Ibrahim Muhammad

ISOLATION, CHARACTERIZATION AND BIOACTIVITY STUDIES OF ETHANOLIC EXTRACT OF ASPARAGUS AFRICANUS (AFRICAN ASPARAGUS) CHAPTER ONE 1.0Introduction Natural products are the chemical compounds fount in nature that usually has a pharmacological activity for use in drug discovery and drug design. About 70% of drugs are derved from plants, leaves and herbs which are known to have medical values.in Africa, traditional medicine is practiced and plants have been exploited for the treatment of many disease (majorie, 1999). Many plant extract owe their potenly to the presence of bioactive substances such as anthraquinone, alkaloids flavonoids, interperiods etc these substances are usually sound in various part of plant foot stem, leaves and which have physiological actions in human body. Desception & habitat The plant iselonged the asparagacene family of genus asparagus. Its specie is asparagus africanus with a common bane of asparagus. The plant asparagus africanus known as dangalin biri, marin, kusu or kayar bera in Hausa & wild asparagus in English. The plant is widely distributed in eastern & sub-saharen Africa and is used locally. It is found countries like Egypt, eritria & Nigeria. It has been widely cultivated as a garden plant particularly in eastern Australia (Stanley, 1994). Ethno medicinal uses of asaparagus africanus. The plant contain a certain variety of phytonutrients known as saponins studied have demanstarted that the saponins obtained from asparagus posses anti-cancer and anti-inflammatory properties (www.anniesemedy.com) The plant can also be used for almost all conceivable ailments. The fibrous stalk help to reduce cholesterol. It is high in lycopene, a powerful antioxidant that is protective against many degenerate disease (www.organicfact.net) A number of bioactive compounds have been isolated from asparagus africanus including furostanol type and spirostanol type, steroidal saponins and flavonoids proteins, starch tannin, racemosal, sapogenins and phenolic compounds There has been a rapid increase in the use of herbal medicines as complements to orthodox medicines. The reasons are ease of access without long waiting times for medical prescriptions, cheaper and lack of regulation by international health authorities on the use of herbal medication (Saad & Said, 2011). There are many plants-derived bioactive constituents of medicinal importance for human and animal health, for instance; traditionally, a mixture of powdered roots of Cassia occidentalis, and Derris brevipes is used to control female fertility. Preliminary phytochemical studies indicated the presence of steroids, terpenoids, flavonoids and glycosides in the ethanolic extracts of these two plants. These compounds are known to exhibit anti-fertility activity (Badami et al., 2003). Principal chemical compound reported from Dendrophthoe falcata for example contains cardiac glycosides, flavonoids and pentacyclic triterpenes. D. falcata has been utilized as an anti-fertility agent for women. The aerial part is also used to induced abortion (Pattanayak & Mazumder, 2009). Asparagus africanus Lam belongs to family liliaceae and is widespread in the drier parts of tropical Africa (Hassan et al., 2012) where it has been widely used for medicinal purposes (Bonjar & Farrokhi, 2004). There are many ethno-pharmacological claims on various parts of A. africanus (Asfaw et al., 1999), such as treatment of immune disorders (Ahmad et al., 2006), anti-fertility and its health related problems (El-Ishaq, Alshawsh & Chik, 2019a; El-Ishaq et al., 2019b; Geremew, Yalemtsehay & Eyasu, 2006). In addition, its root aqueous extract has been taken regularly as an anti-depressant and to ease childbirth (Hamill et al., 2003), treat hypertension, cancer and epilepsy (Moshi & Mbwambo, 2005), applied externally for treating chronic gout (Lohdip & Tyonande, 2005). The main phytoconstituents that may be responsible for the pharmacological effects of A. africanus are saponins, flavonoids and tannins such as spirostanosides, furostanol, sapogenin and lignin (Hassan, Ahmadu & Hassan, 2008). In addition, our previous study confirmed the presence of steroidal saponins such as stigmasterol and sarsasapogenin (El-Ishaq, Alshawsh & Chik, 2019a). Isolation and Characterization of Bioactive Compounds Ethanolic extracts of Asparagus africanus have been subjected to various isolation and characterization techniques to identify and purify the bioactive compounds present in the extract. This technique separates compounds based on their polarity, allowing for the isolation of individual compounds from the complex extract (Chireka et al., 2015). This technique separates compounds based on their interaction with a stationary phase, providing high-resolution separation and identification of individual compounds (Mbamalu et al., 2015). This technique determines the molecular mass of compounds, aiding in their structural identification (Kiranmai et al., 2014). This technique provides detailed information about the structure of compounds, including their molecular connectivity and functional groups (Kiranmai et al., 2014). Asparagus africanus Lam., also known as African asparagus, bush asparagus, wild asparagus, climbing asparagus fern, ornamental asparagus, and sparrow grass, is a versatile plant with a wide range of uses and characteristics. It is a spiny shrub or climbing plant native to Africa, the Arabian Peninsula, and India. It belongs to the Asparagaceae family, known for its edible asparagus species. This plant is characterized by its slender, green stems that can grow up to 3 meters (9.8 feet) long, supporting delicate, fern-like cladodes (modified branchlets). The cladodes, measuring 6 to 15 millimeters long and about 0.5 millimeters wide, are arranged in bunches along the stems, giving the plant a feathery appearance (Van Wyk et al., 2009). Asparagus africanus thrives in a variety of habitats, including rainforests, grasslands, and semi-deserts. It is widely distributed across Africa, particularly in eastern and southern regions, and has also been introduced to other parts of the world, including Australia and parts of Asia (Orchard, 1990). Asparagus africanus has been utilized for various purposes, both traditional and modern. In traditional medicine, it has been employed to treat a range of ailments, including fever, stomachaches, and skin conditions (Watt & Breyer-Brandwijk, 1962). The roots and stems are considered diuretic and anti-inflammatory, while the berries are used as a laxative (Hutchinson & Dalziel, 1954). 1.1Antioxidant Asparagus africanus (A. africanus) extracts have demonstrated significant antioxidant activity, which is attributed to the presence of various bioactive compounds, including phenolic acids, flavonoids, and saponins. These compounds have the ability to scavenge free radicals and prevent oxidative damage to cells. For instance, a study by Adebayo and Afolayan (2008) found that A. africanus extracts exhibited strong antioxidant activity in DPPH and FRAP assays, comparable to the activity of synthetic antioxidants such as BHT and Trolox. Asparagus africanus, also known as African asparagus, bush asparagus, wild asparagus, climbing asparagus fern, ornamental asparagus, and sparrow grass, is a plant with antioxidant properties. Several studies have investigated the antioxidant activity of Asparagus africanus and its potential health benefits. 