EFFECTS OF HEAVY METALS ON THE AIR POLLUTION TOLERANCE INDICES ( APTI ) OF FIVE MEDICINAL PLANTS GROWING WITHIN QUARRY SITE IN ISHI-AGU, EBONYI STATE, NIGERIA.

BY

UGWU PATIENCE NGOZI PG/M. PHARM/08/49752

DEPARTMENT OF PHARMACOGNOSY AND ENVIRONMENTAL MEDICINES, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA

OCTOBER, 2014

TITLE PAGE

EFFECT OF HEAVY METALS ON THE

AIR POLLUTION TOLERANCE INDICES ( APTI ) OF

FIVE MEDICINAL PLANTS GROWING WITHIN QUARRY SITE IN

ISHI-AGU, EBONYI STATE, NIGERIA

BY

UGWU PATIENCE NGOZI PG/M. PHARM/08/49752

BEING REPORT SUMITTED TO THE DEPARTMENT OF PHARMACOGNOSY AND ENVIRONMENTAL MEDICINE, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA, IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER OF PHARMACY (M.PHARM) DEGREE.

PROF.( MRS) .S.I. INYA-AGHA (SUPERVISOR)

DEPARTMENT OF PHARMACOGNOSY AND ENVIRONMENTAL MEDICINES FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA

OCTOBER 2014 i

CERTIFICATION

This project report titled ”Effect of heavy metals on the Air Pollution Tolerance Indices (APTI) of five medicinal plants:Vitex doniana (Lamiceae ), Anthocleista djalonensis (Gentianae), Ricinus communis (Euphorbiacea ), Cnestis ferruginea(Connaraceae) and Manihot esculenta (Euphorbiacea ), growing within quarry site in Ishi-Agu, Ebonyi State Nigeria” is hereby certified as meeting the requirements for the award of Masters of Pharmacy (M.Pharm) degree in the Department of Pharmacognosy and Environmental Medicines, Faculty of Pharmaceutical Sciences,University of Nigeria, Nsukka.

______Supervisor Head of Department

______Date Date

______External Examiner

______Date

ii DEDICATION

This work is dedicated to my lovely, caring husband (Ugwu Benjamin

Sunday) and our children, Ebuka, Ugochukwu and Munachimso.

iii ACKNOWLEDGEMENTS My gratitude goes to God Allmighty, whose unfalling love saw me through this programe. I return all the glory to him. My thanks to my supervisor Professor (Mrs) S.I Inya- Agha whose professional advise and support came handy in the period of this duty. I would like to express my warmest gratitude to Dr (Mrs) U.E Odo who graciously attended to my problem when needed. I appreciate the Head of Dept., Professor C.O Ezugwu who provided professional advice and accommodation that facilitated this work. I wish to thank Rev. Dr S.C. Ezea, Mr Otuu Fred. and Pharm M.Ezejiofor, who have always given me their support and assistance. I wish to recognize my classmates, Pharm Edith Atamah , Pharm (Mrs). Okonta, Pharm Okwubie, Pharm Calister Ugwu, Pharm S.A Chime, Pharm Charity Eze for their encouragement. My appreciation goes to the Technical staff of the Department of Pharmacognosy and Environmental Medicines whose numerous technical supports were reassuring in the course of this study. My deepest gratitude goes to my father, Francis Omeje, whose prayerful support gave me strength. My warmest greeting to Franca, Chioma, Kenneth and Chika for their help to see that this work was successful. Our Almighty God will surely reward them.

Ugwu patience Ngozi University of Nigeria, Nsuka, 2014.

iv ABSTRACT

Effects of heavy metals on the Air Pollution Tolerance Indices (APTI) of five medicinal plants growing within quarry site in Ishi-Agu, Ebonyi State, Nigeria were studied. Following laboratory analysis of the leaves of the plants for metal load and four biochemical parameters of APTI computation. The metal load was determined by the Atomic Absorption spectrophotometric method, while the ascorbic acid was determined by the titremetric method, leaf extract pH by the electro-chemical techniques, relative water content by plant turgidity mass and the total chlorophyll, by spectrophotometric method. Foliar photomicrography of the leaf was also studied. Results of Plants from experimental site showed changes in physical and internal structures of the leaves that lead to closure of some stomata pores in test plants and damage of some epicelluler cells.

Quantification of phytochemical contents of the test plants showed higher values than that of control which may be due to the multiplication of some organic substances caused by oxidative stress, presence of inorganic subtances like heavy metals and lime stone dust.The result of elemental analysis of both experimental and control plants gave the range of concentration of the metals in parts per million (ppm) as follows: - lead ( 2.75 – 14.13 ± 0.02); Nickle

(0.27 - 0.54 ± 0.01) ; Cadmium ( 2.25 - 24.30 ± 0.03); Zinc (0.11- 0.03 ±

0.02); and Arsenic (0.10 - 0.70 ± 0.04). Control –Lead ( 0.00 – 5.52 ± 0.01);

Nickle (0.13 – 0.23 ± 0.01); Cadmium (0.00 - 12.00 ± 0.03); Zinc ( 0.06 – 0.10

± 0.03); and Arsenic (0.02 – 0.20 ± 0.02). There was slight increase of

v Cadmium above maximum daily permissible intake in C. ferruginea , A, djalonensis, and R. communis, also there is high content of Arsenic in

R.communis which is the cause of oxidative strees in test plants that result in the elicitation of antioxidants from plants and multiplication of ascorbic acid content. The values for the percentage computation of APTI in test plants are

V.doniana (11.03), C.ferruginea (7.62), A.djalonesis (9.94), R. communis (11.5) and M. esculenta (8.60). Control pants gave V.doniana (11.86), C.ferruginea

(8.21), A.djalonesis (11.86), R. communis (14.39), and M. esculenta (10.21) that result in reduction of Relative water content, Ph value, Total chlorophyll content and Ascorbic acid content.There is relative high content of heavy metals, physiological and visible changes in some sensitive test plants. Owing to information gotten from the result of this research it is not recommended that plants from the quarry sites be used in herbal formulation because of possible metal intoxication and attenuation of their phythopotency.

vi TABLE OF CONTENTS

Title page: i Certification: ii Dedication: iii Acknowledgements: iv Abstract: v Table of contents: vii List of Tables: x List of Figures: xi CHAPTER ONE: 1 1.1 Introduction 1 1.2 Quarry 2 1.2.1 Effect of quarry on environments 3 1.2.2 Quarry waste 4 1.2.3 Land pollution 5 1.2.4 Noise pollution 5 1.2.5 Damage to biodiversity 6 1.3 Pollution 7 1.3.1 Pollution and vegetation 7 1.3.2 Effect of pollution on leaf anatomy 9 1.4 Air Pollution 11 1.4.1 Air pollution and medicinal plants 13 1.4.2 Types of air pollution, and their sources 14 1.4.3 The threshold which air pollution affects medicinal plants 16 1.4.4 Effect of air pollution on leaf morphology 17 1.4.5 Effect of air pollution on plant sugar content 18 1.4.6 Effects of air pollution on leaf proline 19 1.4.7 General effects of air pollution on medicinal plants 20 1.4.8 Ways of controlling air pollution 23 vii 1.5 Heavy Metals 26 1.5.1 Effect of heavy metals on medicinal plants 27 CHAPTER TWO 29 2.1 Medicinal plants 29 2.2 Pharmacognostic profile of selected medicinal plants 30 2.2.1 Pharmacognostic profile of Cnestis ferruginea 30 2.2.2 Pharmacognostic profile of Ricinus communis 34 2.2.3 Pharmacognostic profile of Vitex doniana 38 2.2.4 Pharmacognostic profile of Anthocliestia djalonensis 41 2.2.5 Pharmacognostic profile of Manihot esculenta 45 2.3 Previous pharmacological invetigation of plants studied 48 2.4 Aim and objective 55 2.5 Significance of study 55 2.6 Definition of terms 56 CHAPTER THREE: MATERIALS AND METHODS 66 3.1 Collection and preparation of plants materials 66 3.2 Chemicals, reagents and equipments 67 3.3 Morphological and microscopic analysis 68 3.4 Qualitative Phytochemical analysis 69 3.5 Quantitative Phytochemical analysis 74 3.6 Determination of Analytical standard 76 3.7 Elemental analysis 80 3.8 Air pollution Tolerance index analysis 84 3.9 Statistical Analysis 86 CHAPTER FOUR: RESULT 87 4.1 Macroscopic /Morphological examination of the leaves of test and control samples 87

4.2 Microscopical examination of the leaves of test and control samples 90

viii

4.3 Summary of the morphological features and microscopic results

of leaf from experimental and control sites respectively. 95

4.4 Phytochemical analysis of the leaves of test and control samples 96 4.5 Analytical standard of the plants leaves of the test and control samples 98 4.6 Elemental analysis of the leaves of test and control samples 99 4.7 Relative water contents of the leaves of the leaves of test and control samples 101 4.8 Chlorophyll contents of the leaves of the leaves of test and control samples 102 4.8 pH of the leaf extract of leaves of test and control samples 102 4.9 Ascorbic acid contents of the leaves of test and control samples 103 4.10 Air pollution tolerance indices (APTI) of the leaves of test and control samples 103 CHAPTER FIVE: DISCUSSION AND CONCLUSION 104 5.1 Discussion 104 5.2 Conclusion 116 Recommendation 117 Reference 118 Appendix 127

ix LIST OF TABLES

Table1 : Summary of the morphological and microscopic features of leaves of test and control samples 95

Table 2: Qualitative phytochemical analysis of the five medicinal plants 96

Table 3: Quantitative analysis of the five medicinal plants 97

Table 4: Analytical Standard of the five medicinal plants 98

Table 5 : Elemental analysis of the five medicinal plants 100

Table 6 : Relative water contents (RWC) of the five medicinal plants 101

Table 7: Chlorophyll contents of test and control samples 102

Table 8: pH of leaf extract of the five medicinal plants 102

Table 9: Ascorbic acid contents of the five medicinal plants 103

Table 10 : Air pollution tolerance indices (APTI) of the five medicinal plants 103

x LIST OF FIGURES Fig 1 : Photograph of Cnestis ferruginea 31

Fig 2 : Photograph of Ricinus communis 36

Fig 3 : Photograph of Vitex doniana 39

Fig 4 : Photograph of Anthocliestia djalonensis 43

Fig 5 : Photograph of Manihot esculenta 46

Fig 6: Macroscopic features of the leaves of test and control samples of Vitex doniana 87

Fig 7: Macroscopic features of the leaves of test and control samples of Cnestis ferruginea 88

Fig 8 : Macroscopic features of the leaves of test and control samples of Anthocliestia djalonensis 88

Fig 9 : Macroscopic features of the leaves of test and control samples of Ricinus communis 89

Fig 10 : Macroscopic features of the leaves of test and control samples of Manihot esculenta 89

Fig 11 : Microscopic view of stomatal occurrence at lower and upper epidermis of the leaf of Vitex doniana of both test and control 90

Fig 12 : Microscopic view of stomatal occurrence at lower and upper epidermis of the leaf of Cnestis ferruginea of both test and control 91

Fig 13 : Microscopic view of stomatal occurrence at lower and upper epidermis of the leaf of Anthocliestia djalonensis of both test and control 92

Fig 14 : Microscopic view of stomatal occurrence at lower and upper epidermis of the leaf of Ricinus communis of both test and control 93

Fig 15 : Microscopic view of stomatal occurrence at lower and upper epidermis of the leaf of Manihot esculenta of both test and control 94

xi Fig 16 (A 1): Graph of the concentration of lead content of the five medicinal plants 127

Fig 17 (A 2): Graph of the concentration of Nickle contents of the five medicinal plants 128

Fig 18 (A 3 ): Graph of the concentration of Cadnium contents of the five medicinal plants 129

Fig 19 (A 4): Graph of the concentration of Zinc contents of the five medicinal plants 130

Fig 20 (A 5): Graph of the concentration of Arsenic contents of the five medicinal plants 131

CHAPTER ONE

1.1 INTRODUCTION

Plants occupy vital position in the ecosystem because of their role as primary producers.They are also the major recipients of environmental pollution owing to their static disposition. While men and other animals move away from the immediate vicinity of compromised environmental status, plants remain relatively immobile receiving environmental pollutants within the ecosystem.

The effect of particulate air pollutants on vegetation have been studied always before now (Manning,1971). Air Pollution Tolerance Indices (APTI) of plants is an important measure to check the effect of air pollution on medicinal plants and other plants generally. Nigerian as with orther developing country suffers pollution burden associated with the ever increasing human population, vehicular traffic, infrastractures and industries, with negative implications for the sustainability of humans, animals and plants communities (Choudhury and

Banerjee, 2009). Since plants major systems and organs are exposed to the atmosphere, any air pollution around the environments reflects on the plant health making some plant show sensitivity, some show minimal or no effect while some are tolerant. The plants response to air pollutant varies from species to species, type of pollutant, its reacting mechanism, and duration of exposure.

World Health Organization (WHO, 1989) recommends that medicinal plants which form the raw materials for the finished products may be checked for the presence of heavy metals. This is because after collection and transformation into final dosage form, the heavy metals resident in these plants may find its way into human body and may inhibit or impair the normal functions of central nervous system the liver, lungs, heart, kidney and brain, leading to hypertension, abdominal pain, skin eruptions, intestinal cramp and different types of cancers(Shad et al. , 2008).

This work is to investigate on the effects of lime Stones dust from quarry site in

Obegu Amaeze village at Ishiagu, Ebonyi state, Nigeria, on some medicinal plants ( Vitex doniana, Anthocliestia djalonensis, Ricinus communis , Cnestis

Ferruginea and Manihot esculenta ). For the fact that single parameter cannot provide clear picture of the pollution induced changes, different measures like

APTI, Phytochemical analysis, plant leaf macroscopy and microscopy, and

Elemental analysis were used to authenticate, compare and relate the result.

1.2 Quarry

Quarry is the process of obtaining quarrying resources, usually rocks, found on

or below the land surface. The difference between mining and quarrying is that

quarrying extracts non metallic rocks and aggregates while mining executes the

site for mineral deposits. Some of the stones extracted was sandstone, lime

stones, perlite, marble, ironstone, slate, granite, rock salt and phosphate. They

are cheap and always ready for conveyance to a large market and its inclination

and depth are below the surface. The two principal branches of the industry are

the so called dimension-stone and crushed-stone quarrying. In the former, blocks of stone, such as marble, are extracted in different shapes and sizes for

different purposes. In the crushed-stone industry, granite, limestone, sandstone,

or basaltic rock is crushed for use principally as concrete aggregate or road

stone (James, 2012).

1.2.1 EFFECT OF QUARRY ON ENVIRONMENT

Quarrying carries the potential of destroying habitats and species they support.

Even if the habitats are not directly removed by excavation they can be indirectly affected and damaged by environmental impacts such as changes in ground water or surface water that cause some habitats to dry out or others to become flooded. Even noise pollution can have a significant impact on some species and affect their successful production . Quarries can also provide a good opportunity to create new habitats or to restore existing ones. Both positive and adverse societal impacts of modern manufacturing technologies have great consequences on economic, health, safety and environment in general.

Limestone quarrying can have many effects on environment, like dirtying of the environment and roads that lead to quarry. Also there are more serious consequences that quarrying portrays, quarrying releases toxic mercury into the air and surrounding soil and water. Even when mercury is not present in significant quantities, the air and water can be polluted by the dust generated by quarry activities (Vincent et al, 2012). Quarry has the ability of destroying habitants and the species they depend on both directly or indirectly. Quarry causes damage to the living species by causing some unfavorable changes to the ground water, noises that prevent them from having sucessesfull reproduction.

Moreover, there is still potential for damage to the environment particularly with water contamination. For example, suspended particles though chemically inert, may imbalance freshwater ecosystem. (Omosanya and

Ajibade 2011). The green plants (machine) mostly with explosives in order to extract material for processing gives rise to noise pollution, air pollution, damage to biodiversity and habitat destruction.

1.2.2 QUARRY WASTE

Quarrying involves the production of significant amounts of waste. Some types of quarries do not produce large amounts of permanent waste, such as sand and gravel quarries, whereas others will produce significant amounts of waste material such as clay and silt. The good news is that they are generally inert and non-hazardous, unlike the waste from many other processes. However, there is still potential for damage to the environment particularly with water contamination. Plants are major components of the ecosystem, a complex interaction between the biotic and abiotic entities of the environment. The industry, unfortunately discharge dust that settles not only on land, plants and trees but also on surface waters used for drinking and other domestic chores by the community. All quarry activities produce enough waste on the process of production, though some quarry do not produce large amount of waste like sand and gravel while some produce permanent waste material like silt and clay crushed for use principally as concrete aggregate or road stone ( Fisher, 1944). Again, like many other man made activities, quarrying involves the production

of significant amounts of waste. For instance, lead content in water will prevent

plants from getting their nutrients from the soil. Also suspended particles may

imbalance fresh water ecosystem. Large amount of solid can lead to flooding if

it is dumped on flood plains. This waste has to be monitored and kept where it

will not affect the environment in a harsh way (Lameed, 2010 ).

1.2.3 LAND POLLUTION

Land pollution, is pollution of earth natural land surface by

industrialization, commercial, domestic and land agricultural activities. Some of

the factors that contribute to land pollution are; chemical and nuclear plants,

mining littering, deforestations, construction etc. (James, 2012 ).

1.2.4 NOISE POLLUTION

Unfortunately, quarrying involves several activities that generate significant amounts of noise. It starts with the preparatory activities, such as establishing road or rail access, compound and even mineral processing facilities .

The excavation of the mineral itself involves considerable noise, particularly if blasting methods are used. Following this, the use of powered machinery to transport the materials as well as possibly processing plants to crush and grade the minerals, all contribute even more noise to the environment (Lameed, 2010).

Such extraction of raw materials from their natural habitats by mining, drilling, harvesting and those that relate to large scale water resources development projects, construction, agriculture, energy, industry and development projects, considerably affect the natural environment.

The process of exposing the mineral to be extracted is usually done by

removing the top soil and other soft layers using a scraper or hydraulic

excavators and dump trucks. The excavations of the minerals itself will involve

considerable noise, particularly when blasting methods are used.

