Asian Pacific Journal of Tropical Medicine (2010)101-104 101

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Document heading The phytoconstituents and the comparative effects of aqueous extract of gabonensis and proviron on the biochemical parameters of male guinea pigs

1 2 Obianime AW , Uche FI * 1Department of Pharmacology, University of Port Harcourt, 2Department of Pharmacognosy and Phytotherapy, University of Port Harcourt, Nigeria

ARTICLE INFO ABSTRACT

Article history: Objective: ToIrvingia investigate gabonensis the phytochemical screening and the effects of the aqueous extractsMethods of Received 21 August 2009 the seeds of on the biochemical parameters of male guinea pigs. : Received in revised form 20 September 2009 The biochemical parameters were assayed using Randox Diagnostic kits, Phenolphthalein Accepted 11 November 2009 method and Resultscolorimetric method. The phytochemical screening was carried out using standard Available online 20 February 2010 procedures. : Phytochemical investigations revealed the presence of flavonoids, tannins, carbohydrate, alkaloids,Irvingia terpenoids, gabonensis steroids, volatile oils, saponins and cardiac glycosides. TheP aqueous extract of seeds (50 - 400 mg/kg) caused a statistically significant ( < Keywords: 0.05 ANOVA) decrease in the levels of total cholesterol, urea, uric acid, total protein, prostatic, alkaline, and Irvingiaacid phosphatases. gabonensis The highest reduction effect was obtained with uric acid at Phytoconstituents 400 mg/kg of extract while the Conclusionsleast effect was observed in total cholesterol. These effects were dose- and time- dependent. : This shows that the seeds of Biochemical parameters Irvingia gabonensis Proviron have hepatoprotective, nephroprotective and cardio protective properties. The Male guinea pigs study therefore, supports the claims on the use of the seeds of this by traditional medicine practitioners as a hepatoprotective and nephroprotective agent. Although further studies need to be done to isolate, identify and characterize the active principles in the seeds of this plant.

