Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 1(8), 72-75, Nov. (2011) Res.J.Chem.Sci. International Science Congress Association 72 Short CommunicationChemical Composition and Acute Toxicity Studies on the Aqueous Extract of Combretum dolichopentalum Leaf in Swiss Albino Mice Kalu F.N. *1, OguguaV.N. 1, Ujowundu C.O.2 and Chinekeokwu C.R.K.Department of Biochemistry, University of Nigeria, Nsukka, Enugu State, NIGERIA Department of Biochemistry Federal University of Technology Owerri, Imo State, NIGERIAAvailable online at: www.isca.in (Received 10th September 2011, revised 29th September 2011, accepted 4th October 2011) Abstract Studies on the phytochemical and macronutrient composition of the leaves of Combretum dolichopentalum were carried out. The qualitative phytochemical composition shows the presence of alkaloid, flavonoid, saponin, tannin, steroid, glycosides and resins. The quantitative phytochemical composition shows that it contains 5.765 ± 0.002 mg/100g tannin, 80±0.003 mg/100g flavonoids, 0.033±0.003 mg/100g cyanide, 8.717± 0.003 mg/100g alkaloids, 17.86 ± 0.002 mg/100g saponins and 6.969±0.005 mg/100g steroid. The macronutrients obtained include 2.101±0.003 mg/100g soluble carbohydrate and 200.0±0.002 mg/100g reducing sugar. The result of acute toxicity (LD50), showed C. dolichopentalum leaf extract to be lethal at doses 3000 and 5000 mg/kg body weight. This study revealed that C. dolichopentalum is a rich source of phytochemical and reducing sugar, which if adequately processed will not only offer chemoprotective benefits to its users, but could also serve as a good source of nutrient. Keywords:Combretum dolichopentalum, phytochemical, macronutrient, antioxidant, acute toxicity. IntroductionThe importance of biological, chemical and pharmacological evaluation of plant derived agents used in the treatment of human ailments has been increasingly recognized in the last decades. This is due to the presence of bioactive constituents in plants such as alkaloids, tannins, flavonoid, saponins etc2,3,4. Studies have been carried out on possible intervention of herbal preparations with diabetic and arthritic conditions in experimental animals5,6. Combretum dolichopentalum(family - combretaceae) is widely distributed and used from Guinea to Southern Nigeria and Cameroon. It is commonly known as sun-birds wine. In Nigeria the locals of Umuahia call it mmanya n’zeza, Edo’s call it Okoso. C. dolichopentalum is used for the treatment of stomach ache, pains, gastrointestinal disorders, such as dysentery, passage of bloody stool, diarrhea and stomach ulcer in and around Ogwa and Mbaise in Imo State Nigeria. C. dolichopentalumis also used as antibiotics and for treatment of stomach upset by nursing mothers after delivery in Mbaise in Imo state of Nigeria. This study aims to determine the fundamental and the scientific bases for the use of C. dolichopentalum. This was achived by quantifying the phytochemical and the macronutrient constituents and also estimation of the acute toxicity (LD50) value. This is vital because of the increasing demand for medicinal plants and plant products as alternative to orthodox medicines especially in developing nations. It is hoped that this study will enhance interest in them. Material and MethodsPlant sample collection and separation: C. dolichopentalum leaves were collected from the premises of the University of Nigeria, Nsukka, Enugu State. They were authenticated by Mr. A. Ozioko of the Bioresource Development and Conservation Program (BDCP), research centre in Nsukka. The fresh leaves were air dried for 2 weeks and ground with a grinding mill. Packaged in air-tight glass jar and stored in at room temperature until analysis was carried out. Phytochemical Test: The presence of alkaloids, flavonoids, tannins, saponins, cyanides, glycosides, resins and steroids were determined qualitatively8,9. The concentrations of the phytochemicals were determined quantitativelyMacronutrient constituent: Macronutrient composition of the leaf extract for soluble carbohydrate, reducing sugar, fats and oil were estimated quantitatively10. Animals for Acute Toxicity Studies: The animals used in this acute toxicity study (LD50) were Swiss albino mice (22-28 g) of either sex obtained from the animal house of the Faculty of Veterinary Medicine, University of Nigeria, Nsukka. They were acclimatized at the animal house of the Department of Biochemistry, University of Nigeria, Nsukka for seven days before the experiments. The mice were maintained adlibitum on water and growers mash (Pfizer Feeds, Aba, Nigeria). The acute toxicity test of C.dolichopentalum extracts was carried out by a modified Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 1(8), 72-75, Nov. (2011) Res.J.Chem.SciInternational Science Congress Association 73 method acute toxicity testing11, to define the range of lethal dose and safe dose for the extract. Swiss albino mice were grouped into 6 with three mice per group. They were treated orally with 250, 500, 1000, 2000, 3000 and 5000 mg/kg body weight respectively of the C.dolichopentalum extract. The animals were then observed over a 24 hr period for nervousness, dullness, in-coordination and or death. Oral acute toxicity dose was calculated using geometric mean of LD50Statistical Analysis: The results obtained were presented as mean ± standard deviation Results and DiscussionThe result of the qualitative phytochemical composition of C. dolichopentalum is shown in table 1. It showed the presence of flavonoids, alkaloids, saponins, steroids, glycosides and resins in the leaves. Phytochemicals are known secondary metabolites of plants which exhibit device pharmacological and biochemical effects on living organisms12. These secondary metabolites in C. dolichopentalum contain appreciable concentrations of flavonoid, saponins, alkaloid and saponins as shown in table 2. It contains Tannin, 5.765± 0.002 mg/100g; flavonoids, 8.0 ± 0.003 mg/100g; alkaloids, 8.717 ± 0.003 mg/100mg; Saponins, 