Pharmacognostic and Acute Toxicity Study of Burkea Africana Root

Burkea africana is a plant that belongs to then family Fabaceae; it is widely spread in tropical Africa including Nigeria. It is of valuable in ethnomedicine especially in the treatment of antidote for venomous stings and bites, cutaneous and sub cutaneous parasitic infection, convulsion and pulmonary troubles. Despite the fact that roots of Burkea africana have several medicinal properties, no standardization parameter has been assessed. Due to lack of standard parameters, proper identification and ascertaining quality and purity in the events of adulteration has been thwarted. The objective of the study was to establish some important pharmacognostic profile and safety margin of Burkea africana root with the hope of assisting in its standardization for quality, purity and safety. Elemental analysis was carried out using acid digestion method and phytochemical composition of the plants was evaluated using standard method. Acute toxicity was achieved using Lorke method to determine the LD50. Chemomicroscopical evaluation revealed the presence of cellulose, tannins, starch, lignin, calcium oxalate, suberin, aleurone grain and mucilage with the exception of calcium carbonate. The average moisture contents, total ash, acid insoluble, water soluble ash, alcohol extractive value and water extractive values in the powdered plant material were 3.8%, 7.5%, 4.43%, 8.07%, 25.0% and 20.33% respectively. In addition, Fe, Mn, Ni, Pb, Cd and Cu were found to be within the safety limit. Phytochemicals which include alkaloids, flavonoids, saponins, tannins, carbohydrates and triterpenes were detected in both aqueous and methanolic extracts. The LD50 of Burkea africana was found to be greater than 5000 mg /kg and could be considered safe for consumption. DOI: https://dx.doi.org/10.4314/jasem.v24i4.4 Copyright: Copyright © 2020 Namadina et al. This is an open access article distributed under the Creative Commons Attribution License (CCL), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dates: Received: 14 February 2020; Revised: 21 March 2020; Accepted: 29 March 2020

Ever before the advent of orthodox medication, plants and plant parts in forms of squeezed, aqueous (cold, warm or hot) and illicit gin extracts have served as dependable sources of medicine in traditional African societies and beyond. Indeed, according to WHO (2002) report, traditional medicine using plant extracts will continue to provide health coverage for over 80% of the world's population, particularly the developing countries. The emergence of hitherto unknown disease causing microbes is of immense public health concern. This requires renewed strategies on treatment and prevention, essentially through the development of new antimicrobials (Iwu et al., 1999). Thus, further drug research and development from any viable source is imperative, and the investigation of indigenous plants may yield positive results. A number of medicinal plants have been found and put into use in ethnomedicine by traditional healers in the management of many ailments for many years (Sofowora, 1993). Many medicinal plants are believed to be important source of new chemical compounds with potential therapeutic effects (Eisner, 1990). The stem bark of Burkea africana is used as antidote for venomous stings and bites, cutaneous and sub cutaneous parasitic infection, convulsion, pulmonary troubles. The plant is indigenous to Tropical Africa including Nigeria and commonly known as Bakin makarho and Apasa among the Hausas and Yoruba, respectively (Burkill, 1985). Previous works revealed that the stem bark extract of the plant possess  (Mathisen et al., 2002); antidiarrhoeal activity (Tanko et al., 2011); anticonvulsant activity (Yaro et al., 2010;Mahdi et al., 2013) and antioxidative stress of the polyphenolic-rich stem bark fractions in cell lines (Cordier et al., 2013). The aim of this work is to carry out pharmacognostic standardization and toxicity analysis on Burkea africana root.