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical is ability of a compound to scavenge DPPH free radicals, which are highly reactive and can damage cells. Extracts of Asparagus africanus have been shown to scavenge DPPH radicals effectively (Kouam et al., 2013). 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical is similar to the DPPH assay but measures the ability of a compound to scavenge ABTS radicals. Extracts of Asparagus africanus have also shown significant ABTS radical scavenging activity (Madjouli et al., 2013). Ferric reducing antioxidant power (FRAP) is ability of a compound to reduce ferric iron (Fe3+) to ferrous iron (Fe2+). Extracts of Asparagus africanus have demonstrated high FRAP values, indicating strong reducing power (N'Guessan et al., 2009). 1.2Anti-inflammatory Anti-inflammatory refers to anything that reduces inflammation. Inflammation is a natural response of the body's immune system to injury or infection. While inflammation is important for healing, it can also become chronic and lead to serious health problems such as heart disease, cancer, and arthritis. Asparagus africanus extracts have demonstrated anti-inflammatory properties by reducing the production of inflammatory mediators (Madjouli et al., 2013). A. africanus extracts have also shown anti-inflammatory potential, suggesting their potential therapeutic use in conditions associated with chronic inflammation. The anti-inflammatory activity of A. africanus extracts is attributed to their ability to suppress the production of pro-inflammatory mediators, such as prostaglandins and nitric oxide. For example, a study by Ezeonu et al. (2013) demonstrated that A. africanus extracts significantly reduced the production of prostaglandins and nitric oxide in LPS-stimulated macrophages, indicating their anti-inflammatory properties. 1.3Statement of the Problem The plant asparagus africanus have been reported to posses numerous pharmacological properties such as antioxidant anti-cancer and anti-inflammatory effects results. This bioactivities are associated with the of different classes of phytonutrient such as triterpenoids, stercidal saponins, flavonoids, furustotiol type & spirostanol-type, racemosol and phenolic compounds hence, there is need for this plant to be further analysed in order to determine its major active principles and evaluated for it could be the future pronishing anti-cancer/antioxidant drug. The is lack of active anticancer drug also there is paucity of on research on new drugs. The recent study shows 50-50 compounds has pronshing potential as anticancer. 1.4Aims and objectives 1.4.1Aim The aim of this research work is to study the antioxidant and antiflammatory potenitals of phytochemicals from asparagus africanus twig. The aim s set to be achieved through the following objectives; 1.4.2Objectives To collect prepare & extract the plant material using organic solvent in polarity gradient Isolate and purify the plant phytochemicals using available chromatographic techniques (TLC, & column chromatography) To evaluate the antioxidant capacity by spectrophotometric determination of total phenotic content (TPC)total flavonoid content *TPC), free radical scavenging ability (via DPPH radical scavenging assay) ferric reducing power (FRP) To investigate the anti-inflam|matory potentials using protein dinature Characterise the isolated compounds using spectroscopic technologies likeIR, UV NMR (ID x 2D NMR) MS (GC-MS) & (LC-MS) CHAPTER TWO LITERATURE REVIEW 2.0Introduction Asparagus africanus (A. africanus), also known as wild asparagus, is a perennial herb native to Africa. It has been traditionally used for the treatment of various ailments, including malaria, diabetes, and hypertension (1). Recent studies have demonstrated the presence of various bioactive compounds in A. africanus, including saponins, flavonoids, and phenolics, which have exhibited antioxidant, anti-inflammatory, and anti-cancer activities (2, 3). This literature review aims to summarize the current knowledge on the isolation, characterization, and bioactivity of ethanolic extracts of A. africanus. 2.1Isolation and Characterization of Bioactive Compounds Several studies have investigated the isolation and characterization of bioactive compounds from A. africanus extracts. Ethanol is a commonly used solvent for extracting bioactive compounds from plants, and it has been shown to be effective in extracting a wide range of compounds from A. africanus (4). Various chromatographic techniques have been employed to isolate and purify the bioactive compounds from A. africanus extracts. Column chromatography, thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC) have been used successfully to separate the different compounds present in the extracts (5, 6, 7). Spectroscopic techniques, such as nuclear magnetic resonance (NMR) and mass spectrometry (MS), have been used to characterize the structure of the isolated compounds. These techniques have identified a variety of saponins, flavonoids, and phenolics in A. africanus extracts (8, 9, 10). 2.1.1Anti-Inflammatory Activity of Crude Saponin Extracts Inflammatory diseases are a major cause of morbidity world-wide. Non-steroidal anti-inflammatory drugs and steroids are the most common drugs used to treat inflammation. Gastrointestinal side effect is a major side effect associated with the currently available non-steroidal anti-inflammatory drugs which limit their application. This may be contributing to the current move by large proportion of world population towards herbal remedies for the treatment of inflammatory diseases. Herbal medicine is still the mainstay of about 75–80% of the whole population, mainly in developing countries, for primary health care because of better cultural acceptability, better compatibility with the human body and fewer side effects. However, the last few years have seen a major increase in their use in the developed world (Parekh et al., 2005). A number of medicinal plants are used in developing countries for the management of a number of disease conditions including pain and inflammatory conditions. The validation of the folkloric claims of these medicinal plants will provide scientific basis for the conservation of tropical medicinal resources, the deployment of the beneficial ones as phytomedicine in the primary health care and the development of potential bioactive constituents as novel lead compounds or precursors in drug design. One of such phytoconstituents is saponins. Saponins are heterogeneous group of naturally occurring surface active glycosides produced mainly by plants and also by lower marine animals and some bacteria (Francis et al., 2002). They are composed of triterpenoid or steroid aglycone moiety and complex oligosaccharide substituent. The hydrophilic properties of the sugar part and lipophilic properties of the aglycone part give saponins their amphiphilic or surfactant properties which in turn give rises to their ability to form stable aqueous foams as well as forming complexes with membrane steroids and lipid compounds (Hostettmann and Marston, 1995). The mounting demand for natural products coupled with their physicochemical properties and numerous biological activities has led to the emergence of saponins as commercially significant