Phytochemical and anti-nutritional constituents of Usivi (Guibourtia coleosperma) seeds from Kavango East Region of Namibia. Chionga Songwe Student Number: 201054779 Supervisor: Dr Kathithileni Martha Kalili Co-Supervisor: Dr Jane Misihairabgwi Research Projects Coordinator: Mr. Niclaas Gariseb

Contents Introduction Rationale Objectives Research Methods Results and Discussion Conclusion References Acknowledgements

Introduction The phytochemical composition of traditionally used plants has become an important research area as it determines their potential as sources of new drugs. Only a small fraction of plant species has been subjected to phytochemical analysis, and of these, only 5-15% have been investigated for biological or pharmacological activity (1). The lesser-known food crops which remain outside the scope of science have not been rejected because of any inferiority. They have simply been overlooked because they are native to the tropics, a region generally neglected because the world’s research resources are concentrated in the temperate zones (2).

Introduction Guibourtia coleosperma commonly known as Usivi in the Kavango east region of Namibia, is an indigenous tree to the North-Eastern parts of Namibia, belonging to the Fabaceae family. The arils of this plant are eaten both raw and cooked and have saved the lives of people in times of famine (2). This seed is so valued by the !Khu Bushmen people for its variety of uses, so they consider it as second only after mongongo (Schinziophyton rautanenii) seeds in terms of importance. They use the leaves of the plant for medicinal purposes such as application to wounds to promote healing whilst a root decoction is used as cure for venereal diseases (3).

Figure 2: Ripe Usivi fruits (4) Figure 3: Soaked Usivi seeds ready to be eaten (4) Figure 1: Usivi habitat (2)

Rationale To the best of our knowledge, the phytochemical profile of the Usivi seeds was not known and no study on the anti-nutritional composition of extracts from Guibourtia coleosperma had been done. Therefore, this research aimed to contribute to the preservation of indigenous knowledge and add scientific knowledge to the profile of Guibourtia coleosperma.

Objectives The specific objectives of the study were: To qualitatively and quantitatively determine the phytochemical constituents of crude extracts of Guibourtia coleosperma seeds. To perform anti-nutrient testing on extracts of the seeds of Guibourtia coleosperma.

Research Methods Collection and pre-treatment of plant material Fresh seeds were collected from an open market in Rundu, Kavango east region of Namibia. The seeds were air-dried and then ground to a uniform powder in a blender before use. Qualitative Analysis of Phytochemicals and Anti-nutrients Phytochemical screening A methanol extract of the seeds was used to test for the presence of nine phytochemical classes i.e. tannins, saponins, resins, alkaloids, terpenoids, phlobatannins, flavonoids, cardiac glycosides and phenols.

Research Methods Quantitative Analysis of Phytochemicals Quantitative Analysis of Anti-nutrients Total Flavonoid Content (TFC): Aluminium chloride assay. Total Phenolic Content (TPC): The Folin-Ciocalteau assay. Tannins: Spectrophotometric method. Free cyanide: Spectrophotometric method (5). Oxalates: Determined by titration. Saponins: Spectrophotometric method. Alkaloids: Gravimetric method. Antioxidant capacity assay: DPPH radical scavenging activity assay.

Results and Discussion Table 1: Results for the phytochemical screening of the methanol extract of G. coleosperma seeds. Phytochemical Class Inference Phlobatannins Absent Cardiac glycosides Tannins Present Phenols Terpenoids Saponins Flavonoids Alkaloids Resins Phytochemical screening allows for the semi- quantitative analysis of phytochemicals and aids in locating potentially pharmacologically active compounds. The results showed the presence of flavonoids, saponins, phenols, tannins and alkaloids in the methanol extract of Guibourtia colesperma seeds.

Results and Discussion Table 2: Quantitative phytochemical analysis of G. coleosperma seeds. The quantitative phytochemical estimation of Guibourtia coleosperma seeds showed that the seeds are very rich in saponins and alkaloids. Parameter Composition (mg/g) Saponins 5.65 Alkaloids 104.7 Total Flavonoids 0.164 Total Phenols 0.1255 Saponins help lower cholesterol and reduce the risk of heart disease. Ones immune function benefits from these plant compounds as well.  Many alkaloids have found use in traditional and modern medicine, or as starting points for drug discovery. Other alkaloids possess psychotropic and stimulant activities (e.g. cocaine, caffeine).

Results and Discussion Table 3: Anti-nutrient analysis of G. coleosperma seeds. The amount of oxalates measured was 196.60 mg/g, this value is way above the permissible levels of 250 mg/100 g fresh weight. Parameter Composition (mg/g) Hydrogen cyanide 0.278 Oxalates 196.6 Tannins 0.7443 Oxalates are extremely painful when deposited in the body. About eighty percent of kidney stones are caused by oxalates and they are by far the most common factor in kidney stone formation.  However, studies show that boiling the vegetable for five minutes and discarding the water eliminates most of the oxalates.

Results and Discussion Cyanide is acutely toxic to humans. The acute lethal dosage of hydrogen cyanide (HCN) in most animal species and humans is ~2 mg/kg. Whilst tannins are touted as excellent antioxidants, their bitter taste, as well as interference with starch digestion, tends to make the edible part of a plant quiet unpalatable. The antioxidant activity results in this study were found to be 49.88%. The seed extract of G. coleosperma exhibits a good potential to act as a free radical scavenger. It is important that food rich in antioxidants is taken so that the antioxidants can neutralise the free radicals in case of enzymatic mechanisms failure or inadequate efficiency.

Conclusions The seeds of this plant are fairly rich in phytochemicals. This provides a rationale for the traditional uses of this plant in the Namibian traditional healing system to successfully treat different ailments. Since its anti-nutrient content is appreciably low, it can therefore be suggested that Guibourtia coleosperma seeds can contribute significantly to the nutrient requirements of man. It is estimated that consumption of foods containing antioxidants reduces the risk of cancer and heart diseases by up to 25%. The data obtained in this study which suggests that consumption of these seeds has the potential to improve the health status of its users as a result of the presence of various compounds vital for good health.

References (1) Nyambe MM. Phytochemical and Antibacterial analysis of indeginous chewing sticks, Diospyros Lycioides and Euclea Divinorum (Masters dissertation, Department of Chemistry and Biochemistry, University of Namibia). (2) Van Wyk BE, Gericke N. People's plants: A guide to useful plants of Southern Africa. Briza Publications; 2000. (3) Curtis B, Mannheimer C. Tree atlas of Namibia. Windhoek: Namibian Botanical Research Institute. 2005;1. (4) Thaipong K, Boonprakob U, Crosby K, Cisneros-Zevallos L, Byrne DH. Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. Journal of food composition and analysis. 2006; 19(6):669-675. (5) Cheikhyoussef A, Shapi M, Matengu K, Ashekele HM. Ethnobotanical study of indigenous knowledge on medicinal plant use by traditional healers in Oshikoto region, Namibia. Journal of Ethnobiology and Ethnomedicine. 2011; 7(1):1.

Acknowledgements Firstly, I would like to express my sincere gratitude to my supervisor Dr Martha Kalili, my co-supervisor Dr Jane Misihairabgwi, as well as Mr Anthony Ishola for the continuous support throughout my study and related research, for their patience, motivation, and immense knowledge. I would also like to thank My immediate family and friends for their insightful comments and encouragement. My sincere thanks also goes to the department of Chemistry and Biochemistry and the School of Pharmacy who gave me access to the laboratory and research facilities. Without their support it would not have been possible to conduct this research.