1.2.5 DAMAGE TO BIODIVERSITY

One of the biggest negative impacts of quarrying on the environment is the damage to biodiversity. Biodiversity essentially refers to the range of living species, including fish, insects, invertebrates, reptiles, birds, mammals, plants, fungi and even micro-organisms. Biodiversity conservation is important as all species are interlinked, even if this is not immediately visible or even known, and our survival depends on this fine balance that exists within nature.

Nevertheless, with careful planning and management, it is possible to minimize the effect on biodiversity. This is one of the disadvantages of quarry on the environment, its negative effect on living organisms like fish, insects, invertebrates, reptiles, birds, mammal, plants, fungi, and micro-organisms. and

1.3 POLLUTION

Pollution is the introduction of a contaminant into the environment. It is created mostly by human actions, but can also be as a result of natural disasters.

Pollution has a detrimental effect on any living organism in an environment, making it virtually impossible to sustain life. It is the human introduction into the atmosphere of chemicals, particulate matter, biological materials that cause harm to human or other living organism, or damage the environment

(Anonymous, 2008). The atmosphere is a complex dynamic natural gaseous system that is esential to all living things. There are some substances in the atmosphere which may impair the health of plants and animals. Pollutants could be classified as either secondary or primary. Pollutants that are formed in the atmosphere and directly pollute the air are called primary Pollutants, while those that are formed in the air when primary pollutant react or interact are known as secondary pollutants(Vincent, et al., 2012)

1.3.1 POLLUTION AND VEGETATION

Industrialization areas suffer pollution because of the chemical emission

and others like oil spillage, Noise etc. These also affect the weather of that

zone which leads to increase in temperature that might result to climate

change. Climate change has become increasingly recognized as one of the

greatest challenges to human and other living things on earth. Worldwide

changes in seasonal patterns, weather events, temperature ranges and other

related phenomena have all been reported and attributed to global climate

change. Numerous experts in a wide range of scientific disciplines have

warned that negative impacts of climate change will become much more

intense and frequent in the future particularly if environmentally destructive

human activities continue unabated. Like all living members of the biosphere,

medicinal and aromatic plants (MAPs) are not immune to the effects of climate change. Climate change is causing noticeable effects on the life cycle and

distributions of the world's vegetation including wild MAPs. Some MAPs are

endemic to geographic regions or ecosystems particularly vulnerable to climate

change, which could put them at risk. The possible effects on MAPS may be

particularly significant due to their value within traditional systems of

medicine and as economically useful plants. The changing temperature and

wind patterns associated with climate change are causing precipitation factors

and giving some trees and shrubs the ability to grow taller in more open areas.

Some production of plants secondary metabolites are influenced by multiple

factors including disease, competition between plants, animals grazing, light

exposure, soil moisture and those other factors that may mitigate the effects of

climate change on plants secondary metabolites (Herbal , 2009).

1.3.2 EFFECT OF POLLUTION ON LEAF ANATOMY

Developments of stomata are often considered as one of the most important developments in plants evolution. By being environmentally controlled gateways into the plants controlling Co 2 uptake and transpiration they are central determinants of photosynthesis, cooling and nutrient uptakes (Arvel,

2009). To be able to balance Co 2 uptake and water transpiration through stomatal movements is an important response to changes in the environmental conditions. Low transpiration due to stomatal closure means less cooling of the leaves and less uptake and transportation of nutrients. Stomatal closure occurs when the guard cells surrounding the stomatal opening lose turgor pressure and close the opening ( Arvel, 2009) . There are many signals that induce stomatal

2+ closure, eg Absciscic Acid (ABA), Secondary messengers like Ca and H 2O2.

When there is an air pollution or oxidative stress in an environment, there are pathwaysb that leads to stomatal closure. Hydro passive stomatal closure occurs when water evaporation from the guard cells is too low to be balanced by water movements into these cells. The water is reduced to the extent that osmotic pressure is reduced and cell loses tugor pressure and shrink (Lucan,

2002).When this happens the guard cells are unable to maintain the shapes and stomatal pores is covered. Active stomatal closures occur when there is increase in ABA Co 2 level. This activates signaling pathways leading to stomatal closure. ABA is produced in the roots and leaves during water stress and is transported to the guard cell by ATP binding cassette (ABC) transporters that are located in the plasma membrane, but when the transporters are knocked out during oxidative or water stress, the ABA uptake is low. The size of the stomatal opening is regulated by the tugor pressure cell volume of the guard cells. Regulation of stomatal opening is linked to transport of ions and water through channel proteins across the plasma and vacuole membrane.

ABA induces the production of reactive oxygen species eg. H 2O2 which in turn acts as a trigger for low production, inhibition of membrane proton pumps and

Ca 2+ across both the plasma and vacuole membranes. H + AT-pase that are hyperpolarizing the plasma membranes must be inhibited to induce ABA mediated stomatal closure (Merlot et al., 2007).The increased ca 2+ level activate slow and rapid type of anion channels, generating an anions efflux from the cells. The anion efflux depolarizes the membrane, which in turn causes k + channel across both the vacuole and the plasma membrane.

Simultaneously Ca 2+ also inhibits k + in channel. Malate is also converted to starch reducing the osmotic potential and turgor pressure further (Kim and

Van, 2011).The plasma membrane is thus depolarized, the turgor pressure and cell volume reduced, then the stomata closed.

Stomata must open to admit Co 2 into the leaf for photosynthesis, but when they open, they allow water vapour to diffuse out of the leaf .Thus, stomata opens enough to support photosynthesis, but they must at the same time prevent excessive water loss. This is called constrained optimization problem (Michael, et al., 1997).

Plants grown under high relative air humidity caused by pollutants leads to the malfunctioning stomata. This stomata are unable to close in response to the darkness, that result in high stomatal conductance and frequent leaf drying in other research done in different humidity it has also been found that the number of stomata per leaf increased with development in higher soil humidity. The stomatal index, the number of stomata relative to the number of epidermal cells was also found to increase with soil moisture. The stomata density has been found to increase in plant with decreased ABA concentrations, which have increased transpiration (Atssman, et al ., 2002). 1. 4 AIR POLLUTION

Air pollution is one of the severe problems the world is facing today. It deteriorates ecological conditions and can be defined as the fluctuation in any atmospheric constituent from the value that would have existed without human activity (Tripathi and Gautam, 2007). In recent past, air pollution is responsible for vegetation injuring and crop yield losses. The increasing number of industries and automobile vehicles are continuously adding toxic gases and other substances to the environment (Jahan and Igbal, 1992). All combustion releases gases and particles into the air. These can include sulphur and nitrogen oxides, carbon monoxide and soot particles as well as smaller quantities or toxic metals, organic molecules and radioactive isotope (Agbaire and

Esiefarienche, 2009). Over the years, there has been a continuous increase in human population, road transportation, vehicle traffic and industries which has resulted in further increase in concentration of gaseous and particulate pollutants (Joshi, et al . , 2009).

Adverse effects of air pollution on biota and ecosystems have been demonstrated worldwide. Environmental stress, such as air pollution is among the factors that limits plants productivity and survivorship (Woo, et al., 2007)

Dust from quarry sites is a major source of air pollution, though the severity will depend on factors like the local microclimate conditions, the concentration of dust particles in the ambient air, the size of the dust particles and their chemistry. For example, lime stone quarries produce highly alkaline and reactive dusts, whereas coal mines produce acidic dust. The air pollution is a nuisance in terms of deposition on surfaces and its possible effects on health, especially for those with respiratory problems. It produces dust that can also have physical effects on the surrounding plants, such as blocking their photosynthetic activities that occupy an important position in the existence of life because of their ability to maintain a balance in the volume of oxygen and carbon dioxide which leads to the purification of the environment.

1.4.1 AIR POLLUTION AND MEDICINAL PLANTS

Higher temperature causes heat stress in plants. This means they grow less and produce fewer crops. In some cases, the plants do not reproduce at all since excessive heat causes sterility of the pollen grains. A temperature increase may be beneficial in areas which are very cold at present. Changes to our climate are happening more quickly now than they have ever done before in the world due to different industries and developments. Though as natures may have it some plants due adapt to the conditions often. Some studies have demonstrated that temperature stress can affect the secondary metabolites and other compounds that plants produce. Water availability directly affects the growth of plants and how much crop they produce. In most part of Africa there is not enough water even in normal conditions for high crop yields for instance, in contrast in Nigeria most plant are grown with enough water and difference in crop growth between the continents can be clearly seen. So changes in temperature and precipitation patterns as a result of climate change are likely to be bad for large areas of the world but may increase crop production in oth er regions. However, one of the likely outcomes of climate change is also an increase in severity of rain storms and drought. These are likely to have large devastating effects on agriculture and medicinal plant plantation.

The increase in atmospheric carbon dioxide (C0 2) levels resulting from

Fossil fuel combustion has fertilizing effect on most plants since C0 2 is needed

for photosynthesis. Conversion of carbon dioxide and water into the simple

sugar (glucose) emits oxygen making it possible for animals to lives on Earth.

Sunlight is the energy that powers this reaction. Scientific experiment has

shown that increasing atmospheric CO 2 levels leads to an increase in plant

growth (shad, et al., 2008 ).

1.4.2 TYPE OF AIR POLLUTANT AND THEIR SOURCES

There are many atmospheric pollutants that have been in existence but few of them are to be discussed here. The sources of air pollutants include human activities , domestic sectors, industry, agriculture, transport and nature as well.

Here, the pollutants, their sources and toxic effects to the environment are being discussed.

1. Solid particles; These are mostly produced by combustion, plants

industrial processes like mineral extraction, cement work, steel works,

foundries, glass works, gypsum quarries and fine chemistry. They have

carcinogenic and mutanogenic effect. 2. Sulphur dioxide (So 2):This is released by coal mining, lignites,

petroleum coke, and heavy fuel oil, domestic heating oil, diesel oil,

production of paper pulp, oil refining, nature and fire wood.

3. Nitrogen oxide (No 2): This is produced by combustion of fossil fuel and

from new industrial processes like fertilizer production, surface

treatment and phytochemical oxidants. In the presence of oxygen, it is

converted to No 2 in the furnace. The reaction continues slowly in the

atmosphere and gives a brownish color of the layer of polluted air 100

meters above town and causes green house effect.

4. Carbon monoxide: It is gotten from incomplete combustion,

agglomeration of mineral steel work, incineration of waste and car

exhaust fumes.

5. Volatile organic compounds: These are released from methane, solvents,

paint application, printing glues and adhesives, rubbers, biomass,

perfumes, cosmetic, News papers and tobacco.

6. Heavy metals: There are various sources of heavy metals which

contaminate the atmosphere. Examples of some of them are:

(a). Arsenic (As) comes either from trace of this metal in solid mineral fuels

and in heavy fuel oil , or else from certain raw materials used in

processes such as glass making, non-ferrous and ferrous metals working.

(b) Cadmium (Cd) is formed mainly through the manufacture of zinc and

the incineration of waste .Burning solid mineral fuel, heavy fuel oil and biomass account for a significant proportion of emission.

(c) Chromium (Cr) comes essentially from the production of glasses,

cement, ferrous metallurgy and foundries.

(e) Copper (Cu) comes from the erosion of over headed cables by railway

traffic. In addition, as for the other heavy metals, ferrous and non-ferrous

metals from metal production processes, the treatment of waste, and

combustion are all, to varying degree major sources of copper emission.

(f) Mercury (Hg) is emitted in small but still excessive quantities by the

combustion of coal and oil, the production of chlorine, and also by the

incineration of household, hospital and industrial waste. Preventive

action has considerably reduced mercury emission in recent years. Other

type of pollutants are Nickel, Lead, Selenium, Zinc, carbon dioxide,

methane, Nitrous oxide, Chlorofluorocarbon, Hydroflurocarbons,

Perflurocarbons, Sulphur hexafluroxide , Hydrofluoric acid , Ozone, and

Hydrogen sulphide (Citepa, 2012).

1.4.3 THE THRESHOLD AT WHICH AIR POLLUTANT EFFECT

MEDICINAL PLANTS AND THEIR SYMPTOMS.

This is injury or symptoms shown by impact of metallic object on some plant by pollutants. These symptoms are always triggered at some threshold extent. The presence of pollutants like, Sulphur dioxide causes intervenial necrotic blotches, red brown die back or banding in pines, in plants. Nitrogen dioxide causes interveinial necrotic blotches similar to those symptoms produced by Sulphurdioxide. Fluoride produces red brown distal necrosis in pines. Ammonia leads to tip margin necrosis at the threshold dose of 55pm

(38×10µg/m 3) for 1 hour. Chlorine causes interveinial necrotic blotches

3 similar to So 2 at the threshold of 0.5-1.5ppm (1400-4530µg/m ) for 0.5-3hr.

Ethylene leads to chlorosis, necrosis, abscission, dwarfin, premature defoliation of medicinal plants but the threshold variably undetermined. Ozone causes upper surface broncing chlorosis and early senescence at the threshold of (157µg/m -3) for 12-13 hrs. Acidic rain shows necrotic sports, distal necrosis pines on medicinal plants at the pH < 3.0 (Citepa, 2012).

1.4.4 EFFECT OF AIR POLLUTION ON LEAF MORPHOLOGY

Pollutants can cause leaf injury, stomata damage, premature senescence, decrease photosynthetic activity, disturb membrane permeability and reduce growth and yield in sensitive plant species (Tiwari, et al., 2006). Reductions in leaf number may be due to decreased leaf production rate and enhanced senescence. The reduced leaf area result in the reduced absorbed radiations and subsequently in reduced photosynthetic rate.

Air pollution stress leads to stomatal closure, which reduces co 2

availability in leaves and inhibits carbon fixation. Plants that are constantly

exposed to environmental distress absorb, accumulate and integrate these

pollutants into their systems. Reports have shown that depending on their

sensitivity level, plants shows visible changes which would include alteration in

the biochemical processes or accumulation of certain metabolites (Agbarie and Esiefarienrhe, 2009).

Sulphur dioxide, nitrogen oxides, acid co 2 as well as suspended particulate matter, when absorbed by the leaves may cause a reduction in the

Concentration of photosynthesis pigment like chlorophyll and carotenoids which directly affected metabolites. One of the major impacts of air pollution is the gradual disappearance of chlorophyll that leads to yellowing of leaves, which may be caused by decrease in the capacity for photosynthesis.

Chlorophyll is the most photoreceptor in photosynthesis, the light driven process in which carbon dioxide is fixed to yield carbohydrates and oxygen.

When plants are exposed to environmental pollution above the normal physiological acceptable range, photosynthesis gets inactivated. What happen to leaf morphology, pigment content also affects the efficacy of the crude extracts

(Joshi and Swami, 2007).

1.4.5 EFFECT OF AIR POLLUTION ON PLANT SUGAR CONTENTS

Mainly soluble sugar from plants and vegetation are source of energy for living organisms. Studies have revealed that there is always loss of soluble sugar in all tested species at all polluted sites. Loss of soluble sugar content in polluted area can be related to increased respiration and decreased CO 2 fixation because of chlorophyll deterioration (Tripathi and Guatam, 2007). Reseachers have found out that pollutants like SO 2, NO 2, and H 2S under hardening condition can lead to loss of soluble sugar in the leaves of plant grown in polluted Area. They have also shown that even in sensitive trees loss of sugar has occurred and is probably due to photosynthetic inhibition or stimulation of

respiration rate.

Increase in amount of soluble sugar is a protecting mechanism of leaves.

Soluble sugar in pine needle decreased on ozone exposure and the more a plant

is resistant to air pollution the more the increase in soluble sugar level eg

Dodonea viscoss and prosopis juliflora (Abedi, et al., 2009 ).

1.4.6 EFFECT OF AIR POLLUTION ON PLANT PROLINE

Proline is an osmotic accumulation in response to several stresses that may have a role in defending plants life, pigment destruction, depletion of cellular lipids and peroxidization of polyunsaturated fatty acid.

Many researchers have shown that environmental stress, causes increase in tree proline Contents. Environmental stresses like high temperature, low temperature, drought, air pollution and soil pollution can lead to reaction of oxygen species in plant cells which is always cytotoxic to all organisms when a plant is exposed to high environmental stress, this forces the chloroplast into an excessive excitation energy level which in turn causes increase in generation of reactive oxygen species and lead to oxidative stress. The proline is known as free radical scavenger to protect plants. Some other amino acids such as tryptophan, tyrosine etc, do the same but proline is counted more important because of its high Concentration on a plant during environmental stress

(Tiwari, et al. , 2006).

1.4.7 GENERAL EFFECT OF AIR POLLUTION ON MEDICINAL

PLANTS

Much experimental work has been conducted on the analysis of air pollutant effects on crops and vegetations as various levels ranging from biochemical to ecosystem levels. Environmental stress, such as air pollution is among the factor that limits plants productivity and survivorship (Woo, et al.,

2007). When exposed to air bone pollutants, most plants experienced physiological changes before exhibiting visible damage to leaves (Liu and

Ding, 2008). The atmospheric S0 2 adversely affects various morphological and physiological characters of plants. High soil moisture and high relative humidity aggregate S0 2 injury in plants.

Industrialization and the automobile are responsible for maximum amount of air pollutants and the crop plants are very sensitive to gaseous and particulate pollutions (Joshi, et al ., 2009). Vegetation is an effective indicator of the overall impact of air pollution and the effect observed is a time averaged result that is more reliable than the one obtained from direct determination of the pollution in air over short period. Although, a large number of trees and shrubs have been indentified and used as dust filters to check the rising urban dust pollution level (Rao, 1979).

Plants provide an enormous leaf tree impingement, absorption and accumulation of air pollutants to reduce the pollutant level in the environment with a various extents for different species. The use of plants as monitors of air pollution has long been established as plants are the initial acceptors of air pollution. They act as the scavengers for many air borne particulates in the atmosphere (Joshi and Swami, 2009). Increase in chlorophyll a, b, total chlorophyll and caroteniods has been shown and proved that chlorosis is the first indicator of flour effect on plant which shows reduction in photosynthesisis because of the damage in sensitive specie tobacco plant. Air pollutants can directly affect plants via leaves or indirectly via soil acidification (Steubing et al , 1989). It has also been reported that when exposed to air pollutants, most plant experience physiological changes before exhibiting visible damage to leaves (Dohmen, et al. , 1990). Previous studies also showed the impact of air pollution on the air ascorbic acid contents, chlorophyll contents of leaf extract, relative water contents (Rao, 1979), and temperature and humidity of a plant.