compounds with expanding applications in food, cosmetics and pharmaceutical industries (Guclu-Ustundag and Mazza, 2007). Steroidal saponins are important raw materials for the production of steroidal hormones and drugs (Brain et al., 1968). Saponins are used as immunological adjuvants in the formulation of vaccines due to their immune enhancing properties (Francis et al., 2002). Information on the biological activities of saponins from variety of sources provide lead for the development and design of new drugs. An example is the chemotherapeutic activity of the ginseng saponins prompted the development of anticancer drugs (Huang and Qi, 2005) and a new class of HIV drugs called maturation inhibitors (PA — 457) developed from betulinic acid derivatives (Panacos, 2005). The five medicinal plants used for this study are locally used in traditional medicine as anti-inflammatory agents. Schwenkia americana Linn family Solanaceae is a slender erect herb, woody at the base, grows up to 1m tall, common in waste places and widespread in tropical Africa and America. Asparagus africanus Lam family Liliaceae (Asparagaceae) is an erect plant that grows up to 1.75m tall with numerous wiry, spiny branches. It is annual from a woody perennial root-stock and generally widespread in the drier parts of tropical Africa. Dichrostachys cinerea (Linn) Wright & Arn family Leguminosae-Mimosoideae is a shrub or small tree that grows up to 12m high or 2–5m in the drier areas and is widespread across tropical Africa. Ficus iteophylla Miq family Moraceae is a tree that grows up to 12m high, with the trunk about 4m in girth, widespread in the savannah region. Indigofera pulchra Willd family Leguminosae-Papilionoideae is a small under shrub or semi-woody erect herb that grows up to 1.5m high widespread in the savannah region of West Africa. These plants provide common remedy for rheumatic pain, arthritic conditions and swellings in Northern Nigeria (Burkill, 1995, 1997, 2000). Some previous studies have reported the anti-inflammatory activities of the crude extracts of these plants (Hassan et al., 2008a; Hassan et al., 2008b; Musa, 2008). The survey shows that S. americana contains steroidal saponins (Kapunda and Delaude, 1988) and the isolation of monodesmodic and bidesmodic saponosides from the roots A. africanus collected in Ethiopia (Debella et al., 1999). Saponins have also been detected to be present in D. cinerea (Kuber and SanthRani, 2009) and also the isolation of free triterpenes such as alpha-amyrin from the plant (Joshi and Sharma, 1974). Some free triterpenes and sterols have been identified in the roots and leaves of different species of Ficus (Lansky et al., 2008) and also in some species of Indigofera (Leite et al., 2006). The anti-inflammatory and other effects of saponins from various plants are well documented (Lacaille-Dubois and Wagner, 1996; Francis et al., 2002). The numerous biological activities associated with saponins have lead to great interest in their characterization and in the investigation of their pharmacological and biological properties. This study was under taken to investigate the anti-inflammatory activity of the crude saponins extracts of the various parts of the above mentioned medicinal plants. 2.1.2Identification of phenolic compounds, antibacterial and antioxidant Bioactive compounds extracted from natural sources can benefit human health (Samavardhana et al., 2015). Plants are natural source of antimicrobial agents with great therapeutic values. According to world health organization (WHO) about 80% of the population in developed countries use plants and their extracts as medicine. Many drugs used in modern medicine are originated from natural sources (Okoko and Oruambo, 2008). Similarly, many researchers have demonstrated the usefulness of different plant compounds as effective antimicrobial agent (Szeto et al., 2002; Jimoh et al., 2008). Raisins (dried grapes; Vitis vinifera), ), are very popular plant in Mediterranean area and are widely used as a traditional and natural biomedicine in several countries where it is well adapted to the climate (Di Lorenzo et al., 2016; Cordero-Bueso et al., 2017). Raisins are considered to be a good source of monomeric catechins, epicatecthins, gallic acids and polymeric procyandins which have an overall beneficial effect on human health (Monagas et al., 2005). The sweetness of raisins was attributed to the presence of glucose and fructose (Winkler, 1962). Previous studies demonstrated that the various phytochemical compounds in raisin such as polyphenol have both antioxidant and antimicrobial activities (Jayaprakasha et al., 2003; Baydar et al., 2004). Raisins contain the highest concentration of total phenolic compounds and the highest antioxidant activity among dried fruits (Karakaya et al., 2001). The antioxidant property of raisins was also attributed to phenolic compounds (Yeung et al., 2006). These findings supporting the idea of using raisin extracts as antioxidants in food systems. The phenolic contents and antimicrobial activities of raisins have previously been studied and found to be highly correlated (Bower et al., 2003). The average distributions of polyphenolic compounds in raisins were estimated to be about 5 per cent in juice, 1 per cent in pulp and the remaining 62 per cent in the seeds (Singletary et al., 2003). It is also well known fact that spoilage of foodstuff due to microbial contamination is a serious problem. Generally, chemical additives are employed to prevent spoilage of food but many of them are carcinogenic. Recent studies, reported that some plant compounds can safely be used for preventing microbial spoilage of food, thus ensuring quality, safety and shelf life of foods. Therefore, the application of plant extracts to prevent food spoilage has been increased throughout the world (Baydar et al., 2004). 2.2BIOACTIVITY STUDIES OF ASPARAGUS AFRICANUS 2.2.1Nephroprotective Effect of Asparagus africanus Lam. Root Extract against Gentamicin-Induced Nephrotoxicity Acute kidney injury is a syndrome characterized by the rapid loss of the kidney excretory function [1]. Acute renal failure refers to the sudden and usually reversible loss of renal function, which develops over a period of days or weeks [2]. Among the causes of acute renal failure, acute tubular necrosis, which occurs due to ischemia or nephrotoxins such as gentamicin (aminoglycoside), is most common, accounting for 85% of the incidences [3]. Nephrotoxicity has been reported in 1.7% to 58% of patients receiving aminoglycoside therapy [2, 3]. Gentamicin is an effective antibiotic that has been used worldwide for many years. While considered an essential medicine by the WHO, gentamicin can also lead to severe kidney damage [1, 2]. The recommended routes of administration of gentamicin are intravenous, intramuscular, intraperitoneal, or topical as it is not sufficiently absorbed by the intestinal tract [4, 5]. However, the potential clinical use of gentamicin is limited due to gentamicin-induced toxicity [4]. Gentamicin can cause tissue injury such as nephrotoxicity, ototoxicity [3, 4], and liver toxicity [5], possibly through the generation of free oxygen radicals. Nephrotoxicity of gentamicin arises due to its accumulation in renal cortical tubular epithelial cells [6]. Although the pathogenesis of gentamicin-induced