This was an experimental study that took place in the following laboratories; the chemistry laboratory of natural substances in the Faculty of Medicine and Pharmaceutical sciences of the University of Douala (FMPS): for the assistance in concentration of the plant extract: the pharmacy technology laboratory of the University Institute of the Gulf of Guinea (IUG); for the phytochemical screening and anti-inflammatory activity.

2.2.1. Material for Phytochemical Screening

Test tubes for holding solutions; Plant extracts; Reactive (methanol, acetic anhydride, acetone, wagner reagents, iron (III) chloride 2% and 0.1%, magnesium fillings, copper (II) solution, sulphuric acid and hydrochloric acid at 25%), dragendorff reagent, chloroform, naphthol, distilled water.

2.2.2. Material for Anti-Inflammatory Activity

Glass tubes; Albumin (egg white); Phosphate Buffer Solution (Psb); Distilled water; Aspirin tablet; Micropipette; UV-vis Spectrophotometer; Round bottom flask (100 mL).

2.2.3. Material for Suppository Formulation

Suppository moulds; Excipient e.g. (cocoa butter); Spatula; Electronic balance; Heating mantle/water bath; beaker; Round bottom flask; Sheet of paper; Refrigerator; Aluminum foil.

The preparation of the plant extracts commenced from the harvesting procedure onto obtaining the methanol extracts of the separate samples.

3.1.1. Harvesting and Treatment of Voucher Specimen

The voucher specimens of Ageratum conyzoides and Cymbopogon citratus were harvested in the (Camrail camp of “cite de la paix”) in the month of November 2020 at 10:20 a.m. Plant stems were then immediately separated from the leaves and other parts after harvesting to optimize the drying process. Thereafter standard procedures for pre-treatment of plant materials were followed. The basic steps include cleaning, air drying under shade at room temperature, grinding into fibers and storage in an airtight container at appropriate temperature (room or refrigerated) [7].

3.1.2. Extraction

After preparing the plant material and transforming it into fibres using a craft mill, leaves of Ageratum conyziodesandCymbopongon citratuswere blended and mixed with 2.5 L of methanol each, for a period of 72 hours at room temperature following an extraction process called maceration which is suitable for preserving thermo-labile compounds. Occasional stirring was done twice a day within the 72 hours to facilitate extraction by increasing diffusion, and removing concentrated solution from the sample surface while bringing new solvent to the menstruum for more extraction yield [8]. The extracts were then filtered using a 0.45 millipore whatmann filter paper associated with cotton wool. Then after, the extracts were concentrated using a rotary evaporator at 60˚C and 102 rotations per minute.

Phytochemical examinations of the stem extracts were carried out applying the standard methods. Hence, the presence or absence of various phyto-constituents was determined [9].

Detection of alkaloids: Wagner’s Test: Filtrates were treated with Wagner’s reagent (Iodine in Potassium Iodide). Formation of brown/reddish precipitate indicated the presence of alkaloids.Detection of phenols: Ferric Chloride Test: Extracts were treated with 3 - 4 drops of ferric chloride solution. Formation of bluish black colour indicated the presence of phenols.Detection of tannins: Ferric Chloride Test: To the alcoholic extract a few drops of 1% neutral ferric chloride solution was added, formation of blue, green or brownish green color indicated the presence of Tannins.Detection of flavonoids:Shinoda Test: To the alcoholic solution of alcoholic extract, a few fragments of magnesium ribbon and concentrated hydrochloric acid were added. The appearance of magenta colour after a few minutes indicates the presence of flavonoids.Test for saponins: Foam Test: Small amount of alcoholic extract was shaken with little quantity of water, if the foam produced persisted for 10 minutes; it indicated the presence of saponins [10].Detection of terpenoids: Salkowski test: The extract was mixed with 2 ml of chloroform and concentrated H₂SO₄ (3 ml) is carefully added to form a layer. A reddish brown coloration of the interface is formed to show positive results of the presence of terpenoids [11].Detection of sugars: Molisch test: Added 2 - 3 drops of alpha-naphthol reagent was added to 2 ml of extract in the test tube. The test tube was inclined very gently and a few drops of concentrated sulphuric acid along the side of the test tube was added. A violet colour ring indicated the presence of carbohydrates in the solution.

Laboratory assays to assess the ability of extracts to inhibit inflammatory processes were carried out using the protein denaturation method from egg white.

3.3.1. Inhibition of Albumin Denaturation Assay

The anti-inflammatory activity of the combined extract was studied by using inhibition of albumin denaturation technique done according to the method described by Mizushima et al., with some few modifications at the level of concentration of the solutions [12].

PREPARATION OF COMBINED EXTRACT (AGERATUM CORNIZOIDES PLUS CYMBOPOGON CITRATUS) AND ASPIRIN SOLUTION

Extracts of Ageratum cornizoides,Cymbopogon citratus and aspirin were weighed to get the same mass of 0.0096 g; with Ageratum cornyzoides and Cymbopogon citratus constituting each half of the desired weight for the mass of the extract. The mass of extract was dissolved in few drops of dimethylsufoxide (DMSO) to have a uniform distribution; equal masses of dissolved extracts and aspirin was diluted in 12 mL of distilled water to have the highest concentration of 800 µg/mL; a serial dilution was made to obtain solutions of decreasing concentration.

