Monday, February 1, 2010

Nutritional and medicinal value of cassava leaves (CHAPTER ONE)

Cassava, Manihot esculenta, also known as manioc or yucca, is a large tuber crop used as a staple in the pacific. Cassava was unknown to the old world before the discovery of America. There are archaeological evidence to two major centres of origin for this crop, one in Mexico and Central America, and the other in Northeastern Brazil. The first Portuguese settlers found the native Indians in Brazil growing the cassava plant and Pierre Martyr wrote in 1494 that the “poisonous roots” of a yucca were used in the preparation of bread. [1].
In Africa, slave merchants introduced the crop to the Western Coast of the continent in about the sixteenth century. Cassava cultivation increased after 1850 in the east African territories because of the efforts of Europeans and Arabs who were pushing into the interior and who recognized its value as a safeguard against the frequent periods of famine. Now grown through out the tropical world, cassava is second only to the sweet potato as the most important starch root crop of the tropics. [1].
Although cassava is an established commercial crop in many tropical countries and hundreds of varieties are in existence, little is generally known of the nomenclature and identification of varieties. Various varieties are usually differentiated from one another by their morphological characteristics such as colour of stems, petioles, leaves and tubers. Moreover, in many instances, the same variety is known in various places by a number of names. The numerous varieties of cassava are usually grouped in two main categories: Manihot Palmata and Manihot utilissima or sweet and bitter cassava respectively. This grouping is a matter of economic convenience as it is difficult to distinguish the two groups by botanical characteristics. However, the distinction rests upon their percentage content of hydrocyanic acid, which causes toxicity in the roots, with the bitter variety having a higher percentage [1].
Cassava is one of the most important stable food crops grown in tropical Africa. It plays a major role in efforts to alleviate the African food crises because of its efficient production of food energy, year – round availability, tolerance to extreme stress conditions, and suitability to present farming and food system in Africa. [1, 2].

A medicinal plant refers to any plant, which contains in one of its organs, substances used for their therapeutic properties. The medicinal plant could also be a precursor for the synthesis of useful drugs. Moringa leaves are good for anaemia, diabetes, high blood pressure (as are cassava leaves), conductivity etc. sesame, baobab, jute, cassia-tree, sorrel, and cowpea leaves cure constipation. Blanched bisaap leaves are used as a malarial prophylactic. [3].
In the form of oils, extracts, gums, waxes, fibres and a vast proportion of natural products, medicinal plants have formed a central point in the living process of man. Indirectly or directly, these serve for prevention and treatment of the simplest to the most complex ailment in human history [4].
However, in modern medicines, medicinal plants have tended to be seen as quaint relics of the past hence next to useless in treatment of serious aliments. To the contrary, about one in four of pharmaceutically prescribed drugs contain ingredients of plant origin. These plants have different medicinal components or compounds, ranging from heart drugs, analgesics, anaesthetics antibiotics, anticancer, anti-parasitic, anti-inflammatory, antioxidant, oral contraceptives, hormones, laxatives etc. tremendous as these may seem, they are derived from different plant species.
Amazingly enough, about 80% of the world’s population rely on the medicinal plants. It is in recognition of this fact that the World Health Organization (WHO) is attempting to incorporate traditional medicine (which is almost completely directly dependent on medicinal plants) into modern health care systems of the developing world.
In such a case where the direct use of these plants to cure physical ailments is, becoming prominent, scientific explanations of their therapeutic activity based on knowledge of their phytochemical constituents become imperative. This relies on phytochemical analysis which no doubt is already being affected for more medicinal plants. [3].

