The presence of bioactive compounds such as saponins, tannins, alkaloids and flavonoids identified in the plant has been suggested to be responsible for the various uses of D. edulis in traditional medicine to cure ringworm, wound, scabies, skin diseases and inflammation [10]. In addition, the potential health-related functions of dietary plants were found to include antibiosis, immunostimulant, nervous system action, detoxification, anti-inflammatory, antigout, antioxidant, glycaemic and hypolipidemic properties [11]. Among the 13 Congolese plants examined for antidrepanocytary activity, the aqueous and ethanol extracts of D. edulis leaves were discovered to normalize the SS blood erythrocytes, following the deoxygenation of haemoglobin in anaerobic condition, thus validating their use in traditional medicine [12].

Peptic ulcer is one of the most common diseases of the gastrointestinal tract (mostly the stomach shown in Figure 1, with an increasing incidence and prevalence. Indeed, it can lead to serious complications and even death in some cases. It affects about 10% of the world’s population with an estimated mortality rate of 15,000 deaths per year. This condition is characterised by an imbalance between factors that damage and those that protect the integrity of the gastric mucosa. It is multi factor, but the highest incidence is usually seen in the context of Helicobacter pilori infection, and the use of non-steroidal anti-inflammatory drugs [13].

Peptic ulcer, including both gastric and duodenal ulcers, have been a major threat to the world’s population over the past two centuries. Chronic inflammation due to colonisation of Helicobacter pylori and nonsteroidal anti-inflammatory drugs account for the large majority of peptic ulcer disease [14]. Peptic ulcer can be delineated as the presence of a deep destruction of the stomach lining or mucosa and/or duodenum, reaching beyond the muscularis mucosa, specifically to the muscle layer owing to the environmental gastric acid synthesis. The two consummate ubiquitous etiological antecedents are the chronic infection with Helicobacter-pylori (Hp) and the use of NSAIDs, involving of course, the ASA. There are distinctive less ubiquitous antecedents that can cause a PU, which are thought-out together, responsible for <5% of cases. Zollinger-Ellison syndrome (ZES) or gastronomy is one amid them which is a neuroendocrine tumor, often located at the head of the pancreas or in the duodenal wall, overactive and gastrin secretory [15].

Figure 1. The human stomach anatomy source: [16].

Percentage composition of moisture, crude fibre, ash, crude fats, crude proteins and carbohydrates were determined for the chloroform and ethanol layers of the chloroform-ethanol extract of the leaves of D. edulis). Phytochemical screening (quantitative and qualitative) of aqueous leaf extract of Dacryodesedulis. Phytochemical screening of the plant extract was carried out using the methods outlined by Harbone [17]. The phytochemical composition was represented using (+) for presence and (-) for absence.

This study was carried out using an in vitro experimental design. Fresh plum leaves (Dacryodesedulis) were gotten from the farm in Kumba and kept in a dark room to dry. Later it was ground using a manual grinding miller to powder and the ground sample was then taken to the Institute Universitaire de Technology (IUT) science laboratory university of Douala where it was analysed for phytochemical and its antioxidant properties.

The Dacryodesedulis extract was prepared by decoction method. 100 g of dried leaf powder was introduced into a 1000 ml volumetric flask containing a volume of 500 ml of distilled water. The mixture was boiled for 30 minutes using a reflux heating system mounted on a heating mantle. After heating, the mixture was filtered through a filter paper No. 1 filter paper. Then, the decoction was dried at 40˚C in a sand bath for 2 days to obtain the crude extract. The crude extract obtained was weighed and the extraction yield was determined using the formula below:

The qualitative analysis of phytochemical constituents was conducted following the methods described by Harbone [17] in a book entitled: “A guide to modern techniques of plant analysis”. The quantitative determination of total phenols, total flavonoids, and flavonol content was evaluated using the methods described by Ramde-Tiendrebeogo et al. 2012, and Chang et al. 2002 [18][19]. These phytochemical analyses were based on the appearance of different colours and the formation of precipitates or products in the final solution.

