Biochemical analysis of selected medicinal plants for value addition in functional food [307606]

“LUCIAN BLAGA” [anonimizat], [anonimizat] / Comisia de evaluare a tezei de doctorat:

[anonimizat], have been used in virtually all cultures as a source of medicine and energy. [anonimizat]. [anonimizat], nutritional, industrial and medicinal properties. It is high value economical and industrial crop. The second focused plant of our research is Plantago ovata L. locally known as Isabgol belonging to the family Plantaginaceae is a [anonimizat], and it is now widely used for its medicinal properties all over the world. Seed husk (rosy-white membranous covering of the seed) traditionally given as a [anonimizat], chronic diarrhea and dysentery.

[anonimizat]. Therefore, in this work we focused to find non conventional source of nutrients by screening and comparative assessment for possible enrichment of functional food to tackle health related problems of daily life. [anonimizat]’s response about utilization of selected medicinal plants. The values of moisture contents for Psyllium seeds from Romania and Psyllium seeds from Pakistan were found as 7.8% and 7.7% whereas percentage of moisture contents for Sweet basil seeds from Romania and Sweet basil seeds from Pakistan were found as 9.7% and 7.0% respectively by using thermo moisture analyzer. The data indicated that seeds of Psyllium from Romania contained low ash content (2.001%) as compared to the seeds of Psyllium from Pakistan (2.41%). Whereas ash contents in the seeds of Sweet basil showed the highest values (7.26 %) as compared to seeds of Sweet basil from Pakistan (2.34%). [anonimizat] (ash content %) [anonimizat] (6.5 %) as compared to psyllium seeds (3.4 %).

From the findings of this study it was observed that Psyllium seeds from Romania contained reasonable amount of fiber content (10.47 %) as compared to Psyllium seeds from Pakistan (9.35 %) whereas sweet basil seeds from Romania contained lowest amount of fiber content (5.88 %) as compared to other ecotype seeds of sweet basil from Pakistan (6.43 %). [anonimizat] (3.2 %) as compared to seeds of sweet basil (2.1%) as in a percentage quantity (%). The results showed that Sweet basil seeds contained high protein contents (29%) as compared to Psyllium seeds (24.5%). These findings suggested that sweet basil seeds with rich source of protein contents might be beneficial as nutritional point of view as compared to seeds of Psyllium. Based on the observations of current study for estimation of fats contents (Lipids), Sweet basil seeds contained low fat contents (15.36%) in comparison with Psyllium seeds (16.55%) but there was not great difference in the values of fat contents in both selected seeds of Sweet basil and Psyllium. The carbohydrate content of analyzed samples suggested that the seeds of Psyllium have the highest amount of carbohydrates (47.19%) as compared to seeds of Sweet basil (42.5%).

We concluded from these observatons, psyllium seeds had highest energy values might be due high percentage of fibers, fats and carbohydrates as compared to sweet basil seeds (424. 24 kcal) that had only protein contents in higher quantity in comparison with psyllium seeds (435.71k cal). Finally, According to the findings of surveyed analysis we concluded that people consumed these plants only based on traditional knowledge and reluctant to used them more frequently. Most of the surveyed people used Psyllium as a source of fibers to cure the digestive problems whereas sweet basil seeds used by the people only for seasonal recreational purposes without knowing its specific worth of efficacy. On the other hand, majority of respondent preferred to use whole grains as compared to refined grains. From these results we found clear idea for novel food product preparations in according to the needs of consumers. Furthermore, bread preparation with psyllium addition and refreshing drink with utilization of sweet basil seeds were our final findings of current study. From the findings of present study we concluded that bread with 3% psyllium husk has more characteristic properties as compared to less amount of psyllium addition 1%. On the other hand drink with 5% basil seeds has acquired thick mucilage as source of fiber.

ACKNOWLEDGMENT

I express my gratitude for Romania Government scholarship and “Lucian Blaga” University of Sibiu, Romania for supporting this research work and facilitating me with all that I needed to complete this research task.

I express thankfulness to my affectionate scientific coordinator Prof. Univ. Dr. Engr, Ovidiu TITA for his keen interest in this work and superb guidance. This work is the fruit of his consistent motivation, guidance, and valuable suggestions. He has always been very helpful in all concerned matters of course work and research.

I am grateful to Dr. Ayub Kholon, Director of Plant Pathology Research institute, Faisalabad, Pakistan, for his support and cooperation to my experimental work in Laboratory. I express my cordial gratefulness to Dr. Daniela Preda, the EMMA coordinator “Lucian Blaga” University of Sibiu, Romania, and Professor Camelia sava for their consistent moral support and encouragement.

Heartfelt and sincerest gratitude must go to Dr. Mihaela Antofie, for her valuable suggestions and guidance. I am thankful to my colleague Roxana for her excellent company and guidance. I also acknowledge the contribution of all those volunteer participants who helped me evaluate this work.

Mental and spiritual supports are the most valuable things for me and for this I am thankful to all my teachers, friends especially Zobia Rehman and Zoya maan. Finally, my deepest dedication is to my beloved Father, mother, brothers especially Mohsin Khalil & sisters (Saira Khaliq and Aisha sadaf) and especially my husband Moazzam Ali for their unconditional love, infinite patience, deep encouragement and every sort of support along my lifetime.

Rehana Khaliq

Sibiu, July, 2017

TABLE OF CONTENTS

TABLE OF CONTENTS vi

LIST OF FIGURES ix

LIST OF TABLES xi

LIST OF ABBREVIATIONS xii

CHAPTER 1 1

1. INTRODUCTION 1

1.1 Background 1

1.2 Problem statement 2

1.3 Scope of the study 3

1.4 Research Objectives 4

1.5 Why Ocimum basilicum and Plantago ovata 5

1.6 Innovative elements of Research 5

1.7 Assumptions 6

1.8 Overview of Proposed Methodology 6

1.9 Thesis Structure 9

CHAPTER 2 11

2 MEDICINAL PLANTS RESEARCH IN PAKISTAN 11

2.1 Geography of Pakistan 11

2.2 Trade, collection and conservation of medicinal plants 12

2.3 SELECTED MEDICINAL PLANTS FOR CURRENT STUDY 14

2.3.1 Ocimum basilicum 14

2.3.2 Plantago ovata 17

2.4 BIOACTIVE COMPOUNDS AND EXTRACTION TECHNIQUES 23

2.4.1 Total Proteins 23

2.4.2 Phenolic Compounds 23

2.4.3 Total soluble Fibers 23

2.4.4 Essential oils 24

2.4.5 Lipids (Fats) 24

2.4.6 Total carbohydrates 24

2.4.7 Extraction Techniques 25

2.4.8 Conventional methods of extraction 25

2.4.9 Non-conventional methods of extraction 26

2.5 FUNCTIONAL FOOD AND SOLUBLE FIBERS 28

2.5.1 Introduction 28

2.5.2 Concept of functional foods 28

2.5.3 History of uses 29

2.6 Impacts on human health with scientific evidences 30

2.7 Plants as a source of functional foods 31

2.7.1 Garlic (Allium sativum) 32

2.7.2 Tomatoes (Lycopersicon esculentum) 33

2.7.3 Others 33

2.8 Safety Issues and considerations 34

2.9 Soluble fibers 35

2.10 Conclusion 35

CHAPTER 3 36

3 PROPOSED METHODOLOGY OF RESEARCH 36

3.1 Proximate Analysis of selected medicinal plants 36

3.2 Determination of morphological characteristics of seeds by Digital microscope 36

3.3 Determination of moisture content by Oven dry method 37

3.4 Determination of moisture content by Thermo moisture analyzer 38

3.5 Determination of Ash content of selected medicinal plants 38

3.5.1 Determination of total mineral matter (Ash) 38

3.6 Determination of crude fiber content of selected medicinal plants 39

3.6.1 Principle: 39

3.6.2 Procedure: 40

3.6.3 Determination of crude fiber from seeds 40

3.7 Determination of Protein of selected medicinal plants 42

3.7.1 Principle: 42

3.7.2 Reagents: 42

3.7.3 Description of the Kjeldahl Method 43

3.7.4 Methodology of analysis 44

3.8 Determination of Fats of selected medicinal plants 45

3.8.1 Methodology of analysis 45

3.9 Determination of Total carbohydrate 46

3.10 Determination of Total Energy values 46

3.11 Survey analysis of selected medicinal plants for consumer response 46

3.12 Novel Product preparation by using mucilage from seeds of selected medicinal plants 47

3.12.1 Bread preparation 47

3.12.2 Drink preparation 51

CHAPTER 4 54

4 RESULTS AND DISCUSSION 54

4.1 Physical characteristics of seeds of selected medicinal plants 54

4.2 Moisture content by Oven dry method 58

4.3 Moisture content by Thermo moisture analyzer 61

4.4 Ash content of selected medicinal plants 63

4.4.1 Mineral matter of selected medicinal plants 66

4.5 Crude fiber content of selected medicinal plants 66

4.6 Protein of selected medicinal plants 70

4.6.1 Nitrogen free extract evaluation 72

4.7 Fats of selected medicinal plants 72

4.8 Total carbohydrate of selected medicinal plants 73

4.9 Total Energy values of selected medicinal plants analyzed 74

4.10 Conclusion on the basis of nutrient analysis of seeds 76

4.11 Survey results of selected medicinal plants for consumer response 77

4.12 Conclusion on the basis of survey analysis 82

4.13 Novel Product presentation by using seeds of selected medicinal plants 82

4.13.1 Evaluation after Bread preparation 82

4.13.2 Evaluation after Drink preparation 85

4.14 Conclusion on the basis of fibrous food preparation 87

CHAPTER 5 89

5 CONCLUSION, CONTRIBUTIONS AND FUTURE DIRECTIONS 89

5.1 Research overview 90

5.2 Research findings 91

5.3 Research contributions 93

5.3.1 Theoretical Contributions 93

5.3.2 Practical Contributions 94

5.3.3 Scientific Contributions 95

5.4 Future directions 95

6 REFERENCES 96

7 ANNEXES 105

ANNEX 1. Survey Questionnaire 105

ANNEX 2. Mucilage from Psyllium and Sweet basil seeds 107

ANNEX 3. List of Publications 108

ANNEX 5. Curriculum Vitae 110

LIST OF FIGURES

Figure 1.1Proposed model of research work 9

Figure 1.2 Thesis organization and structure 10

Figure 2.1: Plantago ovata native to India 18

Figure 2.2: Psyllium husk, mucilage, tasteless material with health benefits 19

Figure 2.3: Processing of isabgol seeds for marketable product 21

Figure 2.1: The flow chart of medicinal plant study and position of extraction techniques (Source: adapted from (Farnsworth, Akerele, Bingel, Soeja, & Guo, 1985). 27

Figure 3.1 Zeiss axiostar plus microscope 37

Figure 3.2 Oven for ash determination (Controller B170 Nabertherm) 38

Figure 3.3 Oven (ESAC-50a Electronic April) Multifunction 39

Figure 3.4 Bread preparation with different concentration of psyllium husk 48

Figure 3.5 Bread preparation with 0 % addition of psyllium husk 48

Figure 3.6 Bread preparation with 1 % addition of psyllium husk 48

Figure 3.7 Bread preparation with 1.5 % addition of psyllium husk 50

Figure 3.8 Bread preparation with 3 % addition of psyllium husk 50

Figure 3.9 Bread preparation with three fractions of psyllium husk 50

Figure 3.10 Moisture % and weight analysis of bread samples 51

Figure 3.11 Total acidity by titration of bread samples 51

Figure 3.12 Sweet basil seeds and psyllium seeds and husk for refreshing drink preparation 52

Figure 3.13 Refreshing drink preparation with three fractions of basil seeds (0.5%, 2% and 5%) 52

Figure 3.14 Refreshing drink preparation with three fractions of basil seeds (0.5%, 2% and 5%) and combination with Psyllium husk (1.5%, 1% and 0.5%) 52

Figure 3.15 pH determination of refreshing drink with three fractions of basil seeds (0.5%, 2% and 5%) and combination with Psyllium husk (1.5%, 1% and 0.5%) 53

Figure 4.1 Microscopic examination of seeds of Psyllium and Sweet basil 56

Figure 4.2 Size of seeds (length and width) of Sweet basil and Psyllium 57

Figure 4.3 Moisture content (g) of selected medicinal plants 59

Figure 4.4 Moisture content (%) of selected medicinal plants 60

Figure 4.5 moisture content (%) comparison among seeds of selected medicinal plants 60

Figure 4.6 Thermo moisture analyzer 61

Figure 4.7 Sweet basil seeds in moisture analyzer 62

Figure 4.8 Psyllium seeds in moisture analyzer 62

Figure 4.9 Moisture content (%) of selected medicinal plants by thermo moisture analyzer 63

Figure 4.10 Oven for ash determination (Controller B170 Nabertherm) 64

Figure 4.11 Ash content (g) of selected medicinal plants 65

Figure 4.12 Ash content (%) comparison among seeds of selected medicinal plants 65

Figure 4.13 Ash content (%) of selected medicinal plants 66

Figure 4.14 Mineral matter percentage (%) comparison between seeds of Sweet basil and Psyllium 66

Figure 4.15 Crude fiber (g) of selected medicinal plants 67

Figure 4.16 Fiber (%) composition of selected medicinal plants 69

Figure 4.17 Fiber percentage (%) comparison among seeds of selected medicinal plants 69

Figure 4.18 Fiber percentage (%) comparison between seeds of Sweet basil and Psyllium 70

Figure 4.19 Protein percentage (%) comparison between seeds of Sweet basil and Psyllium 71

Figure 4..4.20 Percentage (%) comparison among nutrient contents of selected medicinal plants 72

Figure 4.21 Fats (%) comparison between seeds of Sweet basil and Psyllium 73

Figure 4.22 Total carbohydrate percentage (%) comparison between seeds of Sweet basil and Psyllium 74

Figure 4.23 Total Energy values percentage (%) comparison between seeds of Sweet basil and Psyllium 75

Figure 4.24 Proximate composition (%) of selected medicinal plants 76

Figure 4.25 What is source of traditional knowledge among surveyed respondents? 77

Figure 4.26 Surveyed respondents awareness of fibrous food intake in relation to age factor 78

Figure 4.27 What is preference of fibrous food intake among surveyed respondents? 79

Figure 4.28 Relationship between respondents for what purpose they used seeds of selected medicinal plants 79

Figure 4.29 Frequency (%) distribution of usage of selected medicinal plants among respondents 80

Figure 4.30 Respondents opinion about gardening of selected medicinal plants in home 80

Figure 4.31 Respondent opinion about what type of grains they consumed as a source of fiber 81

Figure 4.32 Moisture % of bread with three fraction of psyllium husk (1%, 1.5% and 3%) 83

Figure 4.33 Total acidity of bread with three fraction of psyllium husk (1%, 1.5% and 3%) 84

Figure 4.34 pH determination of refreshing drinks with three fraction of sweet basil seeds (0.5%, 2% and 5%) and combination with three fractions of psyllium husk (1%, 1.5% and 3%) 86

LIST OF TABLES

Table 2.1: Traditional Knowledge related to Sweet Basil 15

Table 2.2: A brief summary of the experimental conditions for two non-conventional methods of extraction for plants material 26

Table 2.3 Strength of evidence for functional foods currently on the U.S. market* 31

Table 2.4: Phytochemicals Associated with Health Promotion and Disease Prevention 33

Table 4.1 Morphological characteristics of seeds of selected medicinal plants (Sweet basil and Psyllium) 55

Table 4.2 Taxonomy of selected medicinal plants in order to find relationship between both species 58

Table 4.3 Moisture content analysis of seeds of Ocimum basilicum (Basil) and Plantago ovata (psyllium) through oven dry method 59

Table 4.4 Moisture content analysis of seeds of Ocimum basilicum (Basil) and Plantago 62

Table 4.5 Moisture content (%) comparison between seeds of Ocimum basilicum 62

Table 4.6 Ash content analysis of seeds of Ocimum basilicum (Basil) and Plantago ovata (psyllium) through oven dry method 64

Table 4.7 Fiber content analysis of seeds of Ocimum basilicum (Basil) and Plantago ovata (psyllium) 68

Table 4.8 Quality analysis of seeds of Sweet basil and Psyllium 71

Table 4.9 Quality analyses of seeds of Sweet basil and Psyllium 75

Table 4.10 Apparent spoilage (visual observation for mold growth) at room temperature (20-22 OC) 84

Table 4.11 Apparent spoilage (visual observation for mold growth) at refrigerator temperature (4 OC) 85

LIST OF ABBREVIATIONS

AOAC Association of Official Analytical Chemists

A.D Anno Domini

CLA Conjugated Linoleic Acid

FAO Food and Agriculture Organization

FDA Food and Drug Administrations

FOSH Food for Specific Health Use

GAO General Accounting Office

GI Gastrointestinal

GRAS Generally Recognized as Safe

IFT Institute of Food Technologists

LDL Low Density Lipoproteins

TG Triglycerides

WHO World Health Organization

CHAPTER 1

INTRODUCTION

Background

Plants influence our life on earth by providing us basic needs have man ever since from his birth and evolution. With this evolution people identified other plants as a source of food, spices, oil and the selected forage and fodder for the domesticated animals that they have in their houses. Medicinal plants and food crops, since times immemorial, have been used in virtually all cultures as a source of medicine and energy. Plants utilize simple substance such as water, carbon dioxide, nitrogen and other elements in small amounts for synthesis of primary metabolites (carbohydrates, proteins, fats and nucleic acid) which transformed into secondary metabolites (alkaloids, flavonioids, steroids, saponins terpenoids etc) extensively used in drugs preparation. Herbs not only provide us chemicals of medicinal value but also provide us nutrition required by our bodies for numerous biological and physiological processes that are necessary for the maintenance of health . Nowadays, the science of ethnobotany and ethnopharmacognosy are playing immense role for searching new molecules from different sources of plants. The material extracted from natural plants through chemical and biotechnological methods has nutritional value which plays great role in human life to form major portion of the diet (. At present, medicinal plants still play important role to generate income to the people of many developing countries of Asia and Europe such as: Ocimum basilicum and Plantago ovata.