acute kidney injury has been the focus of a large number of studies, the underlying mechanisms are not yet fully elucidated [7]. Recent studies suggest that gentamicin nephrotoxicity is a complex and multifaceted process in which gentamicin triggers cellular responses involving multiple pathways that culminate in renal damage and necrosis [8]. Several agents and strategies have been attempted to ameliorate gentamicin nephrotoxicity [7, 8], with the main focus on the use of various antioxidant agents, including the extracts from medicinal plants with antioxidant properties [9]. This biological activity may be attributed to its constituents obtained from plants, mainly phenolic compounds such as flavonoids. Flavonoids are well-known antioxidants possessing free radical scavenging and metal chelating activity [10]. Approximately 20% of all plants discovered on the planet have been subjected to pharmacological or biological testing, and a significant proportion of novel antibiotics are derived from natural or semisynthetic sources [11]. This fascinated the researchers to search out alternative sources of natural products with wide spectra of biological activities [12]. Asparagus africanus, locally called “Saritti” in (Afaan Oromo) and “Kestencha” in (Amharic), belongs to the family Asparagaceae, is a medicinal shrub valued for its medicinal properties. It is widely distributed throughout Africa, including Ethiopia, and parts of Europe, Asia, and Australia. A. africanus, with the common name “African Asparagus,” is a perennial shrub with stems up to 6 m high, growing between 700 and 3,800 m above sea level [13]. However, it is widely distributed and suitably grows higher, up to 6 m at an altitude range of 1,450–2,900 m [14]. A. africanus is known as a folk medicine in Ethiopia, and it is traditionally used for the management of kidney disease in the form of juice, tea, or soup [15]. Various studies also showed that A. africanus plant extract has different biological activities such as anti-bacterial, sexual impotency, gonorrhea, and syphilis [16]; hepatoprotective [17]; and antimalarial and insecticidal repellent properties [18]. In addition to this, A. africanus is used for pharmacological activities such as anti-diabetic [16, 19, 20], anti-protozoal [21], anti-inflammatory and analgesic [22], anti-fertility [19], anti-microbial [23], and anti-oxidant [24] activities. The genus Asparagus contains various phytochemicals such as tannins, alkaloids, terpenoids, steroids, and flavonoids that may cause a definite physiological action in the human body [25]. Chemical constituents of A. africanus plant extract revealed that the presence of several bioactive constituents, such as flavonoids, tannins, steroids, terpenoids, and saponin contained in A. africanus Lam. steroidal saponin, are the major active components of the genus Asparagus [26]. In this study, the nephroprotective activity of the root of A. africanus ethanolic extract was evaluated in two separate doses (200 and 400 mg/kg). In addition to that, the plant extract was compared with gentamicin (100 mg/kg) and silymarin (200 mg/kg) as negative and positive controls, respectively. Phytochemical screening and anti-implantation activity of Asparagus africanus It is extremely obvious that plants play cogent role in the well-being of humans and animals. Hundreds, if not thousands of plant materials have been reported to possess medicinal properties owing to their phytochemical constituents. In addition, many plants have been traditionally explored as remedy for various pathologic conditions such as: burning sensation, fever, nephropathy, hepatopathy, bladder irritation, throat infections, tuberculosis, cough, bronchitis, leucorrhoea, hemorrhoids, hypertension, abortion, and cardiac problem (Warrier et al., 1993). One of such medicinal plants is Asparagus africanus Lam., Asparagaceae, a climbing shrub, which has been used traditionally to treat diverse ailments such as: peptic ulcer, diarrhea (Hutchinson et al., 1963), headache, backache, stomach pain and for easing childbirth (Maroyi, 2011). A. africanus has also been used for other nontherapeutic usage such as food and ornamental purposes (Ribeiro et al., 2010). Despite all these aforementioned uses of this plant, there are still insufficient data regarding its abortifacient and toxicological effects. Geremew et al. (2006) showed that the root of A. africanus possess hormonal properties which can modulate the reproductive function of the experimental rats and suggested that the contraceptive activity of A. africanus needs further exploration to elucidate the bioactive constituents. In line with this, a previous study has been reported two compounds from the roots of A. africanus namely, spirostanosides and furostanol glycoside using combining MS with two-dimensional NMR technique (Asfaw et al., 1999). Furthermore, the structure of isolated compound, sapogenin, from A. hormonal signaling pathway which may be responsible for certain reproductive function (Geremew et al., 2006). Asparagus africanus has been reported to ease childbirth and expulsion of placenta after birth (Hamill et al., 2003), whereas its aqueous extract had inhibited conception in a dose-dependent manner in Sprague–Dawley (SD) rats (Okidi et al., 2019). Owing to the fact that chromatography couple to mass spectrometry method is a direct and fast analytical approach for identification of phytocomponents (Goyal, 2014), this study aimed to screen and identify the major phytochemicals in the aqueous extract of A. africanus root using LC/MS. Subsequently, to determine the in vivo anti-implantation activity and acute toxicity of the aqueous extract of A. africanus in SD rats. Evaluating the oestrogenic activities of aqueous root extract of Asparagus africanus Lam According to the World Health Organisation (WHO), approximately 80% of the world’s population relies on traditional medicine which involves the use of plant extracts (Wachtel-Galor & Benzie, 2011; WHO, 2013). This practice is more common among villagers where modern drugs are not available or are too expensive (Adamu et al., 2005). Asparagus africanus Lam is a plant that is used in traditional medicine for contraception and to assist women during parturition. In addition, the aqueous extract of the plant is believed to have cleansing properties, especially after parturition and its roots contain polyphenols, phytosterol, saponins, and tannins (Yared, Mekonnen & Debella, 2012). Other species of Asparagus (Asparagus pubescens) reportedly have the ability to reduce the number of pups output in rats, mice and rabbit species (Nwafor, Okwuasaba & Onoruvwe, 1998). Medicinal plants have various actions on a body’s physiology, and some plants contain phytoestrogen and may cause fertility, whereas others have abortifacient and antifertility activities (Mukta & Nagendra, 2015). Oestrogen is a vital hormone during development and maintenance of normal sexual and reproductive functions. Oestrogen is well known to be a morphogen and plays a vital role during morphogenesis of the uterus (Heldring et al., 2007). Oestrogen signalling pathways are selectively promoted or inhibited depending on the balance between the activities of oestrogen receptors (ERα and ERβ) in target organs. Oestrogen receptors (ERs) belong to the steroidal hormone superfamily