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| PHYTOCHEMICAL AND NUTRITIONAL CONSTITUENT OF Cnidosculus aconitflius LEAVES COLLECTED IN BIRNIN KEBBI, KEBBI STATE |
ACKNOWLEDGEMENT
All praise and glorification be to Almighty Allah the most beneficent the most merciful, He who spared m life to this moment and grant me knowledge, wisdom, guidance, protection, health, ability and success to see the beginning as well as the end of this project research work. May peace and blessings of Allah be upon his prophet Muhammad (S.A.W).
I would like to extent my sincere gratitude and appreciation to my project supervisor, Mrs. Amina Sulaiman for her patience, guidance, suggestion, encouragement to the success to see the success of this work.
I would also thank the Head of Department, Dr. Ibrahim Babangida and the entire academic staff of the Department of Biochemistry for their contributions towards my academic upliftment.
I wish to recognize with gratitude, the assistance and contribution of Mrs. Rebecca Samuel (Chief Technologist), and other Laboratory Staff of the Department for their technical assistance during the experimental work.
I am deeply extending my special thanks and appreciation to my beloved parents, for their love, encouragement and financial assistance toward the success of my programmed.
My special thanks to my auntys, Aishatu Lawal, and my sibling for their encouragement towards the success of my study.
Finally my special thanks to my friends; Kajar Rejoice Ene and Williams Okechukwu for their words of encouragement during my studies. My other friends and collages, especially those in my Department, I say thank you all. Other people that rendered help in one way or the other but could not be mentioned. I remain grateful.
TABLE OF CONTENT
TITTLE PAGE – – – – – – – – i
CERTIFICATION – – – – – – – – ii
DEDICATION – – – – – – – – iii
ACKNOWLEDGEMENT – – – – – – – iv
TABLE OF CONTENTS – – – – – – – v
LIST OF FIGURES – – – – – – – – viii
LIST OF TABLES – – – – – – – – ix
ABSTRACT – – – – – – – – – x
CHAPTER ONE
1.0 Introduction and Literature Review – – – – – 1
1.1 Introduction – – – – – – – – 1
1.1.1 Botanical Description – – – – – – – 1
1.2 Justification – – – – – – – – 2
1.3 Aim and Objectives – – – – – – – 3
1.3.1 Aim – – – – – – – – – 3
1.3.2 Objectives – – – – – – – – 3
1.4. Literature Review – – – – – – – 4
1.4.1 Medicinal Plants and their Pharmacological Properties – – 4
1.4.2 Some Previous Works Done on the Plant – – – – 9
1.4.3 The Proximate System of Analysis – – – – – 14
1.4.4 Phytochemicals – – – – – – – – 17
CHAPTER TWO
2.0 Materials and Methodology – – – – – – 29
2.1 Materials – – – – – – – – – 29
2.1.1 Plant Collection and Identification – – – – – 29
2.1.2 Preparation of Leaves. – – – – – – – 29
2.1.3 Reagents – – – – – – – – – 29
2.1.4 Equipment and Apparatus – – – – – – 30
2.2 Methods – – – – – – – – – 31
2.2.1 Proximate Analysis – – – – – – – 31
2.2.2 Determination of Vitamins – – – – – – 36
2.2.2.1 Determination of Vitamin A (Rutkowski and Grzegorczyk , 2007) – 36
2.2.2.2 Determination of vitamin C (Rutkowski and Grzegorczyk , 2007). – 37
2.2.3 Qualitative Phytochemical Analysis – – – – – 38
2.2.4 Mineral Analysis – – – – – – – 40
2.2.5 Analysis of Metals using AAS – – – – – – 40
CHAPTER THREE
3.0 Result – – – – – – – – – 44
3.1 Proximate Analysis – – – – – – – 44
3.2 Mineral Component – – – – – – – 44
3.3 Vitamins – – – – – – – – – 46
3.4 Phytochemicals – – – – – – – – 47
CHAPTER FOUR
4.0 Discussion, Conclusion and Recommendation – – – – 48
4.1 Discussion – – – – – – – – 48
4.2 Conclusion – – – – – – – – 50
4.3 Recommendation – – – – – – – 52
Reference – – – – – – – – 53
Appendices – – – – – – – – 61
LIST OF FIGURES
Figure 1.1: Leaf of Cnidoscolus aconitifolius – – – – 2
Figure1.2: Basic structures of some pharmacologically important
plant derived alkaloids – – – – – – 19
Figure 1.3: Basic structures of some pharmacologically important
plant derived glycosides – – – – – – 21
Figure 1.4: Basic structures of some pharmacologically important plant
derived Flavonoids – – – 22
Figure 1.5: Basic structures of some pharmacologically important plant
derived Saponins – -not – – – – – 24
Figure 1.6: Basic structure of phenolic compounds – – – – 25
Figure 1.7: Basic structures of some pharmacologically important
plant derived Steroids – – – 26
Figure 1.8: Basic structures of some pharmacologically important plant
derived Tannins. – – – 27
Figure 1.9: Basic structures of some pharmacologically important plant
derived Terpenes – – – 28
Figure 2.1: Atomic Absorption Spectrophotometer – – – – 41
LIST OF TABLES
Table 2.1: List of reagents used – – – – – – 29
Table 2.2: Equipment and apparatus used in the study – – – 30
Table 3.1: Proximate content of Cnidocolus aconitifolius leaf – – 44
Table 3.2: Minerals constituents of Cnidocolus aconitifolius leaf – – 45
Table 3.3: Vitamin constituents of Cnidocolus aconitifolius leaf – – 46
Table 3.4 Phytochemical composition of Cnidoscolus aconitifolius leaves. – 47
ABSTRACT
This study investigated the phytochemical contents and nutritional profile of Cnidoscolus aconitofolius leaf collected from Birnin Kebbi, Kebbi State Nigeria. The phytochemical, proximate and mineral content, the analyses were done using standard methods, whereas vitamin concentrations were evaluated using HPLC method. The preliminary qualitative phytochemical screening revealed the presence of phenols, flavonoids, alkaloids, terpenoids, saponins and absence of tannins, steroids, respectively. Proximate composition (%) showed that carbohydrate, proteins, crude fat, moisture, ash content and crude fibre contains 65.54, 4.96, 13.33, 0.67, 12.67, and 2.83 respectively. The results of vitamin analysis in (mg/100g) of vitamin A, vitamin and vitamin C,18.47 and 115 respectively and concentrations of minerals in (mg/100g) of Phosphorus, Copper,, Iron, Calcium, Potassium Sodium, Magnesium and Manganese were 5.38, 0.25, 0.1502,1.0753 ,1.02, 3766.67,80.83, 1.32, and 0.0730 respectively. This investigation shows that Cnidoscolus aconitifolius leaf contains high medicinal and nutritional compositions which could be exploited beneficially in treatment of diseases as well as nutritional supplements.
CHAPTER ONEq
INTRODUCTION AND LITERATURE REVIEW
1.1 INTRODUCTION
Generally, plants could either be ornamental, medicinal, as well as nutritional, hence there has been a high level of reliance on plants as a whole by both man and animals for survival (Yuan, et al., 2007) Medicinal plants seemed to be the most researched due to its use over the years in rural communities to treat infections and diseases, with a lot of positive results, thus making them the richest bio-resource of drugs in traditional system of medicine. They also function as medicaments. These medicinal values of plants reside majorly in some chemically active compounds that produce a definite physiological action on the human and animal body (Awoyinka, et al., 2007).The use of plants as medicine is the origin of mankind. Even certain animals are known to consume plants when they are ill. For example, it is believed that dogs eat grass Arugampul (Cynodon dactylon) which, they may have to relieve gastric distress or to dislodge parasites. Since the beginning of human kind, people have relied primarily on plants for nourishment. Through trial and error they discovered that some plants are good for food, some are poisonous and some produce bodily changes such as increased perspiration, bowel movement, urination, relief of pain, hallucination and healing. Over the years back these observations were passed orally from generation to generation with each generation adding and refining the body of knowledge (Kumaravel et al., 2014).
1.1.1 Botanical Description
Cnidoscolus aconitifolius is a large, fast growing leafy perennial shrub, it is evergreen or drought deciduous shrubsup to 6 meters in height with alternate palmate lobed leaves, it has a succulent stem which releases a milky sap whencut and small white flowers or dichotomously branched cymes. Leaves are large and chartacious or sometimes succulent, up to 32cm long and 30cm wide on petioles up to 28cm in length (Ross and Molina, 2002). However raw chaya leaves contains toxic cyanide compound, cooking for 20minutes or more is essential prior to consumption, to inactivate the toxic components. People cook for 2 to 3 minutes prior to consumption or consume itraw (Mordi and Akanji 2012). It is also known to contain phenol, tannin etc. A number of studies exist reporting thetoxic effect of herbal medicines (Calixto, 2000). Cnidoscolus aconitifolius shoots and leaves have been taken as laxatives, diuretic and circulatory stimulant, to improve digestion, stimulate lactation and harden the fingernails (Rowe, 1994).