Ocimum basilicum L., commonly known as basil is a foliage rich plant of family Lamiaceae cultivate throughout the world. Basil is an aromatic herb used extensively, from ancient times, to add distinctive aroma and flavor to food. The leaves can be used fresh or dried and the essential oils extracted from fresh leaves and flowers can be used as aroma additives in food, pharmaceuticals, and cosmetics.

Among the plants known for medicinal value, the plants of genus Ocimum are very important for their therapeutic potentials. Because of its popularity basil is often referred to as King of herbs, being widely utilized due to its economic, nutritional, industrial and medicinal properties. It is high value economical and short duration (75–90 days) industrial crop. Now a day, the essential oils have attracted the interest of scientists due to their potential source of natural antioxidants and aroma active compounds. Essential oils of basil exhibit a wide array of chemical compounds, depending on variations in types, leaf and flower, aroma and origin of the plant. Several analytical methods have been developed to determine the volatile compounds from the essential oils present in different spices (. Therefore, there is urgent need for the further analysis of chemical compounds and investigation of traditional knowledge in order to standardize the phytochemicals contents of the plants for safety, seeing that medicinal plants are rich sources of these chemicals, coupled with the fact that they are cheaper and more accessible to the wide population of world.

Plantago ovata L. locally known as Isabgol belonging to the family Plantaginaceae is a stem less Ayurvedic herb, used in health care for many centuries in South Asia, and it is now widely used for its medicinal properties all over the world. Seed husk (rosy-white membranous covering of the seed) mainly given as a safe laxative, particularly beneficial in habitual constipation, chronic diarrhea and dysentery. It is also used in lowering blood cholesterol level, ice cream making and cosmetics (.

In Bulgaria and Romania, leaves of isabgol are used for its antiseptic properties as a folk remedy to prevent infections on the cuts and scratches . It is native to Persia but also widely cultivated throughout Pakistan, Mexico, West Asia, Iran, Mediterranean regions and India which is the largest producer and exporter of isabgol in the world but the productivity of it is far below the desired levels . Therefore, it is necessary to analyze or examine the nutrient content of Psyllium (isabgol) and also promote its commercial cultivation which might be helpful for the wealth of economy as well as provided by the gene pools and then harnessed for crop improvement.

Problem statement

Food is one of the main necessities for human health and mental growth. But the high rate of human population in developing world results in hunger and malnutrition. Deficiency of nutrients is wide spread due to limited resources as well as unawareness of non conventional source of nutrients from economically important but neglected species such as sweet basil and Psyllium. There is need to explore nutritional significant resources to promote health in addition to overcome food related problems of daily life.

Scope of the study

Biochemical analysis of economically important plants such as Ocimum basilicum and Plantago ovata play a crucial role in assessing their nutritional significance. As various medicinal plants species are also used as food supplement, evaluating their nutritional significance can help to understand the worth of these plants. Main goal of my thesis was to explore bioactive compounds of selected medicinal plants. The estimated nutrients could possibly utilize for improvement of functional food and food products and will be good initiative for food industry in future. This work might be helpful for increasing the wealth of medicines along with non conventional source of fibers and fats. By this method of analysis we can successfully contribute to solve food related problems of health in daily life for instance adequate amount of fibers in food can control heart diseases, help to prevent constipation and weight management in women. However little or no work have been done on these selected plants in this respect so far to verify the uses and to determine the nutrient contents of Plantago ovata and Ocimum basilicum from two geographical diverse regions in order to make a comparative analysis of extracted material whose role in improving functional food. . In comparative assessment by using the finding of this study we will be able to make a conclusion which plant and plant parts are rich in nutrition easily available for making healthy food products. It will help to understand that seeds of which plant has valuable source of nutrient and will give potential usefulness as food fiber, proteins, fats and mineral matter. On the other hand, in view of increasing demand of food and energy to support the growing population, this analysis will be an effort to explore new and nonconventional source of food for diverse region of the world. In recent year, consumption of junk food in almost all part of the world has increased manifold which has led to a number of nutritional deficit diseases.

Evaluating nutritional significance of medicinal plants can play an important role in controlling minor ailments and will help to maintain health thus promote the quality of life. As we already know herbs, nutrients and dietary supplement are major contributors of functional food which help to enhance the structure and function of the body. Therefore, in this work we will focus to find non conventional source of nutrients by screening and comparative assessment for possible enrichment of functional food to tackle health related problems of daily life.

Research Objectives

Keeping in mind that Plantago ovata and Ocimum basilicum medicinally important plants are little bit neglected species in this kind of nutritional estimation, it is decided to investigate their nutritional values that might be helpful for increasing the wealth of medicines and enrichment of functional food products. Research on plants species including medicinal, aromatic and staple food plants have been carried out to investigate nutrient constitutes and bioactive compounds providing large portion of daily human food consumption. However, there are little works in literature that concern to determine the worth of these plants together i.e. Plantago ovata and Ocimum basilicum which are native to geographical diverse regions of Asia. The extracted phytochemicals could possibly utilize for improvement of functional food and food products and will be good initiative for food industry in future by introducing novel food products and drinks with special health benefits. This work might be helpful for consumption of mucilage (source of soluble fiber) from seed of basil with non conventional source of fibers and fats. By this research we can successfully contribute to solve minor ailments from seasonal variations for instance adequate amount of basil seeds in water has soothing effects in summer season and basil dried leaves as spices in food can control heart diseases and help to prevent cancer respectively. Furthermore, psyllium husk in bread and yogurt has multiple benefits for gastrointestinal problems in daily life. The finding from the selected medicinal plants will suggest which plant has a promising potential of soluble fibers better maintain a healthy life.

The specific objectives of this research are summarized as follows:

To study the traditional knowledge related to these plants and find effective ways for conservation of this knowledge.

To estimate the Nutrient contents of Plantago ovata and Ocimum basilicum in terms of biochemical analysis under standard methods of investigation.

Components of nutrients of interest in pharmacology as well as for food products are as follow:

Moisture

Ash

Protein

Fats and lipid

Soluble Fiber

Mineral matter

Total carbohydrate

Total energy values

Highlighting the nutritional value of compounds investigated from Plantago ovata and Ocimum basilicum.

To determine the role of biological active nutrients assessed from plant materials in food products.

Undertake a study on the husk and mucilage obtained from the seeds of Plantago ovata and Ocimum basilicum and its consumption as food supplement in daily diet as fiber requirements by innovation of novel healthy food products and drinks from them at preliminary stages on the basis of indigenous knowledge and some sort of quality analysis.

The aim of this study was to obtain an understanding of what is needed to convert uses of medicinal herbs for functional foods. This includes traditional knowledge about selected medicinal plants as well as issues related to cultivation and expectations from the industry regarding quality and safety of such crops. Based on this knowledge, a further aim was to determine how the cultivation practices of the model crops basil and isabgol need to be adapted to meet quality requirements. This would then contribute to a validated production chain of both crops suitable for use as raw material for the extraction of soluble fibers.

In this thesis, an introductory review is presented to provide background knowledge about the cultivation, trade of medicinal plants in general and selected plants particular for industrial purposes. This includes an overview of the use of plants in various industries, as well as trends in the various markets involved.

Why Ocimum basilicum and Plantago ovata

Plants are important for a variety of reasons. Many people use plants as a source of food as well as medicinal purposes. But selected medicinal plants such as Ocimum basilicum and Plantago ovata are little bit neglected species for both collectively therapeutic as well as food supplements. Furthermore, seeds of both plants have same characteristic properties, they become gelatinous and sticky when soaked in water and absorbs water to perform characteristic functioning in terms of mucilage and a true dietary fiber source. Psyllium husk and seeds of sweet basil is considered as mild and natural laxative thereby facilitates digestion. Therefore, there is need to explore such kind of naturally occurring mucilage which are freely available, low cost and non toxic in usage as compared to synthetic materials.

Innovative elements of Research

The proposed research revolves around the nutrients analysis and fiber investigation of selected seeds to assist the consumers for attaining knowledge of non conventional source of fibers in order to guarantee significant high quality nutrients and consumer satisfaction by providing high energy quality food products. This research presents the analysis based means to systematically accumulate traditional knowledge about selected medicinal plants, nutrient analysis and consumer response provides means to better interpret this knowledge, and help to integration of new knowledge to existing one.

The proposed methodology focuses on nutrients content analysis with further elaboration of fiber contents assessment in comparison of Psyllium and Sweet basil seeds used and required in therapeutic and food products preparation and ensures it effectiveness in daily life. A survey analysis is also defined and implemented in this research work which helps to know the clear need of consumers in order to make improvements in existing knowledge for quality products preparations. This work aims to determine the proximate composition of seeds of Psyllium which originally not growing in Romania because of different climatic conditions not appropriate for the productivity of this plants. On the other hand second selected medicinal plant sweet basil more familiar in local community and cultivate in Romania for culinary and traditional purposes. This work can also facilitate the food scientists as well as local community to under the worth of these selected plants in comparison.

Assumptions

The major assumption to conduct this research is that nutrients assessment of selected medicinal plants and consumer response about fibrous food directly related to these seeds. Because traditional knowledge of local community about consumption of these seeds in different modes for fibrous food requirement and for digestive problems in daily life that has been populated in local community with no authentic verification of safety measures. Although traditional knowledge on the basis of nutrient analysis and other analysis of food products would serve more specific and efficient results for enrichment of food industry to make high quality but cheap novel food products with full of energy and nutrients.

Overview of Proposed Methodology

First of all importance of nutrients under research analysis will be discussed for better comprehend the existing knowledge, importance of selected medicinal plants during the analysis, and its impacts on human health and mental growth. Research is going on for morphological characteristics of seeds that can be deployed in scientific community for further elaboration of exiting knowledge. Research on combining proximate analysis and survey analysis on consumer response is also going on.

We will also see how nutrient analysis and fiber contents assessment with two different methods of seeds of both selected medicinal plants are better choice for knowledge accumulation and reutilization for purpose of quality food products. Combination of fibers and functional foods is a current trend in the food industry. Further we will discuss the importance of fiber in daily food consumption.

Quantitative research is most suitable for this kind of project, as it helps in investigating and exploring what percentage of selected biochemicals they have and how they will be available for food products. As this research project is about analysis of nutrients from selected plant extracts, applicable output of these experimental research and finding helpful patterns leading to a improve food products by making strategies to cope with problems related to food deficit of these nutrients as source of non conventional that’s why quantitative methods best fit for this work.

Most of the time biochemical analysis of medicinal plants is done for using them in medicines and pharmaceutical industry that’s why nutritional significance of these plants is neglected. Bioactive compounds such as fat, fibers and proteins from plant materials can be extracted by various classical extraction techniques. Most of these techniques are based on the extracting power of different solvents in use and the application of heat and/or mixing. In order to obtain bioactive compounds from plants, the existing classical techniques are: Soxhlet extraction, (2) Maceration and (3) Hydro-distillation. We are aimed to analyze the moisture, ash content, crude fiber, proteins, fats, total carbohydrate and mineral matter from selected medicinal plants in order to improve current state of knowledge regarding these plants. This study could play major role for further development of food products with health attributes. From the current work we will be able to better comprehend the value addition of fibers in daily food. Research is on going with following analysis:

The moisture content will be determined by oven drying method and also by thermo moisture analyzer (Infrared AND ML.50) at 105°C for 3 h and at 105°C for 47 minutes respectively and both the values will be placed for comparison.

For ash content, crucibles will be washed thoroughly, heat in oven at 100°C and then cool in the desiccators. 2.5 g of each sample will be weighed in the crucible and heat in muffle furnace at 550 șC, until white or grayish white ash will be obtained. After cooling the samples in desiccators, reweigh the samples and percentage of ash will be calculated .

Crude fiber content will determine by treatment of the samples with 1.25% H2SO4, 1.25% NaOH and then 1% HNO3, filtered and wash with hot water after each step. The residue obtain will dry in oven at 105 șC and heated at 600 șC in furnace. The loss in weight on ignition was expressed as content of crude fiber . Other method will also be applied for the estimation of crude fiber in the seeds which is usually applied for the determination of cellulose in the wheat flour. The detail procedure of this method will be discussed in chapter 3 (Proposed Methodology).

Total fats will be extracted from the sample with petroleum ether (60-70 șC) in a Soxhelt apparatus for about 30 minutes. The residual solvent will be evaporated in a pre-weighed beaker and increase in weight of beaker gave total fats. Nitrogen content in the sample will be estimated by using micro Kjeldahl method and crude protein will be calculated by multiplying the evaluated nitrogen by 6.25.

The values of total carbohydrates will be calculated by the difference given by: 100-(percentage of ash + percentage of total fats + percentage of protein + percentage of crude fiber) .

Total energy value will be calculated by multiplying the values of total carbohydrate, lipid and protein by the factors 4, 9 and 4 respectively, taking the sum of the products and expressing the result in kilocalories . Finally consumer’s response will be accessed by querying it via questionnaire. Moreover, this kind of study perhaps will be the most exciting one regarding the medicinal plants seeds collected from Pakistan and Romania for comparative analysis of medicinal values and nutrients.

Figure 1.1Proposed model of research work

Thesis Structure

Chapter 1 explains problem statement, research objectives, and basic elements of the research such as assumptions of current research, reasons behind the selection of analysis and medicinal plants under study and some overview of proposed methodology.

Chapter 2 initially provides some basic information about medicinal plants research in general and selected medicinal plants such as Plantago ovata (Psyllium) and Ocimum basilicum (Sweet basil) more specifically. Later on it describes the importance of selected medicinal with respect to industrial uses. Further in second chapter bioactive compounds are defined along with their essential activities. It has also been discussed how important extraction techniques are and all the benefits that we can get from usability of this conventional and non conventional extraction techniques. In the same chapter basic concepts of functional foods have also been discussed along with its impacts on human health with scientific evidences, light has also been shed on the soluble fiber which are the focus nutrient of our study; and safety issues and considerations have also been discussed.

Chapter 3 discusses proposed methodology in detail. As core of our proposed methodology is the quality analysis of seeds, therefore different nutrients by standard methodologies have been discussed, and on the basis of requirements of this research a survey analysis on consumer response has been proposed. Further in this chapter the morphological characteristics are explained and how this information is integrated to existing knowledge base.

Chapter 4 is about results obtained after evaluating laboratory analysis results and results from respondent of survey. A comparison is carried out between seeds of both selected medicinal plants on the basis of nutrients, energy values they posses and finally fiber contents in detail. Proposed work has also been evaluated for its benefits for the food industry.

Chapter 5 concludes the discussion and sheds light on the contributions of this research and gives some future directions.

Figure 1.2 Thesis organization and structure

CHAPTER 2

MEDICINAL PLANTS RESEARCH IN PAKISTAN

Geography of Pakistan

Pakistan is a developing country of South Asia, has an area of 80,943 km2 (87.98 million hectares). The country is situated between latitude 23° and 37° North and longitude 61° and 76° East. Pakistan has an altitude ranging from 0 to 8611 m, therefore, has a variety of climatic zones and a unique biodiversity. The annual rainfall ranges from 125 mm in the South to 875 mm in sub-mountainous and northern plains. About 70% of the rain falls during the monsoon season (July-September). However, occasional showers also occur during the winter. The summer months, except in mountainous areas, are very hot, while the winter months are mild in the plains and extremely severe in hilly regions. Temperatures fluctuate from below zero in the mountainous range to 50 °C in the southern plains .

Pakistan is the ninth most densely populated country in the world. According to the latest estimates, the population of the country is over 120 million. The state-owned forest area, under the control of the Forest Department, is 4.58 million hectares, only 5.2 % of the total area of Pakistan, Azad Jammu and Kashmir. Annual production of timber and fuel wood is 0.482 million m3 and 0.234 million m3 respectively. Rich natural resources, diverse ecological zones, and rich flora of plant species are in abundance in Pakistan. It has about 6,000 species of higher plants. It has been reported that 600 to 700 species are used for medicinal purposes. In Pakistan, medicinal plants are naturally grown in many ecological zones throughout the country and on small scale some species are also cultivated. The local communities of different regions of Pakistan have centuries old knowledge about traditional uses of the plants occurring in their areas. The use of plants as medicine is an ancient practice and indigenous knowledge of plants has been transferred from generation to generation. The uses of medicinal plants vary in different parts of the country due to indigenous knowledge and method of utilization. Moreover, rich diversity of medicinal plants is an important source of livelihood for majority of the rural and mountainous communities because of low cost treatment for various common diseases .

Trade, collection and conservation of medicinal plants

The demand of medicinal plants is increasing both in the developed and developing countries including Pakistan. Only few medicinal plants are cultivated and most of the raw material of medicinal plants is harvested from forests and rangelands. According to the surveys and naturally available plant wealth of Pakistan shows that medicinal plants grow in abundance in different regions of country such as in Hazara, Malak and Kurram Agency, Murree Hills, Azad Kashmir, Northern Areas and Baluchistan, or are cultivated on farmlands in Punjab, Sindh, Baluchistan, North West Frontier Province and Kashmir. By the Pakistan Forest Institute (1989), 500 tons of medicinal plants are produced in Hazara and Malakand, 16 tons in Murree Hills, 38 tons in Azad Kashmir and about 24 tons in Northern Areas. These plants are collected from the wild, dried and processed and sold in the local markets or exported to other countries. Uses of medicinal plants at national bases are as follow:

[1] Health care products in traditional medicine (either as raw, single herb preparations or as manufactured finished products, including substances of psychotropic and ritual/religious value.

[2] Health foods either distributed through pharmacies under prescription or as "over-the-counter" medicines.

[3] Raw material for the pharmaceutical industry for extraction of essential oils, fixed oils, tannins, gums and resins and pharmacologically active constituents like alkaloids, glycosides, flavonoids, etc.

[4] Culinary additions, spices and colourings; Natural cosmetics and in perfumes. Different plant species have been used to cure a single disease or several diseases at a time. Approximately, 350-400 medicinal plants species are traded in different drug markets in Pakistan. From the above discussion on the present situation of medicinal plants it was concluded that there is global need of cultivation of medicinal plants unfortunately not enough in Pakistan and emphasis has been given on this issues for uplifting of country’s economy on somehow. However, cultivation of medicinal plants is necessary when demand has increased so far at worldwide. For example European Union (EU) uses 3,000 kg of Glycerrhiza each year for which 400 tonne plant roots are needed and In Russia 50,000 tons of medicinal plants are used annually of which, 50% are cultivated. In India (Lucknow) medicinal plants worth Rs.90 million are grown annually. Whereas in China, the total output value of the pharmaceutical industry was 233 billion yuan (28 billion US$) in 2000 and by the year 2010 the share of traditional Chinese medicine in the international market of herbal medicine is projected to improve to 15% from the existing 3% .