These separate parameters have conflicting results for same species.

However, APTI based on all five parameters has been used for identifying tolerance levels of plants species. Several contributors agree that air pollutants effect plant growth adversely (Rao, 2006; Henry and Heinke, 2005;

Horsfall, 1998). Air pollution tolerance index is used by landscapers to select plant species tolerant to air pollution (Yan-ju, and Huiding 2008) Wrightia tomenta plants grown under high relative air humidity in vitro, had 29.4 % malformed stomata. These stomata are described as large, spherical and wide open, lacking the ability to close. The efficiency of stomatal opening for co 2 uptake and water transpiration is not only determined by the size of opening, but also by the number of stomata. More stomata can take up more co 2 and transpire more (Fordham, et al., 2001). Some studies have demonstrated that temperature stress can affect the secondary metabolites and other compounds that plants produce which are usually the basis for their medicinal activity

(Herbal, 2009). Previous researches reported significant reduction in different leaf variable in the polluted environment comparison with clean atmosphere

(Jathan and Igbal, 1992). In their study on Platnus acerifolia showed changes in leaf blade and petiole size in the polluted air. Significant reduction in length and area of leaflets and length of petiole of G. officinale of polluted plants was recorded. Reduction in dimension of leaf blade of fine tree species in the vicinity of heavy dust and S0 2 pollution was also observed (Jahan and Igbal,

1992). Significant effects of automobile exhaust on the phenology, periodicity and productivity of road side once species was also reported. Decrease in leaf area in drought stress had been observed because tolerance of water contents of tissue possible by decrease in leaf area, decrease in length breadth of leaflets has been demonstrated in leaves of Albizia Lebbeck under the stress of air pollution. Cassia siamea plants growing at two different sites (polluted and non-polluted) on two important roads of Agra city exhibited significant differences in their flowering phenology and floral morphology. Really, effect of air pollution is related to morphology of area of leaf visible damage including reduction of leaf area. The effect of air pollution exchange of gases on area of visible damage including reduction of leaf Area changes as compare to unpolluted condition, necrosis and chlorosis on area of leaf of Avicenia Marine decrease.

1.4.8 WAYS TO CONTROL AIR POLLUTION

(a) REGULATION OF STOMATA CLOSURE

The ability of plants to be able to regulate the size of the stomatal opening

is a very important mechanism to control water loss and survive. The ability is

especially important during water losses and water stress can have serious

consequences for the plants. This can be achieved by changing their growth

pattern, producing stress proteins and chaperones upregulation of antioxidants,

accumulation of compatible solutes, increasing the amount of transporters

involved in ion and water uptake and transport by closing the stomata. This will

also prevent changes in phytochemical constituents or fluctuation in quantities

of secondary metabolites (Mohammad, et al 2012)

(b) TREATING THE PLANTS WITH ABSCISCIC ACID (ABA)

Treatments with ABA on periods of high temperature or low relative air

humidity during developments can offset this malfunctioning and produce

functioning stomata even in high humidity. This measure will also serve to

protect the phytochemical constituents of plants, because if the regulation of

stomata is normal, plants will photosynthesizes normally there by bringing the

necessary nutrients needed by the plants to live healthy. When this is done the secondary metabolites responsible for the therapeutic values of the plants will not be impaired (Arvel, et al., 2009).

(c) PLANTATION OF HYPER ACCUMULATING PLANTS

For the heavy metals, plants, mushrooms, or microorganisms are occasionally successfully used to remove some heavy metals such as mercury.

Plants which exhibit hyper accumulation can be used to remove heavy metals from soil by concentrating them in bio matter. Some treatment of mining tailings has occurred where the vegetations is then incinerated to recover the heavy metals. Generally, the effect of pollution on the environments or medicinal plants can also be controlled by reduction of emission, either through curtailments of industrial activities or scrubbing (Yan and Huiding, 2008).

Landscape planting in the factory vicinity, may offer an occasional options.

Some plants called plane trees due to their ability to survive in such an environments are to be planted around factory, these plants provides an enormous leaf area for impingements, absorption and accumulation of air pollutants to reduce the pollutants levels in an environments. Some plants can accumulate remarkable levels of metals. Metal hyperacumnating species have been identified in at least 45 plant families and individual species can accumulate different metals such as zn, cd, cu, co, ni, se, and As. Some of these medicinal plants such as Mint. St John’s Wort , lauander , marigold , hollyhock , caraway , garlic , garden sorrel , common hemp etc can accumulate high amount of toxic heavy metal in their tissue. (d) PLANTATION OF PHYTOREMEDIATORS

This is known as the new technologies that are based on the use of higher

plants for cleanup process in contaminated environment. Some plants can take

part in phytoremediation successfully than other plants grown under the same

condition. Environmental pollution with metals and xenobioticon is a global

problem, and therefore the development of phytoremediation technologies for

plant-based clean-up of contaminated soils is therefore a significant interest.

Phytoremediation technologies are currently available for small subject of

pollution problems. Traditional techniques often used to affect these

remediation processes include: top soiling contaminated soil with

uncontaminated ones, stabilization of the polluting heavy metals to prevent

leaching into ground water, soil washing, excavating top layers of polluted soil

amongst others. Biological techniques used to remediate metal contaminated

soil essentially involve the use of plants and organisms to remediate these soils.

It includes phytovolatization, phytostabilization, phytofiltration, and

phytoextraction processes (Bagdat and Mohamed, 2007).

1.5 HEAVY METALS

Heavy metals are a member of a loosely defined subset of elements that exhibit metallic properties. It mainly include transition metals, some metalloids, lanthanides and actinides. Heavy metals occur naturally in the ecosystem with large variation in concentration. In modern times, anthropogenic sources of heavy metals, ie pollution, have been introduced to the ecosystem through varying amounts of heavy metals like iron, cobalt, copper, manganese, molybdenum, and zinc are required by humans, but excesive level can be damaging to the organism ( Hogan, 2010). Other heavy metals such as mercury, phitonium and lead are toxic metals and accumulation over time in the body of animals can cause serious illness. Heavy metals toxicity can lead to damage or reduced mental and central nervous function, lower energy level and damage to blood composition, lungs, kidneys, liver and other vital organs. Long term expsosure may result in slowly progressing physical, muscular and neurological degenerative process mimic Alzheimer,s disease, Parkinson diseases, muscular dystrophy and multiple sclerosis ( Hogan, 2010). Allergies are not uncommon and repeated long term contact with some metals may cause cancer, they affect the prokaryotic and eukaryotic cells because colloids are sensitive to ion or heavy metals concentration. Heavy metals pollution can arise from many sources but most commonly arise from the purification of metals, eg smelting of copper and preparation of fuels. Electroplating is the primary sources of chromium and cadmium. Cadmium, lead and zinc are realized in tiny particulates as dust from rubber tires on road surfaces, the small size allows these toxic metals to rise on the wind to be inhaled or transported onto topsoil or edible plants. Through precipitation of their compound or by ion exchange into soil and mud, heavy metals pollutants can localize and lay dormant, which have severe effects on the environments. 1.5. 1 EFFECT OF HEAVY MINERALS ON MEDICINAL PLANTS

It is esential that the quality of plant-based drugs should be assured prior to use. When heavy metals contaminate the plants from which herbal drugs are derived, they affect both plants growth characteristicon and production of secondary metabolites. The presence of heavy metals in medicinal plants may stimulate production of bioactive compound in many plants species (Nasim and

Dhir, 2010). The effect of minerals concentration of medicinal plants also is an area of interest to improve health care system and avoid therapeutic failure.

Medicinal plants are affected by heavy minerals through contamination during cultivation, accidental cross contamination occurring during processing and the use of heavy minerals as a therapeutic ingredient. Some essential aromatic and medicinal plants are capable to accumulate heavy metals from contaminated soils. All heavy metals, both essential (cu, zn ) and non-essential (cd, pd) cause toxic effect to plants and humans if found in high concentrations. The level of

Heavy metal content on medicinal plants depends on climatic factors, plants species, air pollution and other environmental factors. Several other factors may influence accumulation of contaminants, including species, level and duration of Exposure to contaminats, and topography. Other reseachers have revealed that uptake and accumulation of heavy metals may have impacts on medicinal plants that are different from their impact on farm crops. Their method of processing and ingestion is not the same, therefore, it is important to improve quality standard for herbal medicine by examining and revising the maximum allowable values of heavy metals in medicinal plants, using reseachers that based on medicinal plants (Mohammad et al ., 2012).

CHAPTER TWO 2.1 MEDICINAL PLANT Medicinal plants are known as any plant which contains in one of its organs, substances that can be used for therapeutic purpose or precursors for the synthesis of useful drugs. The use of plants to treat ailments is as old as antiquity. Records of humans using plants to treat diseases have been recorded as far back as 6000 to 4000 years ago when Ayurvedic physicians started treating tumors with extracts from Vinca roseus . The use of medicinal herb in the treatment and prevention of diseases is attracting attention by scientists worldwide (Okunrobo, et al., 2008). The use of traditional medicine and medicinal plants in most developing countries, as a normotive basis for the maintenance of good health, has been widely observed.

In modern medicines, medicinal plants have tended to be seen as quaint relicon of the past hence next to useless in treatments of serious ailments. To the contrary, about one in four of Pharmaceutical prescribed drugs contain ingredients of plant origin. Those plants have different medicinal compounds ranging from heart drugs, analgesic, anesthetic, antibiotic, anticancer, anti- inflammatory, anti oxidants, oral contraceptives, hormones, laxatives, etc.

Tremendous as these may seem, they are derived from different plant species.

About 80% of the world’s population relies on the medicinal plant. It is in recognition of this fact that the World Health Organization (WHO) is attempting to incorporate traditional medicines into modern health care system of the developing world. In such case where the direct use of these plants to cure physical ailment is, becoming prominent, scientific explanation of their therapeutic activity based on knowledge of their phytochemical constituents become imperative. This relies on phytochemical analysis which no doubt is already being affected for more medicinal plant (udu, 2010) .

The Pharmacist or Pharmacologist describe whole plant or plant that have medicinal properties as crude drugs of natural or biological origin. These medicinal plants can be prepared as vegetable drugs, concoction, decoction infusion, tisane and maceration ( Sofowora, 2008).

2.2 PHARMACOGNOSTIC PROFILE OF SELECTED MEDICINAL PLANTS 2.2.1 PHARMACOGNOSTIC PROFILE OF Cnestis ferruginea

TAXONOMY

This plant has the following taxonomic hierarchy.

Kingdom : plantae

(unranked) : Angiosperms

(unranked) : Eudicots

(unranked) : Rosids

Order : Oxalidales

Family : Connaraceae

Genus : Cnestis

Species : C. ferruginea

Common name : Ogbakpee

Description : This is a tree of about 8 – 15m high, trunk cylindrical, not buttressed to 45cm diameter, but reaching 20m x 1m. Twinges sparsely spined or not leav es variables 15 -40cm by 7 1/2 - 25cm broad, inflorescence terminal with fleshy flower.

Fig 1: Photograph of Cnestis ferruginea

Geographycal distribution

A shrub or tree to 6m high of deciduous forest and secondary scrub, common throught the Region from senegal to West Cameroons and in other parts of tropical Africa.This plants has a wide repartition in west Africa mianly in the Gambia, in Ghana, Guinea - Bi ssale, Ivory Coast, Liberia, Nigeria, Seirra leone, Southern Benin, Southerast Niger, and Gabon particularly in semidecidious forest. It grows mostly in savanna, mostly in secondary regrowths on farm land. In fences, fringing laggons and near the beach. In south west Senegal, it was first collected in casamance in 1900.This plants is now relatively common and has been found several areas of the Oussouge district such as kabrouses, oussouge and cap skirring (Garron, et al ., 2007).

Ethnomedicinal uses of Cnestis ferruginea

The plant with its scarlet fruits has value as an ornamental. The stems are used by Igbo in Southern Nigeria for boils and in Zaïre they are cut to yield a potable sap. The powdered bark is rubbed into gums in Southern Nigeria for pyorrhea.

In Ivory Coast a paste of root-bark is rubbed on the forehead for headache, and with the addition of ash of burnt bark of Calpocalyx Aubrévillei as a vegetable salt that is given as an appetite stimulant in cases of illness. The leaves are laxative and are taken by the Yoruba in decoction. A leaf decoction is given in

Zaïre to treat bronchitis and as an abortifacient. Leaf-sap is placed on the eyelids and instilled into the eyes in Ivory Coast for eye-troubles and the leaves, or the roots, are used for dysmenorrhoea. Also in Ivory Coast sap expressed from leafy twigs is taken by draught for fevers and a paste of young leaves compounded with a little wood-ash is let down with warm water and administered as an enema for heart burn. The leaf is rubbed on the body in

Southern Nigeria for iba (fever), and the pulped plant is similarly used in Ivory

Coast-Upper Volta for all manners of pains, maigrane, anesthestetics and as a sedative in insanity. The roots are recognized in Zaïre as a purgative. They enter into remedies for treating skin-infections in Nigeria, and examination has shown action against Sarcina lutea and Staphylococcus aureus , but no action against

Gram negative organisms, nor fungi. The roots are used by women in Benin

Province as a skin-ointment. Root-decoction is taken by draught in Ivory

Coast-Upper Volta as an aphrodisiac, and by enema for gynecological troubles, and for dysentery and urethral discharge (Garron et al ., 2007). The roots are held to be a remedy against snake-bite in Senegal, though doubt has been expressed on their efficacy in Ghana. Sap from the roots, or root-powder, is applied to the nostrils for migraine and sinusitis in Ghana and in Ivory Coast-

Upper Volta. Powdered roots enter into magical treatments in Ivory Coast-

Upper Volta for anyone suffering madness due to misfortune, and into a philter sprinkled on roads to prevent the spread of small-pox during an epidemic. Igbo of Southern Nigeria use the roots against toothache. The fruit contains a soft, juicy, somewhat bitter and acid pulp. This is widely used to rub on the teeth to clean and whiten them. It leaves a refreshing taste in the mouth in Soudano- guinea. Fruit-pulp is also rubbed on the skin and is used as a medicine for the throat. It is taken as a tonic, and Efik give a medicine of this and allied species to weakly children to encourage them to walk. Through out the Congo the fruits are used to treat bronchial affections, especially whooping-cough and tuberculosis. In Zaïre fruit-juice is applied to wounds. The fruit with seeds are ground up with spirit or boiled in wine to produce a remedy for snake-bite in

Nigeria.It also has an antibacterial or antioxidant effect (Fred, 2012).

2.2.2 PHARMACOGNOSTIC PROFILE OF Ricinus communis

The plant has the following taxonomic hierarchy

Kingdom : Plantae

(uranked) : Angiosperms

(unranked) : Eudicots

Order : Malpighiales

Family : Euphorbiaceae

Subfamily : Acalyphoideae

Tribe : Acalypheae

Subtribe : Ricininae

Genus : Ricinus

Species : communis

Common name: Castor plants

Description: The castor oil plant can vary greatly in its growth habit and appearance. The variability has been increased by breeders who have selected a range of cultivars for leaf and flower colors, and for oil production. It is a fast- growing, suckering, perennial shrub which can reach the size of a small tree but not cold hardy (Duke and Wain, 1981.). The leaves are 15-45cm long, long- staked, alternate and palmate with deep lobes of coarsely tooted segments.

Plants with the dark leaves can be found growing next to those with green leaves, so there is probably only a single gene controlling the production of the pigment in some varieties at least. The stems also vary in pigmentation. The fruit capsules of some varieties are more showing than the flowers.

In some varieties they start off dark reddish purple or bronze when young, gradually changing to a dark green, sometimes with a reddish tinge, as they mature. The leaves of some other varieties are green practically from the start, whereas in yet others a pigment masks the green colour of all the chlorophyll-bearing parts, leaves, stems and young fruit, so that they remain a dramatic purple-to-reddish-brown throughout the life of the plant. Plants with the dark leaves can be found growing next to those with green leaves, so there probably is only a single gene controlling the production of the pigment in some varieties at least. The stems spiny seed capsules also vary in pigmentation. The fruit capsules of some varieties are more shown than the flowers . The green capsule dries and splits into three sections, forcibly ejecting seeds. The flowers are borne in terminal palenic-like inflorescences of green or, in some varieties, shades of red nmooecious flowers without petals. The male flowers are yellowish-green with prominent creamy stamens and are carried in ovoid spikes up to 15 centimetres (5.9 in) long; the female flowers, borne at the tips of the spikes, have prominent red stigmas . The fruit is a spiny, greenish to reddish- purple capsule containing large, oval, shiny, bean-like, highly poisonous seeds with variable brownish mottling (James, 1983. ).

Fig 2: Photograph of Ricinus communis

Geographical distribution of Ricinus communis

Although castor is indigenous to the southeastern Mediterranean Basin , Eastern

Africa , and India, today it is widespread throughout tropical regions. In areas with a suitable climate, castor establishes itself easily where it can become an invasive plant and can often be found on waste land. It is also used extensively as a decorative plant in parks and other public areas , particularly as a "dot plant" in traditional bedding schem es . If sown early, under glass, and kept at a temperature of around 20 °C (68 °F) until planted out, the castor oil plant can reach a height of 2–3 metres (6.6–9.8 ft) in a year. In areas prone to frost it is usually shorter, and grown as if it were an annual . However, it can grow well outdoors in cooler climates, at least in southern England, and the leaves do not appear to suffer frost damage in sheltered spots, where it remains evergreen. It was used in Edwardian times in the parks of Toronto, Ontario, Canada.

(Christopher, 1996).

Ethnomedicinal uses

The castor oil is normally given orally, alone or with quinine sulphate to induce labour in pregnancy at term. It can be used as vehicle for parenthral administration of steroidal hormones. Castor is a mild and most efficient purgative, and is well adapted for infants and young children, the puerperal state irritable conditions of the alimentary canal or genito –urinary organs. It is also used as one of the purgative agent used to reduce constipation. The leaves have been also recommended in form of decoction to increase secretion of breast milk. The decoction has also been reputed to act as a lactogue and emmenagogue when administered internally. Its decoction from the root is given to treat lumbago and paste for tooth ache. Leaves mixed with oil are placed on sore, boils, and swellings. This leaves are commonly applied over the abdomen to give relief in flatulence in children, infusion of the leaves is used for stomach ache and eye lotion. Fresh juice has been reported to be used as an emetic in the poisoning by narcotic like opium and the leaves are also useful in jaundice (Manpret et al. , 2012).