Figure 1.1: Leaf of Cnidoscolus aconitifolius
1.2 JUSTIFICATION
Plant phytochemicals are useful precursors of compounds that possess therapeutic property. The plant Cnidoscolus aconitifolius is used in herbal medicine and recent studies have shown that A wide variety of claims have been made for its medicinal efficacy as a treatment for numerous ailments ranging from its ability to strengthen fingernails and darken gray hair to cure for alcoholism, insomnia, gout, scorpion stings, brain and vision improvement (Jensen, 1997, Atuahene et al., 1999). Moreover the plant is a good sources of food in many part of the country as such, a deep understanding and knowledge of the plant is required to justify its function.
1.3 AIM AND OBJECTIVES
1.3.1 AIM
The aim of this study is to evaluate the phytochemical and nutritional constituents of cnidoscolus aconitifolius leaves extract obtained from Birnin Kebbi, Kebbi state.
1.3.2 OBJECTIVES
To evaluate the proximate content of the plant leaves. Cnidoscolus aconitifolius
Conduct the phytochemical screening of the plant leaves Cnidoscolus aconitifolius.
To evaluate the nutritional constituents present in the plant leaves. Cnidoscolus aconitifolius
1.4. LITERATURE REVIEW
1.4.1 Medicinal Plants and their Pharmacological Properties
Medicinal plants have been identified and used throughout human history. Plants have the ability to synthesize a wide variety of chemical compounds that are used to perform important biological functions, and to defend against attack from predators such as insects, fungi and herbivorous mammals. At least 12,000 such compounds have been isolated so far; a number estimated to be less than 10% of the total (Tapsell et al., 2006).Chemical compounds in plants mediate their effect on the human body through processes identical to those already well understood for the chemical compounds in conventional drugs; thus herbal medicines do not differ greatly from conventional drugs in terms of how they work. This enables herbal medicines to be as effective as conventional medicines, but also gives them the same potential to cause harmful side effects (Lai and Roy, 2004).Traditional African herbs contain many useful compounds which can be used for the treatment of chronic diseases. Numerous reports suggest that traditional herbs have potentials for preventing pathological outcome of some neurodegenerative diseases, cancers, metabolic disorders, among others and most of the active principles of some useful drugs have been initially isolated from plants. Furthermore, most of the herbal drugs are a mixture of a number of plants or parts of the same plant whose cumulative effect increases their efficacy in curing diseases as well as reducing toxicity (Krall et al., 1978; Manomani et al., 1995). Plants contain diverse groups of phytochemicals such as tannins, terpenoids, alkaloids, and flavonoids that possess enormous antimicrobial potentials against bacteria, fungi and other microorganisms. These are much safer than synthetic drugs and show lesser side effects (Ravi, 2011). The search for components with antimicrobial activities has gained increasing importance in recent times, due to growing worldwide concern about the alarming increase in the rate of infection by antibiotic-resistant microorganisms (Davis, 1982; Shittu et al., 2007). Many plants have the potentials as potent remedies for treating different diseases, especially those used by indigenous people. It is therefore pertinent to provide scientific ground for such medicinal plants regardless of their habit, distribution, economic input and the use for which they are employed. From the ancient times, in search for rescue for their disease, the people looked for drugs in nature. The beginnings of the medicinal plants use were instinctive, as in the case with animals (Biljana Bauer Petrovska, 2012). The records revealed that the human use of plants as traditional medicine dated back to middle Paleolithic age, approximately 60,000 years
ago. The plants were used as flavoring agent, food, insect deterrent, ornamental, fumigant, spices and cosmetics (Maridass et al., 2008).It was also reported that, natural products including plants, animals and minerals have been the single most productive source of leads for the development of drugs. A number of compounds derived from natural sources are currently undergoing clinical and preclinical studies, particularly as anti-inflammatory, cardiovascular, antidiabetic, antiobesity, antimalarial, antiviral and antineoplastic agents. Most of these compounds are derived from leads of plant origin (Satish Sardana, 2012).
The first written record of herbs used as medicines was made over 5000 years ago by the Sumerians, in ancient Mesopotamia (present day Iraq). Sumerian prescriptions for healing, using herbs such as caraway and thyme have been found by archeologists on tablets made of clay. At the same time and perhaps even earlier herbal traditions were being developed in China and India. The roots of Chinese medicine which is based largely on herbals also dated back to approximately 5000 years. The Chinese emperor Chien Nung put together a book of medicinal plants called “Pen Tsao”. It contained over 300 herbs including ‘ma huang’ or Chinese ephedra which is still widely used today and is the herb from which western scientists have derived the drug Ephedrine. The Indian system of medicine was called “Ayurveda”. Ayurveda is still practiced today and many authentic, traditional formulations are available outside India (Kumaravel et al., 2014). The Greeks and Romans derived much of their herbal knowledge from these early civilizations. Ancient Greece was influenced by Babylonia (Mesopotamia), Egypt, India and China. The Greek physician Hippocrates (460-377 BC) who is referred as the “Father of modern medicine” was an herbalist. He is credited with having written, “Let your food be your medicine and your medicine be your food” During the middle ages the knowledge of medicinal plants was furthered by monks in Europe who studied and grew medicinal plants and translated the Arabic works on herbals. We have many herbs available to us as a result of their traditions including “Una de Gato” or Cat’s claw herb (Uncaria Tomentosa). Over the past 100 years, the development and mass production of chemically synthesized drugs have revolutionized health care in most parts of the world and however, large sections of the population in developing countries still rely on traditional practitioners and herbal medicines for their primary care (WHO, 2005). In Africa up to 90% and in India 70% of the population depend on traditional medicine to meet their health care needs. In China, traditional medicine accounts for around 40% of all health care delivered and more than 90% of general hospitals in China have units for traditional medicines (WHO, 2005). Plants are rich in a wide variety of secondary metabolites such as tannins, terpenoids, alkaloids and flavonoids which have been known to have in vitro antimicrobial properties, most of these plants are eaten or used for their rich phytochemical constituents, which provide both preventive and curative properties to consumers against diseases most of which have an age long existence (Edeoga et al., 2005). The medicinal value of these plants lies in bioactive phytochemical constituents that produce definite physiological action on the human body. The relatively lower incidence of adverse reaction to plant preparations compared to modern conventional pharmaceuticals, coupled with their reduced cost consequently encouraged both the consuming public and natural healthcare institutions to consider plant medicines as alternatives as synthetic drugs (Nair et al., 2004). Undoubtedly, medicinal plants are relevant in both developing countries and developed nations of the world as sources of drugs or herbal extracts for various chemotherapeutic purposes. The use of and search for drugs and dietary supplements derived from plants have accelerated in recent years. Ethno pharmacologists, botanists, microbiologists, and natural-products chemists are combine together for phytochemicals research which could be developed for treatment of infectious diseases. While 25 to 50% of current pharmaceuticals are derived from plants, none are used as antimicrobials. Traditional healers have long used plants to prevent or cure infectious conditions; Western medicine is trying to duplicate their successes. Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to have antimicrobial properties (Alanis et al., 2005). Thorough scientific investigations of these phytochemicals go a long way in their proper use for treatment of diseases in medicine and new drug development in the pharmaceutical field (Anyakora et al., 2008) On the other hand, nutritional interest in most plants stems basically from their rich contents of essential amino acids, carbohydrate, lipids, vitamins and minerals Carbohydrates and lipids are primary suppliers of energy, amino acids are for growth and repair of worn out tissues whereas vitamins play a very significant role in maintenance of health and making great contributions to a healthy immune system as well as provisions of all the nutrients necessary for good health (Elfalleh et al., 2009). Cnidoscolus aconitifolius (family-Euphorbiaceae), commonly called chaya is a leafy perennial shrub native of Yucatan peninsula of Mexico in Central America (Araújo, et al., 2008). The plant which is also called spinach tree is consumed as vegetable in soups, salads and therapeutically used for a number of ailments such as diabetes, artherosclerosis, gallstone and high cholesterol (Kuti, et al., 2004). Chaya is a good source of protein, vitamins, calcium and iron; and is also a rich source of antioxidants such as Vitamin C and E as well as Flavonoids (Bartlett, and Eperjesi 2008) and have a possible antidiabetic effect (Jack and Wright, 2012). The leaves are large, 32 cm long and 30 cm wide on chartacious and succulent petioles. The crop originated as a domesticated leafy green vegetable in the Maya region of Guatemela, Belize, Southeast Mexico during pre-Cambrian period (Ibarra and Cruz, 2002). It has continued to be used as food, medicine and ornamental plant till date. Due to its ease of cultivation, potential productivity and above all its substantial nutritional value, the plant has spread all over the world including the tropics. Colloquially the plant is referred to as Chaya (Donkoh et al., 1990). Itis often referred to as Chaya, EfoIyanaIpaja and Efo Jerusalem in the western part of Nigeria; Obarandu or Akwukwonriohurun in the eastern part of Nigeria; “Hospital Too Far” in the Southern part of Nigeria -because they believe it gives blood almost immediately even before one can rush to get from the hospital; and “Catholic vegetable” because it was commonly cultivated and used as vegetables in convents (Donkoh et al., 1990; Iwalewa et al., 2005). Although the plant is mainly cultivated as food it has continued to be an important medicinal plant. Much of its recent spread into new areas may likely be attributed to its medicinal value. A wide variety of claims have been made for its medicinal efficacy as a treatment for numerous ailments ranging from its ability to strengthen fingernails and darken gray hair to cure for alcoholism, insomnia, gout, scorpion stings, brain and vision improvement (Jensen, 1997, Atuahene et al., 1999 ) .