This simply means plant chemistry. Phytochemistry is concerned with the enormous variety of organic substances that are elaborated and accumulated by plant and deals with their chemical structures, biosynthesis, turnover, metabolism, natural distribution and biological functions. Therefore, phytochemical study is the study of these organic substances, which occur naturally in the extract of plants. This is accomplished by simple chemical tests (phytochemical screening) to detect their presence, isolate and purify them and determine their chemical structure and biosynthesis. [5].
These organic substances include alkaloids, tannins, saponins etc, all these accumulate in plant extract. Simple standard chemical test have been derived from such screening, although in some situations, its possible to have false positive tests. Test used in phytochemical screening are simple and rapid as many samples will have to be handled. [5]. Recently, approaches to phytochemical screening has developed to use of sophisticated instruments such as gas-liquid chromatography linked to mass spectroscopy (GLC/MS) to study mirror alkaloids of family cactaceae or to characterize the constituents of volatile oils.
Phytochemical screenings are not only used to search for bioactive agents. Plants have provided the partial synthesis of some useful drugs. An example is the steroidal sapongenins produced by Discorea species and Trigonella species [5]. Plants steroidal sapongenins are used as starting products in synthesis of steroid drugs such as corticosteroids, the sex hormones, and oral contraceptives.
The large-scale isolation of a useful constituent discovered through phytochemical screening is usually achieved chemically (i.e. without biological monitoring) through trial and error, various phytochemical separation techniques such as chromatography, ion exchange resins, and acid-base shakeouts are used. However, there are certain set methods for plant constituents such as volatile oils. These are usually obtained by steam distillation and the composition of the oils determined by GLC/MS [5].

A natural product is a chemical compound or substance produced by a living organism found in nature, that usually has a pharmacological or biological activity for use in pharmaceutical drug design. A natural product can be considered as such, even if it can be prepared by total synthesis. Not all natural products can be fully synthesized and many natural products have very complex structures that are too difficult and expensive to synthesize on an industrial scale. These include drugs such as penicillin, morphine, and paclitaxel (Taxol). Such compounds can only be harvested from their natural source-a process that can be tedious, time consuming, and expensive, as well as being wasteful on the natural resources. For example, four mature yew trees have to be cut down to obtain enough paclitaxel to treat one patient. Furthermore, the number of structural analogues that can be obtained from harvesting is severely limited.
Many higher plants contain novel metabolites with antimicrobial and antiviral prosperities. However, in the developed world almost all clinically used chemotherapeutics have been produced by in-vitro chemical synthesis. Exception like taxol and vincristine, were structurally complex metabolites that were difficult to synthesis in vitro. Many non-natural, synthetic drugs cause severe side effects that were not acceptable except as treatment of last resort for terminal diseases such as cancer. The metabolites discovered in medicinal plants may avoid the side effect of synthetic drugs, because they must accumulate within living cells [6].

1.3.1 TANNIN
Tannins are astringent, bitter tasting plant polyphenols that bind and precipitate proteins [7]. Tannins have molecular weights ranging from 500 to over 3,000 [8]. Tannins comprises of a large group of complex substances that are widely distributed in the plant kingdom, almost every family of plant embodies species, which contain tannins. When tannins occur in appreciable quantities, they are usually localized in specific plant parts such as leaves, fruits, barks or stems. Tannins disappear during ripening process; therefore, it is assumed that the biological role of many tannins in the plant is related to protection against infection, insects or animal herbivore.
Tannins are usually divided into hydrolysable tannins and condensed tannins (proanthocyanidins), while hydrolysable tannins and most condensed tannins are water soluble, some very large condensed tannin are insoluble. Based on their structural characteristics, it is possible to divide the tannins into four major groups [9].
i. Gallotannins, here, galloyl units or their metadepsidic derivatives are bound to diverse polyolcatechin or triterpeniod units.
ii. Ellagitannins, here at least two galloyl units are carbon-carbon coupled to each other and do not contain a glycosidically linked catechin unit.
iii. Complex tannins, in this case, a catechin unit is bound glycosidically to a gallotannin or an ellagitannin unit.
iv. Condensed tannins, these are all oligomeric and polymeric proanthocyanidins formed by linkage of C-4 of one catechin with C-8 or C-6 of the next monomeric catechin.

Figure 1: Galloyl moiety.
Tannins may be employed medicinally in antidiarrheal, haemostatic and anti-haemorrhoidal compounds [7]. Recently, tannins have attracted scientific interest, especially due to the increased incidence of deadly illnesses such as AIDS and various causes. In extensive biological tests, many representatives of tannins were found to have antiviral, antibacterial, antioxidant and especially anti-tumour activity [10], for example, certain tannins can selectively inhibit HIV replication [11].