2.2.3 PHARMACOGNOSTIC PROFILE OF Vitex doniana

Classification;

Kingdom : Plantae

(unranked) : Angiosperms (unranked) : Eudicots (unranked) : Asterids

Order : Lamiales

Family : lamiceae

Subfamily : Viticoideae briquette

Genus : Vitex

Species : doniana Common name : unknown Description

Vitex is a genus of shrubs and trees, from 1 to 35m tall. This is a medium sized deciduous tree, 8-19 m high, with a heavy rounded crown and a clear bole up to 5m. Bark rough, pale brown or grayish white, rather smooth with narrow vertical fissues. The bases of old tress have oblong scales, leaves opposite, glass brous 14-34 cm long, usually with 5 leaflets on stalks 6-14 cm long, 2.9 cm wide, leaf tips rounded or emarginated, leaf bases cuneate. Dark green above, pale grayish-green below, thickly leathey, with a few scattered stellate hairs on the upper surface thermise without hairs, flower petals except on largest libe, which is purple, opposite and axillary cymes, flowers small, blue or violet,

3-12cm in diameter, only a few being open at a time. Fruit oblong,about 3cm long. Green when young, turning purplish black on ripening and with a starchy black pulp. Each fruits contains I hard, conical seed, 1.5 cm long, 1-1.2 cm wide (Gemma et al , 2009)

Fig 3: Photograph of the medicinal plant Vitex doniana

Geographycal distribution

Vitex doniana occurs in a variety of habits, from forest to savanna, often in wet localities and along rivers and on termite mounds, up to 2000m altitude.

It occurs in regions with a mean annual rainfall of 750-2000m.It is most commonly found on alluvial soil. In central Africa, it is often the first species to establish where gallery forest are in low lying areas in the savanna. The growth rate of Vitex doniana is moderate in plantation in norethern cotedivore, seedlings were on average 70-90 cm tall after 3 years, the tallest ones reaching

170 cm (Wikpedia, 2014).

Vitex doniana is a savanna species in wooded grassland and can also be found along forest edges. It can be found throughout tropical Africa. The fruit are black, edible, sweet and mealy. It is frequently eaten as a snack and sold in local markets. The fruits are approximately 3cm long and contain one to four seeds.The fruits are collected from April to June. Vitex doniana (Verbernaceae ) commonly known as black plum or “Ori-nla” is wide spread in the southwestern

Nigeria as a perennial trees ( James, 2010).

Ethnomedicinal uses

The leaf sap is used as an eye drop to treat conjunctivitis and other eye complaints. A leaf decoction is applied externally as galactagogue and against headache, stiffnes, measles, rashes, fever, chicken pox, and hemiplegia and internally as a tonic, anodyne and febrituse are used to treat respiratory diseases. Pastes of pounded leaves and bark are applied to wounds and burns.

Leaf infusions are added to alcoholic drinks to make them strongest root decoction is administered orally to treat stomach complaints and kidney troubles. The bark is also used against leprosy and liver diseases and to control bleeding after child birth. Dried and fresh fruits are eaten against diarrhorea and as a remedy against lack of vitamin (Gemma, 2009 ).

2.2.4 PHARMACOGNOSTIC PROFILE 0F Anthocleista djalonensis

Classfication;

Domain : Eukanyota

Kingdom : plantue

Sub : viridaeplantal

Phylum : Tracheophyta

Subphyhum : Euphyllophytina Infraphylum : Radiatopeses

Class : Asteridae

Superorder : Gentianae

Order : Getianales

Family : gentianae

Tribe : Potaliege

Subtribe : Potalinae

Genus : Anthocleista

Species : djalonensis

Common name : Guinea manding maninka

Description of plant

Small tree up to 15m tall, 40cm in diameter, twings sometimes with, 2 erect spines or small cushions above the leaf axis, leaves opposite, simple and entire, petiole 1-9 cm long aunicled, blade oblong-elliptical to obvate-elliptical, 9-35 x

5-17 m, in young plants up to 115cm x 50cm, base condate, rounded or cuneate, apex rounded. Inflorescence, an erect terminal dichasia cyme, 15-50 cm long.

May florered, pedunele and branches greenish white or pale green with darker green dots, thinkened at the nodes. Flowers bisexual, regular, sepals 4, fine, orbicular, 6-10 mm long. Corolla wish, cylindrical tube 20-32 mm long, lobes

11-14, oblong-anceolate, 10-18 mm long, spreading, white or creamy, stamens as may as corolla lobes and alternating with them, exerted, filaments fused into a riny authers creamy or pale yellow, ovary superior, ovioid, 6-7 mm x 3-4 mm, 4-celled stigma obvoid-cylindrical, apically 2-lobed fruit an ellipsoid berry 3.5-

5 cm x 3.5 cm, prounded at the apex ( James, 2010).

Fig 4: Anthocleista djalonensis

Geographical distribution

Anthocleista comprises 14 species and occurs in tropical Africa, including

Comoros and Madagascar. The 4 West African species have the same vernacular names and are used by local practitioners for the same medicinal purposes. In Ghana, Anthocleista djalonensis flowers in April and May, in

Nigeria from March to May. Fruits occur in Nigeria in October and November.

Anthocleista djalonensis occurs in rather dry localities, in savanna or thickets, from sea-level up to 500 m altitude. Anthocleista djalonensis occurs from

Guinea Bissau east to Cameroon (Neuwinger, 2000).

Ethnomedicinal uses Widely used throughout its distribution area as a strong purgative and diuretic. A root decoction is commonly taken to treat constipation, to regulate menstruation and as an abortification. It is used as wash or bath or as a vapour bath to treat leprosy, venereal disease, edema and scrotal elephantiasis. A root infusion is taken to treat intestinal problems, Acute inflammations and boils on the skin. In mali, a root maceration is taken alone or with honey to treat malaria and a root decoction or root powder in porridge is taken to treat abdominal pain.

A root decoction is also taken to treat heria of the grain. In southern Nigeria a decoction of roots with potash is taken to treat fungal skin infections and filarial worm injections, including loo loa filariasis. A tea made from the chopped soft root bark soaked in water is taken to threat thrush; an alcoholic leaf extract is taken to treat broken bones in woman (Neuwinger , 2000). Traditionally, the leaves are reputed to be used for the treatment of malaria and jaundice . The bark is used as a purgative in small doses as large doses are considered toxic.

According to the Mendi ethnomedicine, when the tree is used as firewood, the people sitting around the fire become sick .

The decoction of the leaves is drunk in Sierra Leone as treatment for jaundice, in Ivory Coast the root is used as a diuretic, vigorous purgative, poison antidote, treatment for leprosy, as an emmenagogue and in the treatment of edemas and elephantiasis of the scrotum. The root decoction is taken against chest pain, constipation and stomach pain. The study was therefore aimed at investigating the anti-inflammatory activity of the leaf extracts with a view to justifying the use of the plant in the treatment of oedemas and elephantiasis of the scrotum (Okunrobo et al. , 2008)

2.2.5 PHARMACOGNOSTIC PROFILE OF Manihot esculenta

Classfication

Kingdom : plantae

(unranked) : Angiosperms

(unranked) : Eudicots

(unranked) : Rosids

Order : Moilpighiales

Family : Euphorbiacea

Subfamily : Crotonoidase

Tribe : Manihoteae

Genus : Manihot

Species : Esculenta

Bionomial name : Manihot esculenta

Common name : Cassava plants

Description of plant

Shrub or small tree, 1.3-5m tall with fresh elongated tuberous, roots or rhizomes, these very wordy, only slightly thickened in wild varieties, under cultivation up to 2.5m long and 10-5cm in diameter weighing up to 40kg average 4-7kg, leaves usually deeply 3-7 parded with spatulate to linear – lancolate accumulates lobes 2.5-15cm long, glabrous; glaucous beneath, minutes puberment along vaius, long petiols, flowers in paracle, less than 12cm long, capsule globesea and 12cm across, with winged angess, seed 3 capsules.The cassava root is long and tapered with a firm, homogenous flesh encased in a detachable rind, about 1mm thick rough and brown on the outside.

Commercial varieties can be 5 to 10cm in diameter at the top and around 15cm to 30cm long. A wordy cordon runs along the root’s axis. The flesh can be chalk-white or yellowish( Allem, 1994).

Fig 5 : Photograph of Manihot esculenta

Ethnomedicinal uses

The poisonous juice is boiled down to syrup and given as an aperients, fresh rhizome made into a poultice is applied to sores. The flour cooked in grease, the leaf stened and pulped and the root decocted as a wash are said to be folk medicine for tumors. Cassava is used to folk remedies for cancerous infections, condylomata, excrescence of the eye and tumours (Hartwell, 1967).

Reported to be antiseptic, cyanogenetic, demulcent, divretic and poison, cassava is a folk remedy for abscesses, boils, conjunctivties, diahea, dysentery, flu, hemia, inflamata, marasnucon, prosthtitis, snake bite, sore, spasm, swellings and testiales. The bitter variety leaves are used to treat hypertension, headache and pain. As cassava is a gluten-free, natural starch, its uses in western cuisine as a wheat alternative for suffers of celiac disease is becoming common. This plant can be used as a precusor for synthesis of drugs it is good for treatment of

Anaemia, diabetes, high blood pressure. Manihot esculenta is also used as heart drug, Analgesicon, anaestheticon, antibioticon, anticancer, anit-parasitic, anit hylamatory, antioxidamt, oral contraceptives, hormones, laxatives etc (Duke and Wain, 1987).

2.3 PREVIOUS PHARMACOLOGICAL INVESTIGATION OF PLANTS STUDIED (a) Cnestis ferruginea

Aqueous extract of Cnestis ferruginea root on paroxetine, induced sexual dysfunction in male rat at the dose of 13.26 and 52mg/kg body weight restored sexual competence .This result supports the folkloric claim of the plant for the management of sexual disorder in males (Asian, 2012). Also, study carried out on this plant species to investigate the effect of Amentoflavone isolated from ethanolic extract revealed the anti-neuroinflamatory and antinoceptives effect.

Cnestis Ferruginea produced significant dose dependent inhibition of pain response elicited by acetic acid and increased nociceptive reaction latency in hot plate test. In addition it produced significant dose. Amentoflavone (CF-2) was isolated from the aqueous n-butanol fraction. CF-2 (12.5, 25 and 100 mg/kg) dependent inhibition of oedema in the carrageenan-induced inflammation

(Olayemi et al., 2008). Extracts of this plant from the fruit have been found to have Antimicrobial effect, especially against gram- positive bacteria.

Chemical Constitiuents of Cnestis ferruginea

The chemical constituents may be responsible for the therapeutic function of

Cnestis ferruginea ;

Amentoflavone (CF-2), Tanin and glycoside (Olayemi et al. , 2008)

(b)Anthocleista djalonensis

The effects of the methanol extract (crude) and aqueous fraction of

Anthocleista djalonensis on acute inflammation has been revealed. Oral administration of the methanol extract showed acute anti-inflammatory activity against carrageenan induced paw edema in rats although not too significantly different from the control at 250 mg/kg and 500 mg/kg. The aqueous fraction of the methanol extract significantly inhibited carrageenan induced paw edema in rats at 500 mg/kg. Significant activity against Dextran induced paw edema in rats was exhibited by both methanol extract and aqueous fractions when administered orally at 250 mg/kg and 500 mg/kg. The dry powdered leaf was found to contain alkaloids, glycosides, saponins and tannins. It is revealed from the screening model used that the methanol extract and aqueous fraction of

Anthocliestia djalonensis possesses acute anti-inflammatory activit ( Okunrobo,

2008).

Anthocliestia djalonensis , Nauclea latifolia and Uvaria afzali , which have been used to treat sexual transmitted diseases (STD) were extracted using ethanol, cold, and hot water respectively. Tested by agar diffusion, macrobroth dilution method, for activity against five strains of staphylococus aureus and two

Eschericlea coli isolated from cases of STD or urethritits. Four different type of bacteria strains, staphylococus aureus , Bacillus subtilis , pseudomonas aeruginosa , and Escherichia coli were used as reference organism. Ethanolic and cold water extracts of Anthocliestia djalonensis showed activity against 9 and the 11 test organism. They were bacter atioststatic at minimum inhibition concentration to the gram positive strains but bactericidal to the gram negative strains (Okoli and Iroegbu, 2004).Treatment of Alloxan induced diabetic rats with the extract of Anthocliestia djalonensis root extract of 37-111mg /kg, caused a significant reduction in fasting blood sugar of the diabetic rat both in acute study and prolonged treatment (2weeks). The activity of Anthocliestia djalonensis were more than glibenclamide ( Okolion et al. , 2012) Chemical constituents of Anthocleisia djalonensis chemical constituents discovered by other reseachers which is suspected to be responsible for the therapeutic function of Anthocliestia djalonensis ; saponins, tannins, reducing sugar, phlobabatanins, alkaloids, cardiac glycoside

(Okunrobo et al. , 2008)

(C) Manihot esculenta

Cassava root has been promoted as a treatment for bladder and prostate cancer (USDA NRCON Plants database). Cassava is used in folk remedies for cancerous affections, condylomata, excrescences of the eye, and tumors.

Reported to be antiseptic, cyanogenetic, demulcent, diuretic, and poison, cassava is a folk remedy for abscesses, boils, conjunctivitis, diarrhea, dysentery, flu, hernia, inflammation, marasmus, prostatitis, snakebite, sore, spasm, swellings, and testicles. Leaves of manihot esculenta studied in Nigeria showed that oral addition of an aqueos leaf extracts to rat induced anti inflammatory and

Anagelsic effects. It has also been proved experimenally that methanoic extract of Manihot esculenta showed most portent anthemintic activity. Study conducted by other reseachers revealed that plant extract of this provided first insight into the antioxidant and anti radical properties in a motel of a complete lipid peroxidation (Duke and Wain, 1981). An alcoholic extract of the leaf was shown, in lab rats, to protect the liver from damage from certain poisons. Also extract from the leaves of the plant were found to exhibit broad spectrum antibacterial activity but no specific antibacterial agents were isolated not identified. Due to the presence of various antioxidant, the plants can be used as natural antioxidants and alternatives to synthetic antioxidant (FAOSTAT, 2012)

Chemical Constituents of Manihot esculenta

Milky juice contains an esential oil (0.13%), saponin (1.14%) glucoside and dyes; the Esential oil containing sulfur is organic combination per 100g. the leaves are reported to contain 100 calories 81.0g, H 2O, 69g protein, 1.3g fat,

9.2g total carbohydrate, 2.1g fiber, 1.6g ash, 144mg Ca, 68mg. P.28mg Fe, 4mg

Na, 409mg ka, 8.280mg b-carotene equivalent, 0.16mg thiamine, 0.32mg riboflavin, 1.80mg niacin and 82 ascorbic acid per 100g, the root is reported to contain 35 calories, 65.5g H 2O, 1.0g protein, 0.2g Fat, 32,4g total carbohydrate,

1.0g Fiber, 0.9g ash, 26mg Ca, 32mg P, 0.09mg Fe, 2mg Na, 394mg K, 0.05mg thiamine, 0.04mg riboflavin, 0.6mg niacin and 34mg ascorbic acid. (James,

1983).

(d) Ricinus communis

Methanolic extracts of the leaves of Ricinus communis were used in antimicrobial testing against eight pathogenic bacteria in rats and showed antimicrobial properties. The extract was not toxic. The pericarp of castor bean showed central nervous system effects in mice at low doses. At high doses mice quickly died. A water extract of the root bark showed analgesic activity in rats.

Antihistamine and anti-inflammatory properties were found in ethanolic extract of Ricinus communis root bark 0n a chicken of four weeks old (Lomash 2010).

Extract of the leaves and other parts of the plants are sold as ‘Spra Kast in USA; Also the expressed juice and aqueous, alkaline extracts of the leaves was found to be active against mycobacteria and yeast (Manpret etal., 2012).The Esential oil from the leave that was analysed by bioassay and Gc-Ms carried, revealed five Constituents of the oil and the antimicrobial activity of the oil was investigated in order to evaluate its efficiency against twelve bacterial and four fungi species, using disc diffusion and minimum inhibitory Concentration method. The essential oil showed strong antimicrobial activity against all micro organisms tested with higher sensitivity for bacillus subtilis, staphlococcus aureus , enterobacterial and cloacae. The cytotoxic and apoptotic effects of the enssential oil on Hela cell lives were examined by MIT assay and was quite strong with Lc50 Values less than 263mg/ml for both cells lives. This showed the potential antimicrobial and anticarcinogenic properties of the Esential oil of

Ricinus communis , showing its possibilities and potential use in the formular of root remedies for topical treatment of infection (Olugbade, et al .,(1982).

Chemical Constituents of Ricinius communis :

Data of study revealed that raw material of Ricinus communis posses these chemical constituents that may be responsible for the therapeutic function of this medicinal plant ; 2-0-p-coumaroyl pruning, 3,4-dimethoxy-6, 8- dilydroxy-coumarin, 30-nor-lupan-3-beta-ol-20-one,6,7-dilytoxy-8-methoxy- oumarin, b9, 10dioxystearic acid, agmatine, alpha-tocopherol, beta-umyrin, beta-sitosterol, calcium, cashene, catalase, chlorogenic-acid, corilagin, edestine, ellagic acid, endotrypsin, fenilic-acid, fucose, gallic-acid, gramma-tocopherol, glutamine acid, glyeolic acid, hen, hyperoside, invert-sugar, invertage, isoguercitrin, kaempferol, kaempferol-3-0-beta-d-glucophranoside, etc(Fred ,

2012 )

(e) Vitex doniana

The earlier reseachers have reported the use of the fruits and leaves of Vitex doniana for medicinal purposes. Aqueos extract of leaves and stem on carbon tetrachloride jnducced liver injury in rats was assessed. With 36 albino rats which were given at Concentration dose of 100 and 200mg/kg body weight.

This revealed significant increase in % change in liver, Kidney and Spleen in animals treated with ccl4 was observed compared with control group. The histological section of the organ shows that the level of hepatic, renal and lymphatic damage were higher in ccl 4 treated rats than those rats supplemented with aqueous extracts of Vitex doniana . He suggested that Vitex doniana may have anti- hepatoxic effect against ccl 4 Induced liver injury in rats ( James,

2010).