Collection and Identification of Plant material:
Burkea africana root was collected in March, 2017 from Babura Local Government Area of Jigawa State and conveyed for identification and authentication at the Herbarium unit of Department of Plant Biology, Bayero University, Kano, Nigeria.
Extraction of Plant Materials: Dried plant materials (50 g) were extracted using cold maceration with 500 ml of both distilled water and methanol. The contents were then be filtered using a filter paper (Whatman no.1), the filtrate was concentrated to dryness using water bath which was kept in desiccator.
Chemo-microscopic Studies on the powdered root of Burkea Africana: Powdered sample (5g) of plant species was used for this study to detect the presence of cell wall materials and cell inclusions. Finely ground sample of plant was cleared in a test tube containing 70% chloral hydrate solution. It was then be boiled on a water bath for about thirty minutes to remove obscuring materials. The cleared sample was mounted with dilute glycerol onto a microscope slide. Using various detecting reagents the presence of cell wall materials and cell inclusions was detected in accordance to WHO (2011) guidelines. Cell wall Materials Test for Cellulose A drop or two of iodinated zinc chloride was added to the powdered sample and allowed to stand for a few minutes and observed under a microscope. It stained cellulose cell wall blue to blue-violet.
Test for Lignin: The powdered plant material was moistened on a slide with a small volume of phloroglucinol and allowed to stand for about two minutes or until almost dry. A drop of hydrochloric acid was added and viewed under a microscope. Pink stained or cherry red was observed for the presence of lignin.
Test for Suberized or Cuticular cell walls: A drop or two of Sudan red was added to the cleared powdered sample and allowed to stand for few minutes and observed under a microscope. Orange red or red colour was observed presence of suberin or cutin on the cell.

Test for Gum and Mucilage:
To a small portion of the cleared powdered sample of the plant, a drop of ruthenium red was added. Appearance of pink coloration was considered positive for gums and mucilage.

Cell Inclusions/ Cell Contents: Test for Starch grains:
To a small portion of the cleared powder sample of the plant, N/50 iodine was added. Appearance of blueblack or reddish-blue coloration on some grains would be considered positive for starch.

Test for Calcium oxalates and Calcium Carbonates:
To a small portion of the cleared powdered sample of the plant, HCl was added, dissolution of crystals in the powdered drug without effervescence was considered positive for calcium oxalate while slow dissolution with effervescence was considered positive for calcium carbonate.
Inulin: A drop of 1-naphthol and that of sulphuric acid was added to the powdered sample and viewed under the microscope. Spherical aggregations of crystals of Inulin turned brownish red and dissolve.
Test for Tannins: To a small portion of the cleared powdered sample of the plant, 5% ferric chloride solution was added. Appearance of greenish black colour was considered as positive for tannins.
Determination of Physicochemical Constants of the powdered root of Burkea africana: Some physicochemical parameters of the powdered sample of the plant such as moisture content, total ash, acidinsoluble ash, water-soluble ash, alcohol and water extractive values was determined as described in the updated edition of quality control methods for medicinal plant materials (WHO, 2011).
Moisture Content: Exactly 3 g each of the powdered sample was accurately weighed and placed in some clean, dried evaporating dishes of known weights. They were placed in an oven and heated at a temperature of 105 o C for 1 hour, then cooled in a desiccator and re-weighed. Heating and weighing were repeated until a constant weight was obtained. The weight loss on drying was computed following the formula below: Where AV = Percentage Ash Value Acid-insoluble ash: This was determined for the powdered plant material. 25 ml of dilute hydrochloric acid was added to the crucible containing ash. It was covered with a watch glass and gently boiled for 5mins. The watch glass was rinsed with 5 ml of hot water and the liquid added to the crucible. The insoluble matter was collected on an ash less filterpaper and washed with hot water until the filtrate is neutral. The filter-paper containing the insoluble matter was transferred to the original crucible, dried in an oven and ignited to a constant weight. The residue was allowed to cool in a suitable desiccator for 30 minutes and then weighed without delay (Evans, 2009). The acid-insoluble ash will then be calculated as a percentage for each of the two plants with the formula % =