1.4.2 Some Previous Works Done on the Plant
Cnidoscolus aconitifolius, a cultivated plant known as Chaya, is also frequently consumed. In their study, they found that Chaya was the best source of ascorbic acid (350.83mg/100g), retinol (5.26mg/100 g), iron (7.51mg/100g) and protein (8.15%). Chaya potential as a medicinal and edible plant suggests that this species ought to be cultivated commercially (Donkoh et al., 1999). Kuti et al., (1999) analyzed the proximate composition and mineral content of 2 edible species of Cnidoscolus (tree spinach). In this study, proximate composition and mineral content of raw and cooked leaves of 2 edible tree spinach species (C.chayamansa and C. aconitifolius) known locally as “Chaya” were determined and compared with that of a traditional green vegetable, spinach (Spinicia oleraceae). Results of the study indicated that the edible leafy parts of the 2 Chaya species contained significantly (p≤0.05) greater amounts of crude protein, crude fibre, Ca, K, Fe, ascorbic acid and β carotene than the spinach leaf. However, no significant (p≥0.05) differences were found in nutritional composition and mineral content between the Chaya species, except minor differences in the relative composition of fatty acids, protein and amino acids. Cooking of Chaya leaves slightly reduced nutritional composition of both Chaya species. Cooking is essential prior to consumption to inactivate the toxic hydrocyanic glycosides present in the Chaya leaves. Based on the results of the study, the authors told that the edible Chaya leaves may be good dietary sources of minerals (Ca, K, Fe) and vitamins (Ascorbic acid and β – carotene). Kuti et al., (2004) evaluated the antioxidant capacity and phenolic content in leaf extracts of tree Spinach (Cnidoscolus sp.). Total phenolic content and antioxidant capacity of 2 tree spinach species (Cnidoscolus chayamansa and Cnidoscolus aconitifolius.) were determined in raw and cooked leaf extracts. Antioxidant capacity was assessed by the oxygen radical absorbance capacity (ORAC) assay, and flavanoid glycoside composition was quantified by High performance liquid chromatography and identified by Gas Chromatography. C.aconitifolius leaves contained more varieties of the flavanoid glycosides than C.chayamansa. Cooking reduced antioxidant activity and phenolic content that resulted in losses of some Kaempherol glycoside and quercetin glycoside residues in leaf extracts. The authors reported that tree spinach leaves are a rich source of natural antioxidants for foods. Adeleke et al., (2010) evaluated the chemical composition of 3 traditional vegetables in Nigeria. In their work, the nutritional composition of 3 traditional vegetables in Iree, Osun state was evaluated. The leafy vegetables such as C. aconitifolius (iyana ipaja), S.nodiflorum (Ogumo) and S. biafrae (worowo) were subjected to evaluation. The results showed that C. aconitifolius had higher protein content (5.91%) and carbohydrate content (8.88%) but there was no significant difference (p≥0.05) in the crude fibre value and that of S.biafrae. S.biafrae had higher moisture content (89%) while S.nodiflorum had higher ash and fat content which were significantly different (p≤0.05) from the other vegetables. C.aconitifolius had higher values in all the mineral contents determined and these were significantly different (p≤0.05) from other vegetable. There were no significant difference (p≥0.05) in potassium, calcium and iron contents of S.nodiflorum and S.biafrae. The authors reported that the 3 vegetables are good sources of nutrients which could be consumed for normal growth. Tadeu et al., (2012) conducted the phytochemical screening and evaluation of antibacterial activity of 4 Cnidoscolus species (Euphorbiaceae) against standard strains and clinical isolates. 4 species of Cnidoscolus [Cnidoscolus infestus Pax and K. Hoffman, Cnidoscolus pubescens Pohl, Cnidoscolus quercifolius Pohl and Cnidoscolus urens (L.) Arthur] were selected and the qualitative phytochemical composition was analyzed by thin layer chromatography using eluent and specific revealing. Antimicrobial activity was evaluated using the agar diffusion method and determining the Minimum Inhibitory Concentration (MIC). The results showed that the phytochemical present in all samples were coumarins, phenolic compounds and terpenoids. Alkaloids and naphthoquinones were not observed in the study. The extract of the barks of C. quercifolius was active against Staphylococcus strains, with a MIC between 250 and 500μg/ml and its dichloromethane fraction had MIC between 62.5 and 250μg/ml against methicillin-resistant Staphylococcus aureus (MRSA). The antimicrobial activities of the bark of C. quercifolius indicated that the mechanism of multidrug resistance of Staphylococcus to current antibiotics does not confer resistance to the compounds present in samples. It was concluded that the identification of the chemical constituents responsible for the antimicrobial activities of C.aconifolius may lead to the identification of new antimicrobial drugs against these pathogens. Miranda et al., (2010), conducted a study on the hypercholesterolemic activity from the leaf extracts of Cnidoscolus aconitifolius. In an in vivo model, high-cholesterol diet administered to mice to induce hypercholesterolemia. 3 extracts from C.aconitifolius (ethanol, methanol and an aqueous extract) were tested on hypercholesterolemic mice. Active extracts were assessed against the in vitro inhibitory activity of the same 3 extracts on the HMG-CoA reductase enzyme by using Vero cells. The specific chemical groups present in the phytochemical extracts were also determined. The results (only the aqueous extract at either doses employed) showed a significant cholesterol reduction (27.9 and 31.1%, for 50 and 100mg/kg.bw) respectively (p<0.01). The extract did not inhibit the HMG-CoA reductase enzyme, suggesting that its compounds act at another level in cholesterol metabolism. Reactions to secondary metabolites indicate the presence of alkaloids in the aqueous and ethanol extracts and phenol hydroxyls in the ethanol and methanol extracts. The authors concluded that the Mexican plant tested has important hypocholesterolemic properties and merits further studies on the clinical applications of these plant extracts. Florence O Jimoh et al., (2009) investigated the chemical constituents and antioxidant potential of Cnidoscolus chayamansa (Euphorbiaceae) root on carbon tetrachloride-induced liver damage. Albino rats were grouped into 4: A – D. Groups A and B received one ml/kg.bw of olive oil and one ml/kg.bw of carbon tetrachloride (CCl4) respectively for 8 days while those in C and D received one ml/kg.bw each of CCl4 and 500 and 1000mg/kg.bw of aqueous extract of C.chayamansa root, respectively for the same period. Chemical analysis of the plant root revealed the presence of tannins, phenolics, flavanoids, saponins, Fe, Zn, Mg, Ca, vitamins A and C. Administration of CCl4 resulted in significant increase (p<0.05) in liver malondialdehyde concentration while the activities of liver alkaline phosphatase, superoxide dismutase, glutathione peroxidase and catalase were significantly reduced (p<0.05). Simultaneous administration of CCl4 and the plant extract at 500 and 1000mg/kg.bw produced values of these biochemical parameters that were compared favorably with the control (p>0.05) in addition to increasing superoxide dismutase activity in the liver (p<0.05). It was concluded that the aqueous extract of C.chayamansa root possessed antioxidant activity and protected the hepatocytes against CCl4 induced damage. The antioxidant activity of the plant may be due to its chemical constituents. The awareness among the young people increased today about the traditional uses of wild medicinal and edible plants (Odhav et al., 2007). The total extent of the knowledge of traditional medicinal plant based therapies should be documented through botanical surveys before this knowledge is irretrievably lost to future generations (Angelica Bautista Cruz et al., 2011). It has been revealed that from the literature review on Cnidoscolus species (Euphorbiaceae) for which many researchers have carried out different study on this herb. The phytochemical studies concerning the chemical composition of this plant have allowed the identification of cyanogenic glycosides, amentoflavone, Kaempherol and quercetin glycosides. Raw and cooked ethanol/acetone/water/acetic acid extracts of Chaya leaves