Also back of vitex doniana was extracted in boiled water at 100 0c and the extracted solution tested on uterine muscles strip prepation. It was found that to have much more potassium and phosphate than calcium, magnesium, zinc and iron, which may be responsible for the effect of the crude extract on uterine muscle for the effect of crude graded uterine muscle contractile effects of prostaglandins, ergometrine and oxytocin. Though the potential effect was not significant on contractile response to actylcholine and potassium chloride. He concluded that the effect of the Vitex doniana bark extract may be not only vo tage operated but may act via uterotonic receptors and also that Vitex doniana used to control postpartum after child birth may be justified

Chemical constituents of Vitex doniana

The phytochemical, nutritive and antinutritive composition of Vitex doniana ;

Anthraquinone, saponins, triterpene and steroids, Flavone, Flavoniods,

Phytin,tannin, ascorbic acid, (Agbofor and Nwachukwu 2011).

2.4 AIM AND OBJECTIVE

This study tend to determine the impact and level of air pollution tolerance index and concentration of heavy metals on some medicinal plants located around Quarry site in Ameze at Ishi-Agu Ebonyi state, Nigeria.

In other to actualize the above stated aim, this study employs the following objectives:

(i) Macroscopic and microscopic examination of the medicinal plants.

(ii) Determination of qualitative and quantification of phytochemical

Constituents of the medicinal plants studied.

(iii) Determination of the concentration of heavy minerals on the leaves

of plants studied.

(iv) Determination of the Air pollution Tolerance index of medicinal

plants studied.

2.5 SIGNIFICANCE OF THE STUDY

The knowledge of air pollution tolerant index of medicinal plants situated in Quarry environment where both metallic and non metallic substances may emit to cause Air pollution is pertinent in the following way:

(1) Enables documentation of medicinal plants that are highly tolerant

to Air pollution which may be used as phytoremediators and the sensitive

species may be used as bioindicators in a polluted environment

(2)Improved the quality of final dosage form that uses medicinal plants as

raw materials.

(3)It gives concentration of heavy metal on medicinal plants at present and gives the idea of how the future might be.

(4) shows the level of heavy metal on medicinal plants at present and gives

the idea of how the future might be.

(5) It brings the knowledge of toxic effect of quarry activities on medicinal plant to herb dealers and the public.

(6) It creates standard for evaluating herbs before use and choice of area for herb cultivation.

2.6 DEFINITION OF TERMS

RELATIVE WATER CONTENTS (RWC)

Relative water contents (RWC) is probably the most appropriate measure of plant water status in terms of the physiological consequence of cellular water deficit. Water potential as an estimate of the energy status of plant, water is useful in dealing with water transport in the soil-plant-atmosphere continuum.

It does not account for osmotic adjustment (OA). OA is a powerful mechanism of conserving cellular hydration under drought stress. For the same leaf water potential two different cultivars can have different leaf RWC, indicating a corresponding difference in leaf hydration, leaf water deficit and physiological water status. Hence RWC is an appropriate estimate of plant water status in terms of cellular hydration under the possible effect of both leaf water potential and OA.

CHLOROPHYLL CONTENTS

Chlorophyll is a naturally occurring molecule that gives plants their green color. Chlorophyll is the molecule that is responsible for facilitating one of the most incredibly miraculous processes on earth and the process of photosynthesisis. is Photosynthesis the process that converts sunlight energy, water and carbon dioxide into our primary source of fuel – glucose. All animals and humans obtain their life-sustaining energy supply from plants, making photosynthesisis to be one of the sources of all life. chlorophyll is greenish pigments which contain a porhpyrin ring . This is a stable ring-shaped molecule around which electrons are free to migrate. Because the electrons move freely, the ring has the potential to gain or lose electrons easily, and thus the potential to provide energized electrons to other molecules. This is the fundamental process by which chlorophyll "captures" the energy of sunlight.

ASCORBIC ACID CONTENTS

Ascorbic acid is an abundant component of plants. It reaches a concentration of over 20 mm in chloroplasts and exisit in all cell compartments, including the cell wall. It has proposed functions in photosynthesis as an enzyme cofactor (including synthesis of ethylene, gibberellins and anthocyanins) and in control of cell growth. A biosynthetic pathway through

GDP-mannose, GDP-L-galactose, L-galactose, and L-galactono-1, 4-lactone has been proposed only recently and is supported by molecular genetic evidence from the ascorbate-deficient vitamin c 1 mutant of Arabidopsis thaliana . Other pathways via uronic acids could provide minor sources of ascorbate. Ascorbate, at least in some species, is a precursor of tartrate and oxalate. It has a major role in photosynthesis, acting in the Mehler peroxidase reaction with ascorbate peroxidase to regulate the redox state of photosynthetic electron carriers and as a cofactor for violaxanthin de-epoxidase, an enzyme involved in xanthophyll cycle-mediated photoprotection. The hypersensitivity of some of the vitamin c mutants to ozone and UV-B radiation, the rapid response of ascorbate peroxidase expression to photo-oxidative stress, and the properties of transgenic plants with altered ascorbate peroxidase activity all support an important antioxidative role for ascorbate. In relation to cell growth, ascorbate is a cofactor for prolyl hydroxylase that posttranslationally hydroxylates proline residues in cell wall hydroxyproline-rich glycoproteins required for cell division and expansion. Additionally, high ascorbate oxidase activity in the cell wall is correlated with areas of rapid cell expansion. It remains to be determined if this is a causal relationship and, if so, what is the mechanism. Identification of the biosynthetic pathway now opens the way to manipulating ascorbate biosynthesis in plants, and, along with the mutants, this should contribute to a deeper understanding of the proposed functions of this multifaceted molecule

( Smirnoff and Wheeler 2000) .

STOMATA

Stomata are known as tiny pores on the surface of leaves that permits the exchange of gases between the atmosphere and the inside of the leaf. Each stoma is formed by two specialized epidermal cells, called guard cells. These cells change shape to open and close the pore on a time scale of minutes. In most plants, stomata are between 30 and 60 micrometers long and occur at densities between 50 and 200 per square mm.

PLANT pH

PH is a measure of hydrogen ion concentration or [H+] in an aqueous solution . It is the negative log of hydrogen ion concentration in a water based solution. It is an abrevation for power hydrogen where p” is short for german word for power, potenz and H” is the element symbol for hydrogen. The H is capitalized because it is standard to capitalize . A simple numerical scale is used to express pH. The scale goes from 0.0 to 14.0, with 0.0 being most acid, and

14.0 being most alkaline. The value, 7.0 is neutral--i.e., neither acid nor alkaline. The pH scales is a logarithimic scale that usually runs from 1to 14.

Each whole pH values belows 7 (the pH of pure water) is ten times more acidic than the higher value and each whole pH value above 7 is ten times less acidic than the one below it. A pH of 3 is ten times more acidic than a pH value of

5.80, a strong acid may have a pH of 1-2, while a strong base may have a pH of

13-14 ( H+ or, more precisely, H3O + in a solution . In water, it nomally ranges from -1 to 14, with 7 being neutral. A pH below 7 is acidic and above 7 is alkaline. Soil pH is considered a master variable in soils as it controls many chemical processes that take place. It specifically affects plant nutrient availability by controlling the chemical forms of the nutrient. The optimum pH range for most plants is between 5.5 and 7.0. However many plants have adapted to thrive at pH values outside this range.

Macroscopic examination of the plant leaves

Macroscopic analysis is the establishment of morphological and characteristic of plants products achieved by organoleptic evaluation. This involves recording features noted upon observing the specimen with or without the aid of a magnifying lens, as well as evaluation of the specimen using other sensory characters such as smell and touch.These test provides size, colour and other properties of the specimen being analysed. This identification and characterization processes can be caried out using various botanical examination techniques (Inya-Agha, 2006). These physical measurements may often provide the simplest and quickest indication for its identity, purity or quality when compared with official monograph.

Macroscopic examination of the plant species gives the idea of the condition of a plant before other analysis.The internal structure of a plants in polluted or stressed environment are affected before visible symptoms (Alireza et al.,

2010) .

Microscopic examination of the plant leaves

Microscopic is concerned with the identification of the characters present in the soaked, cleared concised crude drug materials or powdered crude drug with the aid of the microscope. It gives the anatomical characteristicon of the tissue obtained by transverse, radial and longitidunal sections (Inya-Agha, 2006). It produces the number of stomata present in a particular leave . This helps to know the photosynthetic ability of the plant in a particular environment.

Total ash value

The total ash contents of a crude drug is the residue remaining after incineration of the crude drug. This represents the amount of inorganic salts adhering to or occuring naturally in the drug. Ash determination gives basis for evaluation of the identity and purity crudes. It also gives an idea of the crude drugs extent of aldultration in organic matter (Inya-Agha 2006).The water soluble ash is subjected to greater reduction than in the total ash. It is thus used in the detection of material exhausted by water and as an important indication for the presence of materials substituted for genuine article. Acid – insoluble ash values, is ash which is insoluble in dilute hydrochloric acid. Acid –insoluble ash value is often prefered to total ash values. This preference is based on the fact that majority of crude drugs often contain calcium oxalate in large but variable amounts and as such total ash is of no use in detection of earthy matters adherent to such a drug specimen.Total ash value is still of important since its figure are useful in the esclusion of drugs which have been coated with lime, chalk or calcium sulphate to improve their appearance. Since the calcium oxalates is soluble in hydrochloric acid, variable constituents of the ash are thus removed by this means. The residue, known as acid insoluble ash, is then weighed. It is obvious that earthy matter is likely to occur with leaves which are densely pubescent, or are clothed with abundant trichomes, screting resin or with root and rhizomes, or even earthy matters retained on them after heavy rain storms. Evidence of the presence of such execessive earthy matter can thus be obtianed using this means.

Sulphated ash

Sulphated ash is a process that converts all oxides and carbonates to sulphates at hihger tempratures. Inya-Agha (2006) revealed that consistency level of ash value is often higher in sulphated ash.

Determination of extractive Values The determination of ethanol – soluble extractive and water soluble extractive are used as a means of evaluating drugs whose constituents are not readily estimated by other means. Many method has being used for identification and evaluation of crude drugs . In most cases, the amount of drug soluble in a given solvent is an index of its polarity. For this reason, determination of extractive values is important.

Determination of moisture contents

Moisture contents determination looks very simple in concept, but in practice the accurate determination is complicated by a number of which may vary considerablly from one sample to another. The most common method used in moisture contents determination of plants and food material is the gravimetric method, as modified by the association of Official Analytical Chemist(AOAC)

1980. It is the most important and widely used measurement in sample that absorbs water. It has been stated that sample should be dried to constant weight

(Ibitoye , 2005).

AIR POLLUTION TOLERANCE INDICES ANALYSIS

Air pollution tolerance index analysis is a process that involves different kinds of biochemical parameters used to denotes capability of a plant to combat against air pollution. Generally, in plants analysis the chemical determination is more accurate than the sampling . Analysis of plant tissues is an extremely useful tool for growers. Not only can plant tissue testing be used to monitor the nutrient status of plants but it can help identify nutrient deficiencies and imbalances . This analysis is used to find out plants with higher APTI value that is known to be tolerant to air pollution and can be used as sink to mitigate pollution, while plants with low APTI value show less tolerance and can be used to indicate levels of air pollution.

This process is achieved by combining different values gotten from these four biochemical parameters:

(a) Chlorophyll content analysis : This is the aspect of quantifying two components (chlorophyll a and chlorophyll b), which is always in the multicellular terrestrial of plants, in green and yellow green algae. The chlorophyll contents analysis shows the level of plant photosynthetic ability, development of biomass, and plants growth.The lesser the value the more plants are affected Insuficiency of this chlorophyll limits efficiency of photosynthesis and crop or herb productivity.

(b) Plant pH analysis : This is the way of identifying the basic and acidic states of plants which is indirectly related to the pH state of the soil. It shows Plants with lower pH which are more susceptible while those with higher pH are known to improve tolerance to air pollution. The pH level of leaf extract determines wheather the plants are grown in acidic environment (polluted) or not.The pH level from 5 down is termed to be acidic while from 6 up are basic .

However, this analysis is very useful in identifying plant growth problems resulting from nutrient stress if paired plant samples can be taken from both good and poor areas in the field where the same variety, soil moisture and environmental conditions exist.

(c) Relative water content analysis : T he relative water cotent of the plants determines the presence of water in its cell turgidity, which shows the ability of protoplasmic cell to permit water movement in plants parts.The higher the value the more permeable the protoplasmic cell and lesser the value the more the plants is disadvantaged. This analysis is the most important process for evaluating nutrient and water status of plant. Accurate water content estimation is needed to make irrigation decisions, predict crop yields in the field of agriculture and to known area of water stress. This process helps to find out air polluted and stressed environment that is noticed by low contents of water. The relative water content technique formerly known as relative turgidity was originally described by Weatherly and has been widely accepted as a reproducible and meaningful index of plant water status. Leaf tissue is most commonly used for relative water determination. It May be accurately estimated using the ratio of tissue fresh weight to tissue turgid weight, termed as relative tissue weight.

(d) Ascorbic acid analysis: This is known as the quantitative measurement of ascorbic acid content of a plant which is used as stress maker in an environment. Its higher value shows that the environment is stressed or polluted . This value is dependent of pH of particular environment being higher at high pH and lower at lower pH. (Manju et al 2013), (Jissy and Joya 2010).

CHAPTER THREE

MATERIALS AND METHODS

3.1 Collection, Identification and preparation of Plants Materials.

Fresh leaves of the plants species ( Vitex doniana , Cnestis ferruginea,

Anthocleista djalonensis, Ricinus communis, Manihot esculenta ) were randomly selected from the immediate vicinity of the quarry site, designated as experimental site (ES). A site nearby with similar ecological conditions but not near any industry or Road side was selected as the control site (CS). The plants used for the study where those available in the experimental site. A triplicate of fully matured leaves samples of the various plants where then collected in the morning, placed in paper bags with moist paper towels, and immediately transported in a cool dark container to the laboratory for analysis. The fresh weight (Fm) were taken immediately upon getting to the laboratory. Some samples were preserved in a refrigerator for other analysis while other were dried for further analysis.This protocol was observed for both specimen from experimental and control site. These medicinal plants were identified at the international Center for

Ethnomedicine and Drug Development center, Nsukka, Enugu - State,

Nigeria.The voucher specimen of these medicinal plants were deposited in the

Hebarium of the Department of Pharmacognosy and Environmental Medicine.

3.2 Chemicals, Reagents And Equipment.

All the reagents and solvent used under these studies are of analytical grade. Chemicals and all the glassware, containers and tools were washed with liquid detergent first, rinsed with 20 % (v/v) nitric acid and finally rinsed with distilled water. The containers and glassware were kept in oven until needed.

Distilled water was used throughout the work.

Chloral hydrate, hydrochloric acid, phloroglucinol in hydrochloric and sodium hypochlorite. solution of crystalline cuso 4 in sulphuric acid(Fehling’s solution

1), solution of Rochelle salt and potassium hydroxide (Fehling’s solution 11),

Potassium bismuth iodide solution (Dragendorf”s reagents), solution of iodine in potassium iodide (Wagner,s reagent), potassium mercuric iodide solution

(Mayer,s reagent), saturated solution of picric acid (Hagner,s reagent) Million’s reagents, naphtol solution in ethanol (Molich,s reagent), α-naphtol, sulphuric acid (H 2SO 4), ammonium hydroxide (NH 4OH) chloroform, sodium hydroxide

(NaOH), carbon tetrachloride, ferric chloride, ethanol (70%, 90%,) lead subacetate, glacial acetic acid, ethylacetate, aluminum chloride, olive oil.Meter rule, Standard graph, pencil, Calculator, Micro Slide, Flame and

Photomicrogram, concetrated chloric(VII) acid Riedelde-Haen AG, Stock standard solution of lead(II), concentration Img/cm3 30% HCL, Ammonium was (Pb(No 3)2 water,nitric (V) acid), dithizone solution, Ammonia solution of potassium cyanide, NH 4OH solution, 10 % sodium tartrate solution, 25% ammonia, Nitric(V) acid, Hydroksylamine hydrochloride,0.0425g potassium iodate, 2 % Hcl, 14 % Na 2s205, 1.4 % Na 2 S2 O3, 10 % H 2SO 4, 10.8 L-ascorbic acid, 3% ammonium molybdate, 0.56 % of potassium tartrate, 13.98 % H 2SO 4,

Dithizone, NaoH, 4% Soduim sulphate (SDS), H 2SO 4, 0.5 ml of 0.72 M

Hydroxylammonium, Hydrochloride , acetic acid and sodium thiosulphate,

Ammonium hydrochloride, Citric acid, Diethylglyoxime,bromine water.

Conc Hcl, o.1m Hcl, Nitric acid,0.1ml metallic cadium,20ml Nitric acid, dithizone solution, chloroform, potassium cyanide, 40% NaoH solution,

Hydroxinamine hydrochloride,tartaric acid, chloroform, Thymol blue, distilled water, Deionized water and buffer solution, oxalic acid, EDTA,

Orthophosphoric acid, H 2So4, Ammonium molydbate and water. Acetone and spectrophotometer .

Equipment

Petri dish, weighing balance, Digital pH meter,beaker, spectrophotometer, sharp knife, separating funnel, conical flask, water bath, test tubes, nickel crucible, desiccators, and oven.

3.3 Morphological and microscopic features of the leaves i. Macroscopic analysis Leaf area or leaf size, and leaf length was determined by the use of planimetric method. The leaf length and, leaf stalk, were determined by means of a meter rule on a starndard graph. The leaf area was carried out by placing the entire leaf surface upside down on a standard graph paper calibrated in I cm

2 and traced round with a pencil . The leaf surface was traced through the area

3 of the graph covered by the leaf area, the cm not up to ½ cm were discarded in the count and all the leaf areas within counted (Nzekwe, 2002).

Microscopic analysis

One mm square section of the leaf was cleared in a solution of chloral hydrate, after which it was stained with 1% phloroglucinol and mounted on a micro slide using chloral hydrate as a mountant and flamed to boil. The preparation was allowed to cool and then mounted on the photomicroscope, using 4mm objective (x 400 magnification). The upper and lower epidermis were observed, the presence nature of the stomata and other character were also observed and the resultant image captured using a high resolution digital camera. According to Evans (2009), this experiment was carried out on both control and test samples.