Therefore, main reasons for the depletion of natural resources of medicinal plants are deforestation, over exploitation, over grazing, conversion of natural habitats to agricultural fields in Pakistan causes serious threat to the survival and regeneration of many plant species. At the same time the market value of medicinal plants increases 3 to 5 times from village to local shops and the prices at national market may be doubled to triple of the same item. Some species with high market value are over exploited like Glycyrrhiza glabra, Commiphora wightii, Ephedra intermdia, Saussurea lappa, Ferula foetida, Valeriana wallachii, Colchicum luteum, Bergenia ciliate, Paeonia emodi, Morchella species etc. and their productivity is also declining but better ecological conditions play a major role in the cultivation of medicinal plants and their active ingredients.

Pakistan had and has a good potential of biodiversity of medicinal plants due to its varied climatic conditions. Therefore, there is further need of research and development activities of medicinal plants in areas like ethnobotany, biodiversity conservation, productivity, propagation, cultivation, active ingredients, quality control, value addition, value chain and policy both at national and provincial levels. The traditional uses of plants are mainly in the form of crude plant extracts. Basil and Ispaghul are best known in Pakistan now used all over the world due to their proven properties like culinary herbs and curing of ailments such as stomach problems and cholesterol lowering. Besides this, many medicinal plants have great potential in cosmetic industry, culinary herbs, herbal teas, and essential oils. The demand of alternative medicines and natural food products are increasing both in developed and developing countries. Furthermore, based on traditional knowledge of medicinal plants along with current scientific techniques are continually exploring the active constituents in plants and new drug formulation is being made.

Further efforts and research on conservation, management, sustainable utilization, marketing, ethnobotany, chemistry, pharmacology and genomics are required to unlock the treasure and potential of medicinal plants resources of Pakistan. International development community and research organizations need to devote attention and invest some resources for further research and development of apparently neglected medicinal plants/herbs for nutritional and medicinal uses. As a source of economic livelihood and make a major contribution to health and well-being based on natural remedies, investment and funds will find out the ways to reduce poverty in the developing world especially the rural and indigenous people.

SELECTED MEDICINAL PLANTS FOR CURRENT STUDY

Since time immemorial, gathering of medicinal plants by hundreds of millions of people in developing countries continues for subsistence needs and economic reasons .

Ocimum basilicum

Ocimum basilicum or Ar-Rehan (in Arabic) or commonly known as sweet basil, is an important medicinal plant which has received great attention over the past few decades around the world. Sweet basil has a prominent place in Italian and Southeast regions of Asia as a culinary herb in cuisines and frequently examined herb for its health benefits. The most important species of genus Ocimum is Ocimum basilicum L. belonging to the plant family Lamiaceae and its subfamily is Nepetoideae and usually named as common basil or sweet basil . Basil is a popular and an annual herb, 20-60 cm plant height with white and pink flowers. The useful parts of the plants are leaves and seeds, these highly aromatic leaves used either fresh or dried for spice or as a flavoring and for culinary. Different parts of basil plants are commonly used as flavoring in variety of dishes like in tomato, soups, salads, sauce, sausages and minced beef. It comprises 65 species, adapted to growth in warm conditions and originally it is native to India and other countries of Asia . Basil is one of the species used for commercial seasoning and is used commercially in Egypt, France, Hungary, Indonesia and Mexico.

History

Basil has been cultivated for thousands of years and originated in the Middle East and arriving in England and America in the 1500s and the 1600s respectively. It was traded through medieval spice routes in Asia, India, Africa and the Mediterranean and now basil grow all over the world. The ancient Greeks called basil the “herb of Kings”. It has long been used as preserving herb for mummies of ancient Egypt because of its embalming usage, basil was also known as symbol of mourning in Greece as bsilikon phuton, meaning magnificent, and royal. Ancient records from 907 A.D. indicate sweet basil in the Hunan region of China.

It is derived from the Greek ozo which means to smell, in reference to the strong odors of the species within the genus. In French, it is frequently given the name “Herbe Royale,” revealing the positive light in which it is viewed. Basil grows wild as a perennial on some pacific islands .

Traditional uses

Medicinally, Basil is antibacterial, antidepressant, anti-inflammatory, antioxidant, antiseptic, antispasmodic, antiviral, carminative, a circulatory stimulant, diaphoretic, digestive, emmenagogue, expectorant, febrifuge, galactagogue, nervine and sedative. Basil dried leaves are used from years as flavoring for food and variety of dishes . Land used for the cultivation of this plant in villages was usually fields and arable. The people of the villages cultivate and grow them in home garden for their own purposes.

Table 2.1: Traditional Knowledge related to Sweet Basil

Source .

Pharmacological spectrum application

Sweet basil is an important medicinal plant grown as a source of essential oils and aromatic compounds. 150 species of Ocimum basilicum are being utilized as a source of essential oils mainly in industries, perfumery, dental and oral products, and traditional ritual and medicine. The essential oils of the plant can inhibit the growth of fungus , and also antibacterial. It contains insect repellent and nematocidal activities and is used in pharmacy for diuretic and stimulating properties. Other than flavoring of food, Basil’s essential oil possess antimicrobial properties because of variety of components including linalool, 1,8-cineole, estragole, and eugenol made this herb antiviral, and antibacterial antioxidant, antimutagenic, antitumorigenic . It is also used in perfumes and cosmetics for its smell and is a part of many fragrance compositions. Externally it can be used for different skin infections such as treatment of acne, snake bites and insect stings. Hot tea of basil plant leaves is good for treating nausea, dysentery and flatulence. In addition to these, basil has been used as a remedy for an enormous number of ailments, including cancer, convulsion, deafness, diarrhea, epilepsy, insanity, sore throat, toothaches, and whooping cough.

Industrial and Market demand

During the last decades, much interest has been shown by the scientific community in naturally occurring swell-able biomaterial of Plantago ovata and Ocimum basilicum. These biomaterials are being used as folk medicines. Today, species and culinary herbs are often used interchangeably in the scientific and lay literature. Both are increasingly valued throughout the world for their antioxidant properties along with potential health benefits. According to U.S. Food and Drug Administration (FDA) and U.S. National Arboretum species and culinary herbs have significant function in food as flavoring and seasoning rather than nutrition that are fresh or dried leaves from which no volatile oil or other flavoring principle has been removed (Food and Drug Administration 2007; United States National Arboretum 2006). Basil is cultivated as source of essential oil for the fresh market use as food additive, flavor and fragrance. It has a commercial importance for fresh market industry for trade as fresh and dried herb all over the world.

Conclusion and Recommendations

In developing countries agriculture sector has promising effects on economic growth. To raise the cultivation and quality of economically important crop of small scale farmers may build economic opportunities where they can earn sufficient income from the food markets. In this sense, development of staple food products enriched with mucilage by food industry will be a potential contribution to a broader supply of food products with soluble fiber. Regarding these initiatives for building of economic opportunities, government and research institutes can play important role in financing of further research in this domain and communicating progress in other public institutes. Current work can be helpful to better understand the medicinal importance of basil curing various diseases like antimicrobial infections as well as for value additions in food products.

Plantago ovata

Plantago ovata commonly known as ‘psyllium’ in English and ‘isabgol’ in Hindi belongs to the family of Plantaginaceae, is a short stemmed 10-45 cm tall, annual herb. P. ovata is one of major medicinal crops with industrial significance with various applications in food products and drug supporting agent. P. ovata have been extensively used commercially for the production of seed husk; a white thin membrane covering of the seed has higher mucilage content derived from husk when soaked in water . The scope of this study was to summarize the medicinal and health benefit of psyllium in order to make this plant more prominent in market place where it had negligible attention. Mainly in this study, we tried to address the issues of today life style which increases the risk of developing overweight, constipation due to food deficient of dietary fiber in children and adults. In relation to the issues, the objective of this study was to highlight the uses of psyllium in different aspects not only for pharmaceutical use but also for food processing industries. In order to achieve higher goals of success we need to develop efficient technologies for improvement of varieties through breeding and increase the research work for the identification of other phytochemical with unique medicinal properties.

History

The word Plantago is originating from Latin, meaning ‘sole of the foot’, and ovata refers to the shape of the leaves. Psyllium is a Greek origin word, meaning ‘Flea’ regarding to the colour, shape and size of seeds. Isapghol derived from Persian with meaning of ‘horse ear’ because of boat shape of seeds. The species of Plantago is indigenous to India, Pakistan and Iran and some parts of Europe and introduced it as medicinal plant by Muslims. Therefore, first time it was brought under cultivation in cities of Pakistan i.e., Lahore and Multan. Initially the people of those countries used it as a remedy for chronic dysentery and other intestinal problems . Some previous studies showed that isabgol has and had long history of uses for medicinal purposes and cultivated long ago by the people for curing of ailments.

Figure 2.1: Plantago ovata native to India

Source: (http://bestpsyllium.com/)

Traditional uses

Traditionally, psyllium husk has been used in Asia and Europe since 16th century as an herbal medicine for chronic constipation . Plantago ovata has a long history of use as fiber supplement because it is rich source of soluble fiber since seeds constitute the commercially important part in the plant. It has also been used in Chinese and Indian traditional system of medicines as safe laxative and reduces the risk of developing chronic diarrhea, dysentery and other intestinal disorders . The seed husk does not irritate the intestine and has specific curing properties when mucous membrane is disturbed by inflammatory infections. Moreover, plant is regarded as a remedy for various ailments in traditional system of medicine in different parts of the world. Seed husk is the main constituent of a number of laxative preparations containing sodium bicarbonate and various flavours used in modern medicine. In India and Pakistan its seeds are considered as cooling and diuretic in functionality and accredited for their usefulness in healing of diarrhea, constipation and for gastric problems when decoction of dried seeds taken orally and externally .

Furthermore, mucilage of the dried seed is used externally as an emollient in different parts of the world whereas in Iran water extract of dried seeds used externally for inflammation and orally taken seeds used for indigestion associated with bile secretion abnormalities. In Thailand and Spain, seeds of this plant used in different ways for the treatment of ailments like cold, diarrhea and chronic constipation . There is growing body of literature supporting the beneficial effects of psyllium (mucilaginous material prepared from the seed husk of genus Plantago) for optimal health and diseases prevention.

Figure 2.2: Psyllium husk, mucilage, tasteless material with health benefits

Source: (http://kadampsyllium.blogspot.ro/)

Pharmacological spectrum application

Isabgol has not only traditionally application in life of people but also currently it is utilized in pharmaceutical industry because it was found to be most effective to lower blood cholesterol levels, thereby reducing the risk factors of cardiovascular system diseases which affects more than 80 million people in the world and are the leading cause of mortality in United States . It was found from the previous literature that extract of P. ovata seeds significantly affected the immune system of rabbits. It caused a reduction in anti-HD antibodies as well as an increase in the number of white blood cells and spleen leukocytes. Moreover, aqueous extract of P. ovata seeds reduced hyperglycaemia in type 1 and 2 diabetes in rats. These properties showed that this extract can be used in diabetes treatment . Nowadays it gained attention to use as a as a dietary fiber supplement to promote the regulation of bowel function. In pharmaceutical industry, psyllium is used as thickening agent during capsule formulation. Many health benefits are related to dietary fiber obtained from seed husk. For example a sufficient intake of dietary fiber may reduce risk of developing the diseases such as obesity, diabetes and certain gastrointestinal disorders . According to results published in the scientific literature mentioned above clearly stated that organic psyllium has much more advantageous for healthy life. Because it was widely considered in medical as safe and effective for certain type of diseases when taken in recommended doses.

Industrial and Market demand

Because of medicinal and pharmaceutical application of Plantago ovata, now it has a high value of market demand. In view of increasing market demand, cultivation of this plant at country level or worldwide is utmost important for uplifting the economy of a country. According to scientific report, USA is the chief importer of isabgol seeds and husk and consumes annually 8,000 metric tons which indicate the market value of this crop all over the world. This continued expansion of interest and market seems like due to natural dietary fibers. The crop has a large export demand in USA and Western Europe and about 90% of the production is exported to these countries. Currently, India is the largest producer and the main supplier of pysllium seed and husk to the world market .

The seed husk finds variety of applications in food industry because of the remarkable property of mucilage from seed husk as a thickener; it could be used as such in food industries. It is employed as a basic stabilizer in ice-creams and as an ingredient of chocolates and other food products. Psyllium can be used in food and beverages industry as a substitute, thicker and binder such as in health drinks, beverages, ice cream, bread, biscuits, other bakery products, rice, cakes, jams, instant noodles, breakfast cereals etc., in order to improve the fiber content of the food and to increase the bulk of the food with various health benefits. Psyllium Husk may also be added to fresh fruit drinks or flavored drinks to improve the mouth-feel of the drink and make it richer and impart good consistency to it. In food and beverage industry psyllium is use to Improves softness and body texture, to provide strength as binder and stabilizer .

Furthermore, Plantago ovata contained a product which may some effect on the subjective feeling of fullness and may be a useful supplement in weight control diets which is already taken traditionally by the people world-wide to control bowel function .

On the other way, psyllium is used in the pharmaceutical as well as cosmetic industries. The literature confirms that seed husks of P. ovata have found application in the cosmetics industry. It is also used for sizing purpose and as a base in cosmetics. The husk has been found to be served as a good binder and disintegrants in compressed tablets. The isolated mucilage powder of P. ovata exhibits faster drug dissolution and improved bioavailability, and it was stated that the isolated mucilage powder can be effectively used as disintegrants and superdisintegrant in tablet formulations.

The remains of seed husk around 69% by weight of the total seed crop can be used as cattle and a bird feed with no adverse or side effects on them. Because of international acceptance as medicinal plant lot of efforts exercised to improve the yield and quality of isabgol, there has been only limited success in this concern. Therefore intense efforts are needed for the cultivation of better varieties. This may facilitate the production and yield of isabgol with improved quality made it available easily for food products with variety of health comforts.

Figure 2.3: Processing of isabgol seeds for marketable product

Conclusion and Recommendations

As a result of above discussion regarding commercial and medicinal importance of Plantago ovata, it was concluded that the cultivation and the improvement of culture technologies of this crop at large scale is utmost necessary for need of present time. Thus, it may become a profitable agro-practice for the local people and farmers of that area in the context of developing policy for connecting crop production with the suppliers for industry.

The plant has been cultivated in India and Pakistan from century ago and traditional knowledge access is an asset easy to be explored. Keeping in mind the wide industrial application as well as the increased market demands, appropriate method of cultivation and advance techniques needs to be developed to enhance the yield and the quality of seeds and to get over the traditional process of milling the seeds. On the other hand, seeds may be used for preparation of high valued commercial products. It is desirable to explore the possibilities of intensify cultivation and yield of crop other than arable land which is not suitable for the cultivation of food crops.

Moreover, it was concluded from the previous literature that isabgol seeds has the richest fiber contents consumed as natural source of fiber with various health benefits. This is the main cause of increasing interest of scientific community to do more research work in this direction in order to find newer ways to incorporate this fiber into dietary food products with heath protective effects. Psyllium addition in standard diet may support controlling those issues related to food deficient of soluble fiber. From the foregone discussion, we may conclude that Plantago ovata has potential role in industries for marketable product development and for benefit of a healthy society.

However, higher fiber food may play an important role in helping some individuals achieve fiber intakes to control weight enhancement. For all this to happen, health authorities and food producers may be required a strong communication and to educate the public about the health benefits of psyllium. In order to make this process functional we need to introduce innovative ways to educate the consumers about the strong health effects of organic dietary fibers.

BIOACTIVE COMPOUNDS AND EXTRACTION TECHNIQUES

Any plant which has traditional and cultural health attributes or plant parts used for therapeutic or medical benefits known as medicinal plants . Therefore, phytochemicals or bioactive compounds contain those chemicals which found in plants in small quantities as non nutritive constituents of food plants and certain food including fruits, vegetables, nuts, oils and grains that they have action in the body to promote the human health . Every living organism from unicellular to multi-cellular including plants possesses chemical compounds for their growth and survival which are divided into two categories. One is primary chemical substances aimed at growth and development such as proteins, lipids, amino acids and carbohydrates. Another is secondary metabolites help the plants to increase their overall ability to survive and overcome challenges found in their surroundings. Typically, secondary metabolites in other words bioactive compounds have certain effects (pharmacological or toxicological) on biological systems such as human and animals . In this part of study, initially we are aimed to describe the concepts of selected bioactive compound in relation to value addition in commercial food products for healthy life.

Total Proteins

Leaves and seeds of plants are a good source of proteins, fats and fibers and essential for the maintenance of plants and their high nutritive value suggests that they can be use as raw material for industries . Additionally, proteins are composed of essential amino acids and have the nutritional values for human health.

Phenolic Compounds

Phenolics play an important role in the production of functional and nutraceutical foods.They are responsible for structural and protective functions in plants as defense mechanisms to counteract reactive oxygen species. Phenolic compounds mainly contributing to flavor, color and bitterness of fruits and vegetables and is also well known in providing health beneficial effects .

Total soluble Fibers

Plant based dietary fiber is the indigestible part of food and has two components as soluble and insoluble fibers. The fiber which dissolves in water is known as soluble fiber. It is readily fermented in the colon into gases and physiologically active byproducts. This type of fiber can cause an extended feeling of fullness. Insoluble fiber does not dissolve in water and is readily fermented in the large intestine and provides bulking. They move through the digestive tract easily by absorbing the water. Some type of fibers become gelatinous when absorbs the water in the digestive system . For the treatment of various gastrointestinal disorders, certain food supplementary diets are available in the market for reducing the cholesterol levels and help in the losing the weight with nutritional health benefits whereas soluble fibers supplements may be beneficial for alleviating the irritable bowel syndrome such as constipation and diarrhea . All fractions of the fibers including gum, mucilage, pectin, lignin, cellulose and hemicellose play a significant role in curing various ailments such as diabetics, fatness, constipation and cardiovascular diseases .