demonstrated antioxidant activity and the Chaya tea diminished the glucose levels of diabetic rabbits. The most commonly isolated flavanoids from the leaves of Cnidoscolus species are C-glycosyl flavones and flavanoid glycosides (galactosides, glucosides, rhambosides and rhamnosyl glucosides of quarcetin and/or Kaempherol (Kolterman et al., 1982 and Kuti et al., 2004). Plant-derived polyphenolic flavanoids exhibit numerous biological and pharmacological properties that could potentially afford protection against chronic diseases (Cook et al., 1996; Hollmann et al., 1996; Middleton et al., 1992 and Rice Evans et al., 1995). Kolterman et al. (1984) performed a methanolic extraction of C. aconitifolius leaf and analyzed it by gas-liquid chromatography, identifying glucosidic flavonols, such as galactosidated, glucosidized, rhamnosididated and rhamnosylglucosidates of quercetin and kaempferol, and two quercetin triglycosides. Adanlawo and Elekofehinti (2012) performed the proximate analysis and mineral composition of the leaf extract of Cnidoscolus aconitifolius, and the result shows that Cnidoscolus aconitifolius leaf has a high percentage of carbohydrate 35.70±2.00, protein 18.73±0.65, Ash 13.74±0.76, moisture content 9.49±1.20, crude fibre 9.68±0.93. Amino acid concentrations present include Lys. 5.05±0.13, His 2.60±0.37, NH3 is nil, Arg 5.17±0.15, Asp 8.50±0.48, Thr 3.70±0.25, Ser 3.36±0.37, Glu 13.50±0.74, Pro 3.50±0.29, Gly 4.10±0.02, Ala 4.60±0.11, Cys 1.19±0.48, measured in g/100g. Glutamic acid have the highest concentration while cystine have the lowest concentration. Anti –nutrient present are in low concentration, they include phytin phosphorus (2.67mg/100g), phytic acid (9.47mg/100g), oxalate (2.005mg/100g) and tannin(0.24mg/100g). The average values for Ca, Mg, Na, K, Fe, Zn and P were 400.80, 219.96, 725.44, 194.46, 0.57, 437.44, 0.13, 0.03mg/100g dry sample respectively. Sodium was the most abundant mineral in the sample and when consumed, it may aid osmo-regulation of cellular system. According to the authors the amino acid composition revealed the presence of most essential amino acids thus, making C. aconitifolius a potential panacea for kwashikwor and other related protein-deficiency diseases.Otitolaiye and Asokan (2015) perform an aqueous extract of Cnidoscolus aconitifolius leaf obtained from Sokoto, and analysed by gas chromatography-mass spectroscopy.They reported the presence of different phytochemicals which are cardiac glycosides, flavonoids, phenols, anthraquinones and triterpenoids. However the GC-MS revealed the presence of 42 compounds. The major compounds were Borneol (1.41%), Caryophyllene oxide (2.73%), 1H-cycloprop (e) azulene (2.02%), 4-(1,5-Dimethyl hex-4-enyl) cyclohex-2-enone (4.15%), Farnesol (2.51%), Spiro (4.5) dec-6-en-8-one (6.87%), Longipinane (4.18%) and Benzene (13.37%). The authors concluded that theCnidoscolus aconitifolius contain many biologically active compounds in various concentrations which could have been responsible for its numerous biological actions.
1.4.3 The Proximate System of Analysis
The proximate system for routine analysis of animal food stuffs was devised in the mid-nineteenth century at the Weende, Experiment Station in Germany (Henneberg et al., 1864). It was developed to provide a top level, very broad, classification of food components. The system consists of the analytical determinations of water (moisture), ash, crude fat (ether extract), crude protein and crude fibre. Nitrogen-free extract (NFE), more or less representing sugars and starches, is calculated by difference rather than measured by analysis. Although some of the methods used historically in the proximate system of analysis are not recommended for the preparation of food composition databases (e.g. crude fibre), it is useful to consider the concepts involved as they have dominated views on the composition of foods and food analysis. This system was developed at a time when the chemistry of most food constituents was only partially understood, and the growth of nutritional sciences has shown that for nutritional studies a more detailed and biochemically oriented approach to food analysis is needed. Nevertheless, proximate analysis, including the original methods, still forms the basis for feed analysis, and the analysis of foods for legislative purposes in many countries (Stohmann, 1860).
Ash Content
The ash of biological materials is an analytical terms used for the inorganic residues that remain after the organic matter has been burnt away. The ash is not usually the same as the inorganic matter present in the original food since there may be losses due to volatilization or chemical interaction between the constituents.The value is useful in assessing the quantity or grading certain edible materials (Howitz and Josyn, 1970). Ash content is an indication of the total mineral (Donald et al., 1978).
Moisture Content
Values for water remain an essential constituent in food composition databases because water content is one of the most variable components, especially in plant foods. This variability affects the composition of the food as a whole.The methods are based on the direct or indirect measurement of water removed from the food, changes in physical properties that change systematically with water content, or the measurement of the chemical reactivity of water (Egan, Kirk and Sawyer, 1987; AOAC International, 2002; Sullivan and Carpenter, 1993 Southgate, 1999; Bradley, 1998).
Protein
Proteins are large molecules that play a vital role in the body of all organisms. They are polymers and function of living organisms, they are also polymers of amino acid, twenty of which occur naturally, only nine in adult and ten in infants are essential and the other are regarded as non-essential and in all referred to as complete protein (Desrosier,1997).Among the most important functions performed by protein are ;They provide the structural frame work cells and tissues, example is elastin. They act as antibodies in the blood stream to fight against foreign materials such as bacteria and virus. They are also act as a catalyst and enzymes (Crosby, 1993). Crude protein is the expression used to refer to the percentage of nitrogen, nitrate and some other cyclic compounds containing nitrogen. The quality of food may be judge by its protein content, and the number of essential amino acids it contains and the degree to which its protein is digested and absorbance by the body (Helen, 1989).
Lipid Constituents
Lipid is a generic term that includes fats oils and fat related substances. All are usually insoluble in water but soluble in fat solvent. Fat is a solid and liquid at room temperature, both consist of glycerol bonded toone, two or three fatty acids (Peter, 1981). FAO/WHO (1994) recommended that adequate food composition data on fats should be widely accessible and that standard methods and reference materials should be used for the analysis of fatty acids and preparation of nutrient databases. The report provides good coverage of the compounds and nutritional issues of interest (Christie, 2003). The proximate system of analysis, `fat’ is measured as the fraction of the food that is soluble in lipid solvents. The extracted material contains a range of different classes of substances. For nutritional purposes the measurement of `total fat’ has limited value; nevertheless, it still is widely reported and is retained in many requirements for food labelling and the regulation of food composition (Christie, 2003). The calculation of energy from the result of proximate analysis are the % values obtained from available carbohydrate content, crude protein and crude lipid are multiply by factors 4,4 and 9 respectively and products sum op as expressed using the equation below (Hassan et al., 2008).
Carbohydrates
A carbohydrate is one of the major nutrients found in food. It occurs naturally in food as polyhydroxy aldehyde or ketones compounds of carbon, hydrogen and oxygen. Carbohydrates are found either as sugar or as starch and glycogen (Murray et al., 1983).Carbohydrates supply 4 kcal of energy per gram. The body use carbohydrate first as energy source, since some dietery carbohydrates are readily converted to glucose either during digestion (example galctose). Galactose is specifically required by many tissues but does not have to be provided as such in the diet. Glucose can be formed from glycerol and glucogenic amino acid by gluconeogenesis (Hegarthy, 1989).