3.4 QUALITATIVE PHYTOCHEMICAL ANALYSIS

The test carried out was based on procedures outlined by Harborne (1973),

Evans (2002).

Preliminary phytochemical analysis

(1) Test for carbohydrate

Molisch’s test

A 0.1g of the extract was boiled with 2 ml of distilled water and filtered.

To the filtrate, few drops of Naphthol solution in ethanol (Molisch’s reagent) were added, concentrated sulphuric acid was then gently poured down the side of the test tube to form a lower layer.

(2) Test for alkaloids

A 20ml of 3 % sulphuric acid in 50 % ethanol was added to 2 g of the extract and heated on a boiling water bath for 10 minutes, cooled and filtered.

2ml of the filtrate was tested with a few drops of Mayer’s reagent (potassium mercuric iodide solution), Dragendorff’s reagent (bismuth potassium iodide solution), wagner’s reagent (iodine in potassium iodide solution), and picric acid solution (1%). The remaining filtrate was placed in 100 ml separatory funnel and made alkaline with dilute ammonia solution. The aqueous solution was separated and extracted with two 5 ml portions of dilute sulphuric acid. The extract was tested with a few drops of Mayer’s, Wagner’s, Dragendorff’s reagents and Picric acid solution. Alkaloids give milky precipitate with few drops Mayer’s reagent; reddish brown precipitate with few drops of Wagner’s reagent.

(3) Test for reducing sugars

A 5ml of a mixture of equal parts of Fehling’s solution 1 and 11 were added to

5ml of aqueous extract and then heated on a water bath for 5 minutes. A brick red precipitate shows the presence of reducing sugar.

(4) Test for glycosides

A 5ml of sulphuric acid was added to 0.1g of the extract in a test tube and boiled for 15 minutes on a water bath, then cooled and neutralized with 20% potassium hydroxide solution. 10ml of a mixture of equal parts of Fehling’s solution 1 and 11 was added and boiled for 5 minutes.

(5) Test for saponins

A 20ml of distilled water was added to 0.25 g of the extract and boiled on a hot water bath for 2 minutes. The mixture was filtered while hot and allowed to cool and filtrate was used for the following tests.

(a) Frothing Test

A 5 ml of the filtrate was diluted with 15 ml of distilled water and shaken

vigorously. A stable froth (foam) upon standing indicates the presence of

saponins.

(b) Emulsion test

To the frothing solution was added 2 drops of olive oil and contentss

shaken vigorously. .

(c) Fehling’s test

To 5ml of the filtrate was added 5ml of Fehling’s solution (equal parts of

Fehling’s solution 1 and 11) and the contents were heated on a water

bath. A reddish precipitate which turns brick red on further heating with

sulphuric acid indicates the presence of saponins.

Haemolyeis Test

(6) Test for tannins.

A 1 g of the powered material was boiled with 20 ml of water, filtered and used for the following test.

(a) Ferric chloride test

To 3 ml of the filtrate, few drops of ferric chloride were added.

(b) Lead Acetate test

To a little of the filtrate was added lead acetate solution.

(7) Test for flavonoids

A 10 ml of ethy1 acetate was added to 0.2 g of the extract which was used for the following test.

(i) Ammonium hydroxide test

A 4 ml of filtrate was shaken with 1ml of dilute ammonia solution. The layers were allowed to separate.

(ii) 1% Aluminium Chloride solution test.

Another 4ml portion of the filtrate was shaken with 1ml of 1% Aluminium chloride solution. The layers were allowed to separate. 8. Test for resins

(i) Precipitation test

A 0.2 g of the extract was extracted with 15 ml of 96 % ethanol. The alcoholic exract was then poured into 20 ml of distilled water in beaker.

(ii) Colour test

A 0.2 g of the extract was extracted with chloroform and the extract was concentrated to dryness. The residue was redissolved in 3 ml of acetone and another 3ml concentrated hydrochloric acid was added. This mixture was heated in a water bath for 30 minutes.

9. Test for proteins

A 0.5 g of the extract was extracted with 20 ml of distilled and the filtrate was used for the following tests.

(i) Millon’s test

To a little portion of the filtrate in a test tube, two drops of Million’s reagent were added. A white precipitate indicates the presence of proteins.

(ii) Xanthoproteic reaction test

A 5 ml of the filtrate was heated with few drops of concentrated nitric acid.

(iii) Picric acid test

To a little portion of the filtrate was added a few drop of picric acid.

(iv) Biuret test

A crystal of copper sulphate was added to 2 ml of the filtrate, and then 2 drops of potassium hydroxide solution was added. 10. Test for fats and oil

A 0.1 g of the extract was pressed between filter paper and was observed, a control was also prepared by placing 2 drops of olive oil on filter paper.

11. Test for steroids and terpenoids .

A 9 ml of ethanol was added to 1g of the extract and refluxed for a few minutes and filtered. The filtrate was concentrated solution, the mixture was allowed to stand for 1 hour and the waxy matter was filtered off. The filtrate was extracted with 2.5ml of chloroform using separating funnel. To 0.5ml of the chloroform extract in a test tube was carefully added 1ml of concentrated sulphuric acid to form a lower layer. A reddish brown interface shows the presence of steroids.

Another 0.5ml of the chloroform extract was evaporated to dryness on a water bath and heated with 3ml of concentrated sulphuric acid for 10 minutes on a water bath. A grey colour indicates the presence of terpenoids.

3.5 QUANTITATIVE ANALYSIS OF PHYTOCHEMICALS

This Experiment was done according to Horbone (1973).

Flavonoids

A 1 g of the sample was macerated with 20 ml of ethyl acetate filtered and 5 ml of the filtered added to 5 ml of dilute ammonia, shake for 5 minutes and the upper layer was collected the absorbance was taken at 490 nm.

Tannins

A I g of the sample was macerated with 50 ml of methanol filtered , 5ml of the filterate pipetted and 0.3ml of 0.1N ferric chloride was added in 0.1N Hcl with

0.3ml of 0.008m potassium ferric cyanide. The absorbance was taken at 720nm.

Alkaloids

1g of the sample was added macerated with 20ml of 1.1 ethanol sulfuric acid,

filtered and 1ml of the filtrate, was pipette and 5ml o f 60% H 2 SO 4 added with

5ml of 0.5% formaldehyde in 60% H 2 SO 4 , mix and allowed to stand for

3hours and the absorbance was measured.

Saponins

1g of the sample was macerated with 10ml of petroleum ether decant into a beaker and 10ml of petroleum ether was used to wash the residues. The filtrate was combined and evaporated to dryness. 6ml of ethanol was added to the residues, transfer 2 ml into a test tube and 2 ml of color reagent was added and absorbance was taken at 550nm.

Terpeniods

1 g of the sample was macerated with 50ml 0f ethanol, filtered and 2.5ml of the filterate was pipetted into 2.5ml of 5% aqueous phosphomolybdic acid solution and 2.5ml of concentrated H 2so4 was gradually added and mixed well. The solution was allowed to stand for 30minutes cooled and made up to 12.5ml with ethanol. The absorbance measured at 700nm.

Carbohydrates

1g of the samples was macerated with 50ml of distilled water, filtered and 1ml of the filtrate was measured and mixed with saturated picric acid (2ml) then the absorbance was measured at 530nm.

3.6 DETERMINATION OF ANALYTICAL STANDARDS

i. Determination of ash Values

(a) Total ash Values

A tarred nickel crucible was placed in mauffle furnance for about 15 minutes at 35 Oc, cooled in a desiccator for about one hour and the crucible was weighed (W 1), 3.0g (W 2) of the powder material, was placed into the nickel crucible and heated gently until all the moisture has been driven off and the plant material has been completely charred. The heat was slowly increased until the carbon has vaporized and the residue was free from carbon at 650 degree centigrade and sample turns grey (white ash).The crucible was removed with crucible tong, cooled in a desiccator, and reweighed(W 3). The percentage ash contents was determined by the relationship

% Ash = Final weight of crucible (W 3) - Initial weight of crucible (W 1) X100

Weight of sample (W 2) – initial weight of crucible (W 1)

(b) Water soluble ash Values

A nickel crucible was ingnited to a constant weight at 450 o c, cooled and then weighed (W 1).

A 3.0 g (W 2) of the material was placed and spread over the bottom of the crucible and then reweighed. The plant material was incinerated to 450 0c by gradually increasing the heat until it was free from carbon. The crucible cooled in a desicator and then reweighed. The contents of the crucible was transferred into a beaker, 5 ml of water was added into the beaker and the boiled for 5 mins.

The mixture was filtered through ashless filter paper, and both the residue and the filter paper were dried in an oven. The ashless filter paper containing the residue was compressed into the crucible and was subjected to heat until the ashless paper was eliminated. The crucible was reweighed (W 3) and the differences were noted with the formular,

% Water Insoluble Ash= Weight of sample (W 2) –Initial weight of crucible X I00 Final Weight of crucible (W 3)

% Water soluble Ash = % Total ash- % water Insoluble Acid –insoluble ash value The ash obtained above was transferred into a beaker containing 25 ml of dilute hydrochloric acid and was boiled for five minutes. The insoluble matter was collected in a sintered crucible and an ashless filter paper. The beaker and crucible were washed repeatedly through the filter paper with hot water until it was free from acid. It was ingnited to a constant weight at about 500 O C.

(C) Sulphated ash Values

A nickel crucible was ingnited to a constant weight at 450 0 C, cooled and weighed. 3.0g of the dried material was placed over the bottom of the crucible and then incinerated to 450 0 C by gradually increasing the heat until it was free from carbon. The crucible was cooled in a desiccators and more dilute sulphuric acid was added. The heating was continued to about 800 0 C with occasional cooling and reweighing until a constant weight was obtained. The % sulphated ash value was determined by difference of the two weights, thus:

% Sulphated Ash= Final Weight of sample –Initial Weight of sample x 100 Initial weight of sample

(d) Acid insoluble ash Values

The ash obtianed was transferred into a beaker containing 25ml of dilute

Hydrochloric acid and was boiled for 5mins. The isoluble matters was collected in a sintered crucible and an ashless filter paper. The beaker and crucible were washed repeatedly through the filter paper with hot water until it was free from acid. It was ingnited to constant weight at about 500 0 c.

(ii) Determination of extractive Values

(a) Alcoholic Soluble extractive Values A 5.0 g of the material was weighed accurately and placed in a stopped conical flask. 100 ml of 90% alcohol was added and the stopper of the conical flask was replaced firmly. The flask and its contentss were shaken mechanically for about 6 hours and was allowed to macerate for another another 18 hours and then filtered. The filtrate was collected and evaporated to dryness, and then the residue was dried to a constant weight at 105 0 C.

(b) Water soluble extractive Values

A 5.0 g of the material was weighed accurately and placed in a stoppered conical flask. 100 ml of chloroform - water was added and the stopper of the conical flask was replaced firmly. The flask and its contents were shaken mechanically for 6 hours and was allowed to macerate for another 18 hours and then was filtered. The filtrate was collected and evapourated to dryness and then the residue was dried to a constant weight at 105 0 C.

(iii) Determination of moisture contents

A preheated tarred porcelain crucible was weighed and its weight with lid recorded (W 1). A spatula full of the dried sample was introduced into the crucible and was reweighed, W 2. The sample was heated in an oven at the temperature of 65 0c for 12hours, at interval of 6,3,2,1, hours, until a constant weight, followed by cooling in a desicator before reweighing. the constant weight, w 3 was noted. The percentage moisture was calculated from the relationship.

% Moisture = Weight of sample in crucible (W 2) – constant weight (W 3) x 100 Weight of sample in crucible (W 2)- Weight of crucible (W 1) Where W 2 –W1 = Weight of sample

W2 –W3= Weight of moisture to cool, 2ml of 50% nitric acid solution and gently warm, decant into a 50ml graduated flask and repeated with several washings of 50% nitric acid in order to dissolve the nickel. The filtrate was neutralized with 50% ammonia, filtered, washed with 1% ammonium hydroxide and collected the filtrate and washings in the 50ml flask and make up to the mark within 1% ammonia. An aliquot of this solution was measured into a 50ml standard flask using a pippet, a little distilled water and 2ml of 10% citric acid solution was added and freshly prepared saturated bromine water was added dropwise until distined yellow and

2 drops were added in excess.

3.7 ELEMENTAL ANALYSIS

This is a procedure used to find out the presence of metals in the plant tissue against an accepted range or standard raised by World health Organization for maximum daily permissible level (MPL) ( Jankiewiez et al ., 2001).

A 2 g of sample was weighed into a clean dried crucible and transferred to muffle furnance, ashed at 650 0C for 3hours, cooled in a desicator. 5 ml of

30% Hcl and 10ml of distilled water was added and transferred to 50ml volumetric flask and diluted to volume with distilled water and used for the following test.

LEAD 5ml of solution was placed in a separating funnel, then 10ml of sodium citrate solution and 2ml of 25% ammonia were added (pH of the solution was 8-9).

From this mixture metals were extracted by adding consecutively 5ml portion of extraction solution 1 until dithizone became green after extraction.Time per extraction was 1 minute. The chloroform extracts collected in the other separatory funnel were acidified by adding 25ml of nitric acid and after shaking for 1 minute, the chloroform layer was removed. 5ml of 20% Hydroxylamine,

5ml of ammonia solution of KCN and 10ml of the extraction solution were added to the water layer remaining in the separatory funnel. The mixture was shaken for 1 minute. After separating funnel with 10ml of extraction solution II, both chloroform extracts were mixed and chloroform was added to obtain 25ml.

The absorbance was taken at 520nm against a blank .

Cadmium

A 5ml of solution obtained after ashing was measured into a separating funnel,

10ml of distilled water were added and pH was established within the range of

1.5 - 2.5 by means of means of 20% solution of NaoH agar thymol blue as indicator. 1ml of sodium potassium tartarate 5ml of 1% potassium cyanide in

40% solution of NaoH and 1ml of hydroxylamine hydrochloride were added in succession. Fifteen milliliters of dithizone solution in chloroform were added to the mixture and shaken for 1 minute. After separation, the chloroform layer was tapped to another sepratory funnel containing 25ml of refrigerated tartaric acid.

Extraction solution was added again to the solution remaining in the separatory funnel and after shaking for 1 minute, both chloroform extract were mixed.They were shaken for 2 minutes with wa solution of tartaric acid then chloroform and excess of dithizone were rejected. After 5ml of chloroform were added and the solution were shaken again for I minute, the layer were separated and the chloroform layer was removed. The 0.25ml of hydroxylamine hydrochloride,

15ml of dithizone solution in chloroform (extraction solution II) and 5ml of

0.5% of KCN solution in 40% NaoH were added to the tartaric acid solution containing the extracted cadmium and the mixture was shaken for 1 minute.

After sepration of phases the pink chloroform layer was transferred to a 25ml measuring cylinder. Extraction was repeated using 5ml of dithizone solution, the extract was transferred to a cylinder and chloroform was added to 25ml.

Absorbance of the obtained solution was measured against blank at 520nm

(Wieczorek , 2000).

Zinc

2ml of citric acid solution was added to 5ml of sample solution, then 2 drops of phenolphtalin added and neutralized with ammonia with 0.1ml ammonia in excess. 5ml dithizone solution was added, shaken and carbon tetrachloride layer was discarded. 2ml of carbon tetrachloride was added, shaken and carbontetrachloride discarded. 5ml of dilute dithizone was added to the aqueous layer and the absorbance at 532nm was measured (Shar and Bharnger, 2001)

Nickle: A 5ml of sample solution was weighed into a porcelain basin, add 1g of magnesium nitrate and place a small cone of filter paper in the solution. The

Cone of the filter paper was ingnited and allowed to burn away and the temperature was gentiy raised to a dull red heat in order to burn away all the carbon. The solution was allowed to cool ,2ml of 50% nitric acid solution and gently warm, decant into a 50ml graduated flask and repeated with several washings of 50% nitric acid in order to dissolve the nickel.The filtrate was neutralized with 50% ammonia, filtered, washed with 1% ammonium hydroxide and collected the filtrate and washings in the 50ml flask and make up to the mark within 1% ammonia. An aliquot of this solution was measured into a 50ml standard flask using a pippet, a little distilled water and 2ml of 10% citric acid solution was added and freshly prepared saturated bromine water was added dropwise until distined yellow and 2 drops were added in excess, 5ml of concentrated ammonia followed by 2 ml of dimethylglyoxime solution. The volume was made up to 50ml with distilled water, shaked well and the absorbance measured at 455 nm after standing for 10-20 minutes

(Liberman,1955)

Arsenite : 5ml of sample was added to 0.5ml of 0.0425g of potassium iodate

iodate, followed by 0.5ml of solution ( 14% Na 2S2O5, 1.4% Na 2S2O3, 10% H 2SO 4 ), mixed and allowed to stand for 10minutes. 0.5ml of colour reagent was added and allowed to stand for 10 minutes, the absorbance was taken at 880nm (Dhar et al , 2004). 3.8 AIR POLLUTION TOLERANCE INDICES ANALYSIS

(a) RELATIVE WATER CONTENTS

The plant leaf was floated on distilled water inside a closed Petri dish at room temperature for 24 hours. At the end of the incubation period leaf sample was cleaned dry gently with tissue paper and reweighed to obtain the turgid mass

(Tm). It was placed in a preheated oven at 80 c for 48hrs. Thereafter the leaf, were weighed to obtain the dry mass (Dm). The relative water contents was

FM − DM 100 calculated using the formula: RWC = TM − DM x 1

Where FM = Fresh Mass

DM = Dry Mass

TM = Turgid Mass

(b) TOTAL CHLOROPHYLL CONTENTS

A 3g of the leaf sample was blended and then extracted with 10ml of 80% acetone, left for 15mins, and the liquid portion Decanted, centrifuged at 2.500

RPM for 3mins. The supernatant was collected and its absorbance measured at

645nm and 665nm using spectrophotometer.

