In testing for the presence of bioactive agents in a plant, an extract of the plant must first be prepared by macerating a known weight of the fresh plant with redistilled methylated spirit in a blender. Each extract will then be suction-filtered and the process is to be repeated until all soluble compounds had been extracted, as judged by loss of colour of filtrate. The total extract from each plant part is to be evaporated to dryness in vacuo at about 45 0 c and further dried to constant weight at the same temperature in a hot-air oven. The yield of residue is to be noted and a potion of it is to be used to test for the constituents of the medicinal plants.
METHODS OF TESTING FOR BIOACTIVE PROPERTIES OF MEDICINAL PLANTS.
A] Tests for Alkaloids:- In testing for Alkaloids, Fafowora (2008) explains that about 0.5g of each extract will be stirred with 5ml of I per cent aqueous hydrochloric acid on a water bath; 1ml of the filtrate is to be treated with a few drops of mayer's reagent and a second 1ml portion is to be treated the same way with Dragendorff's reagent.
Turbidity of precipitation with either of those reagents was taken as preliminary evidence for the presence of alkaloids in the extract being evaluated (Harborne, 1973; Evans, 2002). Some laboratories also use Wagner's reagent, picric acid solution or tannic acid solution in place of or in addition to, the two reagents mentioned about (Persiones and Quimby, 1967).
A confirmatory test designed to remove non-alkaliodal compounds capable of eliciting "false-positive" reactions is to be carried out as fallous with all extracts which give preliminary positive test for alkaloids.
A modified form of the thin-layer chromatography (TLC) method described by farnsworth and Euler (1962) is to be used. One gramme of the extract will be treated with 40 per cent calcium hydroxide until the extract is distinctly alkaline to litmus paper, and then extract twice with 10ml portions of chloroform. The extracts are to be combined and concentrated in vacuo to about 5ml. The chloroform extract is then spotted on thin-layer plates. Four different solvent systems (of widely varying polarity) are to be used to develop each plant extract. The presence of alkaloids in the developed chromotograms will therefore be detected by spraying the chromatograms with freshly prepared Dragendorff's spray reagent.
A positive reaction on the chromatograms (indicated by an orange or darker-coloured spot against a pale yellow background) is confirmatory evidence that the plant extract contained an alkaloid.
B] Tests for Saponins
The ability of saponins to produce frothing in agueous solution and to haemolyse red blood cells is used as screening test for these compounds.
For the test, the method described by Wall et al. (1952 and 1954) can be used. About 0.5g of each plant extract was shaken with water in a test tuber. Frothing which persists on warming was taken as preliminary evidence for the presence of saponins. In order to remove "False-positive" results, the blood haemolysis test needs to be performed on those extracts that frothed in water.
About 0.5g of each extract is to be boiled briefly with 50ml phosphate butter, PH 7.4, and then allowed to cool and filtered, 5ml of the filtrate is to be passed for 3 hr. through an asbestos disc (1.5mm thick about 7mm in diameter), which has been previously soaked with two or three drops of 1 per cent cholesterol in ether and dried.
After filtration, the disc should be washed with 0.5ml of distilled water, dried and boiled in 20ml of oxylol for 2hrs to decompose the complex formed between cholesterol and any saponins in the extract. The disc should then be washed in ether, dried and placed on 7 percent blood nutrient ager. Complete heamolysis of red blood-cells around the disc after 6hr. is taken as further evidence of presence of saponins.
C] Test for Tannins
About 5g of each portion of plant extract will be stirred with 10ml distilled water, filtered, and ferric chloride reagent will then be added to the filtrate. A blue-black, green or blue-green-precipitate is taken as evidence for the presence of tannins (Evans 2002).
D] Test for Phlobatannins
Deposition of a red precipitate when an aqueous extract of the plant part was boiled with 1 per cent aqueous hydrochloric acid was taken as evidence for the presence of phlobotannins (Evans, 2002).
E] Test for Anthraquinones
Borntrager's test is used for the detection of anthraquinones. 5g of each plant extract is to be shaken with 10ml benzene, filtered and 5ml of 10 per cent ammonia solution added to the filtrate. The mixture is to be shaken and the presence of a pink, red or violet colour in the ammoniacal (lower) phase indicates the presence of free hydroxyl-anthraquinones.