Saponins are glycosides of steroids, steroid alkaloid (steroids with a nitrogen function) or triterpenes found in plants. They are glycosides containing a polycyclic aglycone moiety of either C27 steroid or C30 triterpeniod (collectively known as sapongenins) attached to a carbohydrate. They have a characteristic bitter taste, foaming properties, and can cause injuries to the digestive mucosa and haemolytic changes in blood. [12].
Saponins are believed to be useful in the human diet for controlling cholesterol, but some (including those produced by the soap berry) are poisonous if swallowed and can cause urticaria (skin rash) in many people. Any markedly toxic saponin is known as a sapotoxin.
In particular, the saponins from Quillaia Saponaria are used in veterinary vaccines as adjuvant (e.g. foot-and-mouth disease vaccines, helping to enhance the immune response). Initially the crude fraction was used but later on Dalsgaard developed a purified mixture, called Quil A. This was more effective and caused less local side reactions. Still Quil A is a mixture of more than 25 different saponin molecules, one of them, the saponin Os 21, is being investigated for possible beneficial adjuvant effects on the human immune system.
The medicinal uses of saponins include; hypercholesterolemia, hyperglycaemia, antioxidant, anticancer, anti-inflammatory, weight loss, gentle blood cleanser. Note; the medicinal value of saponins is due to their expectorant effect.

An alkaloid is strictly speaking a naturally occurring amine produced by a plant, but amines produced by animals and fungi are also called alkaloids [13]. The name is derived from the word alkaline; originally, the term was used to describe any nitrogen-containing base. Alkaloids are usually derivatives of amino acids, and have bitter taste.
More than 10,000 different alkaloids have been discovered in species from over 300 plants families. Alkaloids often contain one or more phenolic or indole rings, usually with a nitrogen atom in the ring. The position of the nitrogen atom in the carbon ring affects the properties of these alkaloids. The amazing effect of these alkaloids on humans has led to the development of powerful painkiller medications and spiritual drugs. [14].
Well known alkaloids include morphine, ephedrine, nicotine and codeine, etc. they have profound and variable effects on man and other animals, hence their use in medicine [15].

Figure 2: Ephedrine Figure 3: Nicotine
Alkaloids are usually colourless, crystalline, non-volatile solids, which are insoluble in water but soluble in ethanol, ether, chloroform and many other organic solvents, some like nicotine and cocaine however, are liquids and soluble in water.
Few alkaloids are also soluble in acid media but only slightly soluble in natural or basic media. Most of them are optically active (usually laevorotatory), [16].
There are three main types of alkaloids
Chart 1: Types of alkaloids

Have heterocyclic ring Does not have heterocyclic Have heterocyclic
with nitrogen ring with nitrogen ring with nitrogen

Derived from amino acids. Not derived from amino acids.

- Colchicine is an example of a proto-alkaloid.
- Pseudo-alkaloids can be derived from; terpenoids or purines.
- The basic unit in the biogenesis of the true alkaloids are Amino acids.

Glycosides are certain molecules in which a sugar part is bounded to some other part. They are plant compounds containing glucose (or a different sugar) combined with other non-sugar molecules, such as glucose + terpene or glucose + phenolic compound. The terms glycosides and glucoside are used interchangeable; however, glucoside is generally used if the sugar component is glucose.
The classification of glycosides can be by the glycone, type of glycosidic bond and by the aglycone.
i. By glycone. If the glycone group of a glycoside is glucose, then the molecule is a glucoside; [17] if it is fructose, then the molecule is a fructoside; if it is glucuronic acid, then the molecule is a glucuronide; etc. In the body, toxic substances are often bounded to glucuronic acid to increase their water solubility; the resulting glucuronide are then excreted.
ii. By type of glycosidic bond, depending on whether the glycosidic bond lies above or below the plane of the cyclic sugar molecule, glycosides are classified as α-or β-glycosides.
iii. By aglycone. Glycosides are also classified, according to the chemical nature of the aglycone for purposes of biochemistry and pharmacology; this is the most useful classification:
- Alcoholic glycosides, e.g. salicin it has analgesic, antipyretic and anti-inflammatory effects.
- Anthraquinone glycoside contain aglycone group that is a derivative of anthraquinone. They have a laxative effect.
- Coumarin glycosides, the aglycone is a coumarin e.g. apterin which is reported to dilate the coronary arteries as well as block calcium channels.
- Flavonoids glycosides, the aglycone is a flavonoid, e.g. quercitin. Among the important effects of flavonoids are their antioxidant effects, and their ability to decrease capillary fragility

Figure 4: Salicin

The term flavonoid refers to a class of plant secondary metabolites. They are polyphenolic compounds possessing 15 carbon atoms; two benzene rings joined by a linear three-carbon chain (C6-C3-C6).