Essential oils

Medicinal plants are well known as a potential source of essential oils. It is highly complex and mixture of constituents obtained by distillation or solvent extraction also from herbs and spices. Essential or volatile oils are characterized by antimicrobial activity including antifungal and antiviral. Besides all these properties, it has been extensively used in food industry as food preservative, flavoring, pharmaceuticals as well as in cosmetics as fragrances.

Lipids (Fats)

The fats are subgroup of lipid called trigylcerides. The functional property of lipids is to store energy in the body. Most of fats found in foods are in the form of trigylcerides, phospholipids and cholesterol. Some dietary fats are essential for absorption of vitamins. Human and mammals are required to attain the certain amounts of fats such as linoleic acid because they are not synthesized in the body from simple precursor in the food. Vegetables oils, seeds, nuts and legumes are a rich source of fats .

Total carbohydrates

Carbohydrates form the foundation of daily diet thus needed to fuel the daily activities in human life. Human nutrition largely depends on cereals which obtain mainly from staple crops such as maize, wheat, rice oats and millet. They provide the calories in the form of complex carbohydrates such as starch.

Extraction Techniques

People used medicinal plants since very long for their nutritional purposes but after the discovery of bioactive compound with medicinal properties became useful for health improvement. The history of medicinal herbs uses for mankind is very old as Herodotus (5th century B.C) mentioned in his writings Leonurus cardiaca (Mother wort) was used by the Romanian people living north of the Danube river. In 19th century, herbal products were introduced by Romanian pharmacopoeia and took a step in 1904 for establishment of institute of medicinal herbs in city of Cluj. Furthermore, an Egyptian papyrus known for uses of medicinal herbs such as coriander and castor oil in thousand of recipes as preservatives . Nowadays, intensify use of bioactive compounds in different commercial industries urge the need of most appropriate extraction method. The selection of proper extraction method for the qualitative and quantitative studies of bioactive compounds from plant materials is a crucial step for the final outcomes of any study, though most of researchers believe in the importance of sample preparation during any analytical study . As a result of huge development in modern chromatographic and spectrometric techniques made biochemical analysis easier and better than before. But till now the success of final results depends on extraction methods along with input parameters . This part of the study aimed to present the overview of different extraction techniques in order to better understand the mechanism of extracting bioactive compounds from medicinal plants.

Conventional methods of extraction

Classical extraction techniques have been used for a long time for the extraction of nutraceuticals from the plants. These conventional techniques based on the choice of solvent along with use of heat. Existing classical techniques include: Soxhlet, hydro-distillation and maceration in which Soxhlet is well established technique and still considered as one of the reference method to compare success of newly developed extraction techniques. Enough scientific reports extensively reviewed this traditional method of extraction .

Non-conventional methods of extraction

At the same time various non- conventional or novel techniques including ultrasound-assisted extraction , pulsed electric field , enzyme digestion , microwave heating, supercritical fluids . , and accelerated solvents , have been studied as non-conventional methods. These novel techniques have been developed for the extraction of bioactive compounds from plants in order to increase the extraction yield, shorten the extraction time, decrease the solvent consumption and enhance the quality of extracts.

Table 2.2: A brief summary of the experimental conditions for two non-conventional methods of extraction for plants material

Source: Adapted from .

Figure 2.1: The flow chart of medicinal plant study and position of extraction techniques (Source: adapted from .

FUNCTIONAL FOOD AND SOLUBLE FIBERS

In this modern and industrialized world, significantly changes have occurred in the concepts of nutrition. Former emphasis has been given on survival through hunger satisfaction and food safety to prevent the development of risk of ailments but more recently food sciences now aim at developing foods to health while at the same time reducing the risk of some major and minor diseases. Within the last decades, the term functional foods gained more attention throughout the world. That is because it has additional physiological benefits beyond that of meeting basic nutritional needs which is already supported by scientific data show that food with nutritive and non-nutritive components have the potential to modulate target functions in the body which are relevant to human health and reduction of disease risk. The present study overviewed the concept of functional food in relation to plant sources with strength of evidence supporting their functionality, safety considerations and recommendations intakes.

Introduction

All foods are functional and provide the therapeutic benefits clearly not a new concept. The primary role of food is to provide taste, aroma and sufficient nutritive value to meet the health requirements of an individual. Hippocrates the father of medicine nearly 2,500 years ago adopted that "Let food be thy medicine and medicine be thy food," is gaining renewed interest in health enhancing role of specific foods. From the perspective of healthy living, it is obligatory for all to eat healthy food. It is pointed in this context about knowledge of nutritional composition of food including quality and quantity of foods with recommended values beneficial for a healthy life. However, it is concluded there are no ‘good’ or ‘bad’ foods only ‘bad’ diets may develop the risk of diseases. Although overall message for health promotion is that which types of food to include or avoid achieving a balanced diet for which extensive guidance is available .

Concept of functional foods

Today, concept of functional foods derives from the observation that some foods and beverages have beneficial physiological effects on human health that are not explained by their nutritional content. There is now trend has developed, nutrition science moved on from the classical concepts of avoiding nutrient deficiencies and to support the idea of “positive" or "optimal" nutrition. A comprehensive lifestyle is mandatory for good health which includes not only balanced diet but also regular exercise, stress avoidance and other positive health practices. This could only be possible to maximize health and reduce disease risk when all of these issues are addressed along with concept of functional foods .

There is increasing scientific evidence that nutrition plays crucial role in the prevention of chronic diseases like diabetes, cardiovascular diseases, obesity and cancer most of them are diet related. The food not only necessary for living but also as a source of mental and physical well-being contributing to promote health and reduction of risk of a pathologic process. Generally, those foods are considered to be part of normal diet that contains biologically active components, which offer the potential of enhanced health or reduced risk of disease .

History of uses

Food and nutrition have become challenging issues in modern societies and functional food is now very popular term in this regard. It was first introduced in Japan in the mid-1980s and refers to foods containing ingredients that significantly affects on bodily functions in addition to being nutritious. Functional foods have no universally accepted definition but intensive research efforts in this field made more prominent growing food category which is recognized as “functional food” at global level. Because of growing interest in functional foods, market of these products has been emerging rapidly. To date, the biggest functional food markets are in Japan is the only country that has formulated a specific regulatory approval process for functional foods. It is known as Foods for Specified Health Use (FOSHU), these foods are eligible to bear a seal of approval from the Japanese Ministry of Health and Welfare. Currently, 100 products are licensed as FOSHU foods in Japan. Now functional food market is flourishing in USA and Europe .

Estimates, however, of the magnitude of this market vary significantly, as there is no consensus on what constitutes a functional food. It depends on the consumption and awareness of functional foods which vary from country to country. It is estimated by the market value of functional foods reached at $28.9 billion. More significant, perhaps, is the potential of functional foods to mitigate disease, promote health, and reduce health care costs.

Impacts on human health with scientific evidences

Regarding the concept of functional food in relation to health has increased substantially and consumers take interest in components of foods with added health benefits. Many scientific organizations and academic are actively working on functional foods in order to establish the scientific basis to support claims for functional components. The desire for an improved quality of life, consumers must have a clear understanding of potential health benefits of functional foods and a strong confidence in the light of scientific evidence that are used to document health effects and claims. Moreover, changing trends in population demographics and socio-economic also indication of urgent need for foods with additional health benefits. A number of studies show that socio-demographic characteristics also play an important role .

Research on functional food link to health is assumed to be the main motivation behind the use of functional foods . Although monitoring taste was also found to be a critical factor for the future acceptance of functional foods . An increase in life expectancy as well as improvements in the costs of health care have stimulated researchers, health professionals and the food industry to investigate how these changes can be managed more effectively. Recently a massive body of research on functional foods has been done in different countries to address cognitive, motivational and attitudinal determinants of the public acceptance . Any claims about health benefits attributed to functional foods based on sound scientific evidence but too often only so-called “emerging evidence” is the basis for marketing some functional foods.

In Table1, scientific evidences and the recommended intake levels are summarized in order to support the functionality and variety of functional foods components.

Table 2.3 Strength of evidence for functional foods currently on the U.S. market*

Source

*Foods that have an FDA-approved health claim (sterol/stanol esters, oats, psyllium, soy) generally are supported by two dozen or more well-designed published clinical trials. For example, the soy health claim petition contained _40 clinical trials, while there are only a few clinical trials on cranberry juice and urinary tract infections. TG, triglycerides; CLA, conjugated linoleic acid; GI, gastrointestinal

Plants as a source of functional foods

The use of functional foods for promoting health and specific dietary recommendations for treating or preventing various types of illness is as old as practice of medicine, have been documented in Hippocratic and Vedic texts and the canons of traditional Chinese medicine. Herbs and spices are added to food to enhance flavor. The foods with culinary and non-culinary herbs to produce healing mixtures were the part of Traditional Chinese remedies.

Many of these have documented medicinal uses that render them functional foods. For instance, Peppermint (Mentha piperita) tea, Ginger (Zingiber officinale root), cinnamon (Cinnamomum spp bark) and licorice (Glycyrrhiza glabra root) has a long history of use in Chinese herbal tonics for digestive complaints and disorders. Peppermint oil contains spasmolytic components that block calcium channels in smooth muscle. Thyme (Lamiaceae spp) was used as a vermifuge in ancient Egypt and its oils possess potent antimicrobial properties. Cranberry (Vaccinium macrocarpon) juice has traditionally use for treatment of urinary infection. An overview of specific phytochemicals from edible plants associated with health promotion and disease prevention are described in Table 2.

The most studied food sources of these phytonutrients are soy beans (Glycine max) and tea (Camellia sinensis leaves), but tomatoes (Lycopersicon esculentum), broccoli (Brassica oleracea), garlic (Allium sativum), turmeric (Curcuma longa), tart cherries (Prunus cerasus) and various types of berries are also receiving considerable attention as functional food candidates. Numerous clinical data indicates that plant based diet with physiologically active ingredients known as phytochemicals can reduce the risk of chronic diseases. Unfortunately, only a small number of these plants have had substantive clinical documentation along with significant scientific agreement” required by the FDA for authorization of a health claim which include oat soluble (β-glucan) fiber soluble fiber from psyllium seed husk , soy protein and sterol- and stanol-ester–fortified margarine . Research on some other plant resources for health benefits are discussed briefly below:

Garlic (Allium sativum)

It has a long history of uses along with a wide variety of medicinal purposes. Garlic has been shown to have a modest blood pressure lowering effect in clinical studies due to the presence of numerous physiologically active organo-sulfur components .

Tomatoes (Lycopersicon esculentum)

Tomatoes and tomato products are also being investigated for their unique properties such as their role in cancer chemoprevention and most significant dietary source of lycopene, a non-provitamin A carotenoid that is also a potent antioxidant .

Others

Clinical trials, which have specifically examined the effect of almonds on blood lipids, have found that these tree nuts significantly reduced total cholesterol by 4–12% and low density lipoprotein (LDL) cholesterol by 6–15%. Although, traditionally foods high in fat are not considered heart-healthy except for fatty fish but evidence is accumulating on the cardiovascular benefits of a variety of nuts, when they are part of a diet that is low in saturated fat and cholesterol .

On the other hand, Studies in animals consistently show that consumption of green tea reduces the risk of various types of cancers . Furthermore, food that is a source of polyphenolics and is just beginning to be investigated for its potential benefits to heart health is chocolate .

Table 2.4: Phytochemicals Associated with Health Promotion and Disease Prevention

Source (http://www.mdheal.org/articles/word2/functionalfoods2.htm)

Safety Issues and considerations

Although there is evidence that some or certain functional foods or ingredients can play a role in disease prevention and health promotion, safety considerations is critical. Safety concerns and issues have recently been raised which is relevant to herbs are complex. The Food and Nutrition Board of the National Academy of Sciences has devoted considerable attention to the upper safe limit for intake of essential nutrients . For the safety of public health, an expert panel stated in the IFT “Expert Report on Functional Foods that components already considered through pre-established programs as generally recognized as safe (GRAS) substances and approved food additives should be accepted for functional foods . Also in 2000, the General Accounting Office (GAO) released a report that raised concerns about the safety of certain functional foods . In June of 2001, the FDA issued warning letters to the food industry concerning the use of “novel ingredients” such as St. John’s wort in conventional food due to absence of such safety information poses a significant safety risk to some consumers .

Soluble fibers

Purpose of this part of research was besides using selected plants for medicine, crude fibers and fats along with little fractions of fibers such as gums and mucilage can be essential for dietary intake as commercial food products and might have significant role treating various diseases such as fatness, diabetes and constipation.

Conclusion

In this industrialized world, significantly changes have occurred in the concepts of nutrition but more recently food sciences is developing foods with additional health benefits widely known as functional foods while at the same time reducing the risk of some major and minor diseases develop another concept in food sciences as nutraceutical (combined term from nutrition and pharmaceuticals). Within the last decades, the term functional foods gained more attention throughout the world. Extensive research is currently directed toward increasing our understanding of “functional foods.” Academic, government and private research institutes around the globe are devoting substantial efforts to identifying how functional foods and food ingredients both from plant and animals (phytochemicals and zoochemicals) might help prevent chronic disease or optimize health, thereby reducing healthcare costs and improving the quality of life for many consumers.

On the other hand, consumers must realize that foods are not good and bad, only dietary pattern may be good or bad. Any health benefits attributed to functional foods should be based on sound and accurate scientific criteria and evidence including rigorous studies of safety and efficacy. Further researches are necessary to find out reasons behind the differences and understand consumers’ needs for functional foods in order to meet the greatest challenges to nutritional health are chronic diseases including obesity, heart diseases, diabetes and cancer. When functional foods do provide genuine public health solutions, they contribute immensely to the public welfare. They help to provide the very conditions for life; they help us to increase our capacities, to exercise our rights, and to live well together then use of functional foods under these circumstances is permissible with health safety considerations.

CHAPTER 3

PROPOSED METHODOLOGY OF RESEARCH

The seeds of selected medicinal plants were collected from local market of Sibiu, Romania in month of May 2015 and seeds of selected medicinal were also collected from local market of Multan, Pakistan in the month of August 2015. Clean seeds of both plants were examined under microscope for evaluation of morphological characteristics. Samples were prepared by coarsely ground the seeds of selected medicinal plants to 20 mesh on the basis of analysis requirement and stored in dry clean bottles. Each sample was studied in triplicate. Details of both plant species, in respect of their scientific, family and local names are elaborated in Table 1.

Proximate Analysis of selected medicinal plants

Summary of proximate analysis of seeds samples is given as followed:

AOAC methods were applied to carryout proximate analysis of the samples for moisture, total ash, crude fiber, crude fats, proteins and carbohydrates. The determination of proteins in terms of nitrogen was done by micro Kjeldahl method involving digestions, distillation and finally titration of the sample . The nitrogen value was converted to protein by multiplying to a factor of 6.25. The lipid (fats content) of the samples was done using Soxhlet type of the direct solvent extraction method. The solvent used was petroleum ether. The crude fiber was also determined by the method described by . The energy values (kcal/100 g) were determined by multiplying the values of carbohydrates, lipids and proteins by a factor of 4, 9, and 4 respectively, and taking the sum expressed in kilocalories . The total carbohydrates were determined by difference method [100 – (proteins + fats + moisture + ash in percentage). All the proximate values were reported in percentage .

Determination of morphological characteristics of seeds by Digital microscope

Clean seeds of Psyllium and sweet basil were visually examined under the microscope for morphological study of seeds. It was carried out as per the reported method for its morphological characters such as colour, odour, taste, shape and size. It was also analyzed for 100 seeds weight (g) for each sample separately which are specifically arranged in Table 2.

Figure 3.1 Zeiss axiostar plus microscope

Determination of moisture content by Oven dry method

The moisture content was determined by oven drying method by the following detail procedure. First of all we dried the empty dish and lid in the oven at 105°C for 3 h and transferred to the desiccators to cool. After that we weighed the empty dish and lid. We weighed about 2.5 g of each sample to the empty dish and spread the sample to the uniformity and placed the dish with sample in the oven. We dried the samples in oven for 3 h at 105°C and higher temperature was 144 °C in the oven. At the time of drying the room temperature was 20°C. After drying we transferred the dish with dried sample with partially covered lid to the desiccators in order to cool the dish with samples. Finally we reweighed the dish with dried samples.

Calculation for Moisture contents in seeds

Moisture (%) = (W1-W2) x 100

W1

W1: wt. (g) sample before drying

W2: wt. (g) sample after drying

Determination of moisture content by Thermo moisture analyzer

For moisture analysis by thermo moisture analyzer for seeds samples of selected medicinal plants were prepared by coarsely ground in the grinder and then temperature in the apparatus (Infrared AND ML.50) was set as 105˚C and moisture of the samples were determined in percentage (%) after 47 minutes. After the moisture content in percentage was obtained then both the values were compared.

Determination of Ash content of selected medicinal plants

At the start of ash content analysis of samples we washed the crucible thoroughly and placed the crucible and lid in the furnace at 550 șC overnight to ensure that all the impurities on the surface of crucible were burned off. After that we cooled the crucibles in the desiccators for 30 minutes and then weighed the empty crucible and lid to 3 decimal places. We weighed about 5 g each sample into the crucible. We heated the samples over low Bunsen flame with lid half covered when the fumes were no longer produced we heated the samples in muffle furnace at 550 șC overnight until white or grayish white ash was obtained. After cooling the samples in desiccators, we reweighed the samples and percentage of ash was calculated .

Figure 3.2 Oven for ash determination (Controller B170 Nabertherm)

Ash (%) = Weight of Ash x 100

Weight of sample

Determination of total mineral matter (Ash)

From the literature review, we found that ash is considered as the total mineral matter or inorganic content of the sample analyzed. This method is originally used to determine the ash content in feedstuffs by calcinations. In our study we applied this method for mineral matter estimation from the seeds of selected medicinal plants.

Principle:

The sample is ignited at 600oC to burn off all organic material. The inorganic material which does not volatilize at that temperature is called ash.

Figure 3.3 Oven (ESAC-50a Electronic April) Multifunction

Procedure:

In this method firstly we weighed crucible and then weighed about 2 g each sample into the crucible. We heated the samples over low Bunsen flame with lid half covered when the fumes were no longer produced we heated the samples in muffle furnace at 600 șC until white or grayish white ash was obtained. After cooling the samples in desiccators, we reweighed the samples and percentage of ash was calculated .

