure to a K+ normal, Ca+2 free and EDTA (0.1 mM) containing Tyrode’s solution for removal of calcium from the tissues. The isolated tissue preparation was further incubated for 30 min in a Tyrode’s solution (K+ rich, Ca+2 free and containing EDTA) of the following composition (mM): KC1 (50), NaCl (91.04), MgCl2 (1.05), NaHCO3 (11.90), NaH2PO4 (0.42), glucose (5.55) and EDTA (0.1). Subsequently, Ca+2was added to the tissue bath in a cumulative fashion to obtain the control calcium concentration-response curves (CRCs). The stepwise increase in contractile response of the tissue revealed that its contractions are dependant on K+-induced influx of extra-cellular Ca+2.

On finding the control CRCs of Ca+2 to be super imposable (usually after two cycles), the tissue preparation was then washed and equilibrated with the test material for 60 min. Followed by reconstruction of the concentration response curves for Ca+2 and compared to the control curves. The concentration response curves of Ca+2 were developed in the presence of various concentrations of test material to assess a possible Ca+2 channel blocking effect. The calcium channel blocking activity on the part of test material was confirmed on shifting the concentration-response curves of calcium constructed in calcium-free medium towards right in a dose-dependant manner (Bolton, 1979).

Isolated rabbit tracheal preparations

Rabbits were starved for 24 hrs prior to experiment and were killed by a blow on the back of the head and tracheal tube was dissected out. The trachea was cut into rings about 3-4 mm in width, each containing about two cartilages. Each ring was opened by longitudinal cut on ventral side opposite to the smooth muscle layer, forming a tracheal strip with a central part of smooth muscle sandwiched between cartilaginous portions on the edges. The preparation was suspended in a 20 ml tissue bath containing Krebs physiological salt solution at 37°C aerated with carbogen. A tension of 1g was applied to each of tracheal strip and was kept constant throughout the experiment. The isolated rabbit tracheal preparation was equilibrated for 45 min before isometric tension of tracheal strips were recorded via force displacement transducers (FT-03) connected to Grass Polygraph Model 7.

The broncho-relaxant effect of the test material was studied on carbachol- and high K+(80 mM)-induced contractions in isolated tracheal preparation. Spasmodic contractions are produced in the isolated tissue preparations on exposure to high K+(80 mM) tissue bath concentrations. Afterward, cumulative addition of test material relaxed the isolated tracheal preparation. The process was repeated with carbachol as well.

The isolated rabbit paired atria preparations:

The paired rabbit atria from healthy rabbits were dissected out, cleaned from fatty tissues and mounted in tissue organ baths containing Krebs physiological salt solution. The isolated tissue preparation was aerated with carbogen gas at 37°C. The isolated paired rabbit atrial preparation used to exhibit spontaneous beating under the resting tension of 1g due to intact pacemaker cells. The tissue was allowed to be equilibrated for a period of 30 min before exposure to any test


material. The isolated paired atrial preparation allows evaluation of the possible effect of test material on both rate and force of atrial contractions. The amplitude of the recorded tracings represented the force of atrial contractions, whereas the number of contractions on the recorded chart represented the rate of atrial contractions. Isoprenaline (1 µM) was used as control inotropic agent and its tracings were recorded in duplicate. The contractile response of the isolated tissue preparation was recorded via a Grass force-displacement transducer (model FT-03) linked to the Grass Model 7 Polygraph.

2.14. Statistical analysis

All the data were expressed as mean ± standard error of the mean (S.E.M) and the median effective concentrations (EC50 values) are given with 95% confidence intervals (CI). The statistical parameter applied was the χ

2-test with P < 0.05 was considered as significantly different.

RESULTS

Preliminary phytochemical analysis:

Preliminary phytochemical analysis of the crude methanolic extract of Terminalia arjuna Roxb. (Ta.Cr) bark revealed the presence of saponins and tannins as methanol soluble extractable constituents of Terminalia arjuna Roxb.

Effect of Terminalia arjuna Roxb. on isolated rabbit jejunum preparations

The crude methanolic extract of barks of Terminalia arjuna Roxb. (Ta.Cr) was tested on spontaneously contracting isolated rabbit jejunum preparations for possible relaxant effect. The Ta.Cr exhibited concentration dependent relaxant effect in tissue bath concentration range of 0.01 to 5.0 mg/ml with EC50 value of 2.015 mg/ml (95% CI, 1.473-2.756, n = 5) (Figure 1), whereas in the presence of propranolol the relaxations were achieved at a concentration of 0.1 to 10.0 mg/ml, with EC50 value of 3.454 mg/ml (95% CI; 3.138-3.800, n = 5) (Figure 2). The Ta.Cr caused partial relaxation of low K+ (25 mM; n=5), high K+(80 mM; n=5), and carbachol (1 µM; n=5)-induced contractions in isolated rabbit jejunum preparations (Figure 3).

Effect of crude extract of Terminalia arjuna Roxb on isolated rabbit tracheal preparations

The Ta.Cr while testing on isolated rabbit tracheal preparations, exerted concentration dependent (0.1-10.0 mg/ml) relaxant effect on carbachol (1 µM)-induced contractions with EC50 value of 3.478 mg/ml (95% CI; 2.413-5.011, n = 5), however, relaxation was found to be partial in presence of propranolol (n = 5). The Ta.Cr also partially relaxed the low K+(25 mM; n = 5) and high K+-(80 mM; n=5)-induced contractions (Figure 4&5).

Effect of crude extract of Terminalia arjuna Roxb on isolated rabbit aorta preparations

The Ta.Cr on application to isolated rabbit aorta preparations, partially relaxed the phenylephrine (1 µM)-induced contraction (n=5) (Figure 6).


Figure 1: Tracing showing effect of crude methanolic extract of Terminalia arjuna Roxb (Ta.Cr) on the spontaneously contractions of isolated rabbit jejunum preparations. (a)

(b)

(c)


Figure 2: Tracing showing effect of crude methanolic extract of Terminalia arjuna Roxb (Ta.Cr) on (a) carbachol (1 µM), (b) high K+(80 mM) and (c) low K+(25 mM)-induced contractions in isolated rabbit jejunum preparations.

Figure 3: Effect of crude methanolic extract of Terminalia arjuna Roxb. (Ta.Cr) on the spontaneous contractions in the absence and presence of propranolol, high K+(80 mM), low K+(25 mM), carbachol (1 µM)-induced contractions in isolated rabbit jejunum preparations. Values are shown as mean ± S.E.M., n = 5-6 Effect of the crude extract of Terminalia arjuna Roxb on isolated rabbit paired atria The Ta.Cr while testing in isolated rabbit paired atria, exhibited a positive inotropic and positive chronotropic effects at low tissue bath concentrations (0.01-0.3 mg/ml), which were found to be blocked on adding propranolol. On further increase in tissue bath concentration of Ta.Cr, there was decrease in both in force of contractions as well as rate of hear beat (1.0-3.0; n=5), however, addition of atropine to the tissue bath blunted the inhibitory influences and restored both the force of contractions as well as rate of heart beats (Figure 7). (a)

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Figure 4: Tracing showing the effect of crude extract of Terminalia arjuna Roxb on (a) high K+(80 mM)-induced concentration and (b) carbachol (1 µM)-induced contraction in the absence and presence of propranolol in isolated rabbit tracheal preparations.


Figure 5: Effect of crude extract of Terminalia arjuna Roxb (a) on high K+(80 mM), low K+ (25 mM) and carbachol (1 µM)-induced contractions (in the absence and presence of propranolol) on isolated rabbit tracheal preparations. Values are shown as mean ± S.E.M., n = 5-6.

Figure 6: Effect of crude extract of Terminalia arjuna Roxb on phenylephrine (1uM)-induced contractions in isolated rabbit aorta preparations. Values are shown as mean ± S.E.M., n = 5.

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Figure 7: Effect of crude extract of Terminalia arjuna Roxb on (a) force and (b) rate of contraction in the absence and presence of atropine (0.1 µM) and propranolol (1 µM) in isolated rabbit paired atria preparations.

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DISCUSSION

The preliminary phytochemical analysis of the crude methanolic extract of bark of Terminalia arjuna (Ta.Cr) revealed the presence of tannins and saponins as methanol soluble extractable constituents.

The Terminalia arjuna, Roxb possesses the folkloric repute of antispasmodic activity, hence, it was subjected to pharmacological investigations to explore its possible relaxant effect on spontaneously contracting isolated rabbit jejunum preparations. The Ta.Cr exhibited a concentration dependent (0.01 to 5.0 mg/ml) relaxant effect likely to be mediated either through Ca+2 channel blocking (CCB) mechanism, through K+ channel opening mechanism or muscarinic receptor antagonism (Gilani et al., 2005 a, b & c). The Ca+2 channel blocking activity is used to be determined by depolarization of the isolated tissue preparations following exposure to high K+(80 mM) as described by Farre et al., (1991). Exposure to high tissue bath concentrations, K+(> 30 mM) is reported to cause smooth muscle contraction through opening of voltage-dependent L-type Ca+2 channels, thus allowing influx of extra-cellular Ca+2 and hence, causing contractile effect (Bolton, 1979). The substances exerting inhibition of high K+-induced contractions are considered to be blocker of Ca+2 inlux (Godfraind et al., 1986). The Ta.Cr was also tested on high K+(80 mM)-induced contractions in isolated rabbit jejunum preparations to explore the mechanism of the observed relaxant effect. The Ta.Cr exerted weak inhibitory effect on high K+-induced contractions, hence possibility of mediation through blockade of Ca+2 channels was ruled out and observed relaxation of spontaneously contracting isolated rabbit jejunum was presumed to be mediated via some alternative mechanism(s). The substances that selectively relax the low K+(25 mM)-induced contractions in isolated rabbit jejunum preparations are considered to be potassium channel openers (Kishii et al., 1992), but Ta.Cr did not exert relaxant effect in this case, hence, it was speculated that observed relaxant effect of Ta.Cr was likely to be due to the presence of some anti-muscarinic component(s) and idea was supported by the evidence that Ta.Cr caused partial blockade of the carbachol (1 µM)-induced contractions. The Terminalia arjuna, Roxb has traditionally been used in the management of respiratory diseases, hence Ta.Cr was subjected to further evaluation for possible bronchodilator activity in isolated rabbit tracheal preparations. The Ta.Cr demonstrated weak relaxation of the high K+(80 mM) and low K+(25 mM)-induced contractions, hence, involvement of calcium channel blocking or potassium channel opening activities was not supported (Hamilton et al. 1986; Gilani et al. 2005c).

The Ta.Cr exhibited partial relaxation of the carbachol (1 µM)-induced contractions in isolated rabbit jejunum preparations but exhibited complete relaxation of the carbachol (1 µM)-induced contractions in isolated rabbit tracheal preparations, which may possibly be mediated through muscarinic receptor blockade or due to some alternative mechanism(s) like β–adrenergic agonistic activity as well as phosphodiesterase (PDE) inhibitory activity (Brain and Hoffman, 2001). The mechanism of the observed relaxant effect of Ta.Cr on carbachol (1 µM)- induced-contractions in isolated rabbit tracheal preparations was evaluated further by testing Ta.Cr on carbachol (1 µM)-induced contractions in the presence of propranolol (1 µM); a β–adrenergic receptor antagonist (Westfal and Westfal, 2006). The relaxant effect of Ta.Cr on carbachol (1 µM)-induced contraction was partially blocked by propranolol, thus, indicating that Ta.Cr may possess some β-adrenergic agonistic activity.


The Terminalia arjuna, Roxb is also reputed for its folkloric use in the management of the cardiovascular diseases, hence it was evaluated for its possible effect on isolated rabbit aorta preparations. The Ta.Cr was unable to demonstrate any effect when applied to the isolated rabbit aorta preparations but it partially relaxed the phenylephrine (1 µM)-induced contractions, which is likely to be mediated through ß adrenergic agonist, α adrenergic antagonist or muscarinic agonist activities

The β-adrenergic receptors agonists via stimulation of adrenergic receptors may cause increase in intracellular level of cAMP, whereas the raised intracellular level of cAMP produces relaxant effect in smooth muscles, and increase in inotropic and chronotropic activities of the cardiac tissues (Orallo et al., 2005). The possible presence of β–adrenergic agonistic activity on the part of Ta.Cr, as detected in case of isolated rabbit tracheal preparation was investigated further on isolated rabbit paired atria preparations. The Ta.Cr demonstrated positive inotropic and positive chronotropic effects at lower tissue bath concentrations, which was blocked on addition of propranolol to the tissue bath and was suggestive of the presence of ß-adrenergic agonistic activity. However, the observed decrease in heart rate and force of contraction on elevating the tissue bath concentration of TaCr, which was blocked on addition of atropine to the tissue bath, reflected the presence of cholinergic muscarinic activity. Thus, indicating that there might be presence of multiple components likely to exert β–adrenergic agonistic, cholinergic muscarininc agonist and antagonist activitie in crude methanolic extract of Terminalia arjuna, Roxb.

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EFFECT OF NON-GENETIC AND GENETIC FACTORS ON BIRTH WEIGHT OF MENGALI SHEEP OF BALOCHISTAN

Mohammad Masood Tariq1, Masroor Ahmed Bajwa1, Shakeel Babar1, Abdul Waheed2,

Farhat Abbas Bukhari1, Tahir Hameed1, Illahi Bakhsh Marghazani3, Yasir Javed1

1Center for Advanced studies in Vaccinology and Biotechnology (CASVAB), University of Balochistan, Quetta, Pakistan. 2Faculty of Veterinary Sciences, Bahauddin Zakariya University, Multan, Pakistan. 3Livestock and Dairy Development Department, Balochistan, Pakistan.