Weight of Residual Ash Original Weight of Powder X 100%
Where %AIA = percentage acid insoluble ash Water soluble ash: To the crucible containing the total ash, 25 ml of water was added and boiled for 5 minutes. The insoluble matter was collected in a sintered glass crucible. It was then be washed with hot water and ignited in a crucible for 15 minutes at 105 o C. The weight of the residue was subtracted from the weight of the total ash. The content of water soluble ash per air dried powdered sample was calculated and recorded (WHO, 2011).
Where %ASA = percentage acid insoluble ash; W1 = weight of total ash; W2 = weight of residual ash and W3 = original weight powder Alcohol-Soluble Extractive Value: 4 g of each of the plant material was separately weighed in a conical flask. 100 ml of ethanol was added and macerated for 24 hours, during which the mixture was frequently shaken within the first 6 hours using a mechanical shaker. It was filtered and 25 ml of the filtrate transferred into an evaporating dish of known weight and evaporated to dryness on a water bath. It was dried to a constant weight, the percentage of alcohol-soluble extractive value was then determined for the plant as  (Evans, 1996).
Quantitative Phytochemical Screening of the Methanol extract of Burkea africana root: Preparation of Fat free Sample: About 2g of the sample was weighed and defatted with 100ml of diethyl ether using a soxhlet apparatus for 2hours.
Alkaloid Determination using Haborne (1973) Method: About 5g of the sample was weighed into a 250ml beaker and 200ml of 10% acetic acid in ethanol were added and covered and allowed to stand for 4hours. This was filtered and the extract is concentrated on a water bath to one quarter of the original volume. Concentrated ammonium hydroxide was added drop wise to the extract until the precipitation is completed. The whole solution was allowed to settle and the precipitates were collected and wash with dilute ammonium hydroxide and then filtered. The residue is the alkaloid, which was dried and weighed.
Flavonoid Determination by the Method of Bohm and Kocipal -Abyazan (1994): About 10g of the plant sample was extracted repeatedly with 100ml of 80% aqueous methanol at room temperature. The whole solution was filtered through Whatman filter upper No. 42 (125mm). The filtrate was later be transferred into a crucible and evaporated into dryness over a water bath and weighed to a constant weight.
Saponin Determination: The method of Obadoni and Ochuko (2001) was used. Out of the grinded samples 10g was weighed for each and put into a conical flask and 100ml of 20% aqueous ethanol were added. The samples were heated over a hot water bath for 4hours with continuous stirring at about 55oC. The mixture was filtered and the residue re-extracted with another 200ml, 200% ethanol. The combined extracts were reduced to 40ml over water bath at about 90oC.
The concentrate was transferred into a 250ml separatory funnel and 20ml of diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. The purification process was repeated and 60ml of nbutanol was added. The combined n-butanol extracts were washed twice with 10ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation the samples were dried in the oven to a constant weight; the saponin content was calculated as percentage.   Method: About 500mg of each sample was weighed into a 50ml plastic bottle and 50ml of distilled water was added and shaken for 1hour on a mechanical shaker. This was filtered into a 50ml volumetric flask and made up of the mark. Then 5ml of the filtrate was pipetted out into a test tube and mixed with 2ml of 0.1M FeCl3 in 0.1M HCl and 0.008M potassium ferrocyanide. The absorbance was measure at 120mm within 10min.

Determination of Total
Phenols by Spectrophotometric Method: The fat free sample was boiled with 50ml of ether for the extraction of the phenolic component for 15minutes. About 5ml of the extract was pipetted into a 50ml flask, and then 10ml of distilled water was added. About 2ml of ammonium hydroxide solution and 5ml of concentrated amyl alcohol were added also. The sample was made up to mark and allowed to react for about 30 and weighed to a constant weight.
Acute toxicity studies of methanol extract of Burkea africana root: Lethal Dose (LD50) Determination: This is the determination of the lethal dose known as LD50. The method of Lorke (1983) was employed. The phase I involved the oral administration of three different doses of 10, 100 and 1,000 mg/kg of the crude extract, to three different groups of three adult Wister albino rats. In a fourth group, three adult male Wister albino rats were administered with equivalent/volume of distilled water to serve as control. All the animals were orally administered the extract using a curved needle to which acatheter had been fixed. The animals were monitored closely every 30 minutes for the first 3 hours after administration of the crude extract and hourly for the next 6hours for any adverse effects. Then the animals were left for 72 hours for further observation.
When no death occurred, the phase II was employed, only one animal was required in each group. Groups 1-4 , animals were orally given 1,500, 2,200, 3250 and 5,000mg/kg dose levels of the crude extract. All the animals were left for observation as in stage one.