Energy
The energy value of a food is the measure of the bond energy of the organic constituents that is, fats, protein and carbohydrates along with insignificant inputs from minor constituents such as organic acids which is produced during respiration. The experimental estimation of this energy involves one of the two procedures either the use of a bomb calorimeter or the calculation of energy from the result of the proximate analysis of the foods (James, 1995). The calculation of energy from the result of proximate analysis are the % values obtained from available carbohydrate content, crude protein and crude lipid are multiply by factors 4,4 and 9 respectively and products sum op as expressed using the equation below (Hassan et al., 2008). Energy value (Kcal per 100g) = (% CHO×4) + (% CP ×4) + (% CL×9)
1.4.4 Phytochemicals
Phytochemicals derived from the Greek word ‘phyto’ meaning ‘plant’ are chemical compounds that occur naturally in plants. Phytochemicals are non-nutritive plant chemicals that have protective or disease preventive properties. They are non-essential nutrients, meaning that they are not required by the human body for sustaining life. It is well-known that plants produce these chemicals to protect themselves but recent researches demonstrate that they can also protect humans against diseases. There are more than thousand known phytochemicals. They are not mandatory for humans to consume. However, it is proven that these chemicals protect plants as well as the humans eating them. Phytochemicals are naturally occurring and are believed to be effective in combating or preventing disease due to their antioxidant effect (Halliwell and Gutteridge, 1992; Ejele et al., 2012). The medicinal lie in their component phytochemicals, which produce the definite physiological actions on human body. The most important of these phytochemicals are alkaloids, tannins, flavonoids and phenolic compounds (Iwu, 2000). Some of these naturally occurring phytochemicals are anti-carcinogenic and some others possess other beneficial properties, and are referred to as chemo-preventers. One of the predominant mechanisms of their protective action is due to their antioxidant activity and the capacity to scavenge free radicals. Phytochemicals, the compounds present in plant are valuable source of food and medicine. They are known to have various biological activities such as antimicrobial, antifungal, antioxidant, etc. (Edeoga, et al,. 2005). There may be as many as 4,000 different Phytochemicals. Some are responsible for color and other organoleptic properties, such as the deep purple of blueberries and the smell of garlic.
Alkaloids
These are the largest group of secondary chemical constituents made largely of ammonia compounds basically of nitrogen bases, synthesize from amino acid building blocks with various radicals replacing one or more of the hydrogen atom in the peptide ring. The compounds have basic properties and are alkaline in reaction turning red litmus paper blue (Sarker and Nahar, 2007). They react with acid to form crystalline salt without the production of water (Firn, 2010). Majority of alkaloids exist as solid such atropine, some as liquid containing carbon, hydrogen and nitrogen. Most alkaloids are readily soluble in alcohol and though they are sparingly soluble in water, their salt are usually soluble. The solution of alkaloids is intensely bitter. These nitrogen compounds functions in the defense of plant against herbivores and pathogens, and are widely exploited as pharmaceuticals, stimulants, narcotics and poisons due to their potent biological activities. In nature alkaloids exist in large proportions in the seeds and root of plants in combination with vegetable acid. Alkaloids have pharmacological applications as anesthetics and CNS stimulants (Madziga et al., 2010). More than 12,000 alkaloids are known to exist in about 20% of plant species and only few have been exploited for medicinal purposes. Plant derived alkaloids in clinical uses includes, the analgesic morphine and codeine, the muscle relaxant tubocurarine and the antibiotics sanguinafine and berberine.
Figure1.2: Basic structures of some pharmacologically important plant derived alkaloids
Glycosides
Glycosides in general, are defined as the condensation products of sugars (including polysaccharides) with a host of different varieties of organic hydroxy (occasionally thiol) compounds (invariably monohydrate in character), in such a manner that the hemiacetal entity of the carbohydrate must essentially take part in the condensation. Glycosides are colorless, crystalline carbon, hydrogen and oxygen-containing (some contain nitrogen and sulfur) water-soluble phyto constituents, found in the cell sap. Chemically, glycosides contain a carbohydrate (glucose) and a non-carbohydrate part (aglycone or genin) (Kar, 2007; Firn, 2010). Alcohol, glycerol or phenol represents a glycones. Glycosides are neutral in reaction and can be readily hydrolyzed into its components with ferments or mineral acids. Glycosides are classified on the basis of type of sugar component, chemical nature of aglycone or pharmacological action. The rather older or trivial names of glycosides usually has a suffix ‘in’ and the names essentially included the source of the glycoside, for instance: strophanthidin from Strophanthus, digitoxin from Digitalis, barbaloin from Aloes, salicin from Salix, cantharidin from Cantharides, and prunasin from Prunus. However, the systematic names are invariably coined by replacing the “ose” suffix of the parent sugar with “oside”. This group of drugs are usually administered in order to promote appetite and aid digestion. Glycosides are purely bitter principles that are commonly found in plants of the Genitiaceae family and though they are chemically unrelated but possess the common property of an intensely bitter taste. The bitters act on gustatory nerves, which results in increased flow of saliva and gastric juices. Chemically, the bitter principles contain the lactone group that may be diterpene lactones (e.g. andrographolide) or triterpenoids (e.g. amarogentin). Some of the bitter principles are either used as astringents due to the presence of tannic acid, as antiprotozoan, or to reduce thyroxine and metabolism. Examples include cardiac glycosides (acts on the heart), anthracene glycosides (purgative, and for treatment of skin diseases), chalcone glycoside (anticancer), amarogentin, gentiopicrin, andrographolide, ailanthone and polygalin. Sarker and Nahar (2007) reported that extracts of plants that contain cyanogenic glycosides are used as flavouring agents in many pharmaceutical preparations. Amygdalin has been used in the treatment of cancer (HCN liberated in stomach kills malignant cells), and also as a cough suppressant in various preparations. Excessive ingestion of cyanogenic glycosides can be fatal. Some foodstuffs containing cyanogenic glycosides can cause poisoning (severe gastric irritations and damage) if not properly handled (Sarker and Nahar, 2007). To test for O-glycosides, the plant samples are boiled with HCl/H2O to hydrolyse the anthraquinone glycosides to respective aglycones, and an aqueous base, e.g. NaOH or NH4OH solution, is added to it. For C-glycosides, the plant samples are hydrolysed using FeCl3/HCl, and aqueous base, e.g. NaOH or NH4OH solution, is added to it. In both cases a pink or violet colour in the base layer after addition of the aqueous base indicates the presence of glycosides in the plant sample.
therefore possess ‘soaplike’ behaviour in water, i.e. they produce foam. On hydrolysis, an aglycone is produced, which is called sapogenin. There are two types of sapogenin: steroidal and triterpenoidal. Usually, the sugar is attached at C-3 in saponins, because in most sapogenins there is a hydroxyl group at C-3. Quillaja saponaria is known to contain toxic glycosides quillajic acid and the sapogenin senegin. Quillajic acid is strenutatory and senegin is toxic. Senegin is also present in Polygala senega. Saponins are regarded as high molecular weight compounds in which, a sugar molecule is combined with triterpene or steroid aglycone. There are two major groups of saponins and these include: steroid saponins and triterpene saponins. Saponins are soluble in water and insoluble in ether, and like glycosides on hydrolysis, they give aglycones. Saponins are extremely poisonous, as they cause heamolysis of blood and are known to cause cattle poisoning (Kar, 2007). They possess a bitter and acrid taste, besides causing irritation to mucous membranes. They are mostly amorphous in nature, soluble in alcohol and water, but insoluble in non-polar organic solvents like benzene and n-hexane. Saponins are also important therapeutically as they are shown to have hypolipidemic and anticancer activity. Saponins are also necessary for activity of cardiac glycosides. The two major types of steroidal sapogenin are diosgenin and hecogenin. Steroidal saponins are used in the commercial production of sex hormones for clinical use. For example, progesterone is derived from diosgenin. The most abundant starting material for the synthesis of progesterone is diosgenin isolated from Dioscorea species, formerly supplied from Mexico, and now from China (Sarker and Nahar, 2007). Other steroidal hormones, e.g. cortisone and hydrocortisone, can be prepared from the starting material hecogenin, which can be isolated from Sisal leaves found extensively in East Africa (Sarker & Nahar, 2007).
Figure 1.5: Basic structures of some pharmacologically important plant derived Saponins
Phenolics
Phenolics, phenols or polyphenolics (or polyphenol extracts) are chemical components that occur ubiquitously as natural colour pigments responsible for the colour of fruits of plants. Phenolics in plants are mostly synthesized from phenylalanine via the action of phenylalanine ammonia lyase (PAL). They are very important to plants and have multiple functions. The most important role may be in plant defence against pathogens and herbivore predators, and thus are applied in the control of human pathogenic infections (Puupponen- Pimiä et al., 2008). They are classified into (i) phenolic acids and (ii) flavonoids polyphenolics (flavonones, flavones, xanthones and catechins) and (iii) non-flavonoid polyphenolies. Caffeic acid is regarded as the most common of phenolic compounds distributed in the plant flora followed by chlorogenic acid known to cause allergic dermatitis among humans (Kar, 2007). Phenolics essentially represent a host of natural antioxidants, used as nutraceuticals, and found in apples, green-tea, and red-wine for their enormous ability to combat cancer and are also thought to prevent heart ailments to an appreciable degree and sometimes are anti-inflammatory agents. Other examples include flavones, rutin, naringin , hesperidin and chlorogenic.