ABSTRACT

Data on birth weight of 2377 lambs obtained from 581 dams and 56 sires of Mengali sheep born during 2005 and 2009 were recorded and analyzed to identify the factors affecting birth weight of Mengali lambs. Mengali flocks were kept under semi-intensive condition in four stations at three different locations (Experimental Station Center for Advanced studies in Vaccinology and Biotechnology (CASVAB), University of Balochistan, (ESC), Quetta, Killi Hassni, Quetta; Khadkucha, Mastung and Peer Wala (Mal), Nushki). The lambing occurred spring and autumn, however majority of the Mengali sheep (85 %) lambed during spring between (February and March months). Among the total lambs born, the male to female sex ratio was 49.43: 50.57 respectively and shown no significant difference between two sexes. The overall least-squares means for birth weight of males and females pooled over parity and type of birth were 3.61±0.36 and 3.48±0.39 kg respectively. The estimate of heritability for birth weight was observed as 0.39±0.06. Study on non-genetic factors revealed that the birth weight was significantly (P<0.05) affected by period of birth, season of birth, sex of the lamb, parity and location of flock. Hence, efforts should be made for better management of pregnant ewes when the climatic conditions are not conducive to them.

Keywords: Heritability, lambs, meat, parity, sex ratio

Corresponding Author: Mohammad Masood Tariq, Center for Advanced studies in Vaccinology and Biotechnology (CASVAB), University of Balochistan, Quetta, Pakistan. Email [email protected], Tel.: +9222855679, Fax: +9220913134


INTRODUCTION

Sheep and goat dwell in a special place in the rural economy of Pakistan. Mengali sheep is an important sheep breed of Balochistan. The main features of this breed are compact body with notable height, pendulous belly, fat tail, body color is black/tan or brown with white patches on the belly or vise versa (Kakar and Ahmad, 2004; Khan et al., 2007, Tariq et al., 2011), and ears are usually medium in size, big black face and white spots on the head with Roman nose, both males and females are polled. Mengali sheep are spread in Mustung, Kalat, Khuzdar and Quetta districts. They are reared mainly for meat, milk, wool and the primary by-product is skin. The animals are well adapted to the local conditions of most of the Districts of Quetta, Khuzdar, Chaghi, Kalat, Mastung, Awaran and Kharan of Balochistan Province. Source of origin of Mengali sheep is still unknown. This sheep breed is mostly raised by native Baloch tribe “Mengal” (main tribe of Chaghi area), therefore known as Mengali (Tariq et al., 2011). Sheep raising in Pakistan (especially in Balochistan) and neighboring countries is mostly kept by local pastoralists on extensive production system. In such a system, output is lower than in an intensive system. Within local area natural selection of breeds is a suitable method for genetic improvement in the traditional low input production systems (Khan et al., 2007; Ali, 2008). Performance traits of farm animals are determined not only by an animal's genetic potential for growth but also by maternal genetic and permanent and temporary environmental effects. Hence, to achieve optimum genetic progress in a selection program, both direct and maternal genetic components should be taken into account, especially if there is not an antagonistic relationship between them (Snowder and Van Veilk, 2003). Birth weight is the first observed trait in life of an animal on which growth, production, and reproduction traits are dependent. So the present investigation was carried out to study the various non-genetic factors affecting birth weight of Mengali lambs born under semi-intensive farm conditions.


The data used in this study were scored from 2005 to 2009 on 2377 lambs obtained from 581 dams and 56 sires of Mengali sheep. Mengali flocks were kept in four stations at three different locations (Experimental Station CASVAB, UoB, Quetta (ESC), Killi Hassni, Quetta; Khadkucha, Mastung and Peer Wala (Mal), Nushki). The Mengali sheep were reared under semi-intensive system of management. The animals were grazed from 8.00 to 17.00 hours. In addition ewes were maintained with 150 to 250 g of concentrate mixture. During the shortage of fodder, animals were fed dry roughages (maize and sorghum and orchard waist) and urea and molasses treated wheat straw. The data were analysed by least-squares technique (Harvey 1990) adapting linear model after adjusting the fixed effects of period and season of birth, sex of the lamb and parity, since these being considered as the potential sources of variation. The period of birth has been divided into five years and the season of birth was classified as season 1 (October to March) spring lambing and season 2 (April to September) autumn lambing. The parity has been grouped as 1st, 2nd, 3rd, 4th and 5th and above. The data were subjected to standard statistical analysis as per Snedecor and Cochran (1986). The fixed effects model used for the analysis estimating the least-squares means to find out the non-genetic factors affecting birth weight in the Mengali sheep was as follows:

Y ijlklm = m + Pi +Sj+ Tk + Ol+ eijklm,


Where

m = overall mean when equal subclass frequencies exist, Pi =effect of ith period (i= 1to 5), Sj =effect of jth season (j =1 to 2), Tk =effect of kth sex of lamb (k= 1 and 2), Ol =Effect of lth parity (l = 1 to 5) Qn =Effect of nth flock (1-4) and Eijklmn =Residual error.

The heritability for birth weight was estimated by paternal half-sib method by including the sires in the model after adjusting the data for significant non-genetic factors.

RESULTS AND DISCUSSION

Sex Ratio

Among the total lambs born, the male to female sex ratio was 49.43: 50.57 respectively. The chi-square test (of goodness of fit) revealed no significant difference between two sexes. The result of sex ratio for the present study was in agreement with many researchers (Maria, 1972; Nawaz, 1983; Kakar, 1993; Akhtar, 1996; Memon, 1998; Sharif, 2001; Hussain, 2006). Maria (1972) reported that the sex ratio lambs (Male: Female) in Ossimi was 46.92:53.08, respectively. Nawaz (1985) reported sex ratio in Awassi, Kachhi and Awassi x Kacchi was 49.26; 48.88 and 50.87 in male and 50.74; 51.12 and 49.13 in female, respectively. Kale and Raman (1994) reported a sex ratio of 52.93% males for Madras Red and 50% for Mandya sheep. Akhtar (1996) reported that sex ratio values in Hissardale sheep flock was found 52.5: 47.5 males and females, respectively. Memon (1998) obtained that secondary sex ratio values were (47:50:52.50); (55.30:44.70) and (52.20:47.80) in Kacchi; Kooka and Dumbi sheep (Male: Female) respectively. Secondary sex ratio for Balochi, Rakhshani, Bibrik and Harnai sheep were (39:61); (37:62); (43:57) and (43:57) respectively (Kakar, 1993). Sharif (2001) determined the sex ratio of Balochi and Bibrik sheep breed of Balochistan were 44:56 and 42:58 (Male: Female) respectively. Hussain (2006) also estimated sex ratio 47.4:52.6 in Hissardale flock, respectively. There were many investigators mentioned that sex ratios showed much variation due to breed differences.

Lambing Pattern

The lambing pattern revealed that the lambing occurred in spring (February and March) and 85% and autumn (September and October) 15%. It is in accordance with the information of Akhtar (1996) for Hissardale sheep, Memon (1998) for Kacchi; Kooka and Dumbi sheep Pattanayak et al. (2003) for Ganjam sheep and Hussain (2006) for Hissardale sheep and Thiruvenkadan et al., (2008) for Mecheri sheep. However, Patro et al. (2006) reported that indigenous sheep of Kendraprada district of Orissa were bred throughout the year and they further noted that majority of the ewes (67 per cent) came to heat in April to June months.

Effect of Non-Genetic Factors

The least-squares means of genetic traits of birth weight (kg) of Mengali lamb are presented in Table 1.


Period of Birth

It was observed from the results that the period of birth had significant (P<0.05) effect on birth weight (Table 1). Further it has been noted that the lambs born in period 4 and 5 (2008-2009 and 2009-2010) had higher birth weight (3.58±0.38 and 3.56±0.40 kg, respectively). While lambs born during the period 1 (2005-2006) had lowest birth weight (3.46±0.29 kg). The present findings of significant effect of period are in agreement with the observations made by Bobhate et al. (2003), Nehra and Singh (2006), Jadav et al. (2007) and Thiruvenkadan et al., (2008). The significant differences in birth weight among lambs born in different period may be attributed to difference in management, selection of rams and environmental conditions.

Table 1. Least-squares (± S.E) means of non genetic traits of birth weight (kg) of Mengali lamb

Effect Number of observations Mean ± S.E

Overall 2377 3.54±0.36 Period * P1 (2005-2006) 279 3.46±0.29a P2 (2006-2007) 331 3.51±0.34b P3 (2007-2008) 450 3.55±0.37 c P4 (2008-2009) 584 3.58±0.38 c P5 (2009-2010) 733 3.56±0.40 d Season * Season 1 (October-March) 2026 3.68±0.35 b Season 2 (April –September) 351 3.49±0.33 a Sex of the lamb * Male 1174 3.61±0.36b Female 1203 3.48±0.39a Parity * First 714 3.40 ± 0.35a Second 568 3.49 ± 0.42b Third 520 3.54 ± 0.38bc Fourth 364 3.58 ± 0.33cd Fifth and above 211 3.61 ± 0.39d Flock at different location ESU Quetta 307 3.62±0.33 c Killi Hassni, Quetta 631 3.53±0.38 b Khadkucha, Mastung 763 3.59±0.35 c Peer wala (Mal) Nushki 676 3.47±0.39 a * significant , Means bearing different superscript indicate significant difference (P<0.05).


Season of Birth

The lambs born during spring were heavier (3.68±0.35 kg) than the lambs born during autumn season (3.49±0.33 kg). Similarly, single born lambs were (3.72±0.29 kg) heavier than twin born lambs (3.45±0.34 kg). The season had significant (P<0.05) effect on birth weight of lambs. It is in accordance with the reports of Akhtar et al. (2001) who analyzed performance data on Hissardale lambs and stated that, the lambs born during spring were heavier (3.8±0.01 kg) than the lambs born during autumn season (3.6±0.02 kg). Similarly, single born lambs were heavier than twin born lambs (3.9±0.14 vs. 3.5±0.03 kg). Male lambs were also heavier (3.9±0.02 kg) than the female lambs (3.5±0.02 kg). Sivakumar et al. (2006) also reported that lambs born during September to February were having higher birth weight than those born during March to August. Thiruvenkadan et al., (2008) reported that lambing occurred throughout the year, however majority of the Mecheri sheep (76.2 per cent) lambed between September and February months.

The ewes those conceived during September to October months had lambing during February and March, favourable environmental conditions with good availability of the fodder during the gestation period, which might have been contributed to higher body weight at birth.

Parity

Parity has significant effect (P<0.05) on birth weight of the lambs. The birth weight of the lambs was lowest in the first parity and it increased as the parity advanced with maximum in the fifth and above parity. It is in accordance with the report of Karunanithi et al. (1994) for the same breed, Mandal et al. (2003) for Muzaffarnagari sheep and Thiruvenkadan et al. (2008) for Mecheri sheep.

Sex of the Lamb

The average birth weight of different sexes revealed that the male lambs had higher birth weight than females. The sex of the lambs had significant (P<0.05) effect on birth weight. High birth weight of ram lambs and significant effect of sex was also reported by several authors (Karunanithi et al., 1994, Sivakumar et al., 2006; Ravimurugan et al., 2007 and Thiruvenkadan et al., 2008). Higher growth in prenatal stage under the influence of male sex hormones with anabolic effect (Hafez 1962) might be the reason for higher birth weight of male lambs.

Flock at Different Locations

The flocks kept at different locations had significant (P<0.05) effect on birth weight of lambs. Flock kept at ESC, Quetta and Khadkucha, Mastung location performed significantly better in all traits compared to other flocks. This difference might be due to better management and regular supplementation of ration to this flock. Refiq et al. (2009) reported that flocks raised at different location had significant effect on birth weight of lambs; that result was found similar to the present study under report.


Genetic Parameter

The estimate of heritability for birth weight observed in this study was 0.39±0.06. Heritability estimates for BW were similar to the estimates reported by many researchers; Pollott et al. (1998) in Turkish Awassi lambs (0.44 ± 0.09), Al-Shorepy (2001) in crossbred lambs (0.32) and Ali (2008) in Karakul (0.30). The estimates of the present study were lower than those obtained by many researchers; Mahmoud (2000) in Barki (0.81), Rahmani (0.66) and Ebangi et al. (2001) (0.61) in Fulbe sheep. The estimates were higher than those reported by Yazdi et al. (1997) in Baluchi (0.14), Hussain (2006) in Thalli (0.07 ± 0.02), Thiruvenkadan et al. (2008) for Mecheri lamb 0.061 ± 0.071, Jadhav et al. (2007) for crossbred sheep 0.14 ± 0.02, Refik et al. (2009) in Turkish Merino (0.14 ± 0.02), and Borg (2009) in Targhee (0.19). The relatively moderate estimate of heritability indicated that a greater portion of variation in growth traits was due to non-genetic factors.

These differences in birth weights may be due to breed, size of the data set or method of estimation used in different studies, production system, climatic conditions and ecological zones, where sheep farms were practiced. This wide variation in birth weight indicated that mass selection for higher birth weight could be made in order to improve the birth weight of lambs so that early lamb mortality may be reduced.

CONCLUSIONS

The study revealed that the birth weight of Mengali lambs was influenced by various non-genetic factors viz., period of birth, season of birth, sex of the lamb, parity and locations of flock. Efforts should be made for better management of pregnant ewes when the climatic conditions are not conducive to them. Since the birth weight is the first observational trait on which growth, production, selection and reproduction traits are dependent; efforts have to be made for improvement of the same through better nutrition and management.