Figure 5: skeletal structure of flavonoids
Flavonoid constitutes one of the most characteristic classes of compounds in higher plants.
According to the IUPAC nomenclature [18], Flavonoids can be classified into:
i. Flavonoids derived from 2-phenylchromen-4-one (2-phenyl-1, 4-benzopyrone) structure.
ii. Isoflavonoids derived from 3-phenylchromen-4-one (3-phenyl-1, 4-benzopyrone) structure.
iii. Neoflavoniods derived from 4-phenylcoumarin (4-phenyl-1, 2-benzopyrone) structure.

Figure 6: Molecular structure of the flavone backbone (2-phenyl-1, 4-benzopyrone)

Flavonoids are most commonly known for their antioxidant activity. However, it is now known that the health benefits they provide against cancer and heart disease are the result of other mechanism, [19]. Flavonoids are also commonly referred to as bioflavonoid in the media; the terms are equivalent and interchangeable, for flavonoids are biological in origin.
Flavonoids have been referred to as “natures’ biological response modifiers” because of strong experimental evidence of their inherent ability to modify the body’s reaction to allergens, viruses, and carcinogens. They show anti-allergic, anti-inflammatory [20], anti-microbial and anticancer activity and especially their potential role in the prevention of cardiovascular disease. The beneficial effects of fruits, vegetables, and tea or even red wine have been attributed to flavonoid compounds rather than to known nutrients and vitamins.

A reducing sugar is any sugar that, in basic solution, forms some aldehyde or ketone. This allows the sugar to act as a reducing agent, for example in the Maillard reaction and Benedict’s reaction. Reducing sugars include glucose, glyceraldehyde, lactose, arabinose and maltose. All monosaccharides, which contain ketone groups, are known as ketoses, and those, which contain aldehyde groups, are known as aldoses. Significantly, sucrose is not a reducing sugar. It is infact known as a non-reducing sugar.
A reducing sugar occurs when its anomeric carbon is free. Since sugars occur in a chain as well as a ring structure, it is possible to have equilibrium between these two forms. When the hemi-acetal or ketal hydroxyl group is free, it is not locked, not linked to another (sugar) molecule, the aldehyde (or keto-) form (i.e. the chain-form) is available for reducing copper (II) ions. When a sugar is oxidized, its carbonyl group (i.e. aldehyde or ketone group) is converted to a carboxyl group [21].

Cassava is a common plant cultivated almost everywhere around us. Its botanical name is Manihot utilissima pohl of the family Euphorbiaceae, but in recent publications, the name Manihot esculenta crantz is being increasingly adopted. The plant is popularly known in Africa in recent times and especially in Nigeria as the “green gold of Africa”, due to its vast uses and the income generated from it.
It is well known that the health of a man is dependent on his dietary intakes. As such, an accurate assessment of the nutrient content of food is becoming more important. Because of this, cassava leaves being one of the good sources of protein, calcium, iron, vitamins etc, it is already cultivated through out the tropical world.
Africa production of cassava is estimated to be 125 million tones. When the crop is given more attention, yields of 30-40 tons per hectare are obtained, and it has been reported that it is normal for some varieties, under appropriates cultivation methods, to yield over 60 tons per hectare.
Cassava leaves form a significant part of the diet in many countries in Africa. They are used as one of the preferred vegetables in Zaire, Central African Republic, Liberia, etc. out of the estimated 125 million tons of cassava produced yearly [22], about 90% is used as human food while the remainder is used largely as animal feed and for other industrial products.

In the third world countries, large number of people die daily due to lack of simple health care and lack of adequate dietary intake, as a consequence of high cost of food substances and modern health care. It is therefore necessary to improvise a means through which the people can attain effective health care and dietary requirement. Therefore, the principal aim of this project work is to
i. Identify the presence of some natural products in cassava leaves (e.g. flavonoids, tannins etc).
ii. Determine the number of components in a chloroform extract of the leaves.
iii. Make known the nutritional value by analyzing for carbohydrate, fats, vitamin, fibre and ash in the leaves.
iv. Educate the public on the need for consumption of cassava greens.

The study will use the leaves of cassava for the analysis; even though both the roots (tubers) and the leaves are used as food as well for medicinal purposes. The extraction method that will be employed is Soxhlet extraction and aqueous extraction procedures. Crude extract of the leaves will be obtained which would be used in testing for the naturally occurring components that are responsible for marked physiological responses in man. In addition, compounds that are good for dietary intake.

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