Figure 1.6: Basic structure of phenolic compounds
Steroids
Plant steroids (or steroid glycosides) also refer to as cardiac glycosides are one of the most naturally occurring plant phytoconstituents that was found therapeutically as arrow poisons or cardiac drugs (Firn, 2010). The cardiac glycosides are basically steroids with an inherent ability to afford a very specific and powerful action mainly on cardiac muscles when administered through injection into humans and animal. Steroids (anabolic steroids) have been observed to promote nitrogen retention in animals with wasting illness (Maurya et al., 2008). Caution should be taken when using steroidal glycosides as small amount would exhibit the much needed stimulation on a diseased heart; whereas excessive dose may cause death. Diosgenin and cevadine are examples of plant steroids. (Maurya et al., 2008).
Figure 1.7: Basic structures of some pharmacologically important plant derived Steroids
Tannins
These are widely distributed in plant flora. They are phenolic compounds of high molecular weight. Tannins are soluble in water and alcohol and are found in the root, bark, stem and outer layers of plant tissue. Tannins have a characteristic feature to tan, i.e. to convert things into leather. They are acidic in reaction and the acidic reaction is attributed to the presence of phenolics or carboxylic group (Kar, 2007). They form complexes with proteins, carbohydrates, gelatin and alkaloids. Tannins are divided into hydrolysable tannins and condensed tannins. Hydrolysable tannins, upon hydrolysis, produce gallic acid and ellagic acid and depending on the type of acid produced, the hydrolysable tannins are called gallotannins or egallitannins. On heating, they form pyrogallic acid. Tannins are used as antiseptic and this activity is due to presence of the phenolic group. Common examples of hydrolysable tannins include theaflavins (from tea), daidezein, genistein and glycitein. Tanninrich medicinal plants are used as healing agents in a number of diseases. In Ayurveda, formulations based on tannin-rich plants have been used for the treatment of diseases like leucorrhoea, rhinnorhoea and diarrhea.
Figure 1.8: Basic structures of some pharmacologically important plant derived Tannins.
Terpenes
Terpenes are among the most widespread and chemically diverse groups of natural products. They are flammable unsaturated hydrocarbons, existing in liquid form commonly found in essential oils, resins or oleoresins (Firn, 2010). Terpenoids includes hydrocarbons of plant origin of general formula (C5H8)n and are classified as mono-, di-, tri- and sesquiterpenoids depending on the number of carbon atoms. Examples of commonly important monterpenes include terpinen-4-ol, thujone, camphor, eugenol and menthol. Diterpenes (C20) are classically considered to be resins and taxol, the anticancer agent, is the common example. The triterpenes (C30) include steroids, sterols, and cardiac glycosides with anti-inflammatory, sedative, insecticidal or cytotoxic activity. Common triterpenes: amyrins, ursolic acid and oleanic acid sesquiterpene (C15) like monoterpenes, are major components of many essential oils (Martinez et al., 2008). The sesquiterpene acts as irritants when applied externally and when consumed internally their action resembles that of gastrointestinal tract irritant. A number of sesquiterpene lactones have been isolated and broadly they have antimicrobial (particularly antiprotozoal) and neurotoxic action. The sesquiterpene lactone, palasonin, isolated from Butea monosperma has anthelmintic activity, inhibits glucose uptake and depletes the glycogen content in Ascaridia galli. Terpenoids are classified according to the number of isoprene units involved in the formation of these compounds. The major groups are shown in Table 1.
Figure 1.9: Basic structures of some pharmacologically important plant derived Terpenes
CHAPTER TWO
2.0 MATERIALS AND METHODOLOGY
2.1 Materials
2.1.1 Plant Collection and Identification
The fresh leaves of plant (Cnidoscolus aconitifolius) were obtained from Birnin kebbi Haliru Abdu Quarters Kebbi state Nigeria, and identified at the Department of Biological sciences, kebbi State University of science and Technology Aliero. specimen is kept in the herbarium. The leaves were air dried, blended and extracted using methanol as solvent.
2.1.2 Preparation of Leaves.
The vegetables were washed to remove debris. The leaves were air dried at room temperature and grounded using pestle and mortar to obtain the powder which was used for the analysis.
2.1.3 Reagents
Table 2.1: List of reagents used
Reagent
Specification
Company/Country
Xylene
Analar
England
Ethanol
BDH Chemicals
England
Phosphotungstate reagent
Analar
England
Petroleum ether
BDH chemical
England
FeCl3
BDH Chemical
England
KOH solution
BDH Chemicals
England
Distilled water
Pure
SSU lab
Vitamin A standard
BDH chemical
Tokyo, japan
Vitamin C standard
BDH chemical
Tokyo, Japan
Vitamin E standard
BDH chemical
Tokyo, japan
H3PO4
Analar
England
2.1.4 Equipment and Apparatus
Table 2.2: Equipment and apparatus used in the study
Materials/Apparatus
Specification
Company/Country Name
Weighing balance
PC-4400/Metal
Tokyo/Japan
Beaker
Glass
England
Conical Flask
Glass
Pyrex, England
Measuring cylinder
Glass
Pyrex, England
Spectrophotometer
AE-350
ERMAInc./Tokyo, Japan
Water bath
GD100
Grant Instruments (Cambridge) Ltd./England
Centrifuge Machine
800D/Metal
Pipette
Robber
England
Test tube
Glass
Pyrex England
UV lamp
China
Muffle furnace
Lento Furnace
Gallenkamp USA
2.2 METHODS
2.2.1 Proximate Analysis
The method of Association of Official Analytical Chemistry (AOAC) 1990 was used for general proximate analysis of the plant samples.
Preparation of Leaves
The leaves were washed to remove debris. The leaves were air dried at room temperature and grounded using pestle and mortar to obtain the powder which was used for the analysis.
Determination of Moisture Content
Principle:
This was based on heating the sample to eliminate all water content in the sample. This was achieved by placing the sample in an oven at 1050C up to 24 hours. High temperature is not needed to avoid decomposition of organic matter.
Procedure:
A clean crucible was dried to a constant weight in an air oven, cooled in a desicator and weighed (w1). 2g of the sample was placed in the crucible and weighed (w2) and dried in the oven for eight hours. The crucible and its contents were cooled in a desiccator and weighed (w3). The procedure was continued until a constant weight was obtained out of which the percentage moisture was calculated.
Calculation:
% moisture =loss in weight due to drying x 100
Weight of fresh sample
=W2 – W3 x100
W2 –W1
Where w1 = weight of empty dish
W2 = weight of fresh sample + empty dish
W3 = weight of dry sample + empty dish
Determination of Total Ash Content
Principle:
The principle was based on the fact that minerals are not destroyed by high temperature. The ash content was determined from the loss of weight that occurs during igniting at a high temperature of 6000C for 5 hours in a muffle furnace, all the organic matter is burnt off leaving the inorganic substance in the form of ash.
Procedure:
2g of the finely ground sample was weighed (w2) into a previously weighed clean crucible (w1) which had been ignited in the muffle furnace at 6000C for one hour and cooled in a desiccator. The crucible containing the sample was heated in a muffle furnace at 6000C for five hours to burn off all the organic matter after which the crucible was cooled in a desiccator and weighed (w3).
Calculation:
% ash = weight of ash X 100
Weight of sample
= W3 – W1 X 100
W2 –W1
Where W1 = weight of empty crucible
W2 = weight of dry sample + empty crucible
W3 = weight of ashed sample
Determination of lipid content
Principle:
It was a continuous extraction of fat content from the sample using n-hexane in a soxhlet extractor and non-polar component of the sample was easily extracted into organic solvent.
Procedure:
Soxhlet extractor with reflux condenser and a small round bottom flask were mixed and 2g (w0) of the grounded sample was placed in the thimble which has been dried and weighed (w1).the empty thimble and the powdered sample was weighed (w2) and the mouth of the porous thimble was covered with clean white cotton in order to distribute the draping n-hexane. The thimble was placed in the extractor and n-hexane was added until it was half in the flask. The flask is then heated for five hours. The thimble was removed with care and the n-hexane in the top container was collected. The extract was removed from the water bath when it is almost free of n-hexane. Finally, the extraction flask coating the oil was weighed to know the content of the crude lipid.
Calculation:
% crude lipid = weight of lipid extracted x 100
Weight of dried sample
= w2 – w1 x 100
W0
Where w1 = weight of empty flask
w2 = weight of oil
w0 = weight of sample
Determination of Crude Fiber
Principle:
This was based on the sequential hot digestion with acid and alkaline solution of the defatted sample, followed by thorough washing with boiling water and finally drying off. These ensure the removal of all material.