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INFLUENCE OF HYPERGLYCEMIA ON OXIDATIVE STRESS AND DNAase-MEDIATED GENOMIC FRAGMENTATION COUPLED WITH APOPTOTIC AND NECROTIC CELL DEATHS IN KIDNEY

Mohammed Saleem1, Muhammad Shoaib Akhtar2*Sidharta D. Ray3, Bashir Ahmed4

1Faculty of Pharmacy, G.C. University, Faisalabad, Pakistan, 2Faculty of Pharmacy, University of Sargodha, Sargodha, Pakistan, 3Long Island University Brooklyn, New York, USA, 4College of Pharmacy, University of Punjab, Lahore, Pakistan.

ABSTRACT

Diclofenac (DCLF) is a potent inhibitor of prostaglandin synthesis and an established antipyretic and analgesic agent. It also has a nephrotoxic profile caused by generation of reactive oxygen species and enhanced apoptotic DNA fragmentation. The specific goals of this investigation were to determine: (i) sensitivity of hyperglycemic mice to nephrotoxic doses of DCLF, (ii) whether hyperglycemia modulates genomic DNA-fragmenting potency of DCLF in kidney, (iii) whether hyperglycemia would increase DCF-induced oxidative stress in kidney and (iv) whether hyperglycemia can alter apoptotic and necrotic death of kidney cells. Male ICR CD-I mice were divided into normal and diabetic groups. Diabetes was induced by administering 100 mg/kg streptozotocin. Both normal and diabetic groups were divided into sub-groups on basis of nephrotoxic doses of DCLF i.e., 100 and 200 mg/kg i.p. The mice were sacrificed after 24-hrs and determined blood urea nitrogen (BUN), lipid peroxidation level and caspase-activated DNAase-mediated genomic DNA fragmentation. In addition, kidney tissues were examined histopathologically and checked for DNA damage. The results obtained showed that DCLF is a potent nephrotoxic agent as it increases BUN and powerfully induces oxidative stress depicted by significant increase in malandialdehyde (MDA) level which lead to massive DNA fragmentation coupled with apoptotic and necrotic cell deaths in both diabetic and non-diabetic groups in vivo. In addition, data have indicated that DCF causes greater nephrotoxicity in diabetics as compared to non-diabetics as shown by higher values of BUN, LPG and genomic DNA fragmentation.

Key words: Diclofenac, diabetes, non-steroidal anti-inflammatory drug, BUN.

*Address for Correspondence: Muhammad Shoaib Akhtar, Professor of Pharmacology, Faculty of Pharmacy, University of Sargodha, Sargodha, Pakistan [email protected]


INTRODUCTION

Diabetes mellitus (DM) is an endocrine disorder characterized by inability of pancreas to secrete significant amount of insulin to maintain physiological levels of blood glucose. Long standing DM is associated with a complex array of disorders like neuropathy, cardiomyopathy, retinopathy, hepatomegaly and nephropathy. Among these, however, nephropathy is the most commonly encountered complication in patients with type I and type II DM. It has been reported that this complication is on the rise world wide (Jorns et al., 2001). One possible explanation for this complication involves the hyperglycemia induced formation of advanced glycosylation end-products which generate reactive oxygen species, suggesting that hyperglycemia causes oxidative damage to cells through NF-κβ-dependent pathways (Mohamed et al., 1999). Streptozotocin (STZ) is a powerful inducer of diabetes in experimental animals by destroying pancreatic ß-cells. Diabetes mellitus could also induce polymorphic alterations of metabolic activities of cytochrome P-450-dependent monooxygenases in induced diabetic animals. The diabetic condition is known to increase (Ray et al, 2001) or decrease (Rour et al, 1986) activities of several drug metabolizing enzymes. For example, STZ-induced hyperglycemia is known to aggravate cardiotoxicity of doxorubicin (Al-Shabanah et al, 2000), thioacetamide-induced hepatotoxicity (El-Hawari and Plaa, 1983) and increase cyclosporine-A-induced renal tumorigenicity (Carmo et al., 2000). Increased toxicity in specific target organs is due to the inhibition of specific drug metabolizing enzymes, whereas decreased toxicity may be due to the polyuremia in the diabetics.

DCF is a potent inhibitor of prostaglandin synthesis and an antipyretic and analgesic agent. However, severe renal dysfunctions are occasional consequences of its toxicity. Although DCF is extensively used, mechanisms underlying its potential to cause multi-organ toxicity remain unknown. A majority of the previous publications primarily describe the forms of hepatic cytochrome P-450 that metabolize DCF, and its toxic metabolites (Tang et al., 1999). However, basic biochemical mechanisms underlying DCLF-induced nephrotoxicity remain poorly investigated, particularly in diabetics. Additionally, from a mechanistic standpoint, it is not known as to how diabetics respond to NSAID-induced nephrotoxicity. Previous studies have investigated the mechanisms underpinning the nephrotoxic potential of DCF in experimental animals (Hickey et al., 2001). The nephrotoxic doses (100-300 mg/kg) of DCF induce apoptotic death of kidney cells in addition to necrosis (Hickey et al., 2001). The present investigation was aimed at understanding the modulatory influence of diabetic condition on key events associated with DCF's apoptogenic potential including the induction of caspase-activated DNAase (CAD) which is considered a principal suspect in orderly fragmentation of genomic DNA. Additional focus was placed upon establishing a cause-and-effect relationship between oxidative stress, DNA fragmentation and DCF's apoptogenic potentia1.


Chemicals used

Chemicals were analytical grade or higher and were used without purification and included streptozotocin, diclofenac (DCF) sodium, disodium EDTA, glacial acetic acid, perchloric acid, ethidium bromide, malonaldehyde, N-laurosylarcosine, sulfuric acid, Lysis buffer, 0.5% triton X-I and trichloroacetic acid


Animals used

Adult male ICR (CD-I) mice weighing 35 g each were of Spargue-Dawely strain and were given access to lab chow (Purina Laboratory rodent chow and tap water ad libitum. The animals were allowed to acclimate in an environment of controlled temperature (22-25oC), humidity, and light/dark (l2h/12h) cycle for one week prior to study. Food and water were withdrawn from all the experimental animals for one hour following drug treatments, and returned. All animal procedures received prior approval by the Institutional Laboratory Animal Care and Use Committee, and met all state and federal regulations.

Induction of diabetes

STZ was dissolved in citrate buffer (pH 4.7) and was injected ip to induce hyperglycemia. Five doses of STZ (100 mg/kg) were administered ip every other day for ten days to induce hyperglycemia. Blood glucose level >200mg/dl was considered as hyperglycemic. DCF was dissolved in normal saline (pH 7.4) and was administered ip. All animals were exposed to DCF for 24 hrs before they were sacrificed.

Grouping of animals

The animals were divided into following groups:

Group I (Control): All animals were injected normal saline ip. Group II: STZ-alone - Streptozotocin 100 mg/kg was given ip. Group III: DCF alone 100 mg/kg ip. Group IV: DCF alone 200 mg/kg ip. Group V: STZ +DCF 100 mg/kg ip and Group VI: STZ +DCF 200 mg/kg ip.

Lethality and nephrotoxicity studies

The numbers of deaths were counted in each cage at the end of 24 hours. Nephrotoxicity was determined by measuring BUN (blood urea nitrogen) levels and kidney histopathology. BUN levels with a UV /VIS Beckman DU-640 spectrophotometer using a Sigma kit based on the method of Talke and Scubert (1965). Enzyme activity is calculated from the decrease in NADH absorbance at 340 nm.

Histological studies

Sections of kidneys were collected at the time of sacrifice and preserved in 10% buffered formalin. Tissue specimens were sent to Pathology Associates (MD) for further processing, sectioning and PAS (periodic Acid Schiff) staining. Normal, apoptotic and necrotic cells were identified from 5mm PAS-stained kidney sections using a Carl-Zeiss bright field microscope (Axioskop 20 equipped with a camera). Previously published guidelines to characterize normal, apoptotic, and necrotic cells were followed (Ray et al., 1996).

Oxidative stress and lipid peroxidation

The extent of oxidative stress was inferred from the degree of lipid peroxidation. The amount of lipid peroxidation was inferred from the levels of MDA (malondialdehyde) detected in kidney tissue. MDA levels from kidney homogenates were determined based on the methods of Bagchi et al. (1992) and Ray and Fariss, 1994). Kidney homogenates were reacted with TBA (thiobarbituric acid) to determine TBA-reactive substances. Absorbencies were extrapolated from a MDA standard curve and read at 532 nm.


Quantitative DNA fragmentation

DNA damage was quantified by using one kidney from each animal in all treatment groups. DNA fragmentation assay was based on method of Ray et al. (1996) and color reaction on method of Burton, 1956). To measure kidney DNA fragmentation, frozen kidneys (in liquid nitrogen) were weighed and homogenized in pre-chilled Lysis buffer (10 mM EDTA, 0.5% Triton X-100, pH 8.0). Homogenates were centrifuged at 27,000g for 20 min. at 4°C to separate intact chromatin in pellet from fragmented DNA in a supernatant. Pellets were suspended in 0.5N perchloric acid and supernatants were treated with concentrated perchloric acid to reach a final concentration of 0.5 N. All the samples were capped, boiled at 90°C for 15 min and centrifuged at 1500g for 10 min to co-precipitate protein along with other debris. The resulting supernatants were treated with diphenylamine reagent (1.5 g DPA + 1 ml 36N H2SO4 + 100 ml glacial acetic acid + 50 mM CH3CHO) for 16-20 hrs at room temperature in darkness. Absorbencies were measured at 600nm with a Beckman DU 640 spectrophotometer. DNA fragmentation in the control sample was treated as 100% fragmentation based on the formula [(frag. DNA)/(frag .DNA + intact DNA)] (Ray et al., 1996; Burton, 1956). DNA appearing in supernatant was divided by the total DNA to find percentage fragmentation. Treatment effects were reported as percent control fragmentation.

Quantitative DNA fragmentation assay

Prior to homogenization, portions from four kidneys were collected together in order to minimize statistical variation. Also, the collected kidneys were almost equally damaged based on average BUN levels and visual inspection. Kidneys were treated with chilled Tris- EDTA Sarkosyl buffer (0.1 M Tris HCl/10 mM EDTA/0.5% N-lauroylsarcosine, pH 7.8) and digested with proteinase-K at 50°C (50µg/ml) for 2 hrs. After digestion, DNA was extracted 3 times with phenol-chloroform and precipitated with absolute ethanol and 2.5 M sodium acetate (pH 4.0). After precipitation, DNA was washed 3 times with 66% ethanol, dissolved in 0.1 M Tris-HCl/20 mM EDTA buffer (pH 8) and digested for 15 min at 37°C with DNAase free RNAase-A (l mg/ml). DNA was reprecipitated, dissolved in Tris-HCl/EDTA buffer and then quantified spectrophotometrically from absorbance at 260 nm and loaded onto an agarose gel. The status of integrity of DNA (15µg/lane) was determined by constant voltage mode electrophoresis (in a large submarine at 60 V, Hoeffer Instruments, San Francisco on a 1.2% agarose gel containing 0.4-µg/ml ethidium bromide. A HindIII digest of DNA served as molecular weight size standard on gel. The gel electrophoretogram was illuminated with 300 nm UV light and a photographic record was made (Ray et al., 1986).

Statistical analysis

Data were analyzed for significance (P< 0.05) using ANOVA followed by the Fisher PLSD test (Stat View II for Macintosh, version 1.04, Abacus Concepts, California) or evaluated by linear regression analysis and correlation (Zar,1984). Simple statistical comparison of DNA fragmentation data was determined by Fischer PLSD test and converted to percentages.

RESULTS

Preliminary data showed that 100 mg/kg ip dose of STZ administered every other day was optimal for induction of hyperglycaemia and its doses exceeding l00 mg/kg, or their prolonged


administration in more than 5 doses were lethal. Its administration in excess of l0 days induced severe sickness and that is why these animals were not included in final studies. Consistent loss of weight (Fig. l) was another stringent criterion which was taken into account while developing this protocol. During trial period, DCLF was administered ip or orally and normal saline was used as vehicle for its dissolution for injections whereas it was suspended in corn oil and sonified for oral administration. The degree of nephrotoxicity induced by DCLF was greater via ip route.

Fig. 1. Streptozotocin induced correlation number of doses for STZ and weight loss in

male ICR mice

Fig. 2. Animal lethality due to streptozotocin-Diclofenac interaction in male ICR mice

Fig. 3. Modulation of blood glucose level by DCLF in diabetic and no diabetic mice


Fig. 4. Effect of Diclofenac on streptozotocin-induced nephrotoxicity

Fig. 5. Effect of Diclofenac on streptozotocin induced kidney DNA fragmentation

Fig. 6. Effect of Diclofenac on streptozotocin induced oxidative stress in kidneys


Fig. 7. Agarose electrophoresis assessment of genomic undamaged qualitative

Fig. 8 A. Representative section of mouse kidney treated with vehicle .9% NaCl, no pathologic changes present. Normal architecture of kidney is vivid and there is total

absence of apoptotic and necrotic cells.

Fig. 8-B. Representative section of mouse kidney treated with 200 mg/kb DCLF, showing

random damage of all cellular compartment. Massive damage to macula densa areas.


Fig. 8-C Representative section of mouse kidney treated with STZ+DCLF 200, showing

nominal damage than DCLF alone. The changes were observed only in proximal and distal tubular cells.

Fig. 8-D Representative section of mouse kidney treated with STZ alone, showing overall

loss of glycogen and minor changes in the cortical areas.

Fig. 9 Representative section of mouse kidney treated with DCLF 200, showing random damage of all cellular compartments. Magnified view of a kidney apoptotic nuclei, that are characteristic of an

apoptotic cell.


Dose response of diclofenac-induced nephrotoxicity

Fig. 4 shows the dose-response effect of DCLF on ICR-mice at 24 hours after its administration. The nephrotoxicity was monitored by serum BUN levels. A positive correlation between the DCLF dose and proportionate increase in serum BUN is clear from Fig. 4. These doses of DCLF were highly effective in inducing kidney injury. The lowest dose was the least effective in inducing kidney injury and the highest dose was the most potent in inducing kidney injury.