1. Introduction Irvingia gabonensis bark is given to women to shorten their [7] Irvinga gabonensis breast feeding period. It is also used for colic and dysentery , (family of ) is a large for hernias, yellow fever and as anti- poison[3]. It also has with dense compact crown of evergreen large with antimicrobial properties; the decoction of the bark is used [7] Irvingia edible seeds and sweet edible fruit pulp. It is found in forgabonensis treating scabies, toothache and skin diseases . rainforests, widely distributed in tropical West and has hypoglycemic effect hence its use as an [8, 9] represented by two varieties: the fruit with sweet edible anti-diabetic agentIrvinga gabonensis. scanty fibrous pulp, fluted or cylindrical holes and fruits with bitter Even though has been used extensively [1,2] Irvingia gabonensisin edible, very fibrous pulp with buttressed whole . in Nigeria in the treatment of various diseases traditionally, usually reach maturity and begin flowering there has never been a scientific report on its safety and at Irvingia 10 -15 gabonensisyears old. effects on biochemical parameters. It is on the light of this is commonly known as African , that this study seeks to establish for the first time, scientific Dikanut, or bush Mango. The seeds are known as Ogbuno information on its effects on the biochemical parameters of in Ibo, Apon in Yoruba Nigeria. The paste from the kernel male guinea pigs. [3, 4] is Irvingiaknown as Dikagabonensis bread in Gabon and Etima in Cameroon . Irvingia gabonensis is very useful to man. The bark of 2. Materials and Methods is used for diarrhea or dysentery , the fruit is rich in and is consumed as a desert fruit 2.1. Plant materials throughout Western and Central Africa[5, 6]. The pulp is used for [5, 7] making jelly, jam and juices . The seeds are used as thickener Irvingia gabonensis for soup, stew or as additive for flavouring [6]. Traditionally the The seeds of were collected in June 2008 from the eastern part of Nigeria. The plant was authenticated by H.D Onyeachusim, a taxonomist at Botany *Corresponding author: Uche F. I., Department of Pharmacognosy and Phytotherapy, University of Port Harcourt, Nigeria Herbarium of University of Port Harcourt Nigeria, where Tel: +23480366891 voucher specimen was deposited. All the chemicals used E-mail: [email protected] Obianime AW et al./Asian Pacific Journal of Tropical Medicine (2010)101-104 102 were of analytical grade. each. Group 1-5 were used for time-dependent studies 2.2. Preparation and extraction of plant sample for a period of 28 days. Group 6-10 were used for dose- dependent studies. The animals from group 6-10 were administered different doses of the extract (50-400 mg/kg/ The plant℃ seeds were dried under the oven at temperature day) for 96 hours after they were sacrificed. While group of 28 for 2 hours. The dried seeds were ground with 1-5 were administered a fixed dose of the extract (400 mg/ hammer mill and the fine powder were extracted using kg/day) over a period of 7, 14, 21, 28 days respectively. Soxhlet apparatus. The yields of the extract were obtained At the end of each treatment period, the animals from after removal of solvent. The extracts were stored in the different groups were anesthetized with diethylether. The refrigerator for subsequent reconstitution and use. blood samples were collected by cardiac puncture with 2.3. Animals 21G needle fixed on 5 mL syringe, for serum liver enzyme makers and blood enzyme assays. These were assayed using Randox Diagnostic kits, [17] Phenolphthalein method[15] ,and Adult male guinea pigs of average weight 300-600 g colorimetric method [16, 19]. Sample serum was separated were obtained from the animal house of University of Port from the cells, centrifuged at 3 400 r for 10 minutes and Harcourt. They were housed in a cage of five animals and used for the assays. were allowed to acclimatize to the new environment for 10 2.6. Statistical analysis days. The animals were properly feed on elephant grass throughout the experimental period. 2.4. Phytochemical screening Data were expressed as mean暲standard error of mean (SEM) of five observations. Statistical analysis of data was performed using analysis of variance (ANOVA). Results Chemical tests were carried out on the extracts and on the were subjected to Graph Pad prism 5 demo (software) powdered specimens using standard procedures to identify analyses, theP differences between mean accepted as the constituents[10, 11] by characteristic colour changes as significant at <0.05 (ANOVA). described by Sofowora A. et al [12, 13]. Briefly, formation of brownish green coloration on addition of 3 drops of ferric 3. Results chloride to sample confirmed presence of tannins; formation of yellow coloration which disappears on standing when 5 mL of dilute ammonia solution and concentrated sulphuric acid Phytochemical screening revealed the presence of steroids, were added sequentially to portion of the extract confirms the flavonoids, alkaloids, steroids, cardiac glycosides, volatile presence of flavonoids; presence of steroids was confirmed by oils, terpenoids, tannins and saponins. Irvingia colour change from violet to blue on addition of 2 mL acetic gabonensis This study showed that the aqueous extract of anhydride and 2 mL sulphuric acid to 0.5 g plant extract; causes a dose- and time- dependent decreases terpenoids were confirmed by formation of reddish brown in the biochemical parameters such as: Urea, Uric acid, colouration of the interface on addition of 2 mL chloroform Creatinine, Total cholesterol, Protein, Alkaline, Acid, and concentrated sulphuric acid, 3 mL to 5 mL of the and Prostatic Phosphatases (Figure1 and 2; Table1P and extract; saponins were confirmed in the plant by the frothing 2).These effects were statistically significantIrvinga gabonensisat < 0.05 test; cardiac glycosides were confirmed by formation of (ANOVA). The observed effects of brown ring of interface on addition of 2 mL glacial acetic on the biochemical parameters of male guinea pigs were acid containing 1 drop of ferric chloride solution and 1 mL comparable to that of Proviron (Table 2). concentrated sulphuric acid; presence of alkaloids were The administration of cadmium caused significant confirmed by Dragendorff reagent which formed a reddish increases in the levels of all the biochemical parameters brown precipitate with the sample. assayed except total protein.Irvinga And gabonensis this effect was inhibited 2.5. Evaluation of biochemical parameters by pre-treatment with (Figure 1 and 2; Table 1). But there was no significant inhibitory effect observed in Urea, irvinga Uric gabonensis acid and alkaline phosphate by pre- The animals were divided into ten groups of five animals treatment with (Figure 1 and 2).