Procedure
The residue (2g) obtained from crude lipid extraction was placed in a conical flask; 200ml of distilled water and 20ml of H2SO4 was added and fixed on a heater and boiled for 30 minutes to maintain a constant volume. The sample was filtered in a muslin cloth, rinsed with warm water and spatula was used to scrape the sample into the flask, 20ml of H2SO4and 10% of NaOH was added to the contents. The content was placed 30minutes then filtered with muslin cloth and the sample was rinsed with petroleum ether. It was then allowed to drain and the residue was scraped into a crucible and placed in an oven and was dried for one hour at 1050C and allowed to cooled in a desicator and weighed (W1). It was then placed in a muffle furnace to ash for two hours at 6000C and allowed to cool in a desicator and weighed (W2). Percentage fibre was then calculated.
Calculation
% crude fibre = W1 – W2 x 100
Weight of sample
Where W1 = weight after drying
W2 = weight after ashing
Determination of Crude Proteins
Principle
Kjeldahl digestion involves oxidation of organic matter with conc. H2SO4 and a tablet of kjeldahl catalyst. The sulphuric acid converts all form of nitrogen to ammonium sulphate. Subsequent addition of an excess amount of NaOH neutralizes the acid and release ammonia which is distilled into boric acid solution and titrated against HCl to the end point so that the amount of HCl consumed by the ammonia could be calculated.
Procedure
Digestion: 2g of the grounded sample was collected in a clean dry 500ml Kjeldahl flask. One tablet of the mixed catalyst and 20ml of H2SO4 was added. Little amount of distilled water was also added into the flask to digest the organic matter present. The flask was heated in a fume cupboard until clear solution was obtained. The content was cooled and transferred into a volumetric flask.
Distillation: 10ml of the aliquot was pipette into a Kjeldahl flask and make the volume up to 50ml with distilled water.20ml of 40% NaOH was added and extract the ammonia out of the sample which was evaporated into 20ml boric acid indicator that was used as the receiver of nitrogen extracted. The ammonia was liberated into the boric acid until the volume was made up to 40mls in the conical flask. The color changes from pink to green.
Titration: the collected sample with ammonia was then titrated against 0.1N HCl to end point which gave the actual amount of protein in the sample. The color changes from green to pink at the end point and the titre value was recorded.
Calculation
% nitrogen= Tv x N x 0.014 x dilution factor (50ml) x 100
Weight of sample x mls of aliquot
% crude protein = %N x conversion factor (6.25)
Where Tv = titre value
N = normality of acid (0.1N)
Dilution factor = 50
Determination of Carbohydrates (by difference)
Carbohydrates was not determined directly but was obtained by difference as below:
Calculation
% carbohydrate = 100 – (% ash + % protein + % fibre + % lipid).
2.2.2 Determination of Vitamins
2.2.2.1 Determination of Vitamin A (Rutkowski and Grzegorczyk , 2007).
Principle:
Carotenoids were transported as complexes with lipoprotein. The bonds were broken by the addition of ethanol and the pigments extracted with petroleum ether. Absorbance was determined at 450nm and the concentrations calculated by reference to dichromate standard.
Procedure
Four cleaned dried test tubes were labeled test A and B, standard and blank and the following were pipetted as follows
Reagent Test Standard Blank
Sample A (ml) 1.25 – –
Sample B (ml) 1.25 – –
95% Ethanol (ml) 1.25 – –
Standard – 5 –
P. ether 2.5 – 5
The tubes were shaken thoroughly and centrifuged for 5minutes and the petroleum portion of the tube containing the test solution was pipetted into a cuvette and the absorbance were taken at 450nm against petroleum blank.
Calculation
The value of β-carotene = O.D of test x conc. Of standard
O.D of standard
2.2.2.2 Determination of vitamin C (Rutkowski and Grzegorczyk , 2007).
Principle:
This was based on color reaction with periodically prepared phosphotungstate reagent and absorbance taken at 700nm.
Procedure:
1ml of the analyzed liquid into the centrifugal test-tube, 1ml of the phosphotungstate (PR) was added and was mixed thoroughly and left in a room temperature for 30minutes. The tube was centrifuged (7000xg, 10 minutes) and the whole was collected from separated supernatant with a pipette. The supernatant was the test sample for spectrophotometric measurement. The standard was prepared in the same way without centrifugation. The absorbance of the test samples A and of the standard sample A was measured at 700nmagainst the mixture PR: 50m solution of oxalic acid =1:1 (v/v) was used as the reference sample.
Calculation
Cx =Ax. Cs
As
Where Cs = concentration of the standard solution =56.8μm/L
2.2.3 QUALITATIVE PHYTOCHEMICAL ANALYSIS
The methanolic extract is tested for the presence or absence of secondary metabolites using standard qualitative phytochemical procedures (Harbourne, 1984).
Test for Alkaloids
To 2ml of the methanolic extract of Cnidoscolus aconitifolius, few drops of Wagner’s reagent (a solution of potassium iodide and iodine) were added into the test tube. The formation of orange brown precipitate indicates the presence of alkaloids (Sofowora, 1993).
Test for Flavonoids
To 3ml of the extract, 1ml of sodium hydroxide (NaOH) is added. A yellow colouration indicates the presence of flavonoids (Treas and Evans, 2002).
Test for Saponins
To 2mls of the methanolic extract, 3ml of distilled water were add and shaken vigorously for 5minutes. The formation 2cm layer of foam which in turn persist for 10minutes, indicates the presence of saponins (Harborne, 1973).
Test for Tannins
To 2mls of the extract, 3 drops of 0.1% ferric chloride were added. The formation of brownish green indicates the presences of tannins (Treas and Evans, 2002).
Test for Steroids
To 2ml of Cnidoscolus aconitifolius methanolic extract in test tube, 6mls of chloroform and 4mls of concentrated H2SO4 were add carefully by sliding of the test tube. The upper layer will turn red, which in turns the sulphuric acid layer turns yellow with green fluorescence. This indicates the presence of steroids (Sofowora, 1998).
Test for Terpenoids
To 5mls of the extract, 2mls of chloroform and 3mls of concentrated H2SO4 were add carefully. A reddish colouration at the inter-phase indicates a positive result for the terpenoids (Harborne, 1998).
Test for Phenols
brownish solution indicates the presence of phenols (Harborne, 1973).
2.2.4 Mineral Analysis
Digestion of Sample (Wet Digestion)
The sample (5g) was digested with 20 ml of 1:1 HCL/HNO3 in a digestion unit until evolution of brown fumes was stopped. 5g of both fresh and dried sample was weighed using electric balance and placed in a muffle furnace at 600°C for 2 hours and allowed to cool for one hour,10 ml of Nitric acid was mixed with 10 ml of Hydrochloric acid and the mixture was added to 5g of sample and subjected to electric heat plate until a brown smoke vanished. The solution was then filtered using watman filter No.1 and marked 100ml with distilled water (Walingaet al., 1989). The solution was used for mineral analysis.
2.2.5 Analysis of Metals using AAS
In mineral analysis (except sodium, potassium, Magnesium and Calcium) Atomic Absorption Spectrophotometer (AAS) was used due to its good precision and accuracy (Madison et al., 1971).
Principle
The principle is based on nebulizing sample solution into an air-acetylene flame where it vaporizes. Elemental ions are atomized and the atoms formed absorb radiation of characteristic wavelength from a hollow-cathode lamp. The absorbance measured is proportional to the amount of analytic in the sample.
Procedure
Atomic absorption spectroscopic standard solution (1000ppm) of Al, Cu, Fe, Mn and Zn supplied by the manufacturer of the AAS machine were used to prepare working standard solutions by appropriate dilutions of the stock solutions. The AAS machine (Alpha 4 model) was set up in accordance with the manufacturer’s instruction for each element to be analyzed. These include fuel (acetylene) and oxidant (air) selection, burner type, an optimum wavelength and slit-width settings. The standards, blanks and the samples were aspirated into the flame and their concentration in ppm was recorded automatically. The concentration of each analyte (x) in the sample was calculated as:
X(mg/100g) = x (ppm) x vol. of sample made x100
Weight of sample x 10
Figure 2.1: Atomic Absorption Spectrophotometer
Analysis of Sodium and Potassium Standard Solution
To prepare 1000ppm Na+ and K+ stock solution, 2.54g and 1.91g of pre-dried NaCl and KCl (at 105oc for 2 hrs.) was dissolved respectively to 100 ml with distilled water in volumetric flasks. Working standard of 5, 10, 15, 20 and 25ppm were prepared by diluting 0.5, 1.0, 1.5, 2.0 and 2.5ml of the stock solution in a 100ml volumetric flasks with distilled water.