Diclofenac-induced animal lethalities

The two lowest doses of DCLF induced minimal death whereas the highest dose, 300 mg/kg, exceeded the limit of LD50 and was not used in this investigation. The number of animals that died after DCLF exposure is given in Fig. 2. Based on these data, only the two lower doses of DCLF (100 and 200mg/kg) were used for the further studies.

Modulation of blood glucose by DCLF in diabetic and non-diabetic mice

Fig. 3 shows the modulatory influence of DCLF on normal and diabetic mice. STZ alone was effective in causing severe hyperglycemia. In contrast, DCLF alone was ineffective in elevating blood glucose levels; rather, it induced slight hypoglycemia (blood glucose less than 70mg/dl). Blood glucose levels in animals given DCLF alone were lower than control animals. Administration of DCLF to STZ-exposed mice did not significantly alter blood glucose levels except that these were slightly higher in STZ+DCLF administered animals as compared to STZ alone.

Modulation of nephrotoxicity by DCLF in diabetic and non-diabetic mice

Fig. 4 shows the degree of nephrotoxicity induced by DCLF in hyperglycemic and non-hyperglycemic mice as reflected by serum BUN levels. The toxicity in diabetic and non-diabetic mice did not reflect the trend observed with blood glucose levels (Fig. 3). Diabetic animals were extremely sensitive to DCLF and showed dramatic increase in blood glucose levels (Fig. 4) whereas normal animals were less vulnerable to nephrotoxic potential of DCLF. It is clear from the data that hyperglycemia (or STZ exposure) did not potentiate DCLF-induced nephrotoxicity; rather impact of DCLF was significantly reduced by the presence of excessive glucose in circulation. BUN levels were lower in STZ+DCLF treated mice as compared to DCLF alone. This reduction in hyperglycemic state may due to rapid elimination of the drug. However, in control mice severe kidney injury was observed as reflected by sharp increase in BUN.

DCLF-induced oxidative stress in kidneys of diabetic and non-diabetic mice

Fig. 5 shows DCLF-induced oxidative stress-mediated lipid peroxidation in kidneys. The tissue levels of lipid peroxidation indirectly quantified by determining MDA levels in kidney homogenates (Fig. 5) showed that both doses of DCLF alone (100 mg/kg and 200 mg/kg) were able to induce significant lipid peroxidation and caused about 4-fold increase in MDA level. Similarly, STZ- alone was equally effective and nearly doubled its level in kidneys. However, hyperglycemia did not accentuate the degree of lipid peroxidation and levels of MDA were lower in STZ+DCLF group as compared to DCLF alone. Hyperglycemia exerted an opposing effect on DCLF-induced oxidative stress and pattern of oxidative stress mimicked the nephrotoxicity trend.


Quantitative DNA damage caused by DCLF in kidneys of normal and diabetic mice

Quantitative assay reliably predicts the extent of fragmented and intact DNA (Ray et al., 1994) and Fig. 6 shows quantitative changes in integrity of genomic DNA during exposure to nephrotoxic doses of DCLF. The administration of DCLF alone caused massive kidney DNA fragmentation at 24 hours after the nephrotoxic dose. A 100 mg/kg dose of DCLF caused a 2.53 fold increase and 200 mg/kg dose caused a 3.57 fold increase in DNA fragmentation as compared to control group. Overall, the pattern of quantitative spectrophotometric DNA fragmentation mirrored the profiles of nephrotoxicity and oxidative stress discussed in Figs 4 and 5. In comparison to DCLF alone, STZ-induced hyperglycemia caused a moderate increase in DNA fragmentation (less than 2-fold). However, DCLF exposure to hyperglycemic mice did not potentiate DNA fragmentation. The percent fragmentation observed in STZ+DCLF groups was lower when compared to DCLF alone group. DCLF failed to take advantage of the diabetic condition and aggravated the slightly unstable integrity of genomic apparatus. Collectively, DNA fragmentation data also followed the changes observed in BUN (Fig. 4) and MDA (Fig.5) levels and suggests a link between these three events.

Qualitative DNA damage caused by DCLF in kidneys of normal and diabetic mice

Apoptotic type ladder-like DNA fragmentation dependent on Ca2+-endonuclease activity was evaluated the data obtained are shown in Fig. 7. The electrophoretogram generated by agarose gel electrophoresis showed that DCLF overdoses resulted in degradation of kidney DNA and a typical ladder of DNA fragments consisting of multiple repetitive bands diagnostic of Ca2+ -endonuclease activity (Hickey et al., 2001). In addition, DCLF exhibited a dose-response effect on the damage. DNA isolated from control group (lane 2) remained totally intact and devoid of any fragmentation. Using the l-kb DNA ladder as a marker size (lane 1), this fragmentation pattern presumably represents 200-bp multiples of DNA. The DCLF-induced loss of large molecular weight DNA (see fluorescence intensity at the top of the gel at wells 5 and 7) was readily apparent on the gels. The overall pattern of DNA fragmentation and a clear-cut ladder induced by toxic doses of DCLF suggested involvement of massive cell death via apoptosis. Compared to DCLF alone, STZ alone (lane 3) was far less active in fragmenting DNA. However, mild laddering observed with STZ alone exposed DNA reflected a modest onset of apoptotic programmed death. Likewise, DCLF exposure in hyperglycemic mice resulted in a laddering which was far less pronounced as compared to DCLF alone. These changes agree with biochemical findings reported earlier (Hickey et al., 2001).

Apoptotic and necrotic deaths in normal and diabetic mice

Histopathological examination of kidney sections vividly followed biochemical findings previously discussed. The photomicrographs of representative kidney sections from control and DCLF (200 mg/kg) treated mice at 24 hours are shown in Fig. 8. In order to assess the degree of nephrotoxicity, paraffin-embedded PAS stained mouse kidney sections from control and DCLF (200 mg/kg) treated groups were examined. PAS staining provided insight into the glycogen status of the tissue. Vehicle controlled (Fig 8, panel-A) kidney sections showed normal structure with no identifiable lesions in the absence of apoptotic and necrotic cells. In this section, most cells were uniformly stained, and intensity of coloration signified that nearly every cell was glycogen loaded with intact cytoplasm. A clear-cut undamaged dark blue stained nucleus was found in every cell and indicated an intact nuclear morphology. But sections from kidney tissues exposed to 200 mg/kg DCLF (Fig 8, panel B) demonstrated massive injury in almost all types of


cells. The primary victims were proximal and distal tubular cells and macula densa of these cells sustained massive injury. Some tubular cells totally lost macula densa regions. The glomeruli showed shrinkage, hypervacuolation, and/or abnormal swelling.

Glycogen-depleted cells were scattered predominantly in the cortex. Cells of the ureter also suffered minor to moderate injury. Heavily drug impacted areas also showed the presence of inflammatory cells which signified ongoing necrosis. On a comparative basis, 200 mg/kg (Fig. 8B) was more damaging than 100 mg/kg (data not shown) which corroborated with previous biochemical findings. Representative section of mouse kidney treated with STZ+DCLF200 mg/kg (Fig.8C) showed less kidney cell damage than DCLF alone. STZ alone (Fig 8D) induced only minor changes and upon examination of the entire section, a negligible number of apoptotic cells were encountered. Fig.9 shows several apoptotic nuclei which were randomly present throughout section which reflect potency of DCLF to "turn on" apoptosis in the kidneys. Unlike DCLF, lack of numerous apoptotic nuclei in STZ+DCLF exposed kidney reflected down regulation of apoptotic process. Collectively, these data suggested that STZ-induced hyperglycemia did not potentiate DCLF nephrotoxicity in mice.

DISCUSSION Diabetes mellitus alone ranks among top ten causes of death in western nations with a worldwide prevalence between 4 to 5 % (McGowan et al., 2001). Despite important improvements in its clinical management, it is not yet possible to significantly control its consequences. Diabetes mellitus represents a group of metabolic disorders in which there is impaired glucose utilization manifested by persistent hyperglycemia, a relative or absolute lack of insulin production by the pancreas, and development of microvascular pathology in the retina and glomeruli. The morbidity associated with long-standing diabetes results from complications such as microangiopathy, retinopathy, nephropathy, neuropathy and their combinations. Hence the basis of these chronic long-term complications has been and still the subject of a great deal of research (Raptis and Viberti, 2001). Evidences suggest that complications of diabetes are consequence of metabolic derangements, mainly hyperglycemia, from the finding that kidneys when transplanted into diabetics from normal individuals develop nephropathic lesions within five years after transplantation (Nam et al., 2001). Conversely, kidneys with lesions of diabetic nephropathy demonstrate a reversal of the pathology when transplanted into non-diabetic individuals. In USA alone all end-stage chronic renal failure patients were diabetics (Hricik et al., 1997). STZ-induced-diabetic hyperglycemia has been shown to aggravate cardiotoxicity of doxorubicin (Al-Shabanah et al., 2000), thioacetamide-induced hepatotoxicity (El-Hawari and Plaa, 1983) and cyclosporine-A-induced renal tumorigenicity (Reddi et al., 1991). The diabetes induces isoenzymes (CYP 450) known to participate in drug metabolism (Chang et al., 2000). Since diabetes and NSAIDs, such as DCLF, share a common target in the kidney, the present study tested whether DCLF during hyperglycemia will synergize, potentiate or antagonize nephrotoxicity and the associated events. Thus several biochemical markers were measured including: (i) changes in blood glucose levels; (ii) changes in serum BUN levels, (iii) tissue MDA levels to indirectly determine ongoing lipid peroxidation associated with oxidative stress, (iv) quantitative and qualitative genomic DNA and (v) histopathological changes.

Data obtained showed that five ip doses of 100 mg/kg streptozotocin (administered every other day for 10 days) were sufficient to induce significant hyperglycemia. This condition was coupled


with gradual loss of body weight (total loss of 3.6 grams in 10 days), which signified induction of acute diabetes mellitus. STZ doses in excess of this regimen either caused severe weight loss, sickness and/or death. These findings are similar to Ray et al. (2001). Similarly, DCLF alone was extremely effective in inducing severe nephrotoxicity. Lower doses (100 and 200 mg/kg) were sub-lethal, whereas, the highest dose (300 mg/kg) was extremely lethal and supertoxic. In fact, a 300 mg/kg dose caused 80% animal death. This corroborates with our earlier observations (Ray et al., 1996).

Administration of DCLF to non-diabetics caused slight hypoglycemia but in diabetic animals there was no blood glucose lowering. This slight hypoglycemic effect of DCLF in non-diabetics may be due to nonspecific response of normal beta cells whereas its ineffectiveness in diabetics may be due to loss of beta cell responsiveness. In addition, at the time of sacrifice, DCLF–exposed kidneys were distinguishable from controls as they were pale and abnormally enlarged and significantly heavier. Animals in 100 mg/kg DCLF-treated group were less active than controls, whereas most animals in the 200 mg/kg group appeared sick and showed low level of activity. The appearances and activity levels of animals of STZ alone and STZ+DCLF groups were nearly identical. The color and texture of kidneys of these two groups were very similar. The impacts of STZ and STZ+ DCLF on kidneys were less severe as compared to kidneys exposed to DCLF alone. As regards nephrotoxicity, DCLF treated non-diabetic mice demonstrated massive kidney injury and showed sharp increase in BUN levels. This indicated a normal course of metabolism of DCLF as well as the after effects in vivo. However diabetic animals responded poorly to DCLF's toxic effects. Their BUN levels were lower than non-diabetics. This may be due to several reasons: (i) DCLF might have been excreted in urine very rapidly, (ii) hyperglycemia might have destroyed/inhibited various iso-forms of cytochrome P-450 enzymes responsible for its metabolism (iii) hyperglycemia might have interfered formation of toxic metabolites of DCLF and (iv) high glucose levels may have provided excessive energy and. protected the cells when they were undergoing toxic assaults by DCLF or its reactive metabolites. Since these events were not examined systematically, it is very difficult to pinpoint whether any or all of these contributed in reducing DCLF-nephrotoxicity in diabetic mice.

Histopathology of PAS-stained kidney sections followed the serum chemistry changes and strongly correlated with DNA damage and lipid peroxidation data. Kidney sections of control mice showed normal cellular structure with no evidence of injury. In contrast, DCLF-induced changes as manifested by numerous morphological alterations coupled with a lethal depletion of glycogen. Abnormal swelling of kidney cells and loss of nucleus or macula densa in numerous proximal and distal tubular cells were most commonly encountered. Numerous cells with apoptotic nuclear morphology ranging from fragmented bodies to fragmented nuclei (including fragmented chromatin) were also prominent in severely damaged areas. DCLF was also able to damage glomerular apparatus as its higher doses induced massive vacuolation to this filtration apparatus. In contrast to changes found in cortex area of the sections, medullary rays and ureter areas remained affected to a relatively lesser degree.

Apoptotic nuclei were predominantly localized to heavily injured proximal and distal tubular cells. The other striking feature was presence of numerous inflammatory cells. STZ alone caused depletion of glycogen and minor changes in macula densa. No gross changes were observed in the glomerular apparatus. Some apoptotic nuclei were seen but not many when compared to DCLF alone. Administration of STZ+DCLF combination was also very effective in inducing morphological alterations. However, overall changes were less complex than the changes


exhibited in mice treated with DCLF alone. A significant reduction in both apoptotic and necrotic deaths was observed. Our histological findings suggest the incidence of apoptosis was random and lacked particular loci; most cells that demonstrated different stages of apoptosis were scattered throughout the heavily damaged areas.

