Chemicals

All