2.3.1. Analysis of Polyphenols.

For phenol determination, 0.1 gram of the aqueous extract was solubilized in 3 mL of ethanol. The solution was treated with 3 drops of iron chloride III 10% (V/V), and the appearance of a blue-violet color indicated the presence of polyphenols.

2.3.2. Analysis of Flavonoids

Few drops of 1% NH₃ solution was added to 0.1 gram of the aqueous extract contained in a test tube. The appearance of a yellow color indicated the presence of flavonoid compounds

2.3.3. Analysis of Tannins

For tannins identification, 0.1 gram of the aqueous extract was boiled in 20 mL of distilled water in a test tube and then filtered. The filtration method used here is the normal method, which includes a conical flask and filter paper. Three drops of 0.1% FeCl₂ were added to the filtered samples and observed for a brownish green or a blue-black colour, which indicates the presence of tannins.

2.3.4. Determination of Total Polyphenols

10 mg of extract was diluted in 1 ml of methanol, then 100 µl of the solution was diluted in 900 µl of distilled water and 1 ml of Folin's reagent was added. After incubation for 30 min, the solution was saturated with sodium carbonate and the optical densities were read at 650 nm against a blank consisting of 1 ml of Folin’s reagent in 1 ml of distilled water.

A calibration curve established from a gallic acid dilution series (10.00, 3.33, 1.11, 0.37, 0.12 mg/ml) was treated in the same way as the extracts. The results were expressed in milligram equivalent of gallic acid per gram of extract. The polyphenol content was obtained from the regression line of the gallic acid calibration curve.

2.3.5. Determination of Flavonoids

We proceeded to stabilize tannins with BSA (Bovine Serum Albumin) Thus, in each extract, 30 ml were taken and 3 g of BSA were added to it, then the solutions obtained were stirred and then filtered with cotton. In the filtrates obtained, the flavonoids were assayed according to the same protocol described above for the polyphenols.

2.3.6. Determination of Tannins

The tannin content was obtained by taking the difference of total polyphenols and flavonoids (non-tannin polyphenol), that is total polyphenols.

2.3.7. Antioxidant Activity (inVitro) of aqueous Leaf Extract of Dacryodesedulis

The method used for the extraction of plant materials. D. edulis leaf and were washed, chopped into pieces and dried at room temperature for about 14 days to a constant weight. The samples were coarsely powdered, each of the coarsely powdered samples was weighed, placed in a big glass jar and extracted by maceration with 80% ethanol for 72 hours (h) at room temperature. Also, wet samples were extracted by maceration with water for 8 h. Figure 2 represents the flow process as described above.

Figure 2. Flow chart for plum leaf.

Figure 3. Various stages in the processing of D. edulis extract Redox activity of iron III (Fe³⁺) ions.

The Fe³⁺ reduction test was performed according to the procedure described by Berker et al. 2007 [20] with some modifications. The various stages of the extraction process are shown in Figures 3(a)-(d) respectively. Principle: this method is based on the ability of a substance to reduce Fe³⁺ ions to Fe²⁺ ions which, in the presence of 1,10-phenanthroline, forms a red-orange complex whose optical density can be measured at 505 nm. The colour intensity is proportional to the quantity of Fe³⁺ ions converted by the extract.

2.3.1. Preparation of Solutions

Fe³⁺solution

This was prepared to a concentration of 1.2 mg/ml. To do this, 1.2 mg of FeCl₃ powder was dissolved in 1 ml of distilled water so as to obtain 100 µg of Fe in 120 µl of the collected solution, which was then diluted by the extract solution to 50 µg.

Ortho-Phenanthroline solution

Ortho-Phenanthroline solution prepared at 0.2% was used to measure the amount of Fe²⁺ formed. To do this, 200 mg of powder was weighed for every 100 ml of ethanol.