(c) LEAF EXTRACT pH

The pH was determined by the Direct madding engineering method using a

Digital pH meter. The leaf extract was made by cold maceration of the leaf with de-ionized water, filtered through an ash less filter paper, then the filtrate used for pH Determination. The pH meter was pre calibrated prior to its usage, using buffer solution of pH 4 and 9. The pH electrode was carefully dipped into the filtrate in a 10ml backer. The value displayed on the crystal liquid Paiel (CLD) of the pH water, was taken as the pH value. The exercise was done in triplicate and the average of the three reading used.

(d) ASCORBIC ACID (AA) CONTENTS ANALYSIS

1g of the leaf sample was treated with 4ml of oxalic acid EDTA extracting solution in a test tube. The 1ml of orthophosphoric acid was added followed by

1ml of 5% H 2SO 4, 2ml Ammonium molydable, and then 3ml of water. The solution was allowed to stand for 15min, after which the absorbance at 760nm was measured. The concentration of ascorbic acid was extrapolated from a standard ascorbic acid curve .

DETERMINATIONS OF PERCENTAGE AIR POLLUTION

TOLERANCE INDICES (APTI)

This can be able to factor out after getting the relative water contents value, pH of the leaves, chlorophyll contents and Ascorbic acid contents

APTI = (A (T+P) + R)/10 contents of the plant leaves.

Where A = Ascorbic Acid contents (mg/g)

T = Total chlorophyll contents of the leaf (mg/g)

P = pH of the leaf extract

R = Relative water contents of leaf extract (%) Based on the development and evaluation of APTI Values among the samples they were categorized into three groups namely:

< 10 is sensitive

> 10 < 16 is intermediate

> 17 is tolerant

3.9 STATISTICAL ANALYSIS

The data were based on three replicates and subjected to analysis of variance.

Standard errors of each individual nutrient of the samples were computed and variations among the species were evaluated by least significance difference

(LSD) at 5% level of probability (p<0.5). Result was presented as Mean ± SEM.

CHAPTER FOUR

RESULT:

4.1 MACROSCOPIC / MORPHOLOGICAL EXAMINATION OF THE LEAVES

OF FIVE PLANTS FROM BOTH EXPERIMENAL SITE AND CONTROL SITE.

Microscopic examination of this leaves help to know the features of medicinal plants in healthy and polluted environment.

Test Control

Fig 6: Photograph of the leaf of Vitex doniana from experimenal site and control

Control Test

Fig 7:Photograph of the leaf of Cnestis ferruginea from experimental site and control

Test Control Fig 8: Photograph of the leaf of Anthocleistia djalonensis from experimental site and control

Fig 9: Photograph of the leaf of Ricinus communis from experimental site and control

Test Control Fig 10: Photograph of the leaf of Manihot esculenta from experimental site and control

4.2 Result of Microscopical examination of the leaves of test and control sample

Microscopic Examination of the leaves that reveals the stomatal occurrence at the uper and lower epidermis of the m edicinal plants studied from both experimental and control sites showed that most plants from control has more stomatal occurrence and stomata opening than others from experimental site (Fig 11-Fig 15) .

Upper epidermis control

Upper epidermis Test

Lower epidermis control

Lower epidermis test

Fig. 11: Microscopic view of stomatal occurrence at lower and upper epidermis of Vitex doniana from experimental site and control

Lower epidermis test

Fig 12: Microscopic view of stomatal occurrence at lower and upper epidermis of Cnestis ferruginea from experimental site and control

Upper epidermis control Upper epidermis test

Lower epidermis control Lower epidermis test

Fig 13: : Microscopic view of stomatal occurrence at lower and upper epidermis of Anthocleista djalonensis from experimental site and control

Upper epidermis control Upper epidermis test

Lower epidermis control lower epidermis test

Fig 14: : Microscopic view of stomatal occurrence at lower and upper epidermis of Ricinus communis from experimental site and control

Upper epidermis control Upper epidermis test

Lower epidermis control Lower epidermis test

Fig 15: : Microscopic view of stomatal occurrence at lower and upper epidermis of Manihot esculenta from experimental site and Control . TABLE 1: Summary of the morphological and microscopic features of leaf from experimental and control site respectively. S/ Sample FAMILY Site Leaf Leaf Stalk Stomata N length Area Length no (cm) (cm) (cm) 1 Vitex doniana Vebernaceae Test 10.0±1.0 85.0±2.0 2.5±0.2 3 Control 14.0±1.0 106.0±2.0 4.0±0.3 7 2 Cnestis Connaraceae Test 22. 0±1.0 30.0±3.0 1.7±0.3 3 ferruginea Control 32. 0±1.0 75.0±1.0 3.0±0.3 6 3 Anthocleista Loganiaceae Test 40.0±1.0 870.0±2.0 6.0±0.3 4 djalonensis Control 64.8±1.0 1350.0±2.0 9.0±0.2 I4 4 Ricinus Euphorbiaceae Test 16.4±1.0 450.0±3.0 13.0±0.3 2 communis Control 20.7±1.0 605.0±1.0 19.5±0.2 6 5 Manihot Euphorbiaceae Test 12.0±1.0 370.0±2.0 12.5±0.2 2 esculenta Control 17.0±1.0 420.0±1.0 15.0±0.3 4 Values are ± SEM , n= 3

4.4 PHYTOCHEMICAL SCREENING OF THE PLANTS

(1) The result of the Qualitative phytochemical Analysis of the five medicinal plants is shown in Table 2.

Table 2: Result of the Qualitative phytochemical Analysis of the five medicinal plants.

S/N Constitutes Cnestis Cnestis Manihot Manihot Anthocleista Anthocleista Vitex Vitex Ricinus Ricinus ferruginea ferruginea esculenta esculenta djalonensis djalonensis doniana doniana communis communis test control test control Test control test control test Control 1 Alkaloid +++ ++ ++++ ++ +++ +++ ++++ +++ ++ +++ 2 Glycoside ++++ +++ +++ ++ +++ ++++ ++ +++ ++++ +++ 4 Carbohydrate ++ ++ +++ ++++ ++++ ++++ ++ ++++ +++ ++++ 5 Flavourids +++ ++++ + ++ +++ ++ ++ +++ ++ +++ 6 Fats & Oil ------7 Resin + + ++ +++ + - - + + - 8 Terpenoids +++ + + ++ +++ ++ + + +++ ++ 9 Steroids ++ + + ++ ++ +++ + + ++ ++ 10 Saponins ++ + ++ + + +++ + +++ ++ ++ 11 Tannins + +++ + + + + ++ ++++ ++ + 12 Proteins ++ +++ +++ +++ +++ ++ ++ ++++ +++ ++ The key - = Not present + = present ++ = present in moderately high concentration +++ = present in very high concentration +++ = Abundantly present

Table 3: QUANTITATIVE ANALYSIS OF PHYTOCHEMICALS (g/100g)

S/N Sample Site Tannins Flavoniods Alkaloids Saponins Steriods Carbohydrates Resins

1 Vitex doniana Experimental 5.98 ± 0.06 10.25 ± 0.03 5.36 ± 0.07 0.15 ± 0.06 0.17 ± 0.20 7.09 ± 0.05 0.15 ± 0.06

Control 4.91 ± 0.03 7.53 ± 0.06 5.69 ± 0.02 0.04 ± 0.01 0.06 ± 0.07 4.38 ± 0.06 0.06 ± 0.3 2 Cnestis Experimental 5.77 ± 0.04 10.25 ± 0.06 5.28 ± 0.08 0.15 ± 0.07 0.07 ± 0.10 5.63 ± 0.30 0.14 ± 0.07 ferruginea Control 5.24 ± 0.06 7.84 ± 0.07 3.49 ± 0.04 0.02 ± 0.01 0.02 ± 0.03 4.66 ± 0.02 0.03 ± 0.02 3 Anthocleista Experimental 7.05 ± 0.01 10.49 ± 0.04 4.47 ± 0.05 0.08 ± 0.04 0.15 ± 0.06 0.72 ± 0.10 0.04 ± 0.03 djanolensis Control 5.40 ± 0.07 9.44 ± 0.04 3.82 ± 0.06 0.05 ± 0.06 0.04 ± 0.7 6.00 ± 10.00 0.01 ± 0.04 4 Ricinus Experimeal 6.67 ± 0.01 11.11 ± 0.06 3.90 ± 0.20 1.87 ± 0.1 0.07 ± 0.03 4.91 ± 0.04 0.07 ± 0.02 Communis Control 5.65 ± 0.03 8.60 ± 0.03 3.73 ± 0.04 0.02 ± 0.7 0.02 ± 0.03 6.79 ± 0.07 0.02 ± 0.01 5 Manihot Exprimental 5.61 ± 0.06 9.63 ± 0.04 4.47 ± 0.06 0.03 ± 0.04 0.15 ± 0.04 6.37 ± 0.1 0.05 ± 0.04 esculenta Control 4.50 ± 0.05 6.79 ± 0.05 3.73 ± 0.03 0.01± 0.01 0.07 ± 0.03 5.74 ± 0.04 0.02 ± 0.02

VALUES ARE MEAN ± SEM, n = 3

,

4.5 ANALYTICAL STANDARDS OF THE PLANT SPECIES USED FOR STUDY. The analytical standards of the leaves of the five medicinal plants from experimental site and control site showing the

percentage composition of the total ash, water soluble ash, sulphated ash, acid insoluble ash, alcohol soluble extractive value,

water soluble extractive value and moisture contents is presented in Table 4.

Table 4: Result of analytical standards of the five medicinal plants SN SITE SITE Alchohol soluble Water solublse Moisture Total ash Acid insoluble Sulphated Water soluble extractive value extractive valves content ash ash ash extracts 1 Vitex Experimental 1.5.9 ± 0.01 1.32 ± 0.04 3.09 ± 0.05 4.30 ± 0.03 7.10 ± 0.03 3.05 ± 0.04 2.66 ± 0.04 Doniana Control 1.3.9 ± 0.01 1.26 ± 0.02 3.05 ± 0.01 3.15 ± 0.03 7.10 ± 0 .04 3.05 ± 0.06 2.65 ± 0.03 2 Cnestis Experimental 1.24 ± 0.04 1.08 ± 0.03 6.80 ± 0.02 4.80 ± 0.07 8.80 ± 0.08 4.85 ± 0.03 5.50 ± 0.02 ferruginea Control 1.22 ± 0.02 1.06 ± .02 3.80 ± 0.04 3.80 ± 0.06 5.90 ± 0.06 4.50 ± 0.04 1.30 ± 0.04 3 Anthocleista Experimental 1.90 ± 0.02 1.02 ± 0.03 7.90 ± 0.04 4.55 ± 0.04 6.80 ± 0.05 4.80 ± 0.06 2.65 ± 0.03 Djalonensis Control 1.90 ± 0.03 0.80 ± 0.02 7.90 ± 0.03 3.85 ± 0.02 5.05 ± 0.04 3.90 ± 0.04 2.05 ± 0.02 4 Ricinus Experimental 1.96 ± 0.01 1.04 ± O.03 7.40 ± 0.02 4.45 ± 0.02 8.65 ± 0.02 5.15 ± 0.03 2.80 ± 0.03 communis Ccontrol 1.02 ± 0.03 1.06 ± 0.04 6.90 ± 0.04 4.05 ± 0.05 8.20 ± 0.05 4.20 ± 0.04 2.50 ± 0.04 5 Manihot Experimental 1.88 ± 0.04 1.26 ± 0.02 6.55 ± 0.02 4.30 ± 0.02 7.50 ± 0.03 4.20 ± 0.03 3.50 ± 0.05 esculenta Ccontrol 1.56 ± 0.04 1.20 ± 0.07 3.90 ± 0.03 3.80 ± 0.04 6.85 ± 0.06 3.504 ± 1.85 ± .04 0.04 Values are mean ± SEM , n= 3 4.6 ELEMENTAL ANALYSIS

Elemental analysis revealed the level of heavy mineral contents of the plant species around the quarry environment. All the mineral contents level shown by the plants are within daily maximum permissible level (MPL), except a slight increase in Arsenic and Cadmium in one or two plants as shown in Table 5. Table 5: Result of elemental analysis (ppm) of the medicnal in plant material.

S/N SAMPLE Site Lead Nickle Cadmium Zinc Arsenic 1 Vitex doniana Experimental 2.75 ± 0.02 0.43 ± 0.10 2.25 ± 0.01 0.15 ± 0.11 0.1 ± 0.01

Control 0.00 ± 0.00 0.23 ± 0.01 0.00 ± 0.00 0.08 ±0.12 0.02 ± 0.06

2 Cnestis ferruginea Experimental 3.45 ± 0.01 0.32 ± 0.01 14.30 ± 0.01 0.3 ± 0.01 0.1 ± 0.02

Control 0.0 0 ± 0.00 0.13 ± 0.02 12.00 ± 0.03 0.07 ± 0.01 0.05 ± 0.02 3 Anthocleista Experimental 7.59 ± 0.01 0.5 ± 0.01 24.3 ± 0.01 0.16 ± 0.02 0.3 ± 0.01 djalonensis Control 1.38 ± 0.03 0.18 ± 0.02 5.01 ± 0.02 0.10 ± 0.01 0.2 ± 0.02 4 Ricinus communis Experimental 14.13 ± 0.02 0.27 ± 0.01 10.02 ± 0.00 0.11 ± 0.02 0.7 ± 0.04

Ccontrol 5.52 ± 0.01 0.21 ± 0.04 8.27 ± 0.02 0.06 ± 0.03 0.2 ± 0.03

5 Manihot esculenta Experimental 13.45 ± 0.02 0.54 ± 0.01 7.26 ± 0.03 1.58 ± 0.01 0.1.± 0.01

Control 1.03 ± 0.03 0.18 ± 0.00 4.75 ± 0.02 0.07 ± 0.01 0.1 ± 0.02

MPL 50ppm 110ppm 10ppm 100ppm 0.5ppm Values shown are mean ± SEM , n = 3

Key : MPL = Maximum daily permissible dose

TABLE:6 The Relative water contents (Rwc) of the Leaves of the medicinal plants. S/N Sample Site Fresh Dry Turgid (RWC%) Weight(g) Weight(g) Weight w − w 2 3 × 100 W2-w1 W2-w3 W3 (g) w − w 1 2 1

1 Vitex doniana Experimental 1.43 ± 0.04 1.1 2± 0.01 8.50 ± 0.05 78.57

Control 2.66 ± 0.03 2.21 ± 0.03 8.63 ± 0.04 80.60 2 Cnestis ferrugenia Experimental 0.92 ± 0.03 0.43 ± 0.02 10.00 ± 0.03 44.44

Control 0.80 ± 0.04 0.42 ± 0.02 9.94 ± 0.02 50.0 3 Anthocleista Experimental 2.75 ± 0.02 1.73 ± 0.03 10.33 ± 0.01 80.64

djalonensis Control 3.12 ± 0.01 2.52 ± 0.01 10.05 ± 0.03 80.95 4 Ricinus communis Experimental 2.51 ± 0.01 2.40 ± 0.02 9.20 ± 0.02 80.64

Control 2.5 3 ± 0.05 2.50 ± 0.03 10.52 ± 0.01 96.00 5 Manihot esculenta Experimental 1.52 ± 0.04 0.83 ± 0.02 11.2 2 ± 0.01 53.33

Control 1.93 ± 0.02 0.9 1 ± 0.01 10.53 ± 0.04 69.2

Values are mean ± SEM , n = 3

Table 7: The chlorophyll contents of samples of the medicinal plants

S/N SAMPLE Site TOTAL CHLOROPHYLL (mg/ml) 1 Vitex doniana Experimental 10.12 ± 0.01 Control 11.13 ± 0.02 2 Cnestis ferruginea Experimental 10.06 ± 0.006

Control 10.20 ± 0.06 3 Anthocleista djalonensis Experimental 10.05 ± 0.03 Control 11.1 ± 0.01 4 Ricinus communis Experimental 8.14 ± 0.01 Control 10.18 ± 0.03 5 Manihot esculenta Experimental 10.02 ± 0.017

Control 12.66 ± 0.06

Values are mean ±SEM, n = 3

Table 8: pH of Leaf Extract of Leaves contents of samples of the medicinal plants S/N SAMPLE SITE PH 1 Vitex doniana Experimental 5.58 ± 0.02

Control 5.41 ± 0.01 2 Cnestis ferruginea Experimental 5.41 ± 0.01

Control 6.15 ± 0.01 3 Anthocleista djalonensis Experimental 5.47± 0.04

Control 6.09 ± 0.01 4 Ricinus communis Experimental 3.53 ± 0.02

Control 5.54 ± 0.03 5 Manihot esculenta Experimental 4.5 ± 0.03 6.69 ± 0.01

Values are mean ± SEM, n=3 Table 9: Ascorbic acid contents of leaves of the samples of the medicinal plants

S/N SAMPLE Site ASCORBIC ACID (mg/ml) 1 Vitex doniana Experimental 2.02 ± 0.06 Control 1.96± 0.02 2 Cnestis ferruginea Experimental 2.05 ± 0.02 Control 1.96 ± 0.02 3 Anthocleista djalonensis Experimental 2.06 ± 0.04 Control 1.09 ± 0.01 4 Ricinus communis Experimental 4.10 ± 0.02 Control 2.01 ± 0.02 5 Manihot esculenta Experimental 2.26 ± 0.01 Control 1.69 ± 0.01 Values are mean ± SEM , n= 3

Table 10: The Air pollution tolerance indices of the medicinal plants S/N Species Site Relative Total pH Ascorbic APTI water Chlorophyll values acid contents contents contents (%) 1 Vitex doniana Experimental 78.57 10.12 5.58 2.02 11.03

Control 84.62 11.13 6.19 1.96 11.86 2 Cnestis ferruginea Experimental 44.44 10.06 5.41 2.05 7.62 Control 50.60 10.20 6.15 1.96 8.21 3 Anthocleista Experimental 80.65 11.10 6.09 1.90 9.94 djalonensis Control 80.95 10.05 5.45 2.06 11.86 4 Ricinus communis Experimental 84.00 10.18 5.54 4.10 11.55 Control 96.00 8.14 3.53 1.69 14.39 5 Manihot esculenta Experimental 53.33 12.66 6.69 2.01 8.60 Control 69.20 10.02 4.54 2.26 10.21

Values are mean ± SEM, n= 3

CHAPTER FIVE

DISCUSSION AND CONCLUSION

5.1 DISCUSSION

Quarrying is a recognized polluting activity affecting the environment but plants are the most affected due to their relative static nature. This study examined the effects of quarrying activities on the plants metal load and Air pollution tolerance indices. Results showed significant changes in some physiological and biochemical properties, from where the APTI was calculated.