A= Weight of crucible with sample (g)

B= Weight of crucible with ash (g)

C= Weight of sample (g)

Determination of crude fiber content of selected medicinal plants

Principle:

A moisture free and ether extracted sample is digested first in weak acid solution, then in base solution. The organic residue is collected in a filter crucible. The loss of weight on ignition is called fiber.

Procedure:

We measured the crude fiber by taking 2 g of oven dried and fat free sample in china dish and fatty material was removed by Soxtec Apparatus. Then digested the fat free sample in 200 mL of 1.25% H2SO4 solution in a conical flask and heated it on hot plate for 30 minutes. Then we filtered the contents with the help of thick linen cloth and gave 2-3 washings with distilled water. After that, digested the residues in 1.25% NaOH solution (200mL) and again heated for 30 minutes. After that we filtered the residues with linen cloth. Again we washed the samples two to three times with distilled water and one washing with acetone. Then we transferred the residue in weighed crucible.

We placed the crucible having residue in hot air oven at 105 0C for 24 hours till constant weight was attained. In the next step we recorded the dry weight of residue then ignite this residue by placing in a muffle furnace at 600 0C for 3-4 hours until the grey or white ash was obtained. Then we again recorded the weight of the ash. Calculate crude fiber percentage as follow:

Determination of crude fiber from seeds

This is the other method we applied for estimation of crude fiber but it is originally applied for cellulose extraction from wheat flour . As cellulose originates from the wheat grain shell is a form of fiber. If fiber is present in great quantity worsens the attributes of the dough and the quality of the products.

Principle of the method

Dissolving and removing soluble components from a quantity of material used and gravimetric (by calcination) method is used and starch in the material by boiling with acids turns into glucose. The albumin in an acidic medium, when boiled, is transformed (in part) into the acidic amide; the rest is treated with a base. The largest mineral substances melt. The insoluble residue dries and turns into ashes.

Materials and Apparatus

Analytical balance (accuracy 0.001 g)

Thermoregulable electric oven

Desiccators, containing anhydrous calcium chloride

Spatula

Gas bulb (Bunsen)

Metal tripod

Asbestos sieve

500 ml Erlenmeyer / Berzelius

Water vacuum tube

Refrigerant with reflux / clock glass;

Filter paper with medium porosity

Porcelain crucible,

Capping flap with lid,

Pliers (for calcination)

H2SO4 solution 5 %

NaOH solution 5%

Ethyl ether

Ethyle alcohol C2H5OH

HCl solution 0.5 n

Method of analysis

First of all we weighed about 2 g of ground seeds sample in a vial. Then we passed the contents into an Erlenmeyer. After that we add 50 ml of sulfuric acid boiled, and approximately 150 ml hot (distilled) water in the samples. We adjusted the flask to a reflux cooler to avoid foaming. In the next step we brought the contents of the dish for boiling kept them for exactly 30 minutes. We kept the level of liquid constant in the glass; add hot water from time to time. We then filtered immediately by suction on the vacuum tube (using a filter bowl (10 μm) and then add about 30 ml of hot water for washing, and filter-wash operations repeated three times.

Then the residue on the filter passed quantitatively (with hot water) into an Erlenmeyer / Berzelius and we add 50 ml of sodium hydroxide boiling in the flask is added to the beaker and approximately 150 ml of hot distilled water. And then boiled, filtered, and washed under the same conditions mentioned as above. After the last wash of the residue, we acidified the samples with 1 ml of hydrochloric acid and then we transferred the residue with hot water to a filter paper. We washed thoroughly the filter paper (with residue) with hot water until the chlorine reaction has disappeared with silver nitrate solution. Then we washed twice the residue with 5 ml of ethyl alcohol and then 5 ml of ethyl ether to remove the fatty matter. After that we inserted residual filter into the ampoule, dry in the oven at 130 ° C for about 5 hours. We placed the residual filter in the desiccators for cooling and after cooling put them dry residue filter in a crucible and calcined at 550 oC for 6 hours in the oven to a constant mass. After calcinations we removed the crucible from the oven, placed in desiccators and we weighed as soon as it has cooled at room temperature.

Calculation:

Fiber content (%) = (Wt. of crucible with residue + wt. offilter paper with residue) – (Wt. of crucible + wt of filter paper x 100

Weight of sample

Determination of Protein of selected medicinal plants

Principle:

The nitrogen of protein and other compounds are transformed into ammonium sulfate by acid digestion with boiling sulfuric acid. The aid digest is cooled, diluted with water and made strongly basic with NaOH. The ammonia is released and distilled into a boric acid solution the ammonia in the boric acid solution is titrated with standardized H2SO4.

Reagents:

Catalyst Mixture (K2SO4 – CuSO4.5H2O – Se), 100:10:1 w/ w ratio

We ground reagent-grade chemicals separately and mixed. If caked, grind the mixture in a porcelain pestle and mortar to pass a 60-mesh screen (0.250 mm), we took care not to breath Se dust or allow Se to come in contact with skin.

Sulfuric Acid (H2SO4), concentrated (98 %, sp. gr. 1.84)

Sodium Hydroxide Solution (NaOH), 10 N

We dissolved 400 g NaOH in de-ionized water, transferred the solution to a one-Liter heavy walled Pyrex flask, let it cool, and brought to volume.

Boric Acid Solution (H3BO3), saturated

We added 500 g H3BO3to a five-Liter flask and then add 3 Liter de-ionized (DI) water, and swirl vigorously. We left that solution overnight. The H3BO3 was in solid form on the bottom of flask.

Tris Solution [hydroxymethylaminomethane] (C4H11NO3), 0.01 N

We dried reagent-grade Tris in an oven at 80 °C for 3 hours, and then cooled in a desiccator, and store in a tightly stoppered bottle. We then dissolved 1.2114 g Tris in DI water, and transferred to a one liter flask, and brought to volume.

Sulfuric Acid Solution (H2SO4), 0.01 N

We added 28 mL concentrated H2SO4 to about 600 – 800 mL DI water in a one liter volume, mixed well and left it for cooling, and brought to volume. This solution contained 1 N H2SO4 solution (Stock Solution). We took pipette with 10 mL of Stock Solution to one liter flask, and brought to volume with DI water. This solution contained 0.01 N H2SO4.

Standard Stock Solution

We dried reagent-grade ammonium sulfate (NH4)2SO4 in an oven at 100 °C for 2 hours, cooled in a desiccator, and stored in a tightly stopper bottle. We then dissolved 5.6605 g dried (NH4)2SO4 in DI water, and brought to one liter volume. This solution contained 1.2 g NH4-N / L (Stock Solution).

Nitrogen estimation was carried out by the micro-Kjeldahl (Kjel Flex K-360) method (Pearson, 1976). Crude protein was subsequently calculated by multiplying the nitrogen content by a factor of 6.25.

Description of the Kjeldahl Method

The Kjeldahl method is applied for the quantitative determination of nitrogen and usually performed in brewing industry for the estimation of protein content in gains. There are three basic steps of this method which are as follow.

Digestion of the sample:

In this step, digestion of samples takes much time because the bonds that hold the polypeptides together break down to form the simpler chemicals such carbon dioxide, water and ammonia. Addition of sulfuric acid and heating the sample mixture to about 370șC to 400șC for 60 to 90 minutes facilitate the oxidation of organic material and releases the ammonia ions.

Distillation

This is the second step of Kjedahl method in which separation of ammonia from digestion mixture is done by raising the pH with sodium hydroxide, which converts the ammonium ions into ammonia gas. After that ammonia gas is evaluated through boiling and distillation in the presence trapping solution of hydrochloric acid.

Titration

In this step, dissolved ammonia in the solution of hydrochloric acid is titrated back in order to collect the distilled-off ammonia and then amount of nitrogen in the protein is determined.

Methodology of analysis

Digestion

We weighed about 0.5g of sample on balance and put it in digestion tubes. We added about 4g of digestion mixture in each digestion tube. We added 10mL H2SO4 in each digestion tube. After adding the sample, digestion mixture and sulphuric acid in digestion tubes put the digestion tubes as such for overnight. We placed the tubes rack in the block-digester and slowly increase temperature setting to about 370°C. The H2SO4 was condensed about half-way up the tube neck; and when solution cleared, continue heating for about 3 hours. After that picked the tubes rack out of the block-digester and carefully placed on a rack holder, and then let tubes cooled at room temperature. We slowly added about 15 mL DI water to the tubes, cooled, and brought to volume. If contents were not dissolved and were solidified we heated the tubes again until the precipitate (gypsum) dissolved, then cooled with tap water. Each batch of samples for digestion contained at least one reagent blank (no soil), and one chemical standard (no soil, one mL of the Stock Solution).

Distillation

Before starting a batch for distillation, we steamed out the distillation unit for at least 10 minutes. We adjusted steam rate to 7 – 8 mL distillate per minute. We took care that water should flow through the condenser jacket at a rate sufficient to keep distillate temperature below 22 °C. We calibrated pH meter with buffer solutions of pH 7.0 (buffer), and 4.0 (sensitivity), after setting for temperature. We then standardized the 0.01 NH2SO4 in the Auto-Titrator by titrating three separate 10-mL aliquots of the primary standard, 0.01 NTris solution, to pH 5.0. We followed that the titrations should agree within 0.03 mL, if not; we then performed titration further aliquots until agreement was found.

We carried out distillations as follows:

We dispensed one mL saturated H3BO3 solution and one mL DI water into a 100-mL Pyrex evaporating dish, placed underneath the condenser tip, with the tip touching the solution surface. We placed the pipette with 10-mL aliquot into a 100-mL distillation flask, and added 10 mL 10 N NaOH solution. After that immediately we attached the flask to the distillation unit with a clamp, started distillation, and continued for 3 minutes, lower the dish to allow distillate to drain freely into the dish. After 4 minutes when about 35-mL distillate is collected, we turned off the steam supply, and washed tip of the condenser into the evaporating dish with a small amount of DI water.

Titration

In this step we performed titration of the distillate to pH 5.0 with standardized 0.01 NH2SO4 using an Auto-Titrator. In the each distillation we contained at least two standards and two blanks (reagent blanks). We took care that recovery of NH4-N should be at least 96 %.

After finishing titration, we took care to the following steps. We washed the Teflon-coated magnetic stirring bar, the burette tip, and the combined electrode into the dish. After that we steamed out the distillations between different samples. And then disconnect distillation flasks containing the digest sample and NaOH, and attached a 100-mL empty distillation flask to distillation unit. We placed a 100-mL empty beaker underneath the condenser tip, turned off cooling water supply (drain the water from the condenser jacket), and steamed out for 90 seconds.

Where:

mL of acid=volume of H2SO4used

Acid N=Normality of H2SO4

0.28=Atomic wt. of nitrogen

Crude protein (%) = N x 6.25

(Nitrogen Free Extract) NFE = 100-Crude protein-Crude fat-Crude Fiber-Ash (Mineral matter) %

Determination of Fats of selected medicinal plants

Ether is continuously volatilized, then condensed and allowed to pass through sample, extracting ether soluble materials. The extract is collected in a beaker. When the process is completed, the ether is distilled and collected in another container and the remaining crude fat is dried and weighed.

Lipid (Fats content) extraction was carried out with a soxhlet extractor with petroleum ether and then the solvent was removed by evaporation.

Methodology of analysis

Reagent: Petroleum ether

We weighed about 1.5 to 2.0 g sample into a clean previously extracted alundum thimble. We placed the thimble and sample in a sample container and fixed under the condenser of Soxtec extraction apparatus. We then added 30-40mL of diethyl ether to the solvent beaker and placed on the condenser with screw ring. We turned on water and extracted for half an hour. After the extraction is completed, we removed the samples. We poured ether from reclaiming tubes into a container for used ether. We dried the ether extract in a 700C explosion proof oven for 30 minutes. We then cooled in desiccators and weighed. Percentage of fat was determined by following formulae:

Fat (%) = weight (g) of fat obtained x 100

Weight of sample

Determination of Total carbohydrate

The values of total carbohydrates were calculated by the difference given by: 100-(percentage of ash + percentage of total fats + percentage of protein + percentage of crude fiber) .

Formulae:

Percentage of carbohydrate was given by: 100 – (Percentage of ash + percentage of crude fiber + percentage fat + percentage protein).

The available carbohydrate was estimated by the following

Formulae: Difference of (% total carbohydrate content – % crude fiber).

Determination of Total Energy values

Total energy values were calculated by multiplying the amount of proteins and carbohydrates by a factor of 4 and lipid by a factor of 9 kcal/100g . And sum of these values gave the total energy of the materials used.

Total Energy values = (4x % Protein + 4x % Carbohydrate + 9x % lipid)

Survey analysis of selected medicinal plants for consumer response

For the purpose of this study, people were randomly surveyed according to the requirement of current research work. Sites were chosen on the basis of easy access to the location and the availability of people. Surveys were conducted with people who live in Pakistan. Surveys were distributed through mailed on the months of June and July 2016.

Surveys use closed questions which can be answered with a yes or no, multiple choice, or short answer . They usually last for no more than a minute and do not require extended responses from participants. For the surveys, ten questions were asked and the age and gender were recorded in order to classify and understand the demography of people who use medicinal plants (See Appendix I). The assessment of indigenous knowledge for seeds consumption of sweet basil and psyllium husk as a source of fiber by local community was carried out in different parts of southern Punjab, Pakistan. A structured questionnaire was prepared with close ended questions. The survey information was then transcribed into a worksheet on Microsoft Excel. Data collected were subjected to the descriptive statistic like percentage, frequency, bar and pie chart.

Novel Product preparation by using mucilage from seeds of selected medicinal plants

Pakistan is a developing country depending on the wheat and rice as staple foods. Wheat and rice are rich source of energy and protein. As Pakistan is bestowed with rich diversity of medicinal plants we selected two medicinal plants (Basil and Psyllium) as a source of fiber for enrichment of foods by utilizing the seeds of both plants.

Bread preparation

For the preparation of bread we utilized the following ingredients; Flour (refined wheat flour) 100g, compressed yeast 3g sugar 2.5g, salt 1.5g and distilled water optimum. Refined wheat flour mixed with Psyllium husk at replacement levels of 1%, 1.5% and 3% w/w %. We prepared the dough by mixing (optimum 3 minutes) the above mentioned ingredients with required amount in a baking pan and placed them for fermentation (75 minutes). After that we remixed that dough for 25 second and then placed that dough in the oven for baking (25 minutes at 130oC). The loaves of the bread were packed in polypropylene for shelf life study and kept at room and refrigerator temperature. In the next day we analyzed the moisture of the bread by moisture analyzer and then total acidity of the bread. Sensory panel evaluation for appearance, crust color, aroma, taste and overall acceptability was done next day by the panel of six semi trained judges on five point hedonic scale.

Figure 3.4 Bread preparation with different concentration of psyllium husk

Figure 3.5 Bread preparation with 0 % addition of psyllium husk

Figure 3.6 Bread preparation with 1 % addition of psyllium husk

Moisture analysis of bread

Moisture content of bread samples were analyzed by thermo moisture analyzer. Bread was prepared from refined wheat flour mixed with Psyllium husk at replacement levels of 1%, 1.5% and 3% w/w %. Bread samples incorporation with psyllium husk with different fractions were weighed about 5g of each and then temperature in the apparatus (Infrared AND ML.50) was set as 130˚C and moisture of the samples were determined in percentage (%) after different intervals. After the moisture content in percentage was obtained then values were described on the bar chat.

Total acidity of bread

Total acidity of bread samples were determined by titration method. Bread samples were prepared by weighing about 25g of each sample to be analyzed and placed them in glass jars. After that we added about 30 ml out of 250 ml of distilled water required for determination of acidity. In the next step we mixed the contents very well with glass rod in order to make a homogenized mixture then left this solution for 5 minutes. From this solution we took 50 ml of mixed bread sample solution in a clean glass beaker. We added the 3 drops of phenolphthalen solution and titrate with 0.1 N NaOH until the pink color of solution was obtained. We repeated the process for the same sample and recorded the ml of NaOH used.

Titratable acidity of bread calculated as:

V= volume of NaOH 0.1N used in titration (ml)

m= weight of bread sample (g)

Shelf life of bread

Shelf lives of selected levels of psyllium on the basis of bread making were studied. Bread samples were analyzed for apparent spoilage by visual observation for mold growth at ambient temperature (20-22 OC) and refrigerator temperature (4 OC). Bread samples were packed in polypropylene bags for observation of shelf life study. During storage visual examination was done over the period of seven days at regular intervals.

Sensory evaluation

The bread samples were packed in polypropylene bags for observation of bread quality through sensory evaluation to the next day of bread preparation. Sensory evaluation of bread quality for appearance, crust color, aroma, taste and overall acceptability was done next day by a panel of semi-trained judges.

Figure 3.7 Bread preparation with 1.5 % addition of psyllium husk

Figure 3.8 Bread preparation with 3 % addition of psyllium husk

Figure 3.9 Bread preparation with three fractions of psyllium husk

Figure 3.10 Moisture % and weight analysis of bread samples

Figure 3.11 Total acidity by titration of bread samples

Drink preparation

For the preparation of refreshing drink (Sharbat), Basil seeds, sugar and Psyllium husk purchased from local market and organic shop respectively. The basil seeds carefully cleaned to remove the dust, stones and chaff. To perform a scientific and systematic work with regard to preparation of refreshing drink, the effect of three fractions of basil seeds (0.5%, 2% and 5%) and in combination with psyllium husk fractions (1.5%, 1% and 0.5%) investigated on preparing the desirable drinks. Therefore, refreshing drinks was prepared using the 5% sugar solution in distilled water. Until now we analyzed the pH of the drinks using pH meter (ORION 2 STAR PH Benchtop Thermo Electron Corporation). After the second day of observations we performed the sensory evaluation of drinks by using 5 level from left to right representing unacceptable, relatively acceptable, good, very good and excellent. We presented the prepared drinks for evaluation in front of semi-trained panelists in this field.