Extract stock solution

The extract stock solution was prepared at a concentration of 1 mg/ml. To do this, 10 mg of D. edulis extract was weighed and dissolved in 10 ml of water. In a 96-well plate template, 120 μl of water was introduced into all the wells except those of the first line (A) where 240 μl of each extract was introduced, and a series of dilutions of the order 2 was carried out. Thereafter 120 μl of Fe³⁺ solution was added and the plates were incubated for 15 min at room temperature. After this incubation, 60 µl of the ortho-Phenantroline solution was added and the plates were re-incubated for 15 min, still at room temperature. At the end of this incubation, the optical density of the content of the wells was read at 505 nm with a plate reader (Opus). The test was performed twice. The extract’s blank solution consisted of the extract, ortho-Phenanthroline and distilled water in place of the Fe³⁺ solution, treated under the same conditions. The solvent blank solution consisted of 120 µl distilled water and 180 µl ethanol. The wells containing 120 µl solvent, 120 µl ethanol and 60 µl of ortho-phenanthroline served as an absorbance control for ortho-Phenantroline. The negative control corresponding to 0% reduction consisted of 120 µl solvent, 120 µl Fe³⁺, 60 µl ortho-phenanthroline and the positive control corresponding to 100% reduction was considered for a maximum optical density of 4. The following formula was used to calculate the reduction percentages of Fe³⁺.

2.3.2. To Evaluate the Antioxidant Activity (inVitro) of Aqueous Leaf Extract of Dacryodesedulis

The method is based on the capacity of the antioxidant (AH) contained in the sample to donate a single electron to the synthetic radical DPPH of violet colour to stabilize it to DPPH of pale-yellow colour.

The DPPH solution was prepared by completely dissolving 20 mg of DPPH in 100 ml of 95˚ ethanol. It was stored away from light before use.Stock solution of extracts: the stock solution of extracts was prepared to a concentration of 1 mg/ml. 10 mg of each extract were weighed and dissolved in 10 ml of solvent.

In a 96-well plate template, 50 μl of methanol was introduced into all the wells except those of the first line where 100 μl of each extract or gallic acid were introduced, and a series of dilution of order 2 was carried out ranging from line 1 to line 11. Line 12 constituted the negative control. Then, 150 µl of an ethanolic solution of DPPH was added to all the wells of the microplate. After 30 min of incubation in the dark and at room temperature, the absorbance was measured with a spectrophotometer at 517 nm. Each test was performed twice. The measurement of the optical densities of the different tubes enabled us to calculate the percentage trapping of free radicals of each range of concentration of extracts and of gallic acid. The trapping percentage (SC: scavenging concentration) was calculated using the following formula:

Aref = Absorbance at t = 60 min of the negative control solution (DPPH solution + methanol)

Ames = Absorbance at t = 60 min of the DPPH solution containing the antiradical.

A100 = Absorbance at the end of the reaction (close to 0) for total entrapment.

The data obtained were subjected to one-way analysis of variance (ANOVA). One – way ANOVA is used to determine whether there are statistically significant differences between the means of three or more independent variables. This helps us find out if the independent variables had a significant effect on the dependent variable. Significant differences are observed at p < 0.05. The results are expressed as means ± standard errors of means (SEM). The analysis was done using the computer software known as Statistical Product and Service Solutions (SPSS), version 21.

The extraction yield of the extract of D. edulis obtained by decoction was determined by relating the mass of the extract to the mass of leaves initially extracted. The ratio calculation showed that the extraction yield is 10% in milligrams. To screen aqueous leaf extract of Dacryodesedulis for secondary metabolites, Preliminary phytochemical screening for secondary metabolites of Dacryodesedulis of the 10% extract showed the presence of polyphenols, flavonoids and tannins. The concentrations were determined using optical density techniques and the values present in mean ± standard deviation.

Figure 4 below shows the variation curve of the optical density values obtained at different concentrations of Gallic acid.

Figure 4. The variation curve of the optical density values obtained at different concentrations of Gallic acid.