RESULTS AND DISCUSSION
Chemo-microscopical examination of powdered root of Burkea africana revealed the presence of cellulose, tannins, starch, lignin, calcium oxalate, suberin, aleurone grain, calcium carbonate and mucilage. (Table 1). The average moisture contents in the powdered plant material using loss on drying method was calculated to be 3.8%, the total ash was 7.5% while acid insoluble and water soluble ash were 4.43% and 8.07% respectively. The alcohol and water extractives values were obtained to be 25.0% and 20.33% respectively (Table 2). Trace metals which include Fe, Mn and Ni detected in Burkea africana powdered root were below the FAO/WHO (1984) permissible limit for edible plants. While others, Pb and Zn were found to exceed the safety limit (Table 3). Phytochemical screening of aqueous and methanol extracts revealed the presence of alkaloid, flavonoids, saponins, tannins, anthraquinones and steroid (Table  4). No death was recorded in the first phase of the study in rats. In the second phase, doses of 1500, 2250, 3250 and 5000mg/kg were used and no death was also recorded (table 5). The oral median lethal dose (LD50) for the methanol root-extract of Burkea africana was therefore estimated to be greater than 5000mg/kg and no sign of behavioural changes were also observed ( Table 6). Chemomicroscopical examination of the powdered root of Burkea africana revealed the presence of cellulose cell wall, lignified cell wall, tannins, starch, calcium oxalate, cutin and calcium carbonate. The microscopic structures are most valuable in the identification of powdered drug as their identification is largely based on the form, the presence or absence of certain cell types and cell inclusions (Jeremiah et al., 2019;. The physicochemical parameters including the moisture content, acid insoluble ash, water soluble ash, alcohol extractives value, water extractives and total ash values were determined from the powdered leave of the plant (Kokate, 2003).The result of average moisture contents using loss on drying method was calculated to be 3.8% and the percentage yield of total ash, acid insoluble and water soluble ash were recorded in percentage values as 7.5%, 4.43% and 8.07% respectively. The extractives obtained were 18.33% and 25.68% for alcohol and water solvents respectively. These are values that are very important as basis to judge the identity, purity and in detecting adulterants in a crude drug (Evans, 2009;WHO, 2011). Moisture content was not high which indicated less chances of microbial degradation of the drug during storage. The general requirement of moisture content in crude drug is recommended not to be more than 14 % (Peter, 1990) and the value obtained in this research work was within the accepted range. Determination of the moisture content helps preventing degradation of drug during storage.
The lower the value, the less likelihood of degradation of drug and suggests better stability of the product. Moisture is considered an adulterant because of its added weight as well as the fact that excess of moisture promotes mold and bacterial growth (Prasad et al., 2012;Nuhu et al., 2016). Total ash value represents both the physiological and non-physiological ash from the plant. The non-physiological ash is an indication of inorganic residues after the plant drug is incinerated.  The acid insoluble ash values obtained in this study indicated that the plant was in good physiological condition and contained little extraneous matter such as sand, silica and soil. The total ash value is used as criteria to judge the identity and purity of drugs (WHO, 2011;Prasad et al., 2012). Extractive value is determined when a given amount of plant material is extracted with a particular solvent. When the crude drug is extracted with a particular solvent, it produces a solution that contains several constituents (Evans, 2009;Vyry et al., 2013). The nature of the crude drug and the solvent used determines the constitution of the phyto-constituents present (Rajurkar and Damame, 1997;Nuhu et al., 2016). It also helps to determine if the crude drug is debilitated or not (Tatiya et al., 2012;Adekunle et al., 2014). This study indicated that ethanol gave higher extractive value (25.0% w/w) compared to water which had extractive value of 20.33 % w/w. The elemental analysis revealed some of the elements that are present in the root of Burkea africana. The elements are rich sources of macro and minor elements that aid in the growth of plants, and as well in human body functions such as muscle contraction, bone formations, growth, metabolism, osmotic balance, regulatory processes activation and other organic bimolecular activities (Rabia et al., 2012). The concentrations of elements gotten from this study were below FAO/WHO (1984) permissible limits for edible plants with exception of zinc and lead. Zinc (Zn) is an essential component of a number of enzymes present in animal tissue including alcohol dehydrogenase, carbonicanhydrase, procarboxypeptidase and aids in normal growth, reproduction, tissue repair and wound healing. Zinc deficiency causes growth