Figure 3.12 Sweet basil seeds and psyllium seeds and husk for refreshing drink preparation

Figure 3.13 Refreshing drink preparation with three fractions of basil seeds (0.5%, 2% and 5%)

Figure 3.14 Refreshing drink preparation with three fractions of basil seeds (0.5%, 2% and 5%) and combination with Psyllium husk (1.5%, 1% and 0.5%)

Determination of pH

The pH of drink was determined by using a pH-meter (ORION 2 STAR PH Benchtop Thermo Electron Corporation). The pH of each sample was determined by immersing pH-meter rod in drink samples one by one and after each sample measurement carefully cleaned the rod with distilled water. Before this determination we placed the drink samples for thermostat at 20 °C (room temperature).

Figure 3.15 pH determination of refreshing drink with three fractions of basil seeds (0.5%, 2% and 5%) and combination with Psyllium husk (1.5%, 1% and 0.5%)

CHAPTER 4

RESULTS AND DISCUSSION

From the history to date, plants play the crucial role for both human and animals as source of food, nutrition and health care and also have significant participation in drugs preparation. On the base of present situation of increasing human population day by day and shortage of fertile land, there is a strong need to explore high-quality but cheap sources of protein and energy in order to alleviate hunger and nutrient deficiency of daily life . Now it is responsibility of government agencies make their efforts to search for new resources of food and nutrition in many parts of the developing world. Agricultural lands and forests are important for local community as a source of food products and also take part in social and economic welfare of people of that community. Shortage of food is a serious problem for excessive growing population, what we need for future based on availability of rich resources .

In the current study, it was aimed to determine the proximate composition of the selected medicinal plants cultivated in various parts of Pakistan and Romania and is important for local communities (Table 1). Besides that, we assessed the nutrient contents of these species to know whether the intake of these nutrients from selected medicinal plants is not harmful.

Physical characteristics of seeds of selected medicinal plants

Morphological characters such as colour, odour, taste, shape and size and weight of selected seeds were determined. There was a single characteristic common between both seeds. They become sticky and gelatinous when soaked the seeds in water and milk under the appropriate temperature for the formation of their jelly like material of both seeds. Based on the present observations under the microscope the color of the Sweet basil were found to be black and on the other hand seeds of Psyllium were found to be pinkish gray to brown in color. The visually examined the seeds of selected medicinal plants showed that the shape of both selected seeds are quite different from each other. The shape of seeds of Sweet basil was of ellipsoid/tear like whereas seeds of Psyllium were of ovate type shape as reported in the present study (Table 4.1, Figure 4.1).

Table 4.1 Morphological characteristics of seeds of selected medicinal plants (Sweet basil and Psyllium)

Sweet Basil seed (Ocimum basilicum) and Psyllium (Plantago ovata)

Under the microscope examination (Axiostar plus, Zeiss) there was no significant difference between seeds from two different regions (Sweet basil from Romania and Pakistan and Psyllium from Romania and Pakistan). According to present study on physical characteristics of seeds of selected medicinal plants, the size (length and width) of seeds were varied in terms of length and width as Sweets basil seeds contained length in the range of 2.14 mm to 2.24 mm and width in the range of 1.14 to 1.15 which were quite similar within the ecotypes of Sweet basil seeds from Romania and Pakistan. Whereas in seeds of Psyllium length were found to be the in the range of 2.49 mm to 2.58 mm and width were in the range of 1.29 to 1.36 which were also quite similar range of length and width with the ecotypes of Psyllium from Romania and Pakistan (Figure 4.2).

Figure 4.1 Microscopic examination of seeds of Psyllium and Sweet basil

Moreover the seeds of selected medicinal plants were also analyzed for 100-seeds weight (g) on the weight balance (Partner, WAS220/C/2) which has maximum weight calculation capacity was 220g and minimum was 10 mg. The results for weight were found to be in the range of 100 seeds weigh between 0.1192-0.1603 for Sweet basil from Romania and 100 seeds weigh between 0.1810-0.1708 for Sweet basil from Pakistan whereas in seeds of Psyllium were in the range of 100 seeds weigh between 0.1529-0.1609 for Psyllium seeds from Romania and 100 seeds weigh between 0.1148-0.1107 for Psyllium seeds from Pakistan (Table 4.1). Until now there was no scientific study was found on the physical characteristics of seeds of selected medicinal plants such Sweet basil and Psyllium collectively. But the comparable results with our study for physical characteristics of Basil seeds was also reported by which supported the present study in scientific way.

Figure 4.2 Size of seeds (length and width) of Sweet basil and Psyllium

Furthermore, other parameters of physical characteristics were also found such as taste and odour which were quite similar for both seeds of selected medicinal plants as described in this study. Both seeds had similar characteristics properties when soaked in water formed mucilaginous material with no characteristic odour. Scientific studies have established that both seeds have mucilaginous characteristics with reasonable amount of hemicelluloses and cellulose which were considered as rich source of fiber and other nutritional components essential for human body functions. As mucilage is most commonly found plant ingredient with a wide range of application in food and non food industries . On the basis of scientific studies Psyllium mucilage has a long history of uses as fiber supplement being reported as gel forming natural polysaccharide which is medicinally active material used for the treatment of diabetes, obesity and remediation of constipation and diarrhea .

But there was no proper scientific studies have found in the literature for mucilage from seeds of Sweet basil with nutritional benefits applicable in food industries which was the main concern of our study could be used in replacement of Psyllium mucilage as a source of fiber in the food and non food industries.

Table 4.2 Taxonomy of selected medicinal plants in order to find relationship between both species

From the above taxonomic classification of selected medicinal showed that they have close relationships in terms of functional properties and might be evaluate those properties with careful investigation on scientific basis

Moisture content by Oven dry method

The morphological characters such as texture, taste, appearance and stability of seeds depend on the amount of water they contain. As the moisture content depends on the environmental conditions such as humidity and temperature in growing period, harvest time, climate as well as storage conditions, knowing this is necessary to predict the behavior of seeds during processing. Thus it is important for scientific community to be able to reliably measure moisture contents of the various medicinal plants materials. Among the analyzed samples, was found to contain the lowest moisture content in Basil seeds by dry oven method whereas the highest moisture content was found to be present in seeds of Psyllium (Table 4.3 and Figure 4.5). PR and PP were found to contain comparatively higher values of moisture content and were significantly different from the seeds of Basil plants.

Table 4.3 Moisture content analysis of seeds of Ocimum basilicum (Basil) and Plantago ovata (psyllium) through oven dry method

Moisture (%) = (W1-W2) x 100 * W1: wt. (g) sample before drying W2: wt. (g) sample after drying

W1

Figure 4.3 Moisture content (g) of selected medicinal plants

Among the analyzed samples, seeds of Psyllium (Romania) and Psyllium (Pakistan) were found to contain the highest moisture content (9.7 % and 9.6%) respectively as compared to the Basil seeds (Romania) and Basil seeds (Pakistan) 9.19% and 6.83% respectively by oven dry method whereas these values for moisture contents comparable to those reported for Indian wild legume seeds varied from 5.7 to 8.5% . Basil seeds showed the lowest moisture content (6.83%) as compared to Psyllium seeds which was relatively closer to the reported values in Basil seeds (5.20%) because its seeds retain less moisture content .

Figure 4.4 Moisture content (%) of selected medicinal plants

Moisture content was observed from 6.83 to 9.7%. Sweet basil seeds from Pakistan contained the lowest amount of moisture (6.83%) while Psyllium seeds from Romania contained the highest amount of moisture (9.7%) by oven dry method (Figure 4.5). The variation in moisture content might be due to difference in climatic conditions and storage of seeds. According to the authors of reported study Plantago ovata contained moisture content as 82% on whole plant basis including stem, leaves and roots but there was little evidences were found for moisture contents in the seeds of selected medicinal plant of our study.

Figure 4.5 moisture content (%) comparison among seeds of selected medicinal plants

Based on these observations, the present study was supported by described the moisture contents in seeds of Sweet basil (9.63%) which is comparable with moisture content in seeds of Sweet basil from Romania (9.19%) by oven dry method.

Moisture content by Thermo moisture analyzer

The commonly accepted method for estimation of moisture content is oven dry method at 105°C for 3 hours but it is most lengthy and time consuming for evaluation of moisture contents in seeds. On the other hand use of a moisture analyzer (ML-50, A & D Company, Limited, Tokyo, Japan) give the instantaneous estimation of moisture content as compare to oven dry method. In the present study, we applied both methods for estimation of moisture content in the seeds of selected medicinal plants and from these observation we concluded that there was no significant difference in moisture contents as compared the both methods for clarification of results on the basis of both methods.

Figure 4.6 Thermo moisture analyzer

The results found for moisture contents by use of moisture analyzer were described in Table 4.4. The values of moisture contents for Psyllium seeds from Romania and Psyllium seeds from Pakistan were found as 7.8% and 7.7% respectively which showed that there was no significant difference between seeds of ecotypes for moisture contents (Figure 4.9). On the other hand percentage of moisture contents for Sweet basil seeds from Romania and Sweet basil seeds from Pakistan were found as 9.7% and 7.0% respectively which showed that there was a significant difference between seeds of ecotypes for moisture contents, this might be due to climate condition and way to keep and storage of seeds in Romania.

Table 4.4 Moisture content analysis of seeds of Ocimum basilicum (Basil) and Plantago

ovata (psyllium) through thermo analyzer* method

* Thermo moisture analyzer infrared: AND ML.50 A & D Company Japan

However, no significant difference was observed between the values of moisture content analyzed by both methods (Table 4.5).

Table 4.5 Moisture content (%) comparison between seeds of Ocimum basilicum

(Basil) and Plantago ovata (psyllium) by both oven dry and thermo analyzer* method

* Thermo moisture analyzer infrared: AND ML.50 A & D Company Japan

According to National Research council, moisture content of 5-20 % in dry matter is considered as recommended range and regarded as high. In the present study the moisture content of seeds of selected medicinal plants analyzed by both methods were in the recommended range of moisture contents. On the basis of these results conclusion can be assessed that seeds of these selected plants might be good source of mineral nutrients as additional functionality for food industries.

Figure 4.9 Moisture content (%) of selected medicinal plants by thermo moisture analyzer

Looking at the results of moisture contents in Sweet basil seeds of selected medicinal plants it was comparable with the reported study of . A careful review of scientific literature have shown that little data and information regarding moisture content by both methods (oven dry and moisture analyzer) for seeds of selected medicinal plants cultivated in Romania and Pakistan.. Therefore, the present study was planned to investigate the moisture content in seeds of Psyllium and Sweet basil by both methods (oven dry and moisture analyzer) for comparison analyses.

Ash content of selected medicinal plants

The ash content, which is an index of mineral contents in biota, considered as the most important element of the nutrients under study. The ash contents in different parts of plants represent mineral constitutes which are essential for the human body functions. The ash contents in the seeds of selected medicinal plants were in the range of 2.001% to 7.26% while seeds of sweet basil from Romania contained the highest ash content (7.26%) as compared to seeds of Psyllium from Romania and Pakistan (2.001% and 2.41% respectively). The highest values of ash contents in the seeds of basil might be due to the certain quantity of minerals as compared to seeds of psyllium as reported in the present study (Table 4.6, Figure 4.12).

Table 4.6 Ash content analysis of seeds of Ocimum basilicum (Basil) and Plantago ovata (psyllium) through oven dry method

Ash (%) = Weight of Ash x 100

Weight of sample

The data indicated that seeds of Psyllium from Romania contained low ash content (2.001%) as compared to the seeds of Psyllium from Pakistan (2.41%). Whereas ash contents in the seeds of Sweet basil showed the highest values (7.26 %) as compared to seeds of Sweet basil from Pakistan (2.34%).

Figure 4.10 Oven for ash determination (Controller B170 Nabertherm)

In the determination of ash content in seeds our findings were in according to the results of Mathews et al., (1993) reported ash content in the seeds of basil were 7.70 % which was quite similar to the present study (7.26%). The results of our study were also supported by other scientific study on basil seeds for ash contents (6.23%) which was closed to the present results (Figure 4.12).

Figure 4.11 Ash content (g) of selected medicinal plants

From the comparative assesssment of seeds for ash contents, results showed that sweet basil seeds might be significant source of mineral contents as compared to Psyllium seeds.

Figure 4.12 Ash content (%) comparison among seeds of selected medicinal plants

Although, uptake of medicinal plants as a whole or in parts are of great interest in terms of nutritional point of view. Thus the effectiveness of medicinal plants for curative purposes can be assessed by estimation of organic constituents in treating various diseases. Moreover, nutritional evaluations of such kind of plants are very much important to find out any shortcomings in the daily food.

Figure 4.13 Ash content (%) of selected medicinal plants

Mineral matter of selected medicinal plants

Ash is considered as the total mineral or inorganic contents of the samples analyzed. Total mineral of selected medicinal plant (sweet basil and Psyllim) estimated by standard method through calcinations with sample weight difference we found the same results as sweet basil contained the high amount of mineral (6.5 %) as compared to psyllium seeds (3.4 %) presented in Figure 4.14.

Figure 4.14 Mineral matter percentage (%) comparison between seeds of Sweet basil and Psyllium

Crude fiber content of selected medicinal plants

Ocimum seeds have characteristics properties as a source of mucilage which contains a reasonable amount of hemicellulose and cellulose, accounting for their hydrophilic character. They are high in fiber and associated nutritional properties, and can be considered as a new non-conventional source of fiber. The importance of fiber in the diet is very well established . According to the scientific literature, Basil seeds as new sources of dietary fibre have already been studied in detail . However, there is a continuous search for a newer source of dietary fiber is ongoing. In Pakistan, traditionally seeds of Ocimum basilicum (locally known as Belangu) have been used in the treatment of dysentery and diarrhoea. These seeds are mucilaginous and are more popularly employed as a thickened infusion in water or milk to form a refreshing Preparation called 'Sharbat'. No systematic studies of the major food constituents of these seeds have been so far reported.

The present work reports on the preliminary findings obtained during investigation on constituents. Both the seeds of current study were a valuable source of fiber and energy. Psyllium (Isabgol) Seeds also contains mucilage substance, which is about 30% of its total weight. Mucilage is made of soluble fiber called polysaccharides. Isabgol seeds contain fiber content 19% and proteins 18.8%. Furthermore psyllium husk contains 75 to 78% fiber from which 70% is soluble fiber and 30 % insoluble fiber according to the study of literature. In view of the increasing demand for protein and energy to support the growing world population, researchers have directed their efforts at exploring new and nonconventional sources of food that can cultivate in the arid and semiarid land regions of the world.

Figure 4.15 Crude fiber (g) of selected medicinal plants

In the present research work on seeds of Sweet basil and Psyllium, for the estimation of crude fibers we have applied two methods for comparison analysis of fiber contents in seeds of selected medicinal plants. The first method we applied for estimation of crude fiber which is originally applied for cellulose extraction from wheat flour. As cellulose originates from the wheat grain shell is a form of fiber. If fiber is present in great quantity worsens the attributes of the dough and the quality of the products. This method of fiber analysis worked on the following principle such as dissolving and removing soluble components from a quantity of material used and gravimetric (by calcination) method is used and starch in the material by boiling with acids turns into glucose. The albumin in an acidic medium, when boiled, is transformed (in part) into the acidic amide; the rest is treated with a base. The largest mineral substances melt. The insoluble residue dries and turns into ashes.

Table 4.7 Fiber content analysis of seeds of Ocimum basilicum (Basil) and Plantago ovata (psyllium)

Fiber content (%) = (Wt. of crucible with residue + wt. offilter paper with residue) – (Wt. of crucible + wt of filter paper x100

Weight of sample

From this method of fiber analyses we found crude fibers in seeds of selected medicinal plants in the range of 10.47 % to 5.88 %. From the findings of this study it was observed that Psyllium seeds from Romania contained reasonable amount of fiber content (10.47 %) as compared to Psyllium seeds from Pakistan (9.35 %) whereas sweet basil seeds from Romania contained lowest amount of fiber content (5.88 %) as compared to other ecotype seeds of sweet basil from Pakistan (6.43 %). The data for crude fiber (%) presented in the Table 4.7. According to comparison evaluation of fiber analysis in our present work it was found in the results obtained from the fiber analysis that Psyllium seeds (10.47%) contains the highest content of crude fiber followed by sweet basil (6.43%), on the other hand between the ecotypes of psyllium and sweet basil, there was no significant difference (Table 4.7, Figure 4.16).

Figure 4.16 Fiber (%) composition of selected medicinal plants

As a nutritive value of food, fibers in the diet are necessary for digestion and for effective elimination of wastes, and can lower the serum cholesterol, the risk of coronary heart disease, hypertension, constipation, diabetes, colon and breast cancer . Thus on the comparative assessment Psyllium can be considered as a valuable source of dietary fiber in human nutrition during malnutrition. The higher fiber content in seeds is due to the presence of cellulose which is beneficial for the absorption and passage of food items in alimentary canal.

Figure 4.17 Fiber percentage (%) comparison among seeds of selected medicinal plants

Our findings were supported by the scientific study of . On other hand we applied acid base digestion method for estimation of crude fiber in seeds. Crude fiber was measured by treatment of the samples with 1.25% H2SO4, 1.25% NaOH and then 1% HNO3, filtered and washed with hot water after each step. The residue obtained was dried in oven at 130 șC and heated at 550 șC in furnace. The loss in weight on ignition was expressed as content of crude fiber . From this method, we obtained different results for both seeds of selected medicinal plants but the findings were the same as Psyllium seeds contained highest crude fiber (3.2 %) as compared to seeds of sweet basil (2.1%) presented in the figure 4.17 in a percentage quantity (%).

Figure 4.18 Fiber percentage (%) comparison between seeds of Sweet basil and Psyllium

On the basis of these findings, our results for crude fiber (5.88 % and 2.1%) for basil seeds were in against of reported study on basil seeds as crude fiber were found as 22.6% by both methods were quite different .

Protein of selected medicinal plants

In case of protein content, Sweet basil had higher amount of protein content compared to Psyllium (Table 4.8). It has been reported that protein-calories malnutrition deficiencies is a major factor responsible in nutritional pathology . According to , plant food that provide more than 12% of its calorific value from protein are considered good source of protein. Furthermore, adults, pregnant and lactating mothers required 34-56 g, 13-19 g and 71 g of protein daily respectively . The results of this work showed that adequate amount of protein are present in sweet basil seeds (Figure 4.18).