of nuclear receptors, which act as transcription factors after binding to oestrogen (Pillai, Jones & Koos, 2002). In addition, oestrogen production is vital for proper implantation of the blastocyst with the uterine wall during pregnancy (Jeff et al., 2001). Oestrogens and progestins are established modulators of the reproductive function in normal cycling and remodelling during menses and pregnancy (Crabtree et al., 2006). An uncontrolled population affects the socio-economic development of a country (Crabtree et al., 2006). To control population growth, there is a need for an acceptable female contraceptive (Londonkar & Nayaka, 2013). Family planning has been promoted through several methods of synthetic contraceptives, but synthetic drugs have many adverse effects. Hence, there is a need to search for safe medicinal plants with contraceptive potentials free from adverse effects (Crabtree et al., 2006). However, due to the lack of intensive and comprehensive investigation, no safe and effective oestrogenic plant has been found (Londonkar & Nayaka, 2013). A model used to evaluate the effect of chemicals on estrogen receptors is the uterotrophic assay in which removal of the ovaries reduces endogenous estrogens, causing the uterus to shrink. Replacement of these hormones with external sources of estrogen causes a trophic response of the uterus (Hye-Rim, Tae-Hee & Kyung-Chul, 2012). The current study uses the uterotrophic assay to investigate the oestrogenic activities of aqueous extract of Asparagus africanus (AEAA) in female Sprague-Dawley rats to screen its phytochemical constituents using GC/MS. 2.3 Antioxidant Activity and Bioactive Compounds of Asparagus africanus Asparagus africanus, a wild edible plant native to Africa, has gained attention for its potential health benefits due to its rich content of bioactive compounds and antioxidant activity. Several studies have investigated the antioxidant properties and bioactive compounds of Asparagus africanus, revealing its promising potential in various health applications. The Asparagus genus, belonging to the Asparagaceae family, includes about 300 species mainly distributed in warm temperate and subtropical regions (Negi et al., 2010). Previous investigations on some species of this genus have indicated their various pharmacological activities such as antioxidant, anti-inflammatory, antifungal, immunostimulant, antibacterial, cytotoxic and anticancer effects (Mitra et al 2012, Negi et al., 2010), and the presence of important bioactive compounds such as flavonoids, polysaccharides, steroidal saponins, ascorbic acid and minerals (Yu and Fan, 2021, Fuentes Alventosa and Moreno, 2015). In recent studies, some extracts of Asparagus genus with potential action against various tumor and cancer cells, were detected (Xiao et al., 2022; Xu et al., 2021). Asparagus africanus Lam. is a perennial climbing plant, widely distributed in tropical Africa. The plant has many medicinal properties and its roots are traditionally used to treat malaria, leishmaniasis infections (Oketch-Rabah et al., 1997), headache, backache, stomach pain, hematuria, hemorrhoids, syphilis, gonorrhea, chronic gout, tuberculosis and venereal diseases (Hassan et al., 2008; Mfengwana and Mashele, 2019). The plant is also used to facilate childbirth (Tafesse et al., 2006). In Ivory Coast, A. africanus is used in association with other plants like an improved traditional medicine in the prevention and treatment of tumors and cancers. The decoction of the root of A. africanus is used as a calming, purgative and emetic, as well as in the treatment of bilharzia (Bouquet and Debray, 1974). Previous phytochemical studies have led to the isolation of steroidal sapogenins (Debella et al., 1999), nor-lignans (Oketch-Rabah et al., 1997), sesquiterpenes, alkaloids, stigmasterols and phenolic compounds (El-Ishaq et al., 2019a; 2019b). Despite the extensive uses of A. africanus in traditional medicine, very few chemical studies have been reported on this plant. Thus, the aim of this study is to report the isolation and structure elucidation of specialized metabolites from the roots and the aerial parts of A. africanus. Thirty-three compounds, were isolated, among them two steroidal saponins and eight nor-lignans were newly described. In addition, the antioxidant capacities of phenolic compounds were evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radical antioxidant capacity (HORAC) and cupric ion reducing antioxidant capacity (CUPRAC) tests. The results showed that many of the isolated compounds exhibited antioxidant potential compared to ascorbic acid, quercetin, and Trolox, which were used as controls. Asparagus africanus is a rich source of various bioactive compounds, including: Phenolic acids: Sinapic acid, caffeic acid, ferulic acid, syringic acid, and chlorogenic acid Flavonoids: Quercetin, rutin, kaempferol, and catechin Saponins: Shatavarin I-IV, sarsasapogenin glycosides Vitamins: Vitamin C, vitamin E, and beta-carotene Minerals: Potassium, magnesium, calcium, and zinc These bioactive compounds contribute to the overall antioxidant capacity of Asparagus africanus and may play a role in its potential health benefits. Chemical constituents of Asparagus Asparagus species, belonging to the family Liliaceae, are native medicinal shrubs valued for their medicinal properties. The genus Asparagus includes about 300 species around the world. The roots of Asparagus are the main source of the drug shatawar, the crude drug also used for increasing the secretion of milk and improving appetite in lactating women. Ripe fruits of Asparagus curillus cause abortion, tuberous roots with honey are given in dysuria, diabetes, and dysentery.[1] The roots of Asparagus racemosus are bitter, sweet oleaginous, cooling, and indigestible, appetizer, and are useful in dysentery, tumors, inflammation, biliousness, leprosy, epilepsy, and night blindness.[2] In Unani system, the roots are used as laxatives, tonic, aphrodisiac, galactogogue, and in disease of kidney and liver. Shoots contain thiophene, thiazole, aldehyde, ketone vanillin, asparagusic acid, and its methyl and ethyl esters used as flavors. Flowers and mature fruits contain quercetin, rutin (2.5% dry basis), and hyperoside, and the leaves contain diosgenin and quercetin-3-glucuronide. A. racemosus roots mainly contain 4 saponins, for example, shatavarin I–IV, the glycosides of sarsasapogenin. Roots of A. racemosus are also used against jaundice. The bark exhibited antibacterial and antifungal activity. The powdered roots contain 2.95% protein, 5.44% saponins, 52.89% carbohydrate, 17.93% crude fiber, 4.18% inorganic matter, and 5% oil. The root of Asparagus officinalis is more diuretic than its shoot, and the root is recommended in dropsy and is a powerful cardiac sedative. Its roots have been used as a remedy for schistosomiasis and tuberculosis. The roots of Asparagus filicinus are considered as tonic, astringent, and vermifuge. In India and China, this plant is given as a powerful diuretic in cholera and rheumatism.