From this curve, the regression equation obtained made it possible to determine the total content of polyphenols (meq g of Gallic acid/ml) and of flavonoids (meq g of Gallic acid/ml) contained in the extract of D. edulis. One Way Analysis of Variance (ANOVA) was used to determine if the concentration of polyphenols, flavonoids and tannings were significant at 0.05 significant level in the 10 % extract. The results obtained were presented in Table 1 and Table2 below:

Table 1. Polyphenol content in the D. edulis extract in meq g of Gallic acid/ml.

Table 2. Flavonoid content in D. edulis extract in meq g of Gallic acid/ml.

Determination of tannins

Tannins are obtained by differentiating between the contents of total polyphenols and flavonoids (non-tannin polyphenol). Table3 below presents the value of the tannins present in the extract of D. edulis.

Table 3. Content of tannins contained in the extract of D. edulis in meq g of gallic acid/ml.

The concentration of polyphenols in the extract of Dacryodesedulis is (5.106 ± 0.048 mgGAE/gE) as shown in Table 1. A one-way ANOVA shows that the concentration is significantly high (p = 0.001 < 0.05) to carry out the polyphenol’s activities in Dacryodesedulis. Also, the concentration of flavonoid in the extract is (0.952 ± 0.024 mgGAE/gE) and this amount is significant (p = 0.003 < 0.05) to show the activities of flavonoids in the extract. Moreover, the concentration of tannins in the extract were significantly high (4.154 ± 0.130 mgGAE/gE, p = 0.0015 < 0.05) to carry out the activities of tannings in the extract.

The antiradical activity of the D. edulis extract was determined by its DPPH radical scavenging capacity (DPPH radical scavenging is an accepted mechanism for screening the antioxidant activity of plants in DPPH essays, violet colour DPPH solutions is reduced to yellow colour product Diphenyl hydrazine) by the addition of the extract in a concentration dependent manner. Thus, a free radical scavenger (plat material) reacts to DPPH to form DPPHH) marked by the change of the purple colour to yellow at different concentrations of the extract (Figure 5 below).

Figure 5. Well plate template obtained after incubation of DPPH radical solution in the presence of D. edulis extract and Gallic acid at different concentrations. The content of each cupule read with the spectrophotometer made it possible to obtain the optical density (OD) values which enabled calculation of the percentage (%) trapping of the DPPH radicals at different concentrations of the D. edulis extract and Gallic acid. Figure 6 below presents the curves of variation of DPPH radical scavenging percentages at different concentrations of each antioxidant.

It appears from Figure 6 below that a small increase the concentration of D. edulis significantly affects the DPPH trapping percentage. Further increase in the concentration show no significant increase in DPPH trapping percentage Thus, D. edulis extract has a significant DPPH trapping percentage in their antioxidant properties.

From these different curves, the regression equations were established, which made it possible to determine the IC₅₀s of 0.37 ± 0.00 and 2.76 ± 0.45 µg/ml respectively for Gallic acid and D. edulis extract.

Figure 6. Percentages of DPPH radical scavenging at different concentrations of each antioxidant, with AG = Gallic acid and ST = extract from D. edulis.

Figure 7 below shows the variation curve of the iron III (Fe³⁺) radical scavenging percentages as a function of the concentrations of D. edulis extract and Gallic acid.

Figure 7. Percentage reduction of Iron III ions (Fe³⁺⁾ radicals in the presence of the extract and gallic acid, with AG = gallic acid and ST = extract of D. edulis.

It appears from Figure 7 above that when the concentrations of D. edulis extract increases, the percentage inhibition effect increases exponentially until a point where an increase in their concentration results to no change in percentage inhibition. Thus, D. edulis extract significantly affects the percentage reduction of Fe³⁺ in its antioxidant properties. From these different curves, the regression equations were established, which made it possible to determine the (IC₅₀ It is (depicts) half the maximum inhibitory concentration (IC₅₀). It is the most widely used and informative measure of a drug’s efficacy. Thus, it indicates how much drug is needed to inhibit a biological process by half (Provides measure of potency). IC₅₀ of 29.97 ± 0.05 and 25.03 ± 0.71 µg/ml respectively for Gallic acid and D. edulis extract.