Numerous findings now suggest that DNA becomes an easy target of both apoptosis and necrosis, and stability of the cellular genomic apparatus is constantly challenged by a wide-spectrum of exogenous toxicants and endogenous (oxidative damage, factor imbalance) threats, which generate DNA lesions. The present study also showed that STZ alone induced minor activation of endonuclease resulting in DNA fragmentation detectable by both methods. STZ+DCLF treated groups showed a slightly higher order of fragmentation but significantly lower than DCLF alone. DNA fragmentation assessed in this study is a total picture of mitochondrial and nuclear DNA fragmentation. Nagata et al (2000) have also reported that endonuclease, which is responsible for inducing DNA ladder is a caspase-activated DNAase (CAD) which preexists in living cells as an inactive complex with an inhibitory subunit, dubbed ICAD. Activation of CAD occurs by means of caspase-3-mediated cleavage of the inhibitory subunit, resulting in the release and activation of the catalytic subunit. Although, final activation of CAD may be or may not be Ca2+- dependent, experiments are in progress to unravel DCLF's intervening role at such regulatory pathways. How DCLF modulated STZ-induced DNA fragmentation remains puzzling. Oxidant-induced injury to the plasma membranes of cells can evoke a wide variety of responses, including changes in transmembrane transport, receptor-ligand interactions, signal transduction, and enzyme activity regulation as well as differentiation and proliferation (Emerit et al., 2001). It is suggested that lipid peroxidation plays a critical role in oxidant-induced renal cell injury; therefore, lipid peroxidation has been used as a sensor for renal injury (Nowzari et al., 2000). Cellular macromolecules are highly vulnerable to oxidative injury by ROS, often leading to cell death by apoptosis and necrosis. Studies indicate that ROS or reactive nitrogen species (RNS) can have diverse effects on mammalian cell growth. In some cases, small quantities of oxy radicals induce cells to undergo apoptosis whereas, in other cases, ROS promote cell proliferation and may function as intracellular second messengers. Evidences suggest that oxidative stress is a common mediator of apoptosis, perhaps via the formation of lipid hydroperoxides. It has been suggested that DCLF metabolism yields a very reactive quinoneimine species, which may be the prime suspect in generating oxidative stress (Tang et al., 1999). DCLF is a powerful nephrotoxicant in addition to its hepatotoxic potential. In the present study, kidney MDA levels were determined to measure oxidative stress generated by DCLF. It caused dramatic increases (4 fold over control) in MDA, and STZ alone produced twice the amount detected in control tissues. DCLF-exposed diabetic tissues showed MDA levels close to STZ alone and significantly less compared to DCLF alone. The trends of elevated BUN levels and DNA fragmentation clearly resembled oxidative stress induced MDA accumulation data. How hyperglycemia reduced toxicity remains a question open for future investigation. Some studies have shown that glucose can act as a free radical scavenger. Weakening of DCLF toxicity, observed in these experiments in diabetics may be a reflection of this fact. There are a number of other mechanisms by which DCLF may have produced nephrotoxicity. Collectively, our results have suggested that DCLF is less effective in diabetics than non-diabetics. This implies that therapeutic doses usually prescribed for non-diabetics may not show equivalent analgesic action on diabetics. This conclusion is based on the fact that hyperglycemic condition failed either to potentiate or synergize DCLF-induced nephrotoxicity in vivo; showing that this drug cannot be considered as a problematic NSAID for diabetics.


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DEPRESSION AND LOW SERUM LEVELS OF DOCOSAHEXAENOIC ACID (DH A) ARE ASSOCIATED WITH INCREASING NUMBER OF PREGNANCIES AMON G POSTPARTUM OMANI WOMEN

Lamya Al Kharusi 1, Mostafa I. Waly2*, Hassan Mohammed Othman3 Nejib Guizani2,

Farhat Al Naabi2, Amanat Ali2

1Obstetrics and Gynecology Department, College of Medicine and Health Sciences, 2Food Science and Nutrition Department, College of Agriculture and Marine Sciences,

3Physiology Department, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman

ABSTRACT

Depression is a global public-health burden. It has been suggested that dietary supplementation of docosahexaenoic acid (DHA) during the prenatal stage may lead to an improvement in postpartum depression. No relevant studies have been conducted in the Sultanate of Oman investigating the status of depression and serum levels of DHA during postpartum period. The present was therefore conducted to collect the baseline information about the postpartum depression status, DHA depletion during the postpartum period and to investigate the impact of repeated pregnancies on development of depression and serum DHA levels in postpartum Omani women. This cross-sectional study involved a total of 87 Omani women including 70 postpartum women and 17 women with no history of pregnancy. A depression scale was used to screen all the study participants. Dietary intake was evaluated using a semi quantitative food frequency questionnaire and serum levels of DHA were measured using HPLC-based technique. The depression score among postpartum women who had 4-8 pregnancies was significantly (P < 0.05) higher than those who had 1-3 pregnancies as well as to controls. No significant (P < 0.05) differences were observed between postpartum women and control group with regard to the consumption of foods containing DHA or its precursors. Postpartum women with 4-8 pregnancies had the lowest serum levels of DHA. The results suggest that depression and low serum levels of DHA were found to be associated with higher number of pregnancies (4 to 8) among the postpartum Omani women.

Keywords: Depression, serum DHA level, postpartum Omani women

*Address for Correspondence: Mostafa Waly, MPH, PhD, Food Science and Nutrition Department, College of Agriculture and Marine Sciences, P.O. Box 34, PC 123, Al-Khoud, Muscat, Oman. Email: [email protected]


INTRODUCTION

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid of the omega-3 series and is essential for the proper cognitive functions and normal development of the nervous system (Haag, 2003). Recent studies suggested that depression during pregnancy is associated with low intake of DHA among susceptible subjects (Mischoulon and Fava, 2000, Dennis. 2004). Repeated pregnancy is a physiological stress factor for depleting the body stores of DHA, especially if synergized with short postpartum period and/or low dietary intake of DHA (Peet et al., 1998, Levant, 2010, Parra-Cabrera et al., 2011). Depression is a global public-health burden that it is associated with substantial disability, including depressed mood, loose of interest or pleasure, feeling of guilt or low self-worth, disturbed sleep or appetite, low energy and poor concentration (American Psychiatric Association, 2000). Postpartum depression is associated with disabling symptoms of uneasiness, irritability, confusion and forgetfulness, anhedonia, fatigue, insomnia, guilt, inability to cope, and thoughts of suicide (Dennis, and Creed, 2007).

Long term psychiatric counseling is crucial for managing the postpartum depression (Gard et al., 1986, Monk et al., 2004). Proper nutrition during pregnancy and after giving birth is also an effective strategy for improving the maternal health and well-being (Barger, 2001). Some evidence suggests that there is an improvement in postpartum depression, when dietary supplements containing DHA are taken during prenatal stage (Zlotnick et al., 2001, Nancy et al., 2010), although the results are still not conclusive (Marangell et al., 2004). No relevant studies have been conducted in the Sultanate of Oman investigating the serum levels of DHA during postpartum period. The present was therefore conducted to collect the baseline information about the postpartum depression status, DHA depletion during the postpartum period and to investigate the impact of repeated pregnancies on development of depression and serum DHA levels in postpartum Omani women. The expected outcome of this study will shed light on the DHA status of Omani women and its impact on their health and well-being.


Study Participants and Study Setting. A cross-sectional study approach was used to conduct this study during the period from February 2009 to December 2010 in the Outpatient Obstetrics and Gynecology Clinic at the Sultan Qaboos University (SQUH), a tertiary referral hospital in Oman. All study participants were Omani nationals. They were briefed about the study protocol and on voluntary basis they signed a consent form for enrollment in the study. A total of eighty seven subjects were recruited for this study on voluntary basis. The subjects were divided into three groups, control group with no history of pregnancy (n=17), postpartum women who had 1-3 pregnancies (n=35) and postpartum women who had 4-8 pregnancies (n=35). All the participating postpartum women were not currently pregnant and they were divided into women who had 1-3 pregnancies, n=35, and those who had 4-8 pregnancies, n=35. The controls were selected from those working in the same hospital and had no previous history of pregnancy. All the study participants were free of endocrine, chronic cardiac and gastrointestinal diseases. The study was approved by the Medical Research and Ethical Committee (MREC) of College of Medicine and Health Sciences, Sultan Qaboos University (SQU).


In-person interviews were scheduled for all study participants for completing the study questionnaire and collection of blood samples for serum DHA measurements. The questionnaire was divided into the following sections:

Anthropometric Measurements: For all study participants, the height was measured to the nearest 0.5 cm and weight was recorded to the nearest 0.5 kg using a precision sale. The body mass index (BMI) of individuals was calculated as weight in kilograms divided by height in square meters (kg/m2) and was categorized as normal (BMI=18.5 -24.9), overweight (BMI=18.5 -29.9) and obese (BMI >30) (WHO, 1995).

Dietary Assessment: The retrospective dietary intake of the study participants was estimated using a semi-quantitative food frequency questionnaire (FFQ) that intended to measure the usual food consumption over the previous 12 months. This period was chosen to take into account the seasonal variations in food consumption with specific reference to foods rich in DHA or its precursors (Block et al., 1990, Willet, 1998). The FFQ was adapted according to portion sizes based on commonly used household serving units/utensils in Oman, and was tested for its validity, reliability and reproducibility before conducting the study (Pietinen et al., 1988). The Food Processor software version 10.2 (ESHA Research, Salem, OR, USA) was used to calculate the daily nutrient intakes (total energy intake, protein, carbohydrate and fat) as estimated from the frequency of consumption, reported portion size, and nutrients content for all foods reported by each study participant.

Depression Assessment: Symptoms of depression for all study participants were assessed using the Hospital Anxiety and Depression Scale (HADS). The scores from 0-7 were considered normal whereas the scores higher than 7 were considered having a tendency towards depression and anxiety (Zigmond, and Snaith 1983, Snaith and Zigmond, 1994).

Biochemical Assessment of DHA: Whole blood samples were drawn after overnight fasting by a sterile syringe into a plain tube taking 5 ml samples. The serum was separated by centrifugation and stored in Eppendorf tubes by freezing at - 80˚C until all samples were collected and thawed just before analysis of DHA. DHA was measured as its methyl ester by adding 150 µl of serum to 5 ml of methanol/acetyl chloride (50:1, vol/vol), the mixture was thoroughly mixed and left at room temperature for 45 minutes (Lepage and Roy, 1988). The methylation reaction was stopped by the slow addition of 3 ml of 6% potassium carbonate solution to the mixture. After 5 minutes, 1 ml of hexane was added and the mixture was vigorously shaken. The hexane layer, which contained the methylated DHA, was separated and dried under nitrogen (Wang et al., 1991). The residue was dissolved in 100µl methanol for lipid analysis by high-performance liquid chromatography (HPLC) carried out on a gradient system (Shimadzu C-4A, ODS, Hypersil C18) equipped with a guard column PR 8 or PR 4 and reversed phase column.The separation of the methyl ester derived fatty acid mixture was carried out using methanol/water (90: 10, vol/vol) as a mobile phase at a flow rate of 0.5 ml/min. The eluent was monitored by an UV detector. The wave length was set at 210 nm. A standard of (cis- 4, 7, 10, 13, 16, 19- Docosahexaenoic acid methyl ester) was used (Wang et al., 1991).

Statistical Analysis: Data was expressed as mean ± S.D. (standard deviation) and analyzed using The GraphPad Prism statistical software for personal computer, version 5. Chi-square (χ2) test was used for categorical variables. One way analysis of variance (ANOVA) followed by Tukey's test, Student’s unpaired t-test and correlation coefficients (r) for continuous variables. The P value < 0.05 was considered statistically significant.


RESULTS

The subjects of this cross-sectional study were divided into three groups; controls group with no history of pregnancy (n=17), postpartum women who had 1-3 pregnancies (n=35) and postpartum women who had 4-8 pregnancies (n=35). The educational level, monthly income and occupation status for the enrolled subjects were comparable with no statistical differences (P < 0.05). Table 1 shows the mean age for control group (21.8 ± 4.2 years), the postpartum group who had 1-3 pregnancies (23.31 ± 5.4 years) and the postpartum group who had 4-8 pregnancies (23.94 ± 4.9 years). There was no statistical significant differences between the mean age values (F= 1.055, P = 0.353). The women in the control group had normal BMI (22.65 ± 3.8), whereas the postpartum women who had 1-3 pregnancies and 4-8 pregnancies were overweight (BMI 27.31 ± 6.8 and BMI 28.29 ± 7.1 respectively). The differences were statistically significant as compared to control subjects (F= 4.53, P < 0.01).