Table 1

Irvingia gabonensis The dose-dependent comparative effects of and Proviron on the biochemical parameters of male guinea pigs. Dose (mg/kg) ACP PAP ALP UA TP Urea Creatine TC Control 12.7暲1.1 7.2暲0.2 24.3暲1.2 399.0暲6.0 52.5暲3.8 6.4暲0.3 63.7暲6.1 2.80暲0.01 a a a a 50 12.2暲1.2 5.7暲0.1 16.3暲1.0 390.0暲6.0 55.3暲3.9 5.6暲0.4 48.3暲3.1 3.40暲0.09 a 100 22.2暲2.2 12.9暲1.1 25.0暲2.5 435.0暲7.0 50.3暲2.0 4.9暲0.3 59.0暲4.5 2.80暲0.01 a a 200 13.8暲1.9 7.0暲0.3 23.3暲2.1 435.3暲6.8 52.3暲2.2 4.4暲0.3 48.0暲3.0 2.70暲0.02 a a a 400 27.0暲3.5 16.4暲2.7 28.8暲1.5 446.5暲6.7 55.0暲3.8 6.0暲0.4 58.7暲4.0 2.90暲0.03 a b Cd 24.7暲2.0 19.7暲3.0 25.5暲1.8 510.5暲27.2 51.5暲1.9 4.9暲0.2 136.0暲14.2 2.90暲0.03 a Cd +I.G 16.6暲2.8 8.6暲0.9 28.5暲1.5 482.5暲6.9 51.3暲1.8 16.9暲0.8 66.3暲6.5 2.90暲0.03 n P P Irviniga gabonensis Results expressed as Mean 暲 SEM; = 5; a: < 0.05; b: <0.001 (ANOVA); Cd represents cadmium; I.G ; ACP acid phosphatase, PAP prostatic acid phosphatase, and ALP alkaline phosphatase; UA uric acid; TP total protein; TC total cholesterol respectively. Obianime AW et al./Asian Pacific Journal of Tropical Medicine (2010)101-104 103 Table 2 Irvingia gabonensis The time-dependent comparative effects of and Proviron on the biochemical parameters of male guinea pigs. Time (days) ACP PAP ALP UA TP Urea Creatine TC 0 12.7暲1.1 7.2暲0.2 24.3暲1.2 399.0暲6.4 52.5暲1.5 6.4暲0.5 63.7暲6.2 2.80暲0.01 a a 7: I.G 13.8暲1.3 7.0暲0.2 23.3暲1.2 390.0暲6.0 52.3暲2.2 4.4暲0.3 48.0暲3.2 2.70暲0.02 a PV 14.3暲1.9 9.8暲0.2 22.7暲1.1 418.0暲7.0 55.7暲3.9 7.2暲0.6 59.7暲4.5 2.70暲0.02 a a 14: I.G 10.2暲2.7 5.8暲0.2 25.7暲1.0 399.0暲6.4 49.7暲1.4 9.7暲 0.9 78.7暲13.5 2.70暲0.02 a a PV 9.8暲2.6 4.9暲0.1 26.0暲1.1 383.0暲6.1 50.0暲2.1 15.8暲1.5 70.0暲11.0 2.60暲0.02 a b 21: I.G 16.2暲2.8 9.3暲1.2 27.7暲1.3 372.0暲5.1 50.7暲2.0 15.3暲1.4 80.0暲13.2 2.70暲0.01 PV 14.4暲2.6 9.0暲1.2 31.0暲1.5 403.0暲7.1 53.3暲2.3 67.0暲0.5 84.0暲13.5 2.70暲0.01 a a 28: I.G 19.3暲3.0 11.5暲1.8 26.0暲1.1 504.8暲9.0 58.8暲4.0 5.8暲0.3 64.0暲6.0 3.10暲0.04 a a PV 33.3暲3.9 15.9暲2.2 34.0暲2.0 521.7暲7.0 81.0暲7.1 5.5暲 0.3 56.0暲4.5 3.40暲 0.05 n P P Irviniga gabonensis Results expressed as Mean暲 SEM; = 5; a: < 0.05; b: <0.001 (ANOVA). Cd represents cadmium; I.G ; ACP acid phosphatase, PAP prostatic acid phosphatase, and ALP alkaline phosphatase; UA uric acid; TP total protein; TC total cholesterol respectively.

Figure 1. Irvinga gabonensis The effects of 50 mg/kg of on the biochemical parameters of male guinea pigs.

are used as basic medicinal agents for their antispasmodic [17, 19] 4. Discussion and bactericidal effects . Tannins have astringent properties, hasten the healing of wounds and inflam mucous membranes. This may be responsible for its use in cleansing The flavonoids, as an anti-oxidant in this plant may and treatment of wounds [17-19]. contribute to the effects of this plant as hepatoprotective, nephroprotective, antimicrobial, anti-inflammatory, and By reducing the total cholesterol levels, it indicates that anti-carcinogenic effect[14,17], Alkaloids in this plant may this plant can exert anti-diabetic, anti- hypertensive, and be responsible for its analgesic effects and its use as an anti- lipidemic properties. This is consistent with the past [17] [14, 15, 17] antimicrobial agent . This is consistent with the past works . By lowering the Irvingialevels of gabonensis Phosphatases works [14, 18-20]. Alkaloids and their synthetic derivatives and Creatinine, it indicates that has Obianime AW et al./Asian Pacific Journal of Tropical Medicine (2010)101-104 104

Figure 2. Irvinga gabonensis The effects of 50 mg/kg of on the biochemical parameters of male guinea pig.