For bound anthraquionones, 5g of each plant extract is to be boiled with 10ml aqueous sulphuric acid and filtered while hot. The filtrate was shaken with 5ml of benzene, the benzene layer separated and half its own volume of 10 percent ammonia solution added. A pink, or violet coloration in the ammonia phase (lower layer) indicates the presence of anthroquinones derivatives in the extract (Evans, 2002).
F] Test for Cardiac Glycosides
The extract is to be dissolved in pryridine and a few drops of 2 per cent sodium nitorprusside together with a few drops of 20 per cent NaOH are to be added. A deep red colour which faded to a brownish yellow indicates the presence of cardenoloides.
1ml of an 8 per cent solution of the extract in methanol will be mixed with 1ml of a 2 per cent solution 3, 5-dinitrobenzoic acid in methanol and 1ml of a 5.7 percent aqueous sodium hydroxide. An immediate violet colour will indicate the presence of cardenolides in the extract, the colour fading gradually through reddish-brown to brownish-yellow with the precipitation of a whitish crystalline solid. The test indicates the presence of a lactone ring in the cardenolide.
0.5g of the extract will be dissolved in 2ml of acetic anhydride and cooled well in ice sulphuric acid was then carefully added. A colour change from violet to blue to green will indicate the presence of a steroidal nucleus (i.e. aglycone partion of the cardiac glycoside) (Shoppee, 1964).
0.5 of the extract will be dissolved in 2ml of chloroform. Sulpuric acid is then carefully added to form a lower layer. A reddish-brown colour at the interface will indicate the presence of a steroidal ring (i.e. aglycone portion of the cardiac glycoside).
Keller kiliani test
0.5 of extract will be dissolved in 2ml of glacial acetic acid containing one drop of ferric chloride solution. This will then be underlayed with 1ml of concentrated sulphuric acid.
A brown ring obtained at the interface will indicate the presence of a desoxy sugar characteristic of cardenolides. A violet ring may appear below the brown ring while, in the acetic acid layer a greenish ring may form just above the brown ring and gradually spread throughout this layer (Evans 2002).
ACTIVE CONSTITUENTS OF PLANTS AND THEIR MEDICINAL VALUES
The active ingredients are the main effective compounds of medicinal plants, the presence and quantity vary from one plant to the other.
Some of the main bioactive agents/constituents of medicinal plants are:
(i) ALKALOIDS:- It is a mixed group or compound mostly contain nitrogen-bearing molecules (NH 2 ) that make them to be particularly pharmacologically active. There are different types of alkaloids, the principal among which are tropane alkaloids, sanguinarine, quinine alkaloid, berberine, reserpine, atropine etc. Each form of alkaloids serves specific function in the body system.
(a) Sanguinarine alkaloid: It is an alkaloid found in some plant roots and it has persistently been used for the treatment of tumors. This bioactive chemical possesses antimicrobial properties.
It is not very toxic, but oral administration in large quantity is discouraged. Hence, it should not be frequently used by oral administration.
(b) Quinine Alkaloid: This is good for the treatment of malaria. It is used occasionally for the prevention of nocturnal leg cramps caused by vascular spasms.
(c) Berberine Alkaloid: This is a form of alkaloid used in the traditional medicine for treating diarrhea. It is mostly used topically and good for gastrointestinal complaints. Berberine could have side effects and allergic reactions when taken and could be poorly absorbed by oral administration.
(d) Reserpine: This is an isolate of complex mixture of alkaloids present in some plants. This isolated compound is used as anxiolytic, transquilizer or sedative in several herbal drugs. Any drug containing this property can have calming effects on psychotic or hypertensive patients without necessarily induced sleep. Also, it can be used to treat psychiatric and palpitation.
(e) Tropane Alkaloids: The drug containing these elements are widely used as anesthetics for operations and are used in preoperative medication, partly for their ability to dry bronchial and salivary secretions as well induce a degree of amnesia and sedation.
(f) Astropin: This alkaloid has direct effects on the body, reducing spasms, relieving pain and drying up bodily secretions.