Table 1: Characteristics of study subjects

Characteristics

Controls

(n = 17)

Postpartum

1-3 Pregnancies

(n = 35)

Postpartum

4-8 Pregnancies

(n= 35)

P value

Age (years)

21.8 ± 4.2

23.31 ± 5.4

23.94 ± 4.9

0.353

BMI (kg/m2) 22.65 ± 3.8 27.31 ± 6.8 28.29 ± 7.1 0.01*

Total Energy Intake (Kcal/d) 2194.23 ± 485 2434.17 ± 526 2708.3 ± 608 0.0067*

Carbohydrates (g/d) 331.42 ± 79.8 398.91 ± 85.6 420.4 ± 91.3 0.000*

Protein (g/d) 78.12 ± 24.1 80.01 ± 21.4 85.7 ± 32.0 0.54

Total Fats (g /d) 61.78 ± 14.7 57.61 ± 15.6 75.98 ± 14.2 0.000*

Saturated (g /d)

Monounsaturated (g/ d)

Polyunsaturated (g/d)

38.12 ± 13.4

6.84 ± 3.4

14.61 ± 10.7

36.74 ± 10.6

5.95 ± 3.7

13.92 ± 9.6

50.38 ± 11.2

6.49 ± 4.7

14.68 ± 7.3

0.000*

0.73

0.93

Data expressed as mean ± SD, * significantly different as compared to controls Group, P <0.05

Food groups analysis shows that the daily intake of servings from various food groups based on the USDA Food Guide Pyramid was similar among postpartum and controls subjects with no significant difference (P < 0.05). The study subjects consumed 6-11 servings from bread and cereals group/day, 3-5 serving from vegetables group/day, 2-4 servings from fruits group/day, 2-3 servings of dairy products/day and 2-3 servings of meat and meat alternatives/day. The


frequency of consumption of foods rich in DHA or its precursors such as fresh fish (including sardines, tuna, mackerel, shellfish and herring), canned fish, vegetable oils, eggs, chicken, organ meats, nuts and beans was similar with for all respondents with no significant difference (P < 0.05). The postpartum women who had 4-8 pregnancies had a significantly higher mean total energy intake as compared to controls group and postpartum women who had 1-3 pregnancies, F= 5.32, P < 0.05 (Table 1). The differences between the means with regard to total daily carbohydrate and total fat intakes were also significantly different (F= 425.9 and F = 14.12 respectively, P < 0.05). The mean total daily protein intakes were comparable among postpartum women and control subjects with no significant difference, F=0.54, P>0.05. Although the mean total daily fat intake between the three groups was statistically different (P < 0.05) the monounsaturated and poly unsaturated fatty acids consumption were comparable for all the three groups with no significant differences (P < 0.05). The high total fat intake was attributed to increased saturated fatty acids consumption.

0.0

2.5

5.0

7.5

10.0Control (1-3) Pregnancies (4-8) Pregnacies

*

*

Dep

ress

ion

Sco

re

Figure 1: Depression score among postpartum and control subjects. * Significantly higher as compared to control group, P < 0.05

0

5

10

15

20Control(1-3) Pregnancies(4-8) Pregnacies

*

*

Ser

um

DH

A L

evel

(µµ µµg/

ml)

Figure 2: Serum DHA level among postpartum and control subjects * Significantly lower as compared to control group, P < 0.05


The mean depression score (Figure 1) was 9.13 ± 3.24 for postpartum women who had 4-8 pregnancies, 7.95 ± 1.04 for postpartum women who had 1-3 pregnancies and 2.89 ± 0.74 for control group. The difference between the three means were statically significant (F = 48.11 and P < 0.05). Figure 2 shows the changes in the levels of the DHA according to the number of pregnancies. The highest level of the DHA was observed in the group of women who had no history of pregnancy, with a mean level of 13.6 ± 1.4 µg/ml. The level of serum DHA decreased gradually with the increase in number of pregnancies. The mean serum DHA level in women with 1-3 pregnancies was 6.72 ± 1.68 µg/ml, which was significantly (P < 0.05) higher than that in postpartum women with 4-8 pregnancies who had only 3.28 ± 0.07 µg/ml.

DISCUSSION

This study investigated the changes in the serum levels of the DHA in the postpartum Omani women, a vulnerable group to depletion of the DHA level from body stores as a result of its utilization by the fetal development during previous pregnancies. There is no background information on the normal blood levels of DHA in Omani women. Therefore the results obtained in this study focused on comparing the serum levels of DHA in postpartum women with those who had no history of pregnancy as well as evaluating their depression score.

Overweight and Obesity were observed among the postpartum women, and there was a weak negative, but non-significant correlation between the serum DHA and BMI (r= - 0.98, P= 0.11). This might be attributed to the increased intake of energy dense foods, rich in saturated fats on the expense of foods rich in DHA. The results of this study show that the serum level of DHA was the highest in women with no history of pregnancy and decreased gradually with increasing number of pregnancies in the postpartum period. Although an inverse association was observed between the serum DHA level and depression, the results were statically non-significant (r= - 0.98, P < 0.09).

The Omani women involved in this study had similar daily intake of DHA rich sources of foods such as local fatty fish or the foods containing the DHA precursors (alpha-linolenic acid) vegetable oils, beans and dark green vegetables. We therefore hypothesize that the observed decrease in serum levels of DHA in postpartum women was a result of increased requirements associated with repeated pregnancies without being matched with an increase in the intake of DHA to compensate the loss of body stores as a result of its utilization for fetal development. The repeated pregnancies were also found to be associated with depression which confirms the previously reported data that repeated pregnancies are a contributing factor for DHA depletion from body stores (Makrides, and Gibson, 2000, Brown, 2008). It was quite interesting to note there was a lack of knowledge among the enrolled Omani women about dietary sources of DHA and its health benefits (USDA, 2005). Western studies have shown that supplementation of pregnant women with fish oil reduced the postpartum depression (Olsen et al., 2003, Krauss-Etschmann et al., 2007). It is recommended to supplement the maternal diet with DHA sopurces as a primary prevention to improve the health and well being of women during pregnancy (Lewis et al., 1995, Dunstan et al., 2004, Decsi et al., 2005). Biochemical and pharmacologic studies suggest that omega -3 fatty acids may modulate the neurotransmitter metabolism and cell signal transduction in humans and any abnormality in omega-3 fatty acid and eicosanoids metabolism may play a causal role in depression (Chiu et al., 2003, 2004). Our study findings suggest that repeated pregnancies are a physiological stress factor that contributes


to depletion of body stores of DHA and if the requirement is not matched with an increased dietary intake of DHA, it might ultimately become a risk factor for developing depression among susceptible postpartum women.

CONCLUSION

Depression and low serum levels of DHA were found to be associated with higher number of pregnancies (4 to 8) among the postpartum Omani women. It is therefore recommended that all Omani women in the child bearing age should get dietary supplements of DAH and must be encouraged to use DHA rich food sources. There is need to develop nutritional educational programs for delivering specific information to increase the awareness of the impact of DHA on human health and wellbeing of the newborn babies. Increasing DHA body stores for women should be a primary goal to meet the additional needs in any future pregnancy; this could be accomplished by adopting national policies such as fortification of foods with DHA and disseminating basic information to the whole population to increase consumption of richest dietary sources of DHA.

Authors' Disclosures of Potential Conflicts of Interest

The authors indicated no potential or actual conflict of interest pertaining to this study.

Authors' Contributions

Dr. Lamya Al-Kharusi , Dr. Mostafa Waly, Prof. Mohamed H. Othman and Dr. Nejib Guizani equally contributed to study design, statistical analysis interpretation of data and drafting the manuscript. Mrs. Farhat Al-Naabi made full contribution to data acquisition and collection. Dr. Amanat Ali contributed to interpretation of results, drafting and revising the final manuscript. All authors read and approved the final manuscript.

Limitation of the Study

This study has some limitations and is subjected to some bias as being a hospital based case control study with small sample size and memory issues related to recall of food intake data reported in the FFQ. Further population-based studies have therefore been planned to evaluate the status of DHA among postpartum women in Oman.

Acknowledgements:

The authors deeply extended their appreciation to all the staff at the Obstetrics and Gynecology Department, SQUH for their dedicated effort to complete this study.

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Canadian Journal of Applied Sciences; 2011; 1(3): 153-157, October, 2011 Intellectual Consortium of Drug Discovery & Technology Development Inc. ISSN 1925-7430 Available online http://www.canajas.com Case Report PNEUMONIA THERAPY OF AN INFANT ADMITTED IN A LOCAL HOSPITAL OF RAWALPINDI, PAKISTAN; A CASE REPORT

Mehreen Zeb, Lubna Shaheen

Ripha Institute of Pharmaceutical Sciences, Ripha International University, G-7/4, 7th Avenue, Islamabad, Paksitan.

ABSTRACT

Pneumonia was regarded as "the captain of the men of death", during the 19th century. It was more dreadful illness of infants. However; the advents of antibiotic therapy and vaccines in the 20th century have seen radical improvements in survival outcomes. Nevertheless, in the third world, and among the very old, the very young and the chronically ill, pneumonia remains a leading cause of death. Thus; we have aimed this case study to understand the scientific and therapeutical comprehension of pneumonia. A six month old baby (girl) was presented in a local hospital, Rawalpindi, Pakistan with pneumonia. On basis of her medical investigation the physician prescribed the injection Claforan (cefotaxime) 250mg IV (intra venous) TDS (three time a day) for four days; injection Grasil (amikacin sulphate) 50mg IV BID (two time a day); Ventolin (salbutamol albuterol) nebulisation after every 3 hours; syrup Paramac (paracetamol) 1 TSF (tea spoonful) or 120mg /5ml SOS (as required); injection Lasix (furusemide) 10mg IV stat (immediately); tablet Capoten (captopril) 12.5mg ½ BID; syrup Acefyl (acefylline piperazine) and syrup Phenergan were administered for first two days. Her vomiting and coughing symptoms were settled. Vital signs showed fever of RR 38 breaths /minute, HR 110/minute, CVS= S1+S2+ Ponsystolic membrane and 102°F temperature. No cyanosis and clubbing were observed. There were certain clinical and pharmaceutical inaccuracies were noted during the treatment. Thus; a rational clinical practice needed to implement health care system. Specially; the avoidable clinical errors are required to be addressed to optimize the regimens. Moreover; the substandard pharmaceutical care and poor clinical services are major obstacles to cure this disease. Therefore; the comprehensive clinical examination and therapeutical care will help to avoid the undesired health related consequences.

Key Words; Pneumonia, infant, pharmaceutical care, clinical services.

Correspondign Author; Mehreen Zeb, Ripha Institute of Pharmaceutical Sciences, Ripha International University, G-7/4, Islamabad, Paksitan. Emial: [email protected]


INTRODUCTION:

Pneumonia is a pulmonary inflammatory condition affecting the alveoli or microscopic air sacs. The main symptoms associated with this mortal disease are high fever, cough, shaking chill, shortness of breathing, sharp or stabbing, chest pain, confusion and an increased respiratory rate (RR). Fever, however, is not very specific, as it occurs in many other common illnesses and may not be seen in severe disease or malnutrition. More serious symptoms may include: central cyanosis, decreased thirst, convulsions, persistent vomiting, or a decreased consciousness. The pneumonia caused by legionella associated with abdominal pain, diarrhea, or confusion, streptococcus pneumonia associated with rusty colored sputum; while the pneumonia caused by Kebsiella may have bloody sputum.

The mortality rate in children (less than 5 years of age) is 14 deaths per 1000 children annually before giving any medical treatment in the north-western part of Pakistan (Khan at el., 1990). While; in a village in the Northern Areas, 44% of all deaths in children less than 5 years of age between 1988 and 1991 were occurred because of the pneumonia ( Marsh al el, 1993). Mortality Surveillance by the Aga Khan Health Services, Pakistan (AKHSP) in the Northern Areas, showed that pneumonia causes approximately 33% of deaths in infants and 37% in children aged 1–4 years (Annual report 2001). While Rudan et al., (2004) reported > 95% early childhood pneumonia worldwide occurs in children aged 0–4 years in developing countries at an incidence rate of 0.28 episodes per year (Rudan at el., 2004). A study conducted in Karachi on children aged 2–59 months in 2002, found low pneumonia rates; 8.2 per 100 child-years (Nizami at el., 2006). Unpublished studies in 1990s conducted near Gilgit (capital of the Northern Areas of Pakistan) reported the incidence of 30 cases per 100 child-years of observation in children under 5 years of age (Pechere at el., 1995).

The high incidences of pneumonia were seen in infants and children at high altitudes with rates of 30 episodes per 100 children per years (Lanata at el., 1994). The factors associated with pneumonia include male gender (Monto at el., 1994), malnutrition (Black at el., 1982), micronutrient deficiency (Barreto at el., 1994), low immunization coverage (Oyejide at el., 1990),low household income (Tupasi at el., 1990), overcrowding (Ruutu at el., 1990), poor breastfeeding practices (Victora at el., 1999) and exposure to indoor air pollution (Smith at el., 2000).

CASE REPORT

A six month old baby (girl) was presented in the Pediatric ward of local hospital, Rawalpindi, Pakistan with chief complaints of high fever (since last 2 days), pain, cough vomiting and severe malnutrition. The high grade fever was not associated with chills and rigors. Her physical examination showed temperature 104°F, respiratory rate 60 breaths/ minute, heart rate 130 beats /minute and chest bilateral crepts on auscultation. Her medical showed sub-vaginal delivery (SVD) and up to date vaccination profile. She is under nourished (poor breast and bottle fed) because of her lower middle class family social background.

On basis of her medical investigation (primary diagnosis) the physician prescribed the injection Claforan (cefotaxime) 250mg IV (intra venous) TDS (three time a day) for four days; injection


Grasil (amikacin sulphate) 50mg IV BID (two time a day); Ventolin (salbutamol albuterol) nebulisation after every 3 hours; syrup Paramac (paracetamol) 1 TSF (tea spoonful) or 120mg /5ml SOS (as required); injection Lasix (furusemide) 10mg IV stat (immediately); tablet Capoten (captopril) 12.5mg ½ BID; syrup Acefyl (acefylline piperazine) and syrup Phenergan were administered for first two days. The clinical findings showed the definite symptoms of pneumonia. On 2nd day of therapy, her vomiting and coughing symptoms were settled with loose motion, abdomen discomfort and chest bilateral crepts. Vital signs showed fever of RR 38 breaths /minute, HR 110/minute, CVS= S1+S2+ Ponsystolic membrane and 102°F temprature. No cyanosis and clubbing were observed. While; on 3rd day, her fever reduced to 101°F, nourishment improved, RR become 50 breaths/minute, HR 140 beats/minute, with chest bilateral harsh sound. CVS= S1+S2+Ponsystolic membrane. Thus; the physical continued the current treatment and executed the Echo tests. Whereas; on the 4th day, the temperature became 102°F, RR 54 breaths /minute, HR 140 beats/minute, abdomen soft, chest bilateral clear. CVS S1+S2+Ponsystolic membrane. Treatment continued and oral intake was encouraged. On the 7th day, her temperature became 98°F, RR 30 breaths/minute, HR 120 beats/ minute. Thus; the treatment was continued.