PROCEDURE OF ANTI-INFLAMMATORY ACTIVITY

A reaction mixture (5 mL) consisting of 0.2 mL of egg albumin (from fresh hen’s egg); 2.8 mL of PBS (pH 6.4); 2 mL of varying concentrations of the combined extracts (50, 100, 200, 400 and 800 µg/mL) was incubated at 37˚C in a biochemical oxygen demand for 15 minutes and then heated at 70˚C for 5 minutes. A similar volume (2 mL) of distilled water served as control; after cooling, their absorbance was measured at 660 nm using the vehicle as blank; aspirin was used as a reference drug and treated similarly for determination of absorbance [13]. The percentage Inhibition of denatured proteins was calculated using the formula:

Denaturation % inhibition = (Vt/Vc − 1) × 100% (2)

where:

Vt: The sample absorbance value;

Vc: The distilled water used as negative control.

The IC₅₀ value was determined from the dose response curve by drawing the percentage inhibition values against the varying concentrations.

3.3.2. Formulation of Suppository

Before formulating suppositories with the dried extracts, we proceeded with just the excipients in order to calculate the displacement factor of the dried extracts that served as principal active substance. After this, size separation was performed for size uniformity assurance of the dried extracts, then we progressively added 1/3 of the excipient and then triturated to obtain a homogeneous mixture. The remaining excipient was placed in a beaker then heated using a heat source at a temperature not less than 45˚C. After the fusion of the excipient, we then added the content into the mortar and the mass was gently stirred (constantly) to homogenise the mixture before filling the wells of the molds [14].

Data showing the amount of extract obtained relative to the initial plant material weight.

Amount of water lost for each plant during drying.

Ageratum conyzoide:

Fresh Ageratum mass = 300 g; Dried Ageratum mass = 60 g; Blended dried Ageratum mass = 56 g; Dried methanolic extract of Ageratum yield mass = 29.75 g; Amount of water lost in Ageratum = (fresh Ageratum mass – dried Ageratum mass) 300 g − 60 g = 240 g.

Cymbopongo citratus:

Fresh Cymbopogon mass = 655 g; Dried Cymbopogon mass = 170 g; Blended dried Cymbopogon mass = 174 g; Dried methanolic extract of Cymbopogon yield mass = 15.45 g; Amount of water lost in Cymbopogon = (fresh Cymbopogon mass – dried Cymbopogon mass) 655 g – 170 g = 475 g;

Ageratumconyzoide: 60 g 300 g × 100 = 20 %

Cymbopongo citratus: 170 g 655 g × 100 = 25.95 %

Percentage yield was calculated as from formula 1.

Ageratumconyzoide: 10.71 g 56 g × 100 = 19.13 %

Cymbopogon citratus: 15.45 g 174 g × 100 = 8.83 %

Findings indicating which phytochemicals are present in the extracts, supporting their medicinal potential are as follows (see Table 1).

Table 1. Results obtained from the phytochemical screening of both plants done separately.

+ = positive; – = negative; +/– =colouration expected was not attained but a colour change was observed.

Data demonstrating the extracts’ ability to reduce inflammation in vitro, compared to controls are presented in Table 2 and Figures 1-3 below.

Table 2. Tabulated representation of the percentage inhibition of the control sample (Aspirin) alongside the inhibitory concentrations of the combined extracts with each concentration respected as shown on the table.

The curves in Figure 1 and Figure 2Figure 2 show the inhibition of denaturated heat-induced proteins by aspirin and combined extract of Cymbopogon citratus and Ageratum cornyzoides respectively. The comparison curve in Figure 3Figure 3 reveals that the efficiency of the combined extract increases significantly with an increase in concentration but was less efficient and potent than Aspirin. This is also confirmed by the IC₅₀ value of aspirin (IC₅₀ 350.1 g/ml) which is greater than that of the combined extract (IC₅₀ 292.7 g/ml).

Figure 1. Normalized percentage inhibitions of absorbance of Aspirin with variation in logarithmic concentrations.

Figure 2. Normalized percentage inhibitions of absorbance of combined extracts with variation in logarithmic concentrations.

Figure 3. Comparative percentage inhibitions of absorbance of combined extracts and Aspirin with variations of concentrations.

The low efficiency and potency of the combined extract is probably due to steric hindrance which prevents the pharmacophore group from acting [15]. The dose-dependent efficiency could be due to polyphenols [16]. Studies also done on terpenoids have been reported to have anti-inflammatory properties [17]. Thus the anti-inflammatory properties could either be due to the presence of polyphenols and terpenoids.

According to literature, one of the characteristics of several Non-Steroidal Anti-Inflammatory Drugs NSAIDs is their ability to prevent denaturation of heat-treated albumin at physiological pH [18]. These results can also be supported with those of Mizushima et al. and Sakat et al. which reported valuable anti-inflammatory activity of Enicostemma axillare using inhibition of albumin denaturation technique [19].

Outcomes related to the successful preparation of the plant-based suppositories are presented in the following images for each extract obtained (see Figure 4Figures 4-9).

Figure 4. Upper view of frozen suppository of plant extracts of Ageratum conzoides and Cymbopogon citratus.

Figure 5. Lower view of frozen suppository of plant extracts of Ageratum conzoides and Cymbopogon citratus.

Figure 6. Foiled frozen suppository of Cymbopogon citratus extract.

Figure 7. Frozen suppository of Cymbopogon citratus extract.

Figure 8. Foiled frozen suppository of Ageratum conyzoides.

Figure 9. Frozen suppository of Ageratum conyzoides.