Phytochemistry And Preliminary Toxicity Studies Of The Methanol Extract Of The Stem Bark Of Garcinia kola (Heckel)
H Kagbo, D Ejebe
H Kagbo, D Ejebe. Phytochemistry And Preliminary Toxicity Studies Of The Methanol Extract Of The Stem Bark Of Garcinia kola (Heckel). The Internet Journal of Toxicology. 2009 Volume 7 Number 2.
The use of plants in traditional medical practice has a long drawn history, and remains the mainstay of primary health care in most of the third world (Prescott-Allen and Prescott-Allen, 1982). Traditional medicines are used by about 60% of the world population; in both developing and developed countries (where modern medicines are predominantly used)
The relevance of a plant to pharmacy lies in the ability of the plant to elaborate organic compounds that possess pharmacological properties, or compounds that are of use in pharmaceutical formulations as flavouring agents or formulation aids. For example, some plant secondary metabolites such as alkaloids, phenols, tannins, glycosides, terpenoids, saponins, flavonoids and steroids have been implicated in their ability to inhibit the formation of pro-inflammatory signalling molecules such as prostaglandin or leukotrienes (Polya, 2003).
Recently discovered substances that have analgesic properties included those of the alkaloids, flavonoids and terpenoids phytochemical classes (Musa
Constituents of the seed of
The anti-inflammatory property of flavonoids is believed to result from inhibition of cyclo-oxygenase enzyme (Liang
Earlier Harley (1970) had reported the antitusive, antitumour and aphrodisiac activities of
Furthermore, In Nigeria as in other developing countries, traditional medicines are in widespread use; with the practitioners formulating and dispensing the recipes.
The medicaments are prepared most often from a combination of two or more plant products which many a time may contain active constituents with multiple physiological activities and could be used in treating various disease conditions (Pieme et al., 2006; Ogbonnia
In this study, the stem bark of
Materials And Methods
Preparation of Plant Material
The stem bark of
The dried plant material was pulverised with a manual grinder, packaged and labelled G1, G2, G3 and G4 according to the locations from which the samples were collected (G1- Bori-Ogoni; G2- Warri, G3- Eket and G4- Port Harcourt city).
100g of each pulverised plant material was soaked in 300ml of methanol and intermittently shaken. The mixture was kept for 72 hrs after which it was filtered with Whatman No. 1 filter paper and concentrated. The filtrate was stored in a refrigerator until required for use.
Phytochemical screening was carried out on the methanolic extract of the stem bark of
Test for cardiac glycosides
A brown ring of the interface indicates a deoxysugar characteristic of cardenolides. A violet ring may appear below the brown ring, while in the acetic acid layer, a greenish ring may form just gradually throughout thin layer.
Concentrated Sulphuric acid was carefully added to form a lower layer.
Test for Anthraquinones
Free Hydroxy anthraquinones
The Borntrager’s test for anthraquinones was used. 5mg of the plant extract was shaken with 10ml of benzene, filtered and 5ml of 10% ammonia solution added to the filtrate and the mixture shaken.
5mg of plant extract was 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% ammonia solution added.
Animals and experimental design
Preliminary test, with 4 mice per pilot dose level, was conducted to establish the range of toxicity so that the proper dose levels could be established for LD50 determinations. With the pilot tests it was possible to establish the highest dose of the extract that killed none of the exposed animals (200mg/kg) and the lowest dose that killed all the animals (500mg/kg). The dose levels used in the acute toxicity study ranged between these two dose extremes.
30 mice weighing between 20 - 25 g were kept in 5 cages (6 per cage) and handled according to standard guidelines for the use and care of laboratory animals.
The animals were maintained on standard animal diet and water. However food was withdrawn 18 h before the start of the experiment according to the method of Amresh et al. (2008).
The LD50 value was calculated with Arithmetic Method of Karber (Turner, 1965; Aliu & Nwude, 1982) and Arithmetic Method of Reed & Muench (Reed & Muench, 1938)
The five groups of mice were administered intraperitoneally with 250mg/kg, 300mg/kg, 350mg/kg, 400mg/kg & 450mg/kg of the extract respectively and the groups were observed for mortality for 24 hours; a sixth group (the control) was administered with serially diluted 5% Na2CO3, the solvent in which the extract showed relative solubility.
Assessment of Haematological Parameters
50 male Wister albino rats (weighing 200-250g) used for this study were obtained from the Faculty of Pharmacy Animal house, University of Uyo. They were housed in plastic cages, with food and tap water available
Administration of Garcinia kola stem bark extract.
Animals in all groups except the controls, were given by gavage, different concentrations of the methanol extract of
Collection and Handling of Blood Samples
All animals were sacrificed at the end of 7 and 14 days of treatment with
Estimation of Packed Cell Volume (PCV)
Blood sample anticoagulated with EDTA was made to enter a plain glass capillary tube, one end of which was later sealed with non absorbent sealer clay. The tube was then spun at 11,000 rpm for 5 min in a microhaematocrit centrifuge.
The PCV value was then read, using a microhaematocrit reader.
Estimation of Blood Cell Parameters
Haemoglobin (Hb) concentration and red blood cell (RBC) were estimated using a semiautomatic haematological analyzer (SWELAB IEO Model). The auto counter utilized 20μl of blood in 16ml of a commercially prepared diluent. The machine’s ability to count cells was based on the principle of electronic impedance.
Phytochemical evaluation of the methanolic extract of
Assessment Of Some Hematological Parameters
Treatment, by gavages with
The phytochemistry and median lethal dose (LD50) of the methanolic extract of the stem bark of
Decoction and infusion of
The result of the phytochemical screening indicated that the stem bark of
The stem bark of
The high phlobatannin & tannin content of the extract therefore scientifically justify the use of pulverized
The haematological studies showed that the indices examined (Hb, PCV, RBC and Platelet counts) showed a dose dependent significant reduction for the animal groups tested in the first week, and a dramatic dose-dependent rise in those tested for two weeks. This could be due to complexes which flavonoid forms with reactive metals such as iron, zinc and copper thereby reducing their nutrient absorption in the first week (Siegenberg, 1991). The formation of these complexes might have affected haemoglobin synthesis and erythropoiesis (Fairbanks,
Furthermore, the antioxidant activity of flavonoids (contained in G.kola stem bark powder) which ultimately maintains the haeme iron in its ferrous state (vis-à-vis the ferric state that is associated with production of defective methaemoglobin) seem to enhance erythropoiesis. The link between antioxidant activity and haemoglobin quality and quantity was shown by the observation that ascorbic acid (a standard antioxidant) via its action as a free radical scavenger, increased significantly the haemoglobin levels in children suffering from sickle cell anaemia (Jaja et al., 2002; Ahumibe and Braide, 2009).
In conclusion, the constituent of the methanol extract of G. kola stem bark extract do not impart any significant toxicological implication to erythrocytes, rather it showed the tendency of increasing the erythrocyte number over time, this property has been confirmed by Esomonu et al, 2005, in the seed of G.kola.
It has been argued that even if LD50 values could be measured exactly and reproducibly, the knowledge of its precise numerical value would barely be of practical importance, because an extrapolation from the experimental animals to man is hardly possible (Lorke, 1983), however, it still serve a great purpose as a first pointer to the safety or toxic potential of a substance whose toxicity profile is not yet known.
The part of the plant under investigation (stem bark of
The result in this study validate the traditional medicinal use of the evaluated part of
The authors really appreciate the assistance of Mr. Danladi Bala and Nsikan Malachy Udo of Departments of Pharmacognosy and Pharmacology & Toxicology respectively, University of Uyo