DISCUSSION

The standard recommendations of BNF (British National Formulary) guide the physicians to design the treatment plan of the baby patient under discussion. The dose regimen of injection Claforan, Ventolin nebulization, syrup Paramac, injection Lasix, tablet Capoten and syrup Acefyl prescribed according to the specifications. But; the dose of injection Amikacin was noticed higher than the recommended dose.

While the syrup Phenergen (Promethazine) is contraindicated in children less than 2 years of age (according to FDA) and less than 3 years of age (according to BNF) was recommended as ½ TSF BD for first two days. Promethazine is known to cause respiratory depression in infants especially, because of the under developed CYP2D6 enzyme system; required to convert the drug to non-toxic metabolites. Thus; the child was under the age of six months and should be avoided to administer. That may introduce the serious medical complications.

Besides this, the patient has the symptoms of loose motions and continuously dehydrated since 5 or more days. She needed to be monitored for fluid and electrolytes to maintain an electrolytic balance. But; she was prescribed diuretic that has aggravated her situation; hypokalemia occurs as a result of the salbutamol/albuterol and furosemide. That was counter balanced by the administration of Captopril which produced hyperkalemia.

Moreover; the furosemide and aminoglycosides (i.e., Amikacin in this case) are prone to cause ototoxicity. That markedly increased combination therapy and need intensive therapeutical monitoring. Aminoglycosides also cause visual disturbances and renal function alteration. But; the clinical setting has not arranged the proper auditory or vision examination system. In addition of that; the drugs like Cefotaxime, Captopril, Paracetamol and Phenergen cause blood dyscrasias and should be monitored (CBC-complete blood count) time to time.

This study is substantiated by Khan &Blum (1979) who reported the potential respiratory depression and apnea introduced by promethazine in infants. They reported four victims of sudden infant death syndrome (SIDS) received promethazine in treatment. They evaluated 52


more SIDS cases, found 23% of the SIDS victims and 22% of near-miss SIDS patients received phenothiazine prior to the event (Khan & Blum 1982). While Buck & Blumer (1991) reported respiratory depression in infants, particularly premature neonates, not be able to metabolize promethazine as well as older children and adults due to lower levels of CYP2D6 activity or reduced sulfur stores. FDA (2000) strengthened the warning section of the prescribing information for promethazine to state that it should not be used in children less than 2 years of age. Starke et al. (2005) reported the 22 cases of respiratory depression the FDA between 1969 and 2003 including 7 deaths. As a result, the FDA added a black box warning to promethazine in November 2004, declaring its use in children less than 2 years as contraindicated.

CONCLUSION

The rational therapy of pneumonia is a serious issue and need unusual intentioned of health professionals. Specially; the avoidable clinical errors are needed to be addressed for optimizing the therapy plans. Moreover; the substandard pharmaceutical care and poor clinical services are major obstacles to cure this disease. Therefore; the comprehensive clinical examination and therapeutical care will help to avoid the undesired health related consequences.

REFERENCES

Annual report 2001, Pakistan Northern Areas and Chitral. Islamabad: Aga Khan Health Service; 2002

Barreto ML, Santos LM, Assis AM, Araújo MP, Farenzena GG, Santos PA, et al., et al. Effect of vitamin A supplementation on diarrhoea and acute lower respiratory tract infections in young children in Brazil. Lancet 1994; 344: 228-31 doi: 10.1016/S0140-6736(94)92998-X pmid: 7913157.

Black RE, Brown K, Becker S, Yunus M. Longitudinal studies of infectious diseases and physical growth of children in rural Bangladesh. Am J Epidemiol 1982; 115: 305-14 pmid: 7064969

Black RE. Zinc deficiency, infectious disease and mortality in the developing world. J Nutr 2003; 133: 1485S-9S pmid: 12730449

Buck ML, Blumer JL. Phenothiazine-associated apnea in two siblings. DICP Ann Pharmacother 1991;25:244–7

Khan AJ, Khan JA, Akbar M, Addiss DG. Acute respiratory infections in children: a case management intervention in Abbottabad District, Pakistan. Bull World Health Organ 1990; 68: 577-85 pmid: 2289294.

Khan A, Blum D. Possible role of phenothiazines in sudden infant death. Lancet 1979;i:364.

Lanata CF, Quintanilla N, Verastegui HA. Validity of a respiratory questionnaire to identify pneumonia in children in Lima, Peru. Int J Epidemiol 1994; 23: 827-34 doi: 10.1093/ije/23.4.827 pmid: 8002198.


Lehmann D. Epidemiology of acute respiratory tract infections, especially those due to Haemophilus influenzae, in Papua New Guinean children. J Infect Dis 1992; 165: S20-5 pmid: 1588165

Marsh D, Majid N, Rasmussen Z, Mateen K, Khan AA. Cause-specific child mortality in a mountainous community in Pakistan by verbal autopsy. J Pak Med Assoc 1993; 43: 226-9 pmid: 8114258)

Monto AS. Studies of the community and family: acute respiratory illnesss and infection. Epidemiol Rev 1994; 16: 351- pmid: 7713184

Nizami SQ, Bhutta ZA, Hasan R. Incidence of acute respiratory infections in children 2 months to 5 years of age in periurban communities in Karachi. J Pak Med Assoc 2006; 56: 163-7 pmid: 16711336

Rudan I, Tomaskovic L, Boschi-Pinto C, Campbell H, WHO Child Health Epidemiology Reference Group.. Global estimate of the incidence of clinical pneumonia among children under five years of age. Bull World Health Organ 2004; 82: 895-903 pmid: 15654403.

Oyejide CO, Osinusi K. Acute respiratory tract infection in children in Idikan Community, Ibadan, Nigeria: severity, risk factors and severity of occurrence. Rev Infect Dis 1990; 12: S1042-6 pmid: 2270403.

Pechere JC, editor. Community acquired pneumonia in children. Worthing: Cambridge Medical Publications; 1995.)

Ruutu P, Halonen P, Meurman O, Torres C, Paladin F, Yamaoka K, et al., et al. Viral lower respiratory tract infections in Filipino children. J Infect Dis 1990; 161: 175-9 pmid: 2153734

Smith KR, Samet JM, Romieu I, Bruce N. Indoor air pollution in developing countries and acute lower respiratory infections in children. Thorax 2000; 55: 518-32 doi: 10.1136/thorax.55.6.518 pmid: 10817802

Starke PR, Waver J, Chowdhury BA. Boxed warning added to promethazine labeling for pediatric use. N Engl J Med 2005;352:2653.

Thomas J. Lamb, P.A. Lumina Station, Suite 225 1908 Eastwood Road Wilmington, NC 28403 [email protected]; http://www.druginjury.com/druginjurycom/2006/05/phenergan_and_s.html

Tupasi TE, Leon LE, Lupisan S, Torres CU, Leonor ZA, Sunico ES, et al., et al. Patterns of acute respiratory tract infection in children: a longitudinal study in a depressed community in Metro Manila. Rev Infect Dis 1990; 12: S940-9 pmid: 2270416

Victora CG, Kirkwood BR, Ashworth A, Black RE, Rogers S, Sazawal S, et al., et al. Potential interventions for the prevention of childhood pneumonia in developing countries: improving nutrition. Am J Clin Nutr 1999; 70: 309-20 pmid: 10479192


Canadian Journal of Applied Sciences; 2011; 1(3): 158-167, October, 2011 Intellectual Consortium of Drug Discovery & Technology Development Inc. ISSN 1925-7430 Available online http://www.canajas.com Research Article

OUTCOME OF CONSERVATIVE TREATMENT IN CARIES SPINE

Muhammad Kaleem Shaukat1, Osama Jafar2 and Mubasher Sarfraz2 1Assistant Professor Department of Orthopedics Surgery, Madina Teaching Hospital, Faisalabad, Pakistan, 2 Shaikh Zayed Hospital, Lahore, Pakistan.

ABSTRACT

Bachground: Tuberculosis remains endemic and major public-health problem in most part of the world. Despite the reduction in the prevalence of the tuberculosis and the advent of new drugs for tuberculosis, the incidence of extra pulmonary tuberculosis continues to increase at world level. In spite of all the advancements regarding diagnosis, treatment and total control of many diseases, this slow but grave disease still affects considerable number of cases and makes them disabled and even paralyzed.

Objective: To assess the outcome of conservative treatment in caries spine.

Design: Descriptive case series study.

Duration of Study with Dates: One year (1st March, 2009 to 28th February, 2010)

Methodology: History and clinical examination performed for sinus, abscess, effectiveness of chemotherapy and biplane X-rays of affected area of seventy three patients were taken. Patients followed-up and outcome was ascertain favorable according to British Medical Research Council if there were no symptom of pain, no sinus or abscess on clinical examination and radiological healing of spinal lesion at the end of one year.

Results: Out of 73 patients, 28 were males and 45 were females between 18-60 years. According to site of lesion thoracic spine was commonly involved (36 patients), lumbar spine in 23 patients followed by thorocolumbar junction in 14 patients. At end of follow-up, 55 (75%) patients achieved favourable status and 18 (25%) patients had unfavourable status.

Conclusion: Current study gave uniformly echo the fact that spinal tuberculosis is a “medical condition” and can be effectively treated by conservative treatment. Key words: Caries spine, Conservative treatment, Anti-tuberculous therapy, Radiological healing of spinal lesion, Kyphosis Corresponding authors: Dr Muhammad Kaleem Shaukat, Assistant Professor Department of Orthopedics Surgery, Madina Teaching Hospital, Faisalabad, Pakistan, 2 Shaikh Zayed Hospital, Lahore, Pakistan.


INTRODUCTION

Worldwide tuberculosis (TB) is the most common cause of infection related death. In 1993, the World Health Organization (WHO) declared tuberculosis to be a global public health emergency. 1 Mycobacterium tuberculosis is the causative microorganism of tuberculosis in humans. Three related organisms Mycobacterium tuberculosis, Mycobacterium africanum, and Mycobacterium bovis are the causes of tuberculosis. Mycobacterium tuberculosis is by far the most common. Spinal tuberculosis often called Pott’s disease or caries spine is by definition, an advanced extrapulmonary manifestation of tuberculosis caused by mycobacterium tuberculosis complex affecting vertebral column.2,3

Osteoarticular tuberculosis represents 3-5% of all cases of tuberculosis and between 10-15% of extrapulmonary tuberculosis. Spinal tuberculosis represents 50% of osteoarticular form.4 It was reported that tuberculosis spine is more common in males than females. Percivall Pott gave his name to spinal deformity with curvature due to abscess and vertebral destruction associated with paralysis of lower limbs as Pott’s paralysis.5, 6

Though there has been a remarkable improvement of pulmonary tuberculosis management, but treatment of tuberculous spondylitis whether surgical or conservative, is still controversial. Regarding the aims of treatment, there is a universal consensus that these should be, first to arrest the progression of disease, and secondly to achieve a stable spine. With the advent of the chemotherapy, there has been a general inclination towards conservative form of therapy. Specific and effective conservative treatment is now the mainstay of treatment.7, 8

The dilemma was much solved by Medical and Research Council and Working Party (MRC&WP) trials, which were a series of six prospective studies. They recommended chemotherapy as a standard first line treatment. In selective patients the results were much better with anterior surgery especially during the first 18 months. But the last and latest report of, MRC&WP have shown no statistically significant difference in the outcome of both the procedures 10-15 years after the treatment.9 Currently the trends are towards the less radical middle path regimen. This unclear rather confusing situation leads to the need of a local study to look upon the results of non-operative ambulatory treatment of caries spine.10

Tuberculous spondylitis being endemic in underdeveloped countries including Pakistan and ranks 6th among the 22 high-burden tuberculosis countries worldwide , posing strong economical set back to whole family and state.11

Surgery attempts only to extirpate the complications arising from disease process. In many developing countries it may not be possible to undertake this surgery because of limited facilities, inadequate hospital beds and financial constraints. Under such circumstances the best available method of treatment for spinal tuberculosis appears to be chemotherapy on an ambulant basis. Our study of conservative treatment will help to generate local data to assess its efficacy in treatment of caries spine. 12

Modern treatment of T.B. spine began with Hibbs (1911) who noticed natural ankylosis of spine and advocated deliberate surgical induction, thus inventing first surgically achieved spinal fusion. Albee described spinal fusion with a bone graft in the same year. In 1934, Ito, Tusuchija and Asami reported the first radical debridement and bone grafting procedure. It was


Hodgson & Stock who first time popularized the anterior decompression as the Hong Kong procedure. They aimed their method at the complete surgical excision of diseased and avascular focus to allow the free access of the antitubercular drugs to the site of the lesion. But this left the spine unstable. So a modification was devised to place a tricortical bone graft for the stabilization of the spine and achievement of early arthrodesis, this is known as Modified, "Hong Kong Procedure".13,14

These trials were performed at different centers. Results of up till 15 years of follow up have shown that initial period of POP immobilization has little if any long term benefits. Conservative treatment showed comparable results to that of anterior decompression.15

Objective

The objective of this study was to assess the outcome of conservative treatment in caries spine.

Operational Definitions

Outcome: will be assessed in terms of

a-Symptom of pain (Using numeric rating scale (NRS) If zero (0) means no pain and ten (10) means pain as bad as it could be.

b-Sinus or abscess on clinical examination.

c-Radiological healing of spinal lesion ( bony fusion and sclerosis of the contiguous surfaces of affected vertebral bodies with reduction or disappearance of intervening disc space).

Outcome will be ascertained favorable if there will be

No Symptom of pain, no Sinus or abscess on clinical examination and radiological healing of spinal lesion at the end of one year.