Figure 4.19 Protein percentage (%) comparison between seeds of Sweet basil and Psyllium

Results regarding proximate analysis of seeds of selected medicinal plants are presented in Table 4.8.The results showed that Sweet basil seeds contained high protein contents (29%) as compared to Psyllium seeds (24.5%). These findings suggested that sweet basil seeds with rich source of protein contents might be beneficial as nutritional point of view as compared to seeds of Psyllium. The protein content of Sweet basil and Psyllium seeds was calculated on the basis of the available nitrogen using Kjeldahl method and was observed in the range of 29 % to 24.5 % respectively. According to the daily recommended protein values for men and women are from 14.5 to 53.3 % in the ration. Based on this fact, the seeds of Sweet basil were considered as good source of proteins as compared to Psyllium seeds.

Table 4.8 Quality analysis of seeds of Sweet basil and Psyllium

Information regarding biochemical composition of Basil seed and Psyllium (Plantago ovata)

*NFE i.e. Nitrogen free extract

According to study of literature, we found basil seeds contained crude protein content 11.4 % which were in against to our findings . This difference of protein values in percentage may be due to climatic conditions which greatly affects on nutritional qualities of seeds.

Figure 4..4.20 Percentage (%) comparison among nutrient contents of selected medicinal plants

Moreover, modern food technology is now interested in nutritionally significant plants extracts. Present study was also conducted to enhance traditional knowledge of selected medicinal plants further and is focused to investigate nutritional values in order to fulfill the needs of consumers for preparation of nutritional rich food products. Furthermore, crude proteins for Psyllium seeds were correlated with the findings of . They reported crude protein in Plantago ovata in both leaves and seeds as 21.87 % and 13.12 % respectively.

Nitrogen free extract evaluation

Nitrogen free extract (NFE) was calculated according to the following expression:

NFE % = 100- (moisture contents % + crude protein % + crude fat % + crude fiber % + ash %). We obtained the results for both seeds of sweet basil and psyllium presented in the Table 4.8.

Fats of selected medicinal plants

The samples of seeds (Sweet basil and Psyllium) were analyzed for estimation of chemical compositions in order to find the percentage of protein, fats, fibers and carbohydrate specifically for comparative assessment of both plants under the standard methods of AOAC.

Based on the observations of current study for estimation of fats contents (Lipids), Sweet basil seeds contained low fat contents (15.36%) in comparison with Psyllium seeds (16.55%) but there was not great difference in the values of fat contents in both selected seeds of Sweet basil and Psyllium (Figure 4.20).

Figure 4.21 Fats (%) comparison between seeds of Sweet basil and Psyllium

According to the authors of reported work, seeds of sweet basil possessed fat content in perecentage of 20.2% whereas crude fats was reported in Psyllium seeds as 43.2% which was not in aggreement with our findings .

Moreover results in other scientific study supported our finding for fats content in the seeds. They reported the fat contents in sweet basil seeds as 13.8% which was closer to our findings for fat contents in Sweet basil seeds . Fats (lipids) are a good source of energy in nutritional point of view as one gram of lipids provide 9 Kcal of energy. The resulted indicated that both the seeds of sweet basil and psyllium might be good source of lipids.

Total carbohydrate of selected medicinal plants

Carbohydrates, fats and proteins are the essential nutrients of life. The quality and quantity of proteins in the seeds are basic factors and important for the selection of plants for nutritive values. Carbohydrates are one of the important source of energy and a major component of daily food consumption. The carbohydrate content of analyzed samples suggested that the seeds of Psyllium have the highest amount of carbohydrates (47.19%) as compared to seeds of Sweet basil (42.5%). According to , recommended ration of the carbohydrates to the energy in a food is from 55 to 75%. Although, the range of carbohydrates of both selected seeds were lower than the reported value of , however, seeds of both plants can be used as a source of energy contribution in a food chain.

Carbohydrates are the principle and primary source of energy in the body. Looking at the results of present study, we observed that lower carbohydrate contents of sweet basil seeds might be due to lesser amount of dietary fibers in the seeds. The dietary fibers contain the cellulose, hemicelluloses, gums and mucilage which are considered as indigestible carbohydrates. Human dietary fibers mainly come from plant cell walls in seeds, fruits and vegetables .

Figure 4.22 Total carbohydrate percentage (%) comparison between seeds of Sweet basil and Psyllium

According to scientific work of leaves of basil contained the carbohydrates (66.65%) which were not correlated to our findings. This difference of carbohydrates was due to source of carbohydrates from leaves of plants not from seeds until now there was no proper study was conducted on the seeds of sweet basil for assessment of total carbohydrates whereas Plantago ovata leaves and seeds contained the carbohydrates in the percentage values of 15.9 % and 8.4 % respectively reported by which was not in accordance to our observations.

Total Energy values of selected medicinal plants analyzed

According to the results of the energy calculations based on the values of carbohydrates, proteins and fats, the highest values was found in the Psyllium seeds (435.71 kcal) whereas basil seeds contained energy values (424. 24 kcal) presented in the Table 4.9.

Table 4.9 Quality analyses of seeds of Sweet basil and Psyllium

Information regarding biochemical composition of Basil seed and Psyllium (Plantago ovata)

Figure 4.23 Total Energy values percentage (%) comparison between seeds of Sweet basil and Psyllium

We concluded from these observatons, psyllium seeds had highest energy values might be due high percentage of fibers, fats and carbohydrates as compared to swet basil seeds that had only protein contents in higher quantity in comparison with psyllium seeds. According to the reported work of on different medicinal plants which were commonly used as anti-inflammatory, antiviral, antimicrobial and as laxative like our selected medicinal plants, results for energy values were in the range of 389.20 kcal to 331.50 kcal considered as good source of energy that was close to our findings. Moreover, under the review of literature we could found the exact scientific study related to our current work.

Figure 4.24 Proximate composition (%) of selected medicinal plants

Conclusion on the basis of nutrient analysis of seeds

In addition to the medicinal values of selected plants, aim of current study was to assess the nutrient profile of seeds of selected medicinal plant whether the intake of these seeds is not harmful. In view of nutrient composition of seeds of psyllium and sweet basil as sole source of nutrients and can be used as potential source of nutrient elements in the diet. The seeds of selected medicinal plants analyzed using the standard methods , findings showed that seeds have adequate amount of nutritional qualities. The seeds of sweet basil have sufficient amount of protein and mineral matter as compared to psyllium seeds. These findings seem to be good for health purposes. This study further indicated that seeds of psyllium are considered as rich source of fiber being abundant in energy values whereas excellent source of fiber too as compared to sweet basil. These promising results are encouraging to do further investigation on comparative basis to explore this vast field of interest for scientific community in different sector might be used for various purposes.

Survey results of selected medicinal plants for consumer response

While evaluating results of present study’ opinion and feedback of consumers really matters. Ideal way to collect this feedback is to deploy in a best suited places and make novel products from these seeds to distribute in that places so they can practically experience how new food product effects their everyday life, how much they can get energy and fiber supplement from such food products, what level of ease or difficulty they face while consuming a novel fibrous food, and overall how it can effect human activities. Although it was not possible but as an alternative we conducted a questionnaire based survey by providing respondents with supplementary information about the proposed work and its application, questionnaire is given in annex 1. We distributed this questionnaire in local community and academia people who have knowledge about these plants. Although a few of them responded yet we analyzed their responses and this analysis is given here. On the basis of survey results we assessed the indigenous knowledge of the local community of Southern; Punjab for what purpose they consumed these seeds, what was the source of knowledge for utilization of these selected plants and which age group of people utilized the seeds of sweet basil more frequently as source of fiber supplements according to their opinion. For the purpose of our study, we came to know through this survey analysis the need of consumers for marketable products preparations.

Figure 4.25 What is source of traditional knowledge among surveyed respondents?

Figure 4.25 demonstrated that source of traditional knowledge about selected medicinal plants under surveyed. The results showed that 77 % surveyed people attained the traditional knowledge about sweet basil and psyllium for what purpose these plants used and consumed for health problem due to seasonal variations from their family members and 15 % people gained that knowledge from careful scientific study of selected medicinal those surveyed people might be belonged to that field of interest. We conclude from these observations, traditional knowledge about these selected medicinal plants still in the pipe line of scientific analysis.

Figure 4.26 Surveyed respondents awareness of fibrous food intake in relation to age factor

Figure 4.26 represented surveyed respondents awareness of fibrous intake in relation to age factor. The results showed that most of the people (87.50 %) who used these plants as a source of fiber were above age of 40. Many of the people under the age of 40 were at least aware of their existence but did not use medicinal plant more frequently as compared to other group of people. Some of them were unaware that actually these medicinal plants use as source of fiber which is due to lack of knowledge in this category of surveyed people.

Figure 4.27 What is preference of fibrous food intake among surveyed respondents?

From the above Figure 4.27, we came to know that (87.5%) most of surveyed people used these plants as source of fiber for cure of digestive problems but preference of intake was in the form of drinks which can be easily prepared in the home and has additional property of cooling in the summer season. For example Psyllium husk is commonly used by the people for laxative purpose and basil seeds soaked in water and sugar added drinks traditionally used by the people for cooling effect in summer. But until now there is no scientific study conducted to check the efficacy of these plants in this regards.

Figure 4.28 Relationship between respondents for what purpose they used seeds of selected medicinal plants

From the Figure 4.28, results indicated that 33.3 % surveyed people used the psyllium husk as a medicated purpose whereas sweet basil seeds used by the people (44.2%) for recreation purposes. There is need to make more scientifically proven usage of these plants in daily life for both purposes such as medicinal and food.

Figure 4.29 Frequency (%) distribution of usage of selected medicinal plants among respondents

According to the above Figure 4.29, we concluded that frequency of usage of these selected medicinal was fall in category of once in a month if they feel to have some change from counter the medication for cure of common ailments. Based on these observations, 33.3 % people used these plants once in a month and then 22 % people used these plants once in a week. In our opinion, this long interval frequency of usage might be due to unaware of proper benefits of these plants and they were reluctant to consume them more frequently.

Figure 4.30 Respondents opinion about gardening of selected medicinal plants in home

Accorrding to the findings of above question described in figure 4.30, the respondents have also been asked about gardening of selected medicinal plants. The opinion of respondent was assessed by two catogories i.e. YES or NO. By this binomial type of question, we assumed that only sweet basil plants contain more yes for gardening of this plant in home for that might be due to traditional purposes. As there is huge scietific literature found for useage of sweet basil plant for tradtional and ritual bases recoreded from history to date. This depicted the importance of selected medicinal plant (sweet basil) more specific. According to the results of above question, 33.3 % was in the favour of gardening the sweet basil in home not only for the pleasant fragnance but also for ritual and recreational purposes. On the other hand the less people were agreed to grow psyllium plant in home.

In the last and the foremost question in our survey was to know about the people opinion which kind of grains they preferred to consumed as a source of nutrients. Under surveyed people 66.6 % people preferred to consumed whole grains as compared to refined grains. Many of them preferred to consume both grains as whole and refined. But majority of surveyed people preferred to consume whole grains because of complimentary nutrients are integrated in the whole grains. The purpose of this question was to analyses the response of people for better preparation of novel food products utilizing whole grains more preferably according to the need of consumers.

Figure 4.31 Respondent opinion about what type of grains they consumed as a source of fiber

Conclusion on the basis of survey analysis

According to observations of survey analysis, we concluded that in study area, the indigenous knowledge about consumptions of selected seeds is in the hands of elder community and transfer from them generation to generation. Our study has a contribution in enhancing or documenting the existing indigenous knowledge belonging to this local community because the knowledge is losing day by day due to exposure to modernization. The results show that people over the age of 40 were actively utilizing the seeds both medicinal and creational purposes. In the study area, 87.5% of surveyed people used these plants as source of fiber for cure of digestive problems but preference of intake was in the form of drinks which can be easily prepared in the home and has additional property of cooling in the summer season. There is need to design the research projects to analyze comprehensively the indigenous knowledge to make the better and effective use of the plants on scientific basis.

Novel Product presentation by using seeds of selected medicinal plants

In all over the world including developing countries mostly consumed foods are usually cereal based products such as breads as they are rich source of energy and other essential nutrients. According to these observations we prepared cereal based product enrich with fiber supplements. On the other hand, we prepared refreshing drinks with different fractions of sweet basil seeds and in combination with psyllium husk according to the needs of consumers. The results of our study are given below after performing some required analysis.

Evaluation after Bread preparation

For the preparation of bread we utilized the following ingredients; Flour (refined wheat flour) 100g, compressed yeast 3g sugar 2.5g, salt 1.5g and distilled water optimum. Refined wheat flour mixed with Psyllium husk at replacement levels of 0%, 1%, 1.5% and 3% w/w %. The present study was conducted by incorporating the psylliu husk at levels of 0%, 1%, 1.5% and 3% in refined wheat flour on percentage basis for preparation of bread making. The specific objectives of the study were to analyze the moisture content, total acidity, shelf life and quality of bread by sensory evaluation.

Moisture content

Moisture content of the bread was increased with increase in the levels of incorporation of psyllium husk. This absorption could be attributed to its high hydration capacity. Up to 1.5% the dough handling was smooth but at 3% level of incorporation of psyllium husk the dough handling was difficult as compared to other fractions under the observations of present study. Our findings were supported by the work of they reported that handling of dough was hard due to its gelling and water absorption properties.

Figure 4.32 Moisture % of bread with three fraction of psyllium husk (1%, 1.5% and 3%)

From the results of moisture content analysis we found that addition of psyllium enhanced the moisture content of bread as compared to control. Bread with 3% psyllium husk contained higher moisture (31.4%) than the other fractions 1% psyllium husk bread contained (26.9%) and 1.5% psyllium husk bread contained moisture as 30.4%. According to the observations we concluded that increased moisture was due to psyllium husk water absorptions.

Total acidity of bread

From the observations of total acidity of bread samples we found that with addition of psyllium husk in bread reduced the acidity of bread to some extent as 3% psyllium husk in bread reduced the acidity of bread from 1.4 to 1.2% which might be due to neutralizing characteristics of psyllium husk when combined with other substances. The degree of acidity is directly related with the activity of yeast and control the growth of many other microorganisms. The growth and activity of microorganism in turn influences acidity of dough and of bread making.

Figure 4.33 Total acidity of bread with three fraction of psyllium husk (1%, 1.5% and 3%)

According to the scientific study of , acidity in terms of hydrogen ion concentration of bread is related to acid combing power of cereals used, growth and activity of yeast which varies with the acidity of medium whereas modified the conditions of fermentation.

Shelf life of bread

Bread samples were analyzed for apparent spoilage by visual observation for mold growth under room and refrigerator temperature. During the storage, spoilage of breads was observed due to mold growth. Up to the 5th day of observation there was no spoilage due to mold growth at room temperature. On the 6th day of storage, spoilage of bread was observed in control bread as well as other bread with 1% psyllium husk. On the other hand spoilage due to mold growth was observed on the 7th day of storage study in all fractions of psyllium husk except in 3% psyllium husk bread.

Table 4.10 Apparent spoilage (visual observation for mold growth) at room temperature (20-22 OC)

Moreover, at refrigerator temperature during storage, spoilage due to mold growth was observed on the 7th day of storage study in the control and 1% psyllium bread whereas bread with 1.5% and 3% fractions showed no spoilage due mold growth even at the 7th day of storage observations.

Table 4.11 Apparent spoilage (visual observation for mold growth) at refrigerator temperature (4 OC)

According to the study of , bread made without any preservative spoiled by the fifth day of storage but in our study on the shelf life of bread showed that spoilage of bread due to mold growth was occurred at the sixth day of storage at ambient temperature whereas 3% psyllium incorporation in bread showed no spoilage at both temperature on the seventh day of storage study. According to our observations it might be due psyllium husk incorporation in the bread reduced the growth rate of mold at the storage conditions.

Sensory evaluation of bread

After the bread baking in laboratory oven, we conducted the sensory evaluation of bread samples with three fractions of psyllium husk (1%, 1.5% and 3%), we found that bread with 3% psyllium was full of fiber with soft texture, good in taste. The visual inspection showed that color of bread was dark brown due incorporation of psyllium. The flavor and appearance was not too much different from the control bread. From the findings of our study we concluded that 3% psyllium husk incorporation could produce the acceptable quality of bread which was in according to the results of .

Evaluation after Drink preparation

The effect of three fractions of basil seeds (0.5%, 2% and 5%) and in combination with psyllium husk fractions (1.5%, 1% and 0.5%) investigated on preparing the desirable drinks. Therefore, refreshing drinks was prepared using the 5% sugar solution in distilled water. Until now we analyzed the pH of the drinks using pH meter (ORION 2 STAR PH Benchtop Thermo Electron Corporation). According to findings of pH we concluded that increasing the concentration of sweet basil seeds (5%) enhanced the values of pH (6.9) approaching to neutralize the refreshing drinks whereas Psyllium husk addition up to 1.5% in basil seeds refreshing drinks interfere the pH values and reduced it from 6.9 to 6.5.

Determination of pH

The pH of each sample was determined by immersing pH-meter rod in drink samples one by one and after each sample measurement carefully cleaned the rod with distilled water. Before this determination we placed the drink samples for thermostat at 20 °C (room temperature). On the basis of observations we found different pH values or different hydrogen ion concentration in different fractions of drink preparation.

Figure 4.34 pH determination of refreshing drinks with three fraction of sweet basil seeds (0.5%, 2% and 5%) and combination with three fractions of psyllium husk (1%, 1.5% and 3%)

The results of pH showed that increasing the concentration of basil seeds in the drinks the values of hydrogen ion concentration increasing it reached 6.9 from 6.2 with increasing the levels of basil seeds concentration in the drinks. In view of present study we observed that addition of basil seeds in drink approaching to the neutralizing the drinks by increasing the values of hydrogen ion concentrations.

Sensory evaluation of refreshing drink

According to our preliminary findings, by increasing the concentration of major ingredient (sweet basil seeds), the stability of the resulted drinks also improved but encountered the other issues was unable to flow become fully gelled. Therefore based on preliminary results of study, it was realized that desirable refreshing drink needs to contain the maximum amount of sweet basil seeds 2% wt. which was physically unstable and easily sediment as a matter of few minutes. It needs to be noted that some of our consumers also preferred the presence of different flavor in the basic ingredient of the drinks to make the resulted drinks more exciting and energetic other than fulfill the fibrous requirements on daily basis. As a result, we decided to conduct another study in future to resolve the issues of desirable drink preparation such as to stabilized the precipitation of seeds by using the natural gum with optimum level as well as with different flavor formulation on the aforementioned consumers needs. Further in the study we assessed the storage capacity of the drinks both at room temperature and refrigerator temperature, we found at room temperature the resulted drinks showed spoilage with bad smell on the next day of observation whereas at refrigerator temperature it can stabilized with taste and without spoilage up to 3 days of storage.