These changes affected the values of APTI from test to control. The metal loads ranges from 0.1 to 24.3, with R. communis (14.13ppm) being mostly affected followed by M. es culent a (13.45ppm), A. djalonensis (7.58ppm), C. ferruginea

(3.45ppm) and V. doniana (2.75ppm), in the test samples, while in control samples, lead was detected only in R.communis (5.52ppm), A. djalonensis

(1.38ppm) and M.esculenta (1.03ppm). Though the concentration of lead in both test and control samples were within the maximum daily permissible level

( Jankiewiez et al., 2001). The relatively higher value in the test samples suggested quarry activities as causative factors since lead is associated with oxidative stress in plants as a result of its role in the elicitation of antioxidants from plants. The ascorbic acid content, a stress marker, was correspondingly higher in the plants with higher lead contents (Enete et al.,2013 ). R. communis with lead content of 13.79 recorded ascobic acid content of 4.10. M.esculenta with lead contents of 13.45 recorded ascorbic acid content of 2.27. A. djalonensis with lead content of 7.59 recorded ascorbic acid content of 2.06.

Ascorbic acid is a multiplication factor in the computation of APTI, and so the

APTI values were correspondingly higher in R. communis . The MPL of lead was given as 50.0ppm (Jankiewiez, 2000). Nickle contents in all the plants are disproportionately very low in both experimental and control site, ranging from

0.1 to 0. 5 ppm. From the experimental site, M. esculenta produced the maximum concentration (0.54ppm) followed by A. djalonensis (0.513ppm), V. doniana (0.430ppm), C. ferruginea (0.32ppm) and R. communis (0.27ppm).

When this result is compared with the concentration of test is higher than the

Control though both are within the maximum permissible level. Despite their concentration this has effect on the internal structures and external features of the plants. The control ranges from V. doniana (0.23ppm) followed by R. communis (0.21ppm), A. djalonensis (0.18ppm), M. esculenta (0.18ppm) and C. ferruginea (0.13ppm).The MPL of zinc is 110 (Pendias and Pendias,1992) .

From Figure 19(A iv ), highest concentration of Zn was found in Manihot esculenta (1.58ppm), followed by Cnestis ferruginea (0.3ppm), A.djalonensis

(0.10ppm), V. doniana (0.08ppm) and R. communis (0.06ppm), these are the values of the concentration of Zn gotten from experimental site but at the control site, the highest concentration was found in A. djalonensis (0.16ppm), followed by V. doniana (0.15ppm), R. communis (0.11ppm), M. esculenta

(0.07ppm) and C. ferruginea (0.07ppm). Higher content of Zinc in

M.esculenta explians its low value of APTI, which shows its sensitivity to quarry environment. This observation, may be the cause of its poor stomata occurrence both at the upper and lower epidermis of the plants. Their is yellowing leaf veination of most M. esculenta located around quarry site. The estimated safe and adequate daily intake of Zinc is known as (100ppm) (Jones,

1987). Though the load of Zn found present in all Species and concentrations were within their respective MPL intake levels but its effect on the leaves remain paramount. Plants from experimental site showed reduced leaf area, yellowing leaf, reduced stalk, reduced leaf number, rough and thick leaf surface when compared to control plants, may be due to exposure of medicinal plants to pollutants which result in changes in foliar anatomy and plant morphology, caused by visible injury (Alaimo et al , 1980).

Caduim was also present in all the plant Species from experimenal site and its concentration ranged from 14.3ppm in A. djalonensis to 7.26ppm in

M.esculenta . It has highest concentration on A. djalonensis (14.3ppm) that is higher than MPL. Cadnuim was disproportionately high in few plants, however constitutes only about 70 % of the test plants, but the minority (30 %) had cadmium content within acceptable limits. The MPL of Cadmium is

(10ppm)(Annan, et al., 2010). The values produced from control site ranged from 8.25 to 4.75ppm and not detected in V.doniana.

The (MPL) of Arsenic is 0.5ppm ( Shad, et al., 2008), but in this study, test plants showed Arsenic content ranging from 0.7 to 0.1ppm where the high content of 0.7ppm was only found in Ricinus communis . The values produced from control site ranged from 0.2 - 0.02ppm which is lower than the values gotten from Test samples. C.ferruginea from Test site gave good example of the features leaves covered by quarry particulates, this particulates covers the surface area of the leaf, causing poor leaf expiration there by leading to occlusions, thus leading to reduced photosynthesis (Manning, 1971). This agrees with the findings of other researchers that pollutants can cause leaf injury, stomata damage, premature senescence, decreased photosynthetic activity, disturbed membrane permeability, reduced growth and poor yield in sensitive plants Species (Tiwari, et al., 2006).

Some V. doniana from experimental site has spots on the leaves which is as a result of fungai, bacteria, and insects that always occurs with lime dust. Also the texture of the leaves from site is thougher than one from control site. C. feruginea showed the presence of lime stone dust on the surface area of the leaves that even covers the vienation of the leaves.The leaf as well showed yellowing colouration due to pore photosynthesis because, the leaf area was covered with dust for a long time. A. djalonensis from experimental site has smaller surface area, less greener leave colouration and also reduced length of stalk . R. communis from experimental site has reduced area, bleached patches, and M. esculenta has yellowing of the leave veination, tough texture of the leaves and insects bites.

The result of foliar examination gave detail of the macroscopic features of the plant leaf and anatomical properties of leaf from experimental site. This revealed visible injuries and changes in external structures of the leaf, stress features on their leaf area (size), leaf length, stock length and leaf color, poor leave expansion. This could be due to poor air quality in environment, stress from water loss and changes in air chemistry which affects plants functionality.

Microscopic examination of A. djalonensis from control showed multiple stomata occurrence, smooth wax, big pores that are not swollen when compared with the Test samples, both at the upper and lower epidermis. This could be on the process of the plant trying to control the absorption of metal load (pollutants) and much aspiration of water contents. This will limit photosynthesis at the same time by narrowing of stomata pores. M. esculenta did not show recognized stomata features present both at the upper and lower epidermis from experimental sites but the epithelia cell wall of the test plants were damaged. This suggests its sensitivity to the environment as the APTI values (8.60) is second to the least value. R. Communis did not show difference at the upper and lower epidermis when compared to the control, this could be due to the its tolerant to the environment.

C. ferruginea revealed few stomata opening and closed pores at both upper and lower epidermis when compared with control this is also seen in features of the leaves . V. doniana also proved tolerant to the environment because it has more stomata occurrence density and pores but rough epicellular wax when compared with control. The efficiency of stomatal opening for carbon dioxide uptake and water transpiration is not only determined by the size of opening but also by the number of stomata because more stomata can take up more Carbon dioxide and transpire more (Fordham, et al ., 2001).

The phytochemical analysis revealed the presence of organic substances which occur naturally in plants extracts that include tannins, Flavoid, alkaliods, saponins, steroids, carbohydrates, and resins. The relative abundances of

Phytochemical analysis is illustrated under , this reveals an increase in phytochemical constituents of test sample than the control which may be as a result of water stresss that leads to hydration in leaf matrix and these increase the secondary metabolites in plant tissue and may cause oxidative stress due to the formation of reactive oxygen species and photo inhibitory damage

(Zoyayed, et al,. 2003). As seen in the APTI indices of ascorbic acid, the values are two times what is seen in control sample.

The Phytochemical constituents of test plants and control which were also studied by quantifying them revealed more prominent presence of organic compound in test plants than control.This might be due to high dust collecting capacity which may be one of the reasons for sensitive plant species showing reduction or different in biochemical and physiological parameters.The extractive values showed variety in values but higher values are revealed in Test plants when compared with the control.This might be the presence of inorganic compounds accumulated by leaf area of the test plants and may also be due to water stress in stressed environment which increase the secondary metabolites concentration in plants tissue and oxidative stressed due to formation of reactive oxygen species and photoinhibitory damage ( Zoyayed et al ., 2003). For the fact that medicinal plants studied were sensitive to the environment, the need to check for the presence and concentration of cursative agents that leads this pollution is important we have stated earlier.

The analyzed value of the four biochemical parameters used to calculate APTI of medicinal plant was worked on in this study.The relative water content of plants Species studied from control site is higher than the polluted area generally. The relative water contents (RWC) of a leaf which shows the presence of water in its relative full turgidity in the plant is associated with cell protoplasmic permeability which showed that plants gotten from unpolluted environment retains water contents than those collected from polluted zone

(Simone and lucia.,1999). V. doniana from site reads 78.57 % while control is

84.6 % , followed by C. ferruginea control which gave 50.0 % but the test showed 44.4 %. A. djalonensis control gave 80.94 %, test 80.64 %, R. communis control reads 96.00 % while test is 80.64 %, and M. es culenta 53.33

% and 69.2 % both for test and control respectively. Among the Test plants

Species studied, R. communis (80.64 %) and A. djalonensis (80.64 %) showed higher level of water content, followed by V.doniana (80.60%), M. esculenta

(53.35%), and C. ferruginea (44.44%). Low relative water contents of a leaf is associated with the poor protoplasmic permeability in cell, which causes loss of water and dissolved nutrients, resulting in early senescense of leaves (Agrawal and tiwari 1997).This irregular occurrence in plant tissues reduces plants medicinal potency. The total chlorophyll contents of the selected plants Species from quarry and non quarry were worked on.This chlorophyll contents of a plants, signifies its photosynthetic activity as well as the growth and developments of biomass of plant. It is evident that chlorophyll contents of plants varies with the pollution level as well as with biotic and abiotic condition

(Katiyar and Dubey,2001). The total chlorophyll contents of selected plants

Species from quarry site revealed that V. doniana contian 10.12mg/ml and

11.3mg/ml, C. ferruginea 10.06mg/ml and 10.20mg, A. djalonensis

10.05mg/ml and 11.10mg/ml, R. communis 8.14mg/ml and 10.18mg/ml, then finally M. esculenta 10.02mg/ml, 12.66mg/ml, as Test and Control respectively.

V. doniana showed higher contents of chlorophyll (11.12mg/ml) among the Test plants, followed by C. ferrruginea (10.06mg/ml), also by A. djalonensis {10.05mg/ml},then M. esculenta (10.02mg/ml) and R. communis (8.14mg/ml). Regarding the study area, Chlorophyll contents was highest in Vitex doniana collected from control site and least in Ricinus communis collected from experimental sight. This chlorophyll contents is low where their is poor photosynyhetic ability due to settling of particulates (metals) from quarry on the surface area of the plants leaf. This varies with the tolerance as well as sensitivity of the plants, the higher the sensitivity nature of the plants, the lower the chlorophyll contents and the higher the APTI value of a plant is the more tolerance it is. Degrading of photosynthensis pigment has been widely used as an indication of air pollution (Ninave et al 2001 ). Low content of the chlorophyll always occur in plants found in polluted environment because, the affected plants tries to protect the plant from too much loss of water due to harshness of the environment. This protective measure of the leave at the same time closes their stomata pores that is regarded as the nose of the plants and thereby reducing the chances of photosynthesis to occur which indirectly affects chlorophyll occurrence.

The leaf pH of selected plants Species for different sites are stated in

Table (8). A reduction in leaf pH was observed among the plants studies with respect to the control site. Plants from experimental site, showed lower values level of leaf pH. V. doniana gave (5.58) followed by A. djalonensis (5.47 ) then C. ferruginea (5.41), M. esculenta (4.54) and R. communis (3.53), when compared to the values produced from control site. Plants sensitivity to polluted environments always show lower or decline result of pH. In plants any decline in pH value occurs when there is shift in cell sap pH towards the acidic side in the presence of acidic pollutant and this might decrease the efficiency of conversion of hexo sugar to ascorbic acid thereby reducing the ascorbic acid medicinal potency. In this study, plant leaves pH worked on gave the following result as control: V. doniana (6.19,) , C. ferruginea (6.15,) A. djalonensis

(6.09,). R. communis (5.54, ). M. esculenta (6.69, ). The pH of the medicinal plants studied is affected by air pollution when compared with control. This might be due to the interaction of heavy metals with rain water that result in mixture and solutions with varying pH, depending on the pollutants emitted. Ascorbic acid is pH dependent, being more at higher pH and lesser at lower pH, hence the leaf extracts pH on the higher ( ie above 6 upwards) side gives tolerance to plant against pollution, but reveals plants senstivity to Air pollution at lower values (5 - 0 ). In the selected plant from quarry, there are increasing trend of ascorbic acid concentration when compared to the control area (Tripathi and Guatam 2007). These plants species studied showed V. doniana

(2.02,1.96mg/ml), C.ferruginea (2.05,1.96mg/ml,) A.janolensis ,

(2.06,1.906mg/ml), R. communis (4.10,2.01mg/ml) and M. esculenta ,

(2.26,1.69mg/ml) as the Test and control respectively. Present study showed elevation in the concentration of ascorbic acid with respect to the Test site, in all medicinal plants selected.

Ascorbic acid is a reducing agent and plays a role in the cell wall synthesis , defence and cell division (Conklin, 2010). Pollution load leads to increase in ascorbic acid contents of all the plants species may be due to the increased rate of production of reactive oxygen species during photo-oxidation of So 2 to so 3 where sulphites are generated from So 2 absorbed, so it is as a result of its tolerant against sulphur dioxide pollution. This shows the presence of air pollution in that environment.

The result of Air Pollution Tolerance Indices (APTI) calculated for both test and control samples of medicinal plants studied are depicted in table (10).

R. communis among the test plant exhibited the highest APTI value of about

(11.55, 14.39) followed by A. djalonensis (9.94, 11.29), V. doniana (11.03, 11.86), M. esculenta (8.60, 10.21) and C. ferruginea (7.62, 8.21) for Test and

Control respectively. The most sensitive medicinal plants among the one studied in APTI are M. esculenta and C. ferruginea, they are also impilicated in stomatal occurrence, erosion of the epithenial wax and destruction of epithenial wax. Frequency of epithenial cells existed low in their test plants than control.

The plants with lower APTI values are more sensitive to air pollution while those with higher APTI values are said to be tolerant. Therefore, in the present studies among the medicinal plants, R. communis was found to be more tolerant to particulate pollution because its highest APTI value and C. ferruginea was susceptible to the same environment because of its lowest APTI value. High dust collecting capacity may be one of the reasons for the sensitivity of C. ferruginea studied to become highly susceptible to the metallic and non metallic pollutants, making reduction or increase of different biochemical and physiological parameters (Singh,2005). Based on the tolerance nature, these medicinal plants studied can be arranged in this order as R. communis > A. djanolensis > V. doniana >M. esculenta > C. ferruginea . C. Ferruginea and M. esculenta. C. Ferruginea and M. esculenta falls to the category of sensitive plants because its value is ≤ 10 and V. doniana , A. djalonensis and R. communis falls to inter- mediate because their Values are >10 and <16.

5.2 CONCLUSION

Medicinal plants growing around quarry site at Ishi Agu Ebonyi state are affected by lime stone dust that lead to air pollution. This was noticed by change in morphological features of the plants, increased values of ascorbic acid contents, poor relative water contents, acidic pH, poor photosynthetic ability, and presence of some heavy metal above daily maximum permissible level

(MPL). Bassed on these findings, herb dealers, traditional medicine practitioners and the general public are advised not to cultivate or collect medicinal plants for treatment of any ailment within the quarry sites and environs as long as quarry activities are in progress. Medicinal plants should therefore be collected from the non-polluted area.

RECOMENDATION

Considering the outcome of this research, the following solution is required for its full compliance and implementation:

There is a need to sensitize the ethnomedical practitioners on the necessity of cultivating medicinal plants out side industrial areas in order to minimize effect of pollution which may denature the medicinal plants and possibly affect their therapeutic efficacy.

Plants that have high APTI values are to be cultivated around quarries as phytoremediators of environmental polluted sites, and may not be very useful in preparation of herbal formulation due to the possibility of attenuation of their phytopotency from pollutants.Herbalists should be advised to avoid using plants from the quarry sites, as their plant stand the risk of contamination with heavy metals, particulate matter and gaseous particles. REFERENCE

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APPENDIX I

60

50

40

30 test 20 control Standard

Concentration Leadof in PPM 10

0 Vitex doniala Cnestis Anthocliesta Recinus Manihot Vitex ferrugineaCnestis djalonensisAnthocleista communis Ricinus esculentaManihot djalonensis communis esculenta doniana ferruginea Plant Species

Fig 16: The concentration of lead contents in the medicinal plants studied

APPENDIX II

0,6

0,5

0,4

0,3 test control 0,2

Concentration Nickleof in ppm 0,1

0 Vitex doniala Cnestis Anthocliesta Recinus Manihot Vitex doniana Cnestis Anthocleista Ricinus Manihot ferruginea djalonensis communis esculenta ferruginea djalonensis communis esculenta Fig 17: Plant Species

Fig 17: The concentration of Nickel contents in the medicinal plants studied. (standard= 110ppm)

APPENDIX III

30

25

20

15 test control 10 standard Concentrationof Cadnium in ppm

5

0 VitexVitex doniana doniala Cnestis AnthocliestaAnthocleista RecinusRicinus ManihotManihot ferrugineaferruginea djalonensisdjalonensis communiscommunis esculentaesculenta Plants Species

Fig 18: The concentration of cadmium contents in the medicinal plants studied

APPENDIX IV

1,8

1,6

1,4

1,2

1

0,8 test control 0,6 Concentration Zincof in ppm 0,4

0,2

0 Vitex doniala Cnestis Anthocliesta Recinus Manihot Vitex doniana Cnestis Anthocleista Ricinus Manihot ferruginea djalonensis communis esculenta ferruginea djalonensis communis esculenta Plant Species

Fig 19: The concentration of zinc contents in the medicinal plants studied( standard=100ppm)

APPENDIX V

0,8

0,7

0,6

0,5

0,4 test control 0,3 standard

Concentrationof Arsenic inppm 0,2

0,1

0

V.Vitex doniana doliala CenestisCnestis AnthocliestaAnthocteista RecinusRicinus ManihotManihot ferruginea djalonensis communis esculenta ferruginea djalonensis communis esculenta Plants Species

Fig 20: The concentration of arsenic contents in the medicinal plants studied

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