(ii) FLAVONIODS: This plant compound is recognized as having properties which are beneficial to human health. It possesses antioxidant elements and ensures healthy circulation. Flavonoids help to strengthen capillary walls. The compound at times is referred to as phytoestrogens. Phytoestrogens are associated with relief of menopausal symptoms, reduction of osteoporosis, improvement of blood cholesterol levels and lowering the risk of certain hormone-related cancer and coronary heart disease.
(iii) SAPONINS: These are heterogeneous group of natural products both with respect to structure and properties. There are two types of saponins triterpenoid and steroidal saponins. They are found in many plant-derived foods and medicinal plants. Many plants containing steroidal saponins have a marked hormonal activity while triterpenoid saponins are often strong expectorants and aid the absorption of nutrients.
Saponins are used as anti-inflammatory (glycyrrhizin, aescin), wound healing (asiaticoside), veinotonic (aescin, ruscogenic glycosides), expectorant (senegocides) and spasmolytic (hedera-saponin) products.
In short, saponins has broad spectrum of biological and pharmacological activities such as anti-inflammatory, anti-hepatotonic, hypoglacemic, antimicrobial and anti-viral activities.
(iv) TANNINS: Tannins are produced to a greater or lesser degree by all plants. They draw the tissues closer together and improve their resistance to infection.
(v) PHENOLS: This group of compounds includes salicylic acid, the natural forerunner of aspirin. Another phenol is thymol, a constituent of thyme. Phenols are antiseptic and reduce inflammation when taken internally. These bioactive agents have an irritant effect when applied to the skin.
(vi) POLYPHENOLS: These are found mostly in tea, coffee etc. These chemical elements protect a person against ageing and can inhibit cancer growth.
Polyphenols act as antioxidants, that is, they protect cells and body chemicals against damage caused by free radicals and reactive atoms that contribute to tissue damage in the body. Polyphenols can also block the action of enzymes that cancer need for growth and they can deactivate substances that promote the growth of cancers.
(vii) ANTHRAQUINONES:- These chemical elements have an irritant laxative effect on the large intestine, causing contractions of the intestinal walls and stimulating a bowel movement and make the stool more liquid thereby easing bowel movements.
(viii) CARDIAC GLYCOSIDES: It is found in many medicinal plants and it contains digitoxin, digoxin and ditoxin. Cardiac Glycosides have a strong and direct action on the heart, helping to support its strength and rate of contraction when it is failing.
Cardiac Glycosides are also significantly diuretic. They also help to transfer fluids from the tissues and circulatory system to the urinary tract, thereby lowering blood pressure.
(ix) CYANOGENIC GLYCOSIDES: These glycosides are based on cyanide, a very potent poison. They have a helpful sedative and relaxant effect on the heart and muscles when taken in small doses. These chemical elements can suppress and soothe irritant dry coughs.
(x) ANTHOCYANINS:- These are pigments which give flowers and fruits, a blue, purple or red hue. Anthocyanins keep the blood vessels healthy. Blackberry and grapes contain appreciable quantities of anthocyanins.
(xi) GLUCOSILINATES:- These chemical elements are found exclusively in species of the mustard family. Glucosilinates have an irritant effect on the skin, causing inflammation and blistering. Herbs containing glucosilinates could be applied as poultices in relieving painful joints; they increase blood flow to the affected area and helpful in removing the build-up of waste products, which is a contributory factor in joint problems. Glucosilinates also help to reduce thyroid function.
(xii) VOLATILE OILS: These are important plant constituents and many of them are strong antiseptic. Some volatile oils contain sesquiterpenes. Volatile oils have anti-inflammatory effects.
(xiii) NICOTINE: This is not uncommon in small amounts in plant tissues and are found mostly in tobacco plant, Nicotiana tobacum L. Nicotine appears to improve learning and memory.
(ix) BITTERS: Bitterness increases efficacy of many medicinal plants. Bitterness stimulates secretions by the salivary glands and digestive organs. Indeed, the secretions can dramatically improve the appetite and strengthen the overall functions of the digestive system.
(x) MINERALS AND VITAMINS: Some plants contain significant amount of minerals and vitamins, the presence and quantity depend on plant family, history and phytochemical properties of the plant.
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