Conclusion on the basis of fibrous food preparation

In almost all part of the world, the highly consumed nutritious food is considered as cereal based products. On the basis of this fact, the nutritional experts have paid attention to introduced novel food products with good health profile for human beings. Bread is one of the cereal based food, also part of our daily diet and considered as excellent vehicle for addition of functional ingredient like psyllium husk to increase the fiber content. The major objectives included in this part of study to optimize the level of psyllium husk in bread making process as well as to assess the quality of bread by different parameters. These findings can be easily adopted by the bakeries to prepare value added food required by the health cautious people. Moreover, for preparing the refreshing drink we assessed the pH of the drinks with different fractions of sweet basil seeds. On the other hand, we concluded what percentage of seeds is acceptable for drinking purpose with stable property. According to our observation, 2% wt of sweet basil is optimum for preparation of refreshing drink on daily basis fiber requirement whereas storage capacity was not more than 3 days on the refrigerator temperature. However, this refreshing drink has been traditionally prescribed for digestive problems apart from therapeutic applications, the refreshing drink (Belangu sharbat) are commonly used in hot season as cooling effects.

So we evaluated our work with respect to nutritional analysis of seeds of selected medicinal plants, consumer’s response about utilization of selected medicinal plants. We also compared seeds of both plants on the basis of proximate composition in order to better assess the efficacy of plants. A survey has also been discussed to represent consumers’ opinion about usage of selected medicinal plants in daily life. On the basis of survey analysis we prepared two fibrous enriched products such as bread and refreshing drink with different fractions of seeds and psyllium husk. Next chapter will conclude the current study and will discuss some future work.

CHAPTER 5

CONCLUSION, CONTRIBUTIONS AND FUTURE DIRECTIONS

Human beings benefited from plants in many ways. As plants influence human life on earth by providing basic needs such as food, shelter and basic necessities have man ever since from his birth and evolution. With time of evolution people identified other plants as a source of food, spices, oil and the selected forage and fodder for the domesticated animals that they have in their houses. Medicinal plants and food crops, since times immemorial, have been used in virtually all cultures as a source of medicine and energy.

Among the plants known for medicinal value, the plants of genus Ocimum are very important for their therapeutic potentials. Because of its popularity basil is often referred to as King of herbs, being widely utilized due to its economic, nutritional, industrial and medicinal properties. It is high value economical and industrial crop. The second focused plant of our research is Plantago ovata L. locally known as Isabgol belonging to the family Plantaginaceae is a stem less Ayurvedic herb, used in health care for many centuries in South Asia, and it is now widely used for its medicinal properties all over the world. Seed husk (rosy-white membranous covering of the seed) traditionally given as a safe laxative, particularly beneficial in habitual constipation, chronic diarrhea and dysentery.

As we already know herbs, nutrients and dietary supplement are major contributors of functional food which help to enhance the structure and function of the body. Therefore, in this work we focused to find non conventional source of nutrients by screening and comparative assessment for possible enrichment of functional food to tackle health related problems of daily life. The finding from the selected medicinal plants suggested which plant has a promising potential of soluble fibers better maintain a healthy life. Furthermore, seeds of both plants have same characteristic properties, they become gelatinous and sticky when soaked in water and absorbs water to perform characteristic functioning in terms of mucilage and a true dietary fiber source. Therefore, there is need to explore such kind of naturally occurring mucilage which are freely available, low cost and non toxic in usage as compared to synthetic materials.

Research overview

There is increasing scientific evidence that nutrition plays crucial role in the prevention of chronic diseases like diabetes, cardiovascular diseases, obesity and cancer most of them are diet related. The food not only necessary for living but also as a source of mental and physical well-being contributing to promote health and reduction of risk of a pathologic process. Generally, those foods are considered to be part of normal diet that contains biologically active components, which offer the potential of enhanced health or reduced risk of disease.

This chapter consists of the findings of this research work, contributions, future directions, and an overall conclusion.

The research objectives have been outlined in chapter 1. In the same chapter basic elements of research are discussed also.

Our emphasis during this research was on the comparative study of both plants in terms of nutritional constituents and successfully implemented in food industry for nutritious food products. In this sense, we proposed development of staple food products such as drinks and breads enriched with mucilage by food industry might be a potential contribution to a broader supply of food products with soluble fiber. Regarding these initiatives, we could find economic opportunities; government and research institutes need to play important role in financing of further research in this domain and communicating progress in other public institutes.

From the past literature we concluded, the local communities of different regions of Pakistan have centuries old knowledge about traditional uses of the selected medicinal plants (sweet basil and Psyllium) occurring in their areas. The use of plants as medicine is an ancient practice and indigenous knowledge of plants has been transferred from generation to generation. The uses of medicinal plants vary in different parts of the country due to indigenous knowledge and method of utilization. Moreover, rich diversity of medicinal plants is an important source of livelihood for majority of the rural and mountainous communities because of low cost treatment for various common diseases. Further in second chapter bioactive compounds were defined along with their essential activities with relation to the functional foods and its impacts on human health with scientific evidences, light has also been shed on the soluble fiber which were the focus nutrient of our study.

Chapter 3 discusses about proposed methodology for evaluation of proximate compositions in detail and their associated literature. As core of our proposed methodology is the quality analysis of seeds, therefore different nutrients by standard methodologies have been assessed, and on the basis of requirements of this research a survey analysis on consumer response has been conducted to have clear understanding about success of products in the market place. Further in this chapter the morphological characteristics of seeds of sweet basil and Psyllium are explained in order to succeed in value addition to existing knowledge base.

Chapter 4 was about evaluation of laboratory analysis and valuable opinion and knowledge possessed by respondent of survey. We found from evaluation, seeds of psyllium is a good source of fiber supplements as compared to sweet basil. On the evaluation of comparison analysis we concluded that sweet basil not only have the sufficient fibers but also available source of rich proteins nutrients, energy values they possessed in recommended range of .We found the both plants are rich source of nutrients and can be use in daily food products as a source of supplementary nutrients. We also found that consumers have limited knowledge about utilization of these plants and at least consumed seeds or husk on the basis of traditional knowledge gained through family resources.

Therefore, the objective of this research was not only to enhance the exiting knowledge on scientific basis and further build the idea for utilization of available cheap resources for benefit of society in health and economic point of view.

Research findings

In this section overall summary of all the practical work done and its major findings have been discussed.

Moisture evaluation: Among the analyzed samples, seeds of Psyllium (Romania) and Psyllium (Pakistan) were found to contain the highest moisture content (9.7 % and 9.6%) respectively as compared to the Basil seeds (Romania) and Basil seeds (Pakistan) 9.19% and 6.83% respectively by oven dry method. The values of moisture contents for Psyllium seeds from Romania and Psyllium seeds from Pakistan were found as 7.8% and 7.7% whereas percentage of moisture contents for Sweet basil seeds from Romania and Sweet basil seeds from Pakistan were found as 9.7% and 7.0% respectively by using thermo moisture analyzer.

Mineral evaluation: The data indicated that seeds of Psyllium from Romania contained low ash content (2.001%) as compared to the seeds of Psyllium from Pakistan (2.41%). Whereas ash contents in the seeds of Sweet basil showed the highest values (7.26 %) as compared to seeds of Sweet basil from Pakistan (2.34%). On the other hand, mineral matter (ash content %) determination by sample weight difference, we found the same results as sweet basil contained the high amount of mineral (6.5 %) as compared to psyllium seeds (3.4 %).

Fibers evaluation: From the findings of this study it was observed that Psyllium seeds from Romania contained reasonable amount of fiber content (10.47 %) as compared to Psyllium seeds from Pakistan (9.35 %) whereas sweet basil seeds from Romania contained lowest amount of fiber content (5.88 %) as compared to other ecotype seeds of sweet basil from Pakistan (6.43 %). From the other method of fiber estimation, we obtained different results for both seeds of selected medicinal plants but the findings were the same as Psyllium seeds contained highest crude fiber (3.2 %) as compared to seeds of sweet basil (2.1%) presented in the figure 4.17 in a percentage quantity (%).

Proteins evaluation: The results showed that Sweet basil seeds contained high protein contents (29%) as compared to Psyllium seeds (24.5%). These findings suggested that sweet basil seeds with rich source of protein contents might be beneficial as nutritional point of view as compared to seeds of Psyllium.

Fats evaluation: Based on the observations of current study for estimation of fats contents (Lipids), Sweet basil seeds contained low fat contents (15.36%) in comparison with Psyllium seeds (16.55%) but there was not great difference in the values of fat contents in both selected seeds of Sweet basil and Psyllium.

Total carbohydrate evaluation: The carbohydrate content of analyzed samples suggested that the seeds of Psyllium have the highest amount of carbohydrates (47.19%) as compared to seeds of Sweet basil (42.5%).

Energy values evaluation: We concluded from these observatons, psyllium seeds had highest energy values might be due high percentage of fibers, fats and carbohydrates as compared to swet basil seeds (424. 24 kcal) that had only protein contents in higher quantity in comparison with psyllium seeds (435.71k cal).

Evaluation of surveyed respondents: According to the findings of surveyed analysis we concluded that people consumed these plants based on traditional knowledge and reluctant to used them more frequently. Most of the surveyed people used Psyllium as asource of fibers to cure the digestive problems whereas sweet basil seeds used by the people only for seasonal recreational purposes without knowing its specific worth of efficacy. On the other hand, majority of respondent preferred to used whole grains as compared to refined grains. From these results we found clear idea for novel food product preparations in according to the needs of consumers.

Fibrous food preparation:

On the basis of survey analysis we prepared two fibrous enriched products such as bread and refreshing drink with different fractions of seeds and psyllium husk. From the findings of current study we found 3% psyllium husk bread has more characteristics properties as compared to other fractions and will be source of fiber for consumers. Moreover the drinks with basil seeds 5% contained the thick mucilage as a source of fiber and might be reduced the acidity of stomach in human beings.

Research contributions

The work presented in this thesis is the outcome of almost three years research activities conducted in “Lucian Blaga” university of Sibiu and Plant Pathology Research Institute Faisalabad, Pakistan. The main outcome of this research is quality analysis of seeds of selected medicinal plants performed in two sections. In the first term we assessed the proximate composition of seeds including moisture (oven dry method and moisture analyzer), ash content and then fibers content estimation of seeds by two different methods for comparative study of fibers evaluation performed in Faculty of Agricultural sciences, Food Industry and Environment Protection “Lucian Blaga” university of Sibiu, Romania. In the next section, our experimental work based on protein, fats, fibers and mineral matter analysis conducted in (Ayub Research Center) Plant Pathology Research Institute Faisalabad, Pakistan. All theoretical and practical contributions are summarized in following sections.

Theoretical Contributions

Theoretical contributions include consideration of different extraction techniques in the light of literature for their usability for different purposes, analysis of proximate composition and their methodologies and importance of functional food in respect to human health with associated literature. Highlights of theoretical contributions are given here.

Medicinal plants in Pakistan with application of traditional knowledge in detail in the light of scientific literature.

Study on selected medicinal plants Ocimum basilicum and Plantago ovata with concepts of industrial uses and efficacy of selected plants in medicine and food industry.

Different extraction techniques and importance of bioactive compound from medicinal plants with relation to human life have been described in the light of reviewed literature.

We also analyzed in detail the work of different scientific community for evaluation of functional food products with implementation of medicinal plants extracts under the standard procedures.

In the light of reviewed literature we proposed a conceptual model of research work that helps to understand the work accordingly.

Practical Contributions

Theoretical contributions mentioned above have been followed by some practical contributions. Comparative study between seeds of sweet basil and Psyllim on the evaluation of nutritional analysis is the major contribution of this study. It mainly contains the following elements.

Morphological characteristics such as size, weight, taste odour and color described in detail under the digital microscope for comparative assessment of seeds of both plants sweet basil and psyllium.

Quality analysis of seeds in terms of nutritional components beneficial for human health and further evaluation of Proteins, fats, fibers, moisture, mineral and carbohydrates in order to enhance nutritional capacity of food products also proposed in this study.

Consumers responed through surveyed analysis gave the better idea to proposed traditional consumed food produts to a market base products through scientific approaches.

Fiber analysis perfomed through different method gave the accurate estimate of fiber contents for value addition in scientific literature and might have applications in food industry.

The major objectives included fibrous food preparation study to optimize the level of psyllium husk up to 3% in bread making process as well as to assess the quality of bread by different parameters. These findings can be easily adopted by the bakeries to prepare value added food required by the health cautious people. Moreover, for preparing the refreshing drink we assessed the pH of the drinks with different fractions of sweet basil seeds. On the other hand, we concluded what percentage of seeds is acceptable for drinking purpose with stable property. According to our observation, 2% wt of sweet basil is optimum for preparation of refreshing drink on daily basis fiber requirement whereas storage capacity was not more than 3 days on the refrigerator temperature.

Scientific Contributions

We produced two journal articles (one accepted and one under review) and three international conference articles from this research work.

Future directions

In future this comparative study of seeds (sweet basil and psyllium) on basis of nutritional estimation will help to understand the scientific community the worth of these plants in terms of nutrients constituents step ahead for exploring bioactive compounds other than these evaluated in the present study. This knowledge can be used by the experts of food industries and non food industries for novel functional food preparation with further improvements. We also prepared novel food products as a non conventional source of fiber supplements by actively utilizing selected seeds of plants at preliminary stages in order to test the efficacy of selected medicinal plants (sweet basil and psyllium) in field of functional food research. This work would have been more fascinating if we could have further analysis on fibrous bread and drink with different fractions of seeds and husk of selected medicinal plants in order to better understand the significance of food products beneficial for human health. Traditional Knowledge about these selected plants with such kind of analysis make retrieval of knowledge more clear and efficient for improvement of human health. But it requires some analysis on phytochemicals for comparison of both seeds which could serve in various field of life. It would also better if we could have practically applied those extracted phytochemicals in the daily food products to enrich with healthy beneficial nutrients. It would not only helpful for scientific community to attain more accurate knowledge and results but also increasing wealth of economy in different ways. Right now we keep it for future work.

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ANNEXES

ANNEX 1. Survey Questionnaire

ANNEX 2. Mucilage from Psyllium and Sweet basil seeds

Mucilage in relation to selected medicinal plants with scientific observations

ANNEX 3. List of Publications

Before PhD

JOURNAL PUBLICATIONS

Growth Responses of Plantago ovata L. to varying levels of NaCl by Rehana Khaliq, Maria Zahoor, Zafar Ullah Zafar, Habib-ur-Rehman Athar published in Iranian Journal of Plant Physiology.

Degree of salt tolerance in some newly developed maize (Zea mays L.) varieties by Rehana Khaliq, Maria Zahoor, Zafar Ullah Zafar, Habib-ur-Rehman Athar published in Iranian Journal of Plant Physiology.

Alleviating adverse effects of salt stress on maize (Zea mays L.) by pre-sowing treatment with weak microwaves” by Rehana Khaliq, Maria Zahoor, Zafar Ullah Zafar, Habib-ur-Rehman Athar under review

Physiological and biochemical basis of salt tolerance in Ocimum basilicum L. by Rehana Khaliq, Saira Khaliq, Zafar Ullah Zafar, Habib-ur-Rehman Athar published in Journal of Medicinal Plants Studies.

CONFERENCE PUBLICATIONS

Risks and opportunities for medicinal plants cultivation in the south of Transylvania by Maria Mihaela Antofie, Camelia Sand Sava, Horia Barbu and Rehana Khaliq in International Conference of “Etnofarmacologica in sprijinul sanatatii omului si mediului 21-23 June 2013 (Ethno pharmacology in support of human health and the environment, Brasov Romania)

Traditional Knowledge – the basic for developing the rural community resilience against food and drought by Maria Mihaela Antofie, Camelia Sand Sava, Horia Barbu and Rehana Khaliq in The 4th “Aquatic Biodiversity” International Conference 8th -11th of October, 2013 (Sibiu, Romania)

Means and tools for conservation of agro-biodiversity in Pakistan by Rehana Khaliq and Maria Mihaela Antofie in the Agri-Food, Science, processes and Technologies international conference ( 14- 15 May, 2014) Sibiu, Romania

Enabling environment assessment in supporting long term food security in south east Transylvania-Romania by Maria-Mihaela Antofie, Rehana Khaliq in The 8th International conference, Political Science International Relation and Security Studies (23-25 May, 2014) Sibiu, Romania.

During PhD

CONFERENCE PUBLICATIONS

Industrial applications of Psyllium: An overview by Rehana Khaliq, Ovidiu tita, Maria Mihaela Antofie, Camelia Sand Sava in 2nd international conference for PhD students (IPC) June, 2015 in Sibiu, Romania.

Functional food in relation to human health: A review by Rehana Khaliq, Ovidiu tita, Maria Mihaela Antofie, Camelia Sand Sava in Agri food international conference for May, 2015 in Sibiu, Romania.

Food safety challenges in Pakistan: Legislation and policies by Rehana Khaliq, Ovidiu tita, Maria Mihaela Antofie, Camelia Sand Sava in International conference of political science, international relation and security studies (May 22-24,2015).

JOURNAL PUBLICATIONS

Assessment of salt tolerance in Ocimum basilicum by Chlorophyll fluorescence by Rehana Khaliq and Ovidiu TITA in journal of scientific paper series, management, economic engineering in agriculture and rural development under review.

Survey assessment of indigenous knowledge for seeds consumption of sweet basil and psyllium by local community in southern punjab, Pakistan by Rehana KHALIQ, Ovidiu TITA, Camelia SAVA in journal of scientific paper series, management, economic engineering in agriculture and rural development under review.

Comparative study between seeds of sweet basil and Psyllium on the basis of proximate analysis by Rehana Khaliq and Ovidiu Tita (under review)

ANNEX 5. Curriculum Vitae