Procedure
The flame photometer (model 400l,corning U.K) was stabilized for 30 minutes before the galvanometer reading was adjusted to zero with distilled water and then to full scale with standard solution of 10ppm. The other standard solutions were subsequently aspirated alongside with the blank and samples solutions. The concentration of sodium and potassium were determined as:
X(mg/100g) = x (ppm) x vol. of sample made x100
Weight of sample x 10
Analysis of Phosphorus
The amount of phosphorus in the sample was determined using tin (II) chloride colorimetric method as reported by IITA (1988). The principle of the method is based on combination of hydrofluoric acid and ammonium fluoride to extract acid –solution forms of phosphorus present in the sample. The phosphate ion combines with ammonium molybdate solution under acidic conditions to form a yellow complex compound known as ammonium phosphor-molybdate as in the equation:-
4po34 + 12(NH4)2 Moo4 +24H+4(NH4)3PO4 12MO3 +12NH4+ +12H2O
Upon selective reduction using tin (II) chloride solution, the yellow complex is reduced to a blue compound due to the molybdenum blue as in equation.
4(NH4)3PO4.12MO3+Sn2+ Molybdenum blue + Sn4+
The intensity of the blue colour is proportional to the concentration of phosphate in the sample.
Procedure
Two ml (2ml) of the digest was pipetted into a 100ml volumetric flask and 2ml each of ammonium fluoride and hydrofluoric acid solution were added to the contents. Ammonium molybdate solution (2ml) was added to the contents, mixed properly and then 2ml of stannous chloride solution was added, shaken and made up to the volume. The contents were allowed to stand for 5 minutes for colour development. Similarly, 0, 2.5, 5.0, 7.5, 10 and 12.5ml of standard phosphorus (1.1g of KH2PO4 in 250ml distilled water) were pipetted into a 100ml flask and treated as for the digested samples.
Finally, the absorbance of the standards, blank and samples were measured using colorimeter (Jenway PFP7 Corning 405) at 760nm wavelength. A calibration curve was plotted from which the concentration of phosphorus in the samples was extrapolated as:
X(mg/100g) = x (ppm) x vol. of sample made x100
Weight of sample x 10
CHAPTER THREE
3.0 RESULT
3.1 Proximate Analysis
Table 3.1: Proximate content of Cnidocolus aconitifolius leaf
Proximate Concentrations(100%/100g)
Moisture 0.67±0.58
Ash 12.67±0.58
Fiber 2.83±0.76
Protein 4.96±0.25
Lipid 13.33±1.15
Carbohydrate 65.54±2.34
Mean ±SD, n=3
3.2 Mineral Component
Table 3.2: Minerals constituents of Cnidocolus aconitifolius leaf
Elements Constituents (mg/100g)
Ca 1.02±0.076
Mg 1.32±0.03
K 3766.67±152.75
Na 80.83±3.82
P 5.38±0.05
Zn 0.1502±0.00
Fe 1.0753±0.00
Cu 0.2545±0.00
Mn 0.0730±0.00
Mean SD, n=3
3.3 Vitamins
Table 3.3: Vitamin constituents of Cnidocolus aconitifolius leaf
Vitamins Constituents (mg/100g)
Vitamin A 18.12±0.20
Vitamin C 15.20±2.41
Mean ±SD, n=3
3.4 Phytochemicals
Table 3.4 Phytochemical composition of Cnidoscolus aconitifolius leaves.
Phytochemical
Methanolic extract
Aqeous Extract
Flavonoid
+
+
Tannin
_
_
Saponin
+
+
Glycoside
+
_
Alkaloid
+
+
Terpenes
+
+
Steroids
+
+
Phenolics
+
+
Key; Present (+), Absent (-).
CHAPTER FOUR
4.0 DISCUSSION, CONCLUSION AND RECOMMENDATION
4.1 DISCUSSION
Phytochemical constituents are responsible for medicinal activity of plant species. This study determined the qualitative and nutritional evaluation leaf of Cnidoscolus aconitifolius. The qualitative phytochemical screening of the methanolic extract of Cnidoscolus aconitifolius showed the presence of Phenols, Flavonoids, Alkaloids, Terpenoids and Saponins. This supports previous findings that the plant contains metabolites such as Phenols, Terpenoids, Saponins among others (Peixoto et al., 2012; Orji et al., 2016; Otitolaiye and Asokan (2016). However, tannin and steroids were present which is in contrast to a previous study reported by Peixoto et al., (2012), and Orji et al., (2016), this may be attributed to the type of solvent used during the extraction. This study agrees with the findings of Araújo et al. (2008), who reported the presence of some of these phytochemicals and attributed the healing and anti-inflammatory activities of Cnidoscolus species to its tannin content. Tannins present in the plant also are used as an anti-diabetic, anti-microbial and anti-viral agent (Pinent et al., 2005; Huwel, 2002; and Matsui et al., 2001). Tannins also play a role in treatment of cardiovascular disease and also have anti-ancer activity (Neto, 2007; Dolara et al., 2005 and Rasmussen et al., 2005). The presence of tannins in plants help quicken the healing of wounds and burns (Sodipo and Akiniyi, 2000). Cnidoscolus aconitifolius leaves have high content of phenols. This was quite expected as many species of this genus are known for their high phenolic content. The presence of phenol is a clear indication that Cnidoscolus aconitifolius can be exploited in pharmaceuticals for the treatment of many disease conditions such as cancer therapy because phenols are well known for the enormous ability to combat cancer (Orji et al., (2016). The presence of phenol in the plant suggests the ability to block specific enzymes that cause inflammation (Okwu, 2001).The presence of terpenoids also justifies and supports the use of Cnidoscolus aconitifolius in the treatment of bacterial infections because terpenes are active against bacteria; it also serves as anti-diarrheal agent (Trease and Evans, 1985).The presence of steroids indicate Cnidoscolus aconitifolius is used to treat a variety of conditions in which the body’s defense system malfunctions and causes tissue damage. Steroids are used as the main treatment for certain inflammatory conditions, such as systemic vasculitis (inflammation of blood vessels) and myositis (inflammation of muscle). They may also be used selectively to treat inflammatory conditions such as rheumatoid arthritis, lupus, Sjögren’s syndrome, or gout (Owolagba et al., 2009).
The potential of a food or plant is determined primarily on nutrient composition, the nutritional constituents of Cnidosculus acontofolius leaves shows the presence of proximate contents such as carbohydrate, proteins, crude fats, moisture contents, ash contents and then crude fibre.
The ash content of a plant based food is the function of mineral element present it presence shows that the mineral element are present in Cnidosculus acontofolius leaves the result of mineral content shows that the carbohydrate possess large amount which is 65.54 while the moisture content seem to be the less as 0.67 while the crude lipids is 13.33 ash content 12.67, crude protein 4.96 and crude fibre 2.83. Ash content present is the function of the mineral present goes further to expose the nutritional benefits of the leaves. The vitamin evaluation based on these it reveal the present of vitamin A and vitamin C the result shows that Vitamin C has 115mg/100g while Vitamin A shows 18mg/100g. the presence of Vitamin C indicates that these plant serves as an antioxidant. This vitamin C is the potent antioxidant that facilitates the transport and uptake of non heme iron at mucosa; the reduction of folic acid intermediate and synthesis of cortisol while vitamin A and E are powerful antioxidant which help to protect cells from damage by free radicals and it is a vital for the formulation and normal function of red blood cells and the muscles. The mineral composition reveals the presence of phosphorus, potassium, copper, zinc, iron, calcium, sodium, magnesium and manganese. The potassium shows the highest results which is 3766mg/100g and the lowest was the iron, then the present of sodium and potassium indicates that the plant can be used in the management and treatment of diseases because potassium and sodium and non activator of energy potentials across the nerve membrane, then the calcium serve as fabrinect formation which formed fibrinogen. This fibrin serve as a clotting factor which is responsible for homeostasis potassium and copper help in the brain function, the iron plays a vital part in the blood function and this may explain the traditional used of these plant as a blood boaster, the iron serves as a component of hemoglobin in blood which transport oxygen from the lungs to different parts of the body.
.
4.2 CONCLUSION
The results of the study revealed that the leaves of Cnidoscolus aconitifolius has numerous potential as a medicinal plant. It possess antimicrobial, anti-inflammatory, antioxidant and analgesic properties..
4.3 RECOMMENDATION
The result showing that the leaves of Cnidosculus aconitifolius contain useful chemical compounds that are both medicinal and nutritional which can be utilized for chemotherapeutical purpose and nutritional supplement. However it is recommended that anti-nutritive supplement using anti-nutritive investigation using variety of solvent should further be carried out to ensure it is complete for human safety consumption.
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