MAB Digest 17 timber products. 1994; : 67-8. hepatoprotective and nephro-protective effects. This is also Irvingia gabonensis [14, 17, 20] [7]Okolo CO. The Analgesic effects of stem bark consistent with the past works . J Ethno Pharmacol 45 The Uric acid, total cholesterol and protein lowering extract. 1995; (2):125-9. [8]Adamson I. The Erythrocyte membrane ATPase in diabetes; effect effect of these indicate that the plant can have anti- Irvingia gabonensis Enzyme 36 hypertensive, anti-inflammatory and anti- nociceptive of dikanut ( ) 1986; (3):212-5. effects. This is also consistent with the past works [14, 17-20]. [9]Omoruyi F, Adamson I. Effects of supplement of dikanut and cellulose on plasma pids and composition of hepatic phospholipids This studyIrvingia therefore gabonensis supports the claims on the folkloric - Nutrition Research use of as a hepatoprotective and in streptozotocin14 induced diabetic rat. 1994; (4):537-44. nephroprotective agent. It also supports the potentials of the Trease and Evans Pharmacognosy. [10]Trease GE, Evans WC. plant as anti-carcinogenic, anti-lipidemic, analgesic and A physician guide to herbal medicine. anti-inflammatory agent. Further studies are on the way to 13th ed. London: Ballere Tindal;1989. isolate, identify and characterize the active principles in Phytochemical methods A guide to modern techniques Irvingia gabonensis [11]Harbone JB. : the seeds of as well as identification of of analysis. Irvingia gabonensis 3rd ed. London: Chapman and Hall;1978. mechanism of action of . Medicinal plants and traditional medicine in Africa [12]Sofowora A. . Ibadan: Spetrum Books Ltd;1993. Conflict of interest statement [13]Sofowora OO. Phytochemical screening of Nigerian medicinal Lloydia 41 Plants. 1978; :41,234. We declare that we have no conflict of interest. [14]Adeneye AA, Amole OO, Adeneye AK. The hypoglycemic and hypocholesterolemic activities of the aqueous and extract of Fitoterapia 77 Phyllanthus amarus in mice. 2006; : 511-4. References [15]Babson LA, Greeley SJ, Coleman CM, Phyllips GD. Phenolphthalein Monophosphate as a substrate for serum alkaline Clin Chem 12 [1]Harris DJ. A revision of the irvingiaceae in African bulletin du phosphatase. 1996; : 482-90. National De Vbelgique 65 Jardin Botanigue. 1996; (1-2):143-96. [16]William FH, Harold DM. Determination of serum acid phosphates Biochemical Analysis 4 [2]Okofor JC. Woody plants of nutritional importance in traditional method. 2006; : 257-84. Forest Abstract farming systems of the Nigeria humid tropics. [17]Uche O. The Phytochemical screening and the effects of 44 Phyllanthus amarus 1983: (10): 607, 211-21. methanol extracts of on the Biochemical J Appl Sc Environ Manage 12 [3]Ayuk ET. Uses, management and economic potential of I. parameters of male guinea pigs. 2008; Forest Ecology and gabonensis in humid low lands of Cameroon. (4):73-7. Management 113 1999; :1-9. [18]Okwu DE, Josiah C. Evaluation of the chemical composition of two Afri J Biotechnol 5 [4]Akubor PI. The suitability of African bush mango juice for wine Nigerian medicinal plants. 2006; (4): 357-61. Plant Foods Hum Nutri 49 production. 1996; :213-9. [19]Okwu DE. Phytochemicals and vitamin content of indigenous Nutritional values of Ogbuno(Irvingia gabonensis J Sustain Agric Environ 6 [5]Ejiofor MAN. spices of Southern Nigeria. 2004; (1): 30-37. excelsa var. ). Paper presented at the International Centre for Research [20]Uche FI, Obianime AW. The effects of vitamin E on Vanadium in and International Institute of Tropical Agriculture pentoxide- induced toxicity in biochemical parameters of male Irvingia gabonensis J Appl Sc Environ Manage 2 Conference on . Ibadan, Nigeria;1994. guinea Pigs. 2008; (3):101-5. [6]Leakey R, Newton A. of tropical trees for timber and