MATERIAL AND METHODS

Study design: Descriptive case series study.

Setting: The study was conducted at Department of Orthopaedic Shaikh Zayed Hospital, Lahore and Madina Teaching Hospital, Faisalabad, Pakistan.

Study Duration: One year (1st March, 2009 to 28th February, 2010)

Sample size: The sample size is 73 cases by using WHO sample size calculator for single proportion with absolute precision required 9%, confidence level taking 95% expected percentage of favorable status (No symptom of pain, no Sinus or abscess on clinical examination and radiological healing of spinal lesion) with conservative treatment in caries spine i.e. 81%.

Sampling technique: Non-probability purposive sampling

Sample Selection

Inclusion Criteria: Tuberculosis of spine involving any vertebra in dorsolumbar region, age 18 years or more and patients of both sexes.

Exclusion Criteria: Patients excluded from study with motor deficits at time of presentation on clinical examination, kyphosis >40 degree at the effected level on radiograph at presentation,


deteriorating neurological functions on clinical examination while on antituberculous therapy, involvement of three or more vertebrae, pregnant women and diabetes mellitus.

Data Collection Procedure

Patients attending at Orthopaedics outpatient department, Shaikh Zayed Hospital, Lahore and Department of Orthopedic Madina Teaching Hospital, Faisalabad, for treatment of back pain was our study population. Seventy three patients fulfilling the selection criteria were identified. They were explained about the study and an informed consent got signed about their inclusion in the study. The demographic profile (i.e. age, sex) were noted, clinical examination was performed for sinus or abscess and biplane x-rays of affected area were taken. Conservative treatment was started with initial phase of four drugs.

Patients were followed up in outpatient department and pain was rated using NRS. Outcome of the study was evaluated at the end of one year from start of chemotherapy. Outcome was ascertained favorable if there was no symptom of pain, no sinus or abscess on clinical examination and radiological healing of spinal lesion at the end of one year. Effect modifier like age was addressed using stratification. All this information was collected through specially designed proforma.

Data Analysis: All the collected information was entered into SPSS version 10.0. Qualitative variables i.e. sex, pain (Yes, No), abscess or sinus (Yes, No) and radiological healing (Yes, No) and status of conservative treatment (Favorable, unfavorable) were presented as descriptive statistics, calculating their frequencies and percentages. Quantitative variables i.e. age was presented as numerical statistics, calculating its mean and standard deviation.

Data was stratified for age to address effect modifier.

RESULTS

The study was conducted in Orthopaedics outpatient Department, Shaikh Zayed Hospital, Lahore and Madina Teaching Hospital, Faisalabad. The study was conducted on 73 patients during one year. There were 28 males (38%) and 45 females (62%). Male to female ratio was 1:1.6 (Data presented in table 1).

The patient’s prestnted in Table 1 were also grouped into three age profile. The first age group patients aged ranged 18-32 years (n = 39) 53%, in second age group patients aged between 33-46 years (n = 19) 26% and in the third age group patients aged 47-60 years (n = 15) 21%. The mean±SD between the ages was 34.62±13.95 years. The male and female ratio in frequency and percentage also presented in the same table (1).

According to the site of lesion involvement of thoracic spine was higher involved 36 patients (49%) as compaired to, thoracolumbar junction in 14 patients (20%) and lumbar spine in 23 (31%) [Table 2]. The male and female ratio in frequency and percentage also presented in the same table (2).

All the patients were having pain prior to starting treatment. At the end of study 61 (84%) patients were pain free and did not pain relief in 12 (16%) patients. Seventeen out of 73 patients were having sinus or abscesses at the start of treatment. At the completion of study 6 patients


(35.29%) remained with sinus or abscesses clinically while 11 completed improved. All the patients were having radiological active disease prior to starting treatment. At the end of study 55 patients had radiological healing of spinal lesion [Table 3]. (Figure 1a,b,c,d).

In 18 patients (25%) there is no radiological healing. At the end of one year after start of conservative treatment, favorable status achieved in 55 patients (75%) and 18 patients (25%) remained with unfavorable status. The male and female ratio in favorable and unfavorable status are 23:32 and 5:13 respectably (Table 4).

Table 1. Frequency distribution of patients according age and sex

Groups Age (years)

Frequency (M:F)

Percentage (M:F)

1 18 – 32 39 (14:25) 53.0(36:64) 2 33 – 46 19 (6:13) 26.0(32:68) 3 47 – 60 15 (8:7) 21.0(53:47)

Mean±SD 34.62±13.95 (n = 73)

Table 2. Frequency distribution according to site of lesion Site of lesion Frequency

(M:F) Percentage

(M:F) Thoracic 36(12:24) 49.0(33:67)

Thoracolumbar junction 14(6:8) 19.0(43:57) Lumbar 23(10:13) 32.0(43:57)

(n = 73)

Table 3. Pre and post treatment frequencies of variable Pre-treatment Post-treatment

Frequency Percentage Frequency Percentage Pain (using NRS) 73 100.0 12 16 Sinus or abscess 17 24.0 6 35.29

Radiological healing

- - 55 75

Key: NRS Numerical rating score (n = 73)

Table 4. Frequency distribution according to final outcome Outcome Frequency

(M:F) Percentage

(M:F) Favorable 55(23:32) 75.0(44:56)

Unfavorable 18(5:13) 25.0(28:72)


(a) (b)

1) Represented X-Rays of pre-Treatment AP & lateral views of patients with caries spine D12 – L1

(a) (b)

2)

Represented X-Rays of Post-Treatment AP & lateral views of patients with caries spine D12 – L1

DISCUSSION

Tuberculosis remains a major public-health problem in most of the world especially in developing countries. Though there has been a remarkable improvement of pulmonary tuberculosis management, but treatment of tuberculous spondylitis whether surgical or conservative, is still controversial. With the advent of the chemotherapy, there has been a general inclination towards conservative form of therapy.17

In our study the average age of the patients at the time of the chemotherapy was 34.62 years with range18 to 60 years they were further splitted into three sub groups to uniform the data and study. (Table 1). In a study done by Nene and Bhojraj9 the average age of the patients


was 37 years. In another study reported by Parthasarathy et al17, the mean age of the patients was younger age group. In a study by Park et al18 the average age of the patients was 44.07±16.57 years.

(a) (b)

1) Represented X-Rays of pre-Treatment AP & lateral views of patients with caries spine L1 – L2

(a) (b)

2) Represented X-Rays of post-Treatment AP & lateral views of patients with caries spine L1 – L2

Patients presented at younger age in present study which is good agreement with the study conducted by Nene and Bhojraj19 . In under developed countries it effects the people of the


younger age group. In the developed countries it involves people at the extreme of age mostly old people.

Out of 73 patients 45 (62%) were female and 28 (38%) were male. The male to female ratio was 1: 1.6 (Table 1). In a study done by Nene and Bhojraj19 and reported 71% female and 29% male. In another study by Parthasarathy et al17 reported 47.40% were females and 52.60% males. In a study done by Dharmalingam20 reported 27% women and 73% men. In a study by Kursat et al21 reported 29% were females and 71% males. In a study by Park et al10 reported 69 patients were males (50.40%) and 68 were females (49.60%). In our study, there is female predominance as they constitute 52% population of our society where male gender is given preference over females in all aspect of life. Might be that reason, they could not fulfill their nutritional demands and remained more prone to diseases like tuberculosis.

In our study, the most common vertebral area involved was the thoracic (49.0%) then Lumbar (32%) and thoracolumbar junction (19%). Preponderance in dorsal spine is due to close anatomical relationship with lungs, which is the most common site for tuberculous seeding.22 In a study conducted by Nene and Bhojraj19 they only included cases of thoracic spine. In the study thoracic spine involvement was (84.3%) and thoracolumbar junction in (15.7%). In a study by Parthasarathy et al17 reported that thoracic involvement was (37%) and lumbar involvement (42%) and followed by thoracolumbar junction in (14%). In a study by Park et al18 reported thoracic involvement was (33.1%) and lumbar involvement (44%) and followed by thoracolumbar junction in (9.7%).

In our study 17 patients with sinus and abscesses on admission, 11 patients (65%) resolved in one year. Results of present study were differ to other reported studies which were of long duration and follow-up as abscesses or sinuses required longer duration to resolve. In study by Nene and Bhojraj19 reported the abscesses resolved with medical treatment in follow up of 27 months. In 12th report of the MRCW party on tuberculosis of the spine the rate of resolution was similar in all the series 83% lesions resolved by 24 months.8 In study by Parthrasaranthy12, 80% abscesses resolved in six years.

In our study radiological healing of spinal lesion was observed in 75% patients in one year. Radiological healing was low in present study as study was of short duration. Results may be improved with longer study duration and follow-up. In study by Nene and Bhojraj19 reported the radiological healing in 98% of cases in 27 months. In another study done by Parthrasaranthy17 80% by six years.

In present study, 75% patients achieved favorable status in one year. Our results were not in line to the other reported studies, because short duration of study. Results can be improved with longer study duration and follow-up. In a study by Nene and Bojraj19 reported results of 98% favorable status was achieved with nonsurgical ambulatory treatment in 27 months. In a study by Parthrasaranthy17 who reported favorable status in 94% patients in 10 years. MRC&WP7 reported 77% favorable status in 3 years.

CONCLUSION

With the advent of chemotherapy and understanding the resistance now ATT is the main stay in the treatment of caries spine. Uncomplicated cases of caries spine is a medical condition


and can effectively managed with conservative treatment in a vast majority of cases and indications for surgery are few and specific. So it was concluded that conservative treatment is very effective in the treatment of spinal TB, involving vertebral bodies without paraplegia. It is recommended for better results of such study with longer duration and follow-up needs to be conducted.

REFERENCES

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2. Lesic A, Bumbasirevic M, Savic B, Cobeljic G, Pesut D. Current diagnosis and treatment of the Osteoarticular tuberculosis.srp Arh Celok Lek 2004; 132: 345-51.

3. Govender S. Spinal infections. J Bone Joint Surg [Br] 2005; 87-B: 1454-8.

4. Camillo FX. Infections of the spine. In: Canale ST, Beaty JH, eds. Campbell’s operative orthopaedics. 11th ed. Philadelphia: Mosby 2008; 2238-66.

5. Dunn R, Zondagh I. Spinal tuberculosis: diagnostic biopsy is mandatory. S Afr Med J 2008; 98: 360-2.

6. Rosales-Olivares LM, Valle-Cerna I, Alpizar-Aguirre A, Miramontes-Martínez V, Arenas-Sordo Mde L, Reyes-Sánchez A. 1Percutaneous biopsy evaluation in the diagnosis of thoracic and lumbar vertebral destruction syndrome. Cir Cir 2007; 75: 459-63.

7. MRC&WP on TB spine: A 03 years controlled of short course regimens of chemotherapy in the ambulatory treatment of spinal tuberculosis. J Bone Joint Surg (Br) 1993; 75-B: 240-8.

8. MRC&WP on TB spine: A 05 year assessment of controlled trials of shot course chemotherapy regime for 09 months duration for spinal tuberculosis in patients with ambulatory from standard radical surgery. In J Orthop 1999; 23: 73-81.

9. MRC&WP on TB spine: A 15 year assessment of controlled trials comparing 9 months ambulant chemotherapy with radical and debridement series for spinal TB. J Bone Joint Surg (Br) 1998; 80-B: 456-62.

10. Sriram K, Somasundaram PR, Parthasarathy R, Chandrasekaran V. A reliable and simple method for identifying at start patients with tuberculosis of spine suitable for ambulatory chemotherapy. Indian J Orthop 2006; 40: 160-3.

11. Khushk IA, Ahmed I, Shah SS. Tuberculosis control in Pakistan: current issues and challenges. J Coll Physicians Surg Pak 2006; 16: 387-8.

12. Rajab TK, Barre LJ. Back pain from spinal tuberculosis. J Am Coll Surg 2008; 207: 453.

13. Bak KH, Cheong JH, Kim JM. Titanium mesh cage for anterior stabnilization in truberculous spondylitis: is it safe? J Korean Neurosurg Soc 2006; 40: 412-8.

14. Kanabar P. Tuberculosis of lumbar spine. Indian J Orthop 2005; 39: 81-9.


15. Seliukova NV. Zonography in the diagnosis of spinal tuberculosis at the outpatient stage. Probl Tuberk Bolezn Legk 2008; 11: 21-3.

16. Ahmad S. Treatment of late onset of neurological deficit in caries spine. J Pak Orthoped Assoc 2000; 54: 64-7.

17. Parthasarthy R, Sriram K, Santha T, Prabharkar R. Short course chemotherapy for tuberculosis of the spine, a comparative report between medical treatment and radical surgery, 10 year report. J Bone Joint Surg (Br) 1999; 81: 464-71.

18. Park DW, Sohn JW, Kim EH, Cho DI, Lee JH, Kim KT, et al. Outcome and management of spinal tuberculosis: a retrospective study of 137 adult patients at Korean teaching hospitals. Spine. 2007; 32: E130-5.

19. Nene A, Bhojraj S. Results of nonsurgical treatment of thoracic spinal tuberculosis in adults. Spine J 2005; 5: 79-84.

20. Dharmaligam M. Tuberculosis of the spine – the Sabab experience: epidemiology, treatment and results. Tuberculosis 2004; 84: 24-8.

21. Kursat OM, Reha B, Meltem T, Ilknur D, Dilay D, Sibel A, et al. Extrapulmonary tuberculosis in non-human immunodeficiency virus-infected adults in an endemic region. Ann Thorac Med 2007; 2: 118-21.

22. Nigg AP, Schulze-Koops H, Wirth S, Weiss M, Bogner JR. Tuberculous spondylitis (Pott's disease). Infection 2008; 36: 293-4.

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