retardation and skin lesions (Chatterjee and Shinde, 1995) NAMADINA .Manganese (Mn) is often found in minerals in combination with iron. It is helpful in carbohydrate metabolism and served in the body as a co-factor for the enzymes involved in hydrolysis, phosphorylation, transamination and decarboxylation. It promotes the activities of transferases such as superoxide dismutase and aids as antioxidant to scavenge damaging particles (superoxide) known as free radicals in the body (Dias, 2012). Low levels of manganese can cause infertility, bone malformation weakness and seizures. The main function of iron is the transport of oxygen to the tissues (haemoglobin) and also in cough associated with angiotensin-converting enzyme (ACE) inhibitors, haemopoietic and cell mediated immunity (Faizul et al., 2012). The deficiency of iron has been related to anemia and described as the most prevalent nutritional deficiency. The systemic decrease in Copper levels causes iron deficiency. Therefore it is antianaemic and essential for the formation of iron and haemoglobin. Copper (Cu) play important role in treatment of chest wounds and prevent inflammation in arthritis and similar diseases (Faizul et al., 2012).
Besides macro and trace elements, heavy metals were also present in the root of Burkea africana but in negligible concentrations that will not cause harm when consumed or ingested as prescribed by WHO, (2007). Though, it is advisable not to be taken for a long period of time to prevent untoward effects. Cobalt (Co) is required in very small amounts in all mammals and is used to treat several different types of cancer in humans and to treat anaemia, but the intake of its high amount can cause heart diseases (Faizul et al., 2012). Lead (Pb) which exceed the permissible limit, is toxic and a non-essential element for human body, it causes a rise in blood pressure, kidney damage, miscarriages and subtle abortion, brain damage, declined fertility of men through sperm damage, diminished learning abilities of children and disruption of nervous systems (Khan et al., 2011 andObiajunwa et al., 2002). Concentration of elements in plants varies from region to region due to factors such as environmental, atmospheric, pollution, season of collection of sample, age and soil conditions in which plant grows (Faizul and Rahat, 2011). The preliminary phytochemical screening of the aqueous and methanol root extract of Burkea africana revealed the presence of several constituents. These constituents are known to be responsible for several pharmacological activities. Flavonoids were reported as prostaglandin synthetase inhibitors (Watanebe et al., 2000). This suggests that reduced availability of the prostaglandins by flavonoids might have been responsible for their analgesic activity. The presence of tannins and saponins possibly might have given rise to the observed anti-inflammatory property and contributed to the anti-pyretic activity of the plant extract. Saponins possess a wide range of therapeutic actions in the body including anti-inflammatory, expectorant, diuretic, anti-malarial and haemolytic effects on red blood cells, while tannins are used in compress for cuts and wounds, haemorrhoids, varicose veins and in medicine for diarrhoea, catarrh, heavy menstrual flows and inflammatory conditions of the digestive tract (Evans, 1989). Cardiac glycosides increase the force of myocardial contraction and reduce conductivity within the atrioventricular (AV) node. They are used in the treatment of supraventricular tachycardias, especially for controlling ventricular response in persistent atrial fibrillation (Prassas and Diamandis, 2008). In order to determine the safety margin of drugs and plant products for human use, toxicological evaluation was carried out in experimental animals using Lorke's method to predict toxicity and to provide guidelines for selecting a "safe'' dose in animals and also used to estimate the therapeutic index (LD50/ED50) of drugs (Olson et al., 2000;Rang et al., 2012). In this study, median lethal dose (LD50) of the extracts (aqueous and methanol) of the Burkea africana root was carried out orally in rats. The LD50 was found to be greater than 5000 mg/kg when administered orally in rats and all the animals remain alive and did not manifest any significant visible signs of toxicity at these doses. These studies showed the extracts Burkea africana root of are practically nontoxic when administered using the oral route. This is based on the toxicity classification which states that substances with LD50 values of 5000 to 15,000 mg/kg body weight are practically non-toxic (Loomis and Hayes, 1996).

Conclusion:
The established pharmacognostic standards for the powder of Burkea africana root could be used as a diagnostic tool for the standardization and identification of this medicinal plant for its purity, quality and compilation of a suitable monograph on Burkea africana. Methanolic root extract of Burkea africana was found to be practically safe and is considered safe for use. Nonetheless, further studies are encouraged to evaluate toxicity at much higher doses.