[3] Ancient Grecians and Romans used Asparagus for its diuretic properties. It helps flush out the kidneys and help in the prevention of the formation of kidney stones. Chinese pharmacists save the best Asparagus roots for their families and friends, believing that it will increase feelings of compassion and love. In India, it is used to promote fertility, reduce menstrual cramping, and increase milk production in nursing mothers. Asparagus acts to increase cellular activity in the kidneys and thus increases the rate of urine production. Phenolic Acids as Key Antioxidants In recent decades, an increasing interest has been focused on wild edible plants to broaden the diversity of the human diet because of their nutritional and medicinal values [1,2]. In the Mediterranean basin, wild plants rich in antioxidants have been harvested and eaten seasonally for many generations. They have been used as food, medicine, dye and ornaments and are also an important source of income for local people in different regions. Among edible wild plants, the Asparagus genus has a relevant position, including over 250 species of both food and medicinal interest [3]. Among these, Asparagus acutifolius L., is an herbaceous, perennial species widely distributed in the Mediterranean area, and mainly known for the fine flavor of its spears. It is generally sold in local markets at relatively high prices and used in typical restaurant dishes. From the nutritional point of view, A. acutifolius spear, which is the edible part of the plant, is rich in antioxidant phenolics, Vitamin C, folates, ascorbic acid and dietary fiber [4]. Among phenolics, the major compounds found are Quercitin-3-O-rutinoside and Isorhamnetin-3-O-rutinoside and to a lesser extend Kaempherol-3-O-rutinoside [5]. Aqueous extracts of A. acutifolius, rich in these compounds, have been shown to possess significant antiproliferative and pro-apoptotic activity against bladder and lung cancer lines [6]. Di maro et al. [7] have found rutin (3-O-Quercitin-rutinoside) as the main flavonoid along with high levels of isoquercitrin (Quercitin-glucoside). They have also found important antiproliferative activities against neuroblastoma and liver, cervical and lung cancer cells. This plant is also used for its medicinal properties which are related to the presence of different bioactives in various plant parts including phenolics, saponin and polysaccharides [8]. For instance, a decoction of the root has been used traditionally by the ethnomedicine as a diuretic [9]. However, in recent years, wild edible food sources, particularly wild A. acutifolius, have fallen into neglect since their traditional uses are being lost. The revalorization of these wild vegetables as potential sources of functional food ingredients and/or including their consumption in modern diets could contribute to the preservation of their traditional knowledge and culinary uses, as well as to improve the biodiversity and rural sustainability of the collection areas. Among plant secondary metabolites, phenolic compounds have been extensively studied and are commonly used as antioxidants because they might retard the oxidative degradation of lipids and thereby improve the quality and nutritional value of food [10]. Asparagus contains flavonoids (mainly rutin) and other phenolic compounds which possess strong antioxidant properties [11,12,13,14]. The potential health benefits of the antioxidants in asparagus include reducing the risk of cancer, cardiovascular and cerebrovascular diseases [15,16,17]. As far as we know there is no report on either the biological activity or the phytochemical composition of the leaf, stem, pericarp or rhizome of A. acutifolius. Except for a report on the characterization of the root’s saponin [8], the available studies in the literature describe the nutritional assessment, phenolic composition and antioxidant properties of only the edible portion of this plant [18,19,20]. However, considering the presence of different compounds in different portions of the plant, it seems interesting to study the different organs as possible sources of bioactives with different types of applications in sectors such as food, cosmetics or pharmaceuticals. Therefore, the aim of this study was to characterize the bioactive compounds of the A. acutifolius leaf, stem, pericarp and rhizome, paying special attention to the functional properties (antioxidant, cytotoxic, lipase inhibitory and antimicrobial activities) of their ethanolic extracts. Analgesic and Anti-Inflammatory Activities of Asparagus Africanus Root Extract Asparagus africanus Lam (Liliaceae) is an erect armed herb that grows up to 5ft high. The plant is widely distributed in tropical Africa. In Nigeria, the plants are known as “Shekan bera” in Hausa and “aluki” in Yoruba (Dalziel, 1956). In traditional medicine, the plant is used for the treatment of headache, backache, stomach pain and as an aid in child birth (Msonthi and Magombo, 1983). The plant is also used for heamaturia, heamorrhoids (Desta, 1993), malaria, lishmaniansis, bilharziasis, syphilis and gonorrhoea (Oketch-Rabah et al., 1992). The root extract is applied externally for the relief of pain, rheumatism and chronic gout (Watt and Breyer-Brandurijk, 1962). It is also used as a diuretic, for sore throat and otitis (Oliver, 1960). Three steroidal saponins have been isolated from the roots of A. africanus (Debella et al., 1999). Many plants have shown very effective medicinal values for various ailments of human and domestic animals. Studies have witnessed several of the pharmaceuticals currently available to physicians have a long history of use as herbal remedies. Digoxin and digitoxin, drug for heart failure and atrial dysrhythmias, from digitalis leaves [27]; quinine, antimalarial drug, from cinchona bark [19] and anti-cancer compound bruceatin, from the ethiopian plant, Brucea antidysentrica [14], are examples of the contributions of traditional pharmacopoeia. Despite its being natural, reputation and continued use over many centuries, recent studies on laboratory animals have shown many plants used as medicinal activity have potential toxicity on blood parameters and histopathology of internal organs [5, 7, 18]. In addition, the advancement of technology has enabled to detect minute amounts of carcinogenic and toxic chemicals and recognize potentially hazardous effects of some of the herbs used in traditional medicines [25]. It therefore appears that, although traditional medicine is widely used to treat various diseases and often more available and affordable than modern medicine, it is not without limitations. Asparagus africanus Lam., commonly known with its vernacular name “Seriti” (Afaan Oromo) is one of the most effective medicinal plants widely used to treat malaria [10], impotency [29], diarrhea [1], lishmaniansis, bilharziasis, syphilis and gonorrhoea [26] and fertility [15]. The plant belongs to family Asparagaceae which includes 300 species in the genus Asparagus, widely distributed throughout Africa. It is a perennial shrub or climber with stems up to 6 m high growing between 700 and 3800 m above sea level [11, 17]. Different studies have shown antimalarial activity of the plant with different potential of parasitimal suppression. The crude extracts of A. africanus for instant have shown a parasitimial suppression of 46.1 % (root parts) and 40.7 % (aerial parts) on Swiss albino mice [12]. The methanol extracts of the plant found to have parasitaemia suppression against Plasomodium berghei at a dose of 200 mg/kg [12]. Butanol fractionated extract of A. africanus showed the highest inhibition (85.94 %) of P. berghei parasitaemia in the swiss albino mice [10]. In spite of such widespread use of the plant, its safety based on their effective dose is not yet known. The butanol fractionated root extract of Asparagus africanus Many plants have shown very effective medicinal values for various ailments of human and domestic animals. Studies have witnessed several of the pharmaceuticals currently available to physicians have a long history of use as herbal remedies. Digoxin and digitoxin, drug for heart failure and atrial dysrhythmias, from digitalis leaves [27]; quinine, antimalarial drug, from cinchona bark [19] and anti-cancer compound bruceatin, from the ethiopian plant, Brucea antidysentrica [14], are examples of the contributions of traditional pharmacopoeia. Despite its being natural, reputation and continued use over many centuries, recent studies on laboratory animals have shown many plants used as medicinal activity have potential toxicity on blood parameters and histopathology of internal organs [5, 7, 18]. In addition, the advancement of technology has enabled to detect minute amounts of carcinogenic and toxic chemicals and recognize potentially hazardous effects of some of the herbs used in traditional medicines [25]. It therefore appears that, although traditional medicine is widely used to treat various diseases and often more available and affordable than modern medicine, it is not without limitations. Asparagus africanus Lam., commonly known with its vernacular name “Seriti” (Afaan Oromo) is one of the most effective medicinal plants widely used to treat malaria [10], impotency [29], diarrhea [1], lishmaniansis, bilharziasis, syphilis and gonorrhoea [26] and fertility [15]. The plant belongs to family Asparagaceae which includes 300 species in the genus Asparagus, widely distributed throughout Africa. It is a perennial shrub or climber with stems up to 6 m high growing between 700 and 3800 m above sea level [11, 17]. Different studies have shown antimalarial activity of the plant with different potential of parasitimal suppression. The crude extracts of A. africanus for instant have shown a parasitimial suppression of 46.1 % (root parts) and 40.7 % (aerial parts) on Swiss albino mice [12]. The methanol extracts of the plant found to have parasitaemia suppression against Plasomodium berghei at a dose of 200 mg/kg [12]. Butanol fractionated extract of A. africanus showed the highest inhibition (85.94 %) of P. berghei parasitaemia in the swiss albino mice [10]. In spite of such widespread use of the plant, its safety based on their effective dose is not yet known. With the problems of increasing drug resistance and difficulties in getting affordable effective antimalarial drugs, traditional medicines, mainly of plant sources could be an important and sustainable ways of treatment if supported by clinical evidences of safety and efficacy. Medicinal plants not only complement modern medicine but also are the basis for the development of modern pharmaceuticals. Despite highest (85.94 %) inhibition of P. bergei parasitaemia, no clinical evidence of safety (toxicity) yet found on butanol fractionated extract of A. africanus. This study is therefore aimed to investigate the toxicity of butanol fractionated extract of A. africanus Lam., on some blood parameters and histopathology of liver and kidney in mice. CHAPTER THREE 3.0MATERIAL AND METHOD 3.1MATERIALS Fresh Asparagus africanus shoots 95% ethanol Soxhlet extractor Rotary evaporator Spectrophotometer Fourier transform infrared (FTIR) spectrometer Nuclear magnetic resonance (NMR) spectrometer DPPH (2,2-diphenyl-1-picrylhydrazyl) FRAP (ferric reducing antioxidant power) reagent ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) reagent Bacterial and fungal strains (Staphylococcus aureus, Escherichia coli, Candida albicans, Aspergillus fumigatus) Lipopolysaccharide (LPS) Mouse macrophages (RAW 264.7) Human breast cancer cells (MCF-7) Human colon cancer cells (HCT-116) Human leukemia cells (HL-60) 3.2Methods Extraction of Asparagi africanus ethanolic extract 1. Fresh Asparagus africanus shoots were collected from the wild and washed thoroughly with distilled water. 2. The shoots were then cut into small pieces and air-dried for 7 days until completely dry. 3. The dried shoots were ground into a fine powder using a mortar and pestle. 4. The powder was extracted with 95% ethanol using a Soxhlet extractor for 6 hours. 5. The ethanolic extract was then filtered and concentrated under reduced pressure using a rotary evaporator. 3.2.1Phytochemical screening of Asparagi africanus ethanolic extract Phytochemical screening of the ethanolic extract was performed using standard methods to identify the presence of various secondary metabolites, including phenolic acids, flavonoids, saponins, and alkaloids. Plants have been in existence for a long time. Over 250,000 species of flowering plants are known and estimated 155,000 species can be found in the tropics [1]. South Africa is recognised as one of the countries in the world with the richest biodiversity (http://www.gcis.gov.za/). Apart from fuel, fibre and food, plants have always been known to be a traditional source of medicines, since they contain secondary metabolites of high chemical diversity. Chemical diversity is one among several important factors that has given rise to continuing interest in research into natural [1]. Most medicinal plants contain some organic compounds that cause a definite physiological action in the human body, as a result of the presence of bioactive substances, such as tannins, alkaloids, carbohydrate, terpenoids, steroids and flavonoids [2]. These substances are also reported to be present in the roots of Asparagus species [3]. In view of the reports on bioactive substances in natural plants, phytochemical screening of plants has become more important. Phytochemical analyses are conducted on medicinal plants to ascertain the presence of constituents that are known to exhibit medicinal factors, as well as physiological activities [4]. Furthermore, recent studies have shown that phytochemicals present in leaves and roots have received a great deal of attention, mainly for their role in preventing diseases caused by oxidative stress, which releases reactive oxygen species. Considering the importance of these substances, which fight against various illnesses and are good for human consumption, checking for their presence in any medicinal plant is a necessity. In addition, there is a need to validate the presence of these bioactive constituents through an organised infrastructure (phytochemical screening) if they are to be used as an effective therapeutic means in novel drug discovery [5]. Therefore, in this study knowledge of the chemical constituents of A. africanus is desired and this will be obtained through qualitative phytochemical screening. 2.2 Phytochemical Screening of Asparagus africanus All the chemical tests were carried out on the methanolic and water extracts, using the standard procedure to identify the chemical constituents by colour changes [4, 6, 7]. 2.2.1 Test for Saponins Amounts of about 0.7 g of the extracts were dissolved in 3 mL of distilled water and shaken vigorously. The formation of emulsion was observed. The test was performed in triplicate. 