17.86 ± 0.02 mg/100mg; steroids, 6.969 ±0.005 mg/100g; and 0.033 ± 0.003 mg/100g cyanide. Phytochemicals have been associated with antimicrobial activities and numerous physiological activities in mammalian cells in various studies13-15. The content of alkaloid and flavonoid could be the reason for its use in the treatment of stomach ache, pains and gastro intestinal disorders. These two phytochemicals have been shown to have anti-inflammatory and analgesic effects16. The use of the leaves for the treatment of diarrhea and stomach ulcer in and around Ogwa in Imo State in Nigeria, suggest a combined effect of alkaloid, and tannin content. Tannin, a polyphenol have been shown to exhibit antimicrobial actions and alkaloids, a parasitic repelling effect17. Caution is usually taken in the consumption of plant material, with very high concentration of alkaloids and tannins because they could inhibit certain mammalian enzyme activities such as those of cyclic- AMP. Alkaloids also affect glucagons and thyroid stimulating hormones18. The concentrations of these phytochemicals in C. dolichopentalum are below levels found in some plant materials, which required further treatment to reduce their phytochemical concentration before use15,19. The benefits derived from C. dolichopentalum use by nursing mothers after delivery may be linked to the desire to eliminate or reduce oxidative stress arising from pregnancy and labour. Flavonoid which have been shown to be a constituent of C. dolichopentalum are powerful antioxidant compounds and have also been reported to have antiviral, antiallergic, anti-platelet, antitumour and anti-inflammatory properties20,21. Saponins are known to have hypocholesterolemic activities22and are thus useful in controlling cholesterol level. A decrease in serum cholesterol concentration reduces the metabolic burden that would have been placed on the liver during metabolism23. The cyanide content also support the claim that C. dolichopentalum could be consumed without pretreatment. This is indicated by the very low concentration of cyanide (0.033± 0.003 mg/100g), this is also less than the permissible limits of 10-20 mg/100g 24. Hydrogen cyanide (a product of cyanide) has been shown to be extremely toxic to a wide spectrum of organisms, due to its ability to link with metals (Fe, Mn2+, Cu2+) that are functional groups of many enzymes. Thus, inhibiting processes like the reduction of the oxygen in the cytochrome respiratory chain, electron transport in photosynthesis and the activities of enzymes like catalase and oxidase25,26. Hydrogen cyanide can be significantly reduced by boiling, heating and soaking27. The micronutrient constituent of C. dolichopentalum showed it contains 200.0± 0.002mg/100g reducing sugar, 2.101± 0.003mg/100g soluble carbohydrate as shown in table 2. C. dolichopentalum extract also showed a trace amount of protein. This high content of reducing sugar suggests that C. dolichopentalum leaves may be used as a source of carbohydrate (disaccharide and monosaccharide). Carbohydrates provide energy to the cells of the body, particularly the brain, the only carbohydrate dependent organ in the body28. Fats and oil were not detected. Table – 1 Result of qualitative phytochemical composition of C. dolichopentalum extract PhytochemicalExtract Alkaloid +++ Flavonoid +++ Saponin +++ Tannin ++ Acidic compound - Steroid ++ Glycoside ++ Resins ++ Bioavailability key; – = not detected, = present in low concentration, ++ = present in moderately high concentration, +++ = present in very high concentration Table – 2 Result of macronutrient composition of C. dolichopentalum extract MacronutrientExtract Fat and oil - Protein + Carbohydrate ++ Reducing sugar +++ Bioavailability key: = not detected, = present in low concentration, ++ = present in moderately high concentration, +++ = present in very high concentration Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 1(8), 72-75, Nov. (2011) Res.J.Chem.SciInternational Science Congress Association 74 Table – 3 Result of quantitative phytochemical composition ofC. dolichopentalumSample Concentration Soluble carbohydrate 2.101 ± 0.003 mg/100g Tannin 5.765 ± 0.002 mg/100g Flavonoids 8.0 ± 0.003 mg/100g Cyanide 0.0334±0.003 mg/100g Steroid 6.969 ± 0.005 mg/100g Glycoside 1.0914±0.003 mg/100g Alkaloids 8.717± 0.003 mg/100g Saponin 17.86 ± 0.002 mg/100g Reducing Sugar 200.0±0.002 mg/100g Values are means ± standard deviation of triplicate determinations. Table – 4 LD50 of aqueous extrat of C. dolichopentalum Group(n=3) Plant Extract (mg/kg body weight) Dead Birds 1 250 0 2 500 0 3 1000 0 4 2000 0 5 3000 0 6 5000 0 Geometric mean of LD50 calculated as Square root of the highest non-lethal dose multiplied by the least lethal dose. LD50 = Highest non-lethal dose x Least lethal dose = 2000 x 3000 = 2449.49 mg/ kg body weight Acute toxicity study is an important test in the toxicological investigation of an unknown substance. The index of acute toxicity is the LD50, though not regarded as a biological constant11,29. The acute toxicity (LD50) value of aqueous extract of Combretum dolichopentalum leaves was calculated to be 2449.49mg/kg body weight.The result of the oral acute toxicity (LD50), studies showed C. dolichopentalum leaf extract to be lethal at doses 3000 and 5000 mg/kg body weight, an indication that the LD50 of the plant is less than 3000mg/kg. This result places C. dolichopentalum in category 5(� 2000mg/kg 5000mg/kg) according to the globally harmonized system of classification and labeling of chemicals30. Conclusion This research has revealed that C. dolichopentalum is a rich source of phytochemical and reducing sugar. This indicates that the leaves of C. dolichopentalum will offer medicinal, chemoprotective and nutritional benefit to its users. Acknowledgement Authors are grateful to staff of the Department of Biochemistry, for their advice and support during this research work. References 1. 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