2.2.2 Test for Carbohydrates Benedict’s test was performed using 0.2 g of the extract dissolved in 2 mL of methanol; 1,000 uL of the sample was put into a test tube and the same proportion of Benedict’s solution was added. The test was carried out in triplicate. 2.2.3 Test for Protein The test for protein was carried out measuring out 0.2 g of the extract dissolved in 2 mL of methanol; a few drops of 1% CuSO4 and 4% NaOH were used; these are also Biuret reagents. The test was performed in triplicate. 2.2.4 Test for Flavonoids Three methods were used to determine the presence of flavonoids in the plant sample [4, 8]. Five (5) mL of dilute ammonia solution was added to both the methanol and distilled water extracts, followed by the addition of concentrated H2SO4. Yellow colouration observed in each extract indicated the presence of flavonoids. The test was done in triplicate. 2.2.5 Test for Tannins Amounts of about 0.7 g of the extracts were dissolved in 2 mL methanol and distilled water, then three drops of 0.1% ferric chloride solution were added. The formation of precipitates in the solution was observed; there was a blue-black or brownish green precipitate, which indicated the presence of tannins. 2.2.6 Test for Steroids and Terpenoids (Salkowski Test) About 2 g of each extract was dissolved in 2 mL of both methanol and distilled water. Both extracts were premixed with 1 mL of chloroform and afterwards concentrated sulphuric acid (H2SO4) was added to the sample. A layer was then formed. These experiments resulted in a reddish brown colour precipitate at the border or interface of chloroform and H2SO4, which confirmed the presence of steroids and terpenoids. 2.2.7 Test for Cardiac Glycosides (Keller-Killani Test) About 0.7 g of each extract was dissolved in 2 mL of methanol and distilled water, and then the extracts were both treated with 2 mL of glacial acetic acid containing one drop of ferric chloride solution. This was underlaid with 1 mL of concentrated sulphuric acid. A brown ring at the interface indicated the deoxysugar characteristic of cardenolides. A violet ring appeared below the brown ring, while in the acetic acid layer, a greenish ring formed gradually throughout thin layer. 2.2.8 Test for Alkaloids (Meyer’s Test) Amounts of about 1 g of each extract were dissolved in 3 mL of methanol and distilled water; 1 mL of the Meyer’s solution (potassium mercuric iodide solution) was added to 200 μL of the respective samples. The methanol extracts became creamish in colour, while the water extracts turned orange brownish in colour. 2.2.9 Test for Alkaloids (Dragendorff’s Test) About 1 g of each extract was dissolved in 3 mL of methanol and distilled water and 1 mL of the Dragendorff’s solution (potassium Bismuth iodide solution) was added to 200 μL of the respective samples. Both the methanol and distilled water turned. Spectroscopic analysis of Asparagi africanus ethanolic extract 1. UV-Vis spectroscopy analysis was performed to determine the presence of conjugated double bonds in the ethanolic extract. 2. FTIR spectroscopy analysis was performed to identify the functional groups present in the ethanolic extract, such as hydroxyl, carbonyl, and aromatic groups. 3. NMR spectroscopy analysis was performed to determine the specific compounds present in the ethanolic extract, such as sinapic acid, quercetin, rutin, and kaempferol. Evaluation of antioxidant activity of Asparagi africanus ethanolic extract 2.1 Plant Collection and Identification Asparagus africanus root was collected from a farm in Bauchi road, Jos, Plateau State. The identification of the plant was at the Herbarium, Botany Department, Obafemi Awolowo University (O.A.U.), Ile-Ife, Nigeria. 2.2 Preparation of Aqueous Extract The root of the plant was cleaned by washing it under a running tap water. It was then dried at room temperature. Thereafter, it was ground to powder and macerated in distilled water for three days. It was then filtered after maceration for three days using a sieve and filter paper (Whatman no. 1). Rotary evaporator (at 45°C) was used for concentrating the filtrate. A freeze dryer was used for removing the water in the filtrate in order to obtain a dried sample (extract) [21]. The dried Asparagus africanus root aqueous extract was then preserved by storing it in a refrigerator before using it for the study. 2.3 Animals The animal experiment (PG/Pharmcol/2012/02) was carried out using the guidelines approved by Pharmacology Department, Faculty of Pharmacy, O.A.U., Ile-Ife, Nigeria. Eight – nine weeks old Wistar rats (150-200g) of both sexes (total of 30 males and 30 females) gotten from Animal House, Pharmacology Department, were kept in cages that are ventilated. Broilers mash was used for feeding the animals and they had free access to water. The Wistar rats were allowed to get used to the environment for two weeks prior to commencing the research work. 2.4 Acute Toxicity Studies (Median Lethal dose [LD50] Determination) The median lethal dose of Asparagus africanus root aqueous extract in Wistar rats via oral route was ascertained by carrying out acute toxicity studies [22]. 2.5 Glucose Loading After twelve hours fasting of the animals, 10/g/kg glucose was administered orally to the animals. Blood was taken from the vein of the animal tail after thirty minutes of administering glucose [23,24]. The fasting blood glucose concentration was measured with glucometer [21]. Animals with ≥ 7.0 mmol/l fasting blood glucose concentration were used for the experiment. 2.6 Induction of Diabetes Using Streptozotocin Streptozotocin at 60 mg/kg prepared in distilled water was used to induce diabetes in Wistar rats (via intraperitoneal route) after twelve hours fasting of the animals. After seventy-two hours, blood was taken from the vein of the animal tail [23,24] and the concentration of the fasting blood glucose was determined. Animals with concentration of the fasting blood glucose > 11.1 mmol/l were taken for the research work [25]. 2.7 Administration of Doses There was six groups with five Wistar rats in each group [26,27]. Groups 1 was orally administered distilled water (5 mL/kg) to normoglycemic Wistar rats. Asparagus africanus root aqueous extract at 100, 200 and 400 mg/kg was orally administered to diabetic Wistar rats in groups 2, 3 and 4 respectively. The diabetic animals in group 5 were orally administered glibenclamide (5 mg/kg). Group 6 (control group) was orally administered distilled water (5 mL/kg) to diabetic Wistar rats. In glucose loaded rats (15 males and 15 females) the administration was once. Whereas to Wistar rats (15 males and 15 females) that were induced diabetes with streptozotocin, administration was daily for twenty-one days. 2.8 Antidiabetic and Antioxidant Studies The concentration of the fasting blood glucose in the blood taken from the vein in the tail of the animals was determined in minutes and days [25]. On the 21st day, Wistar rats that were induced diabetes with streptozotocin were sacrificed. The liver of the animals harvested were washed using cold normal saline and thereafter with 0.15 M Tris-HCl (pH 7.4). The homogenate of liver (10% w/v) was used for the determination of the concentration of thiobarbituric acid reactive substance level (absorbance was read at at 532 nm) [28], reduced glutathione (absorbance was read at 412 nm) [29] and catalase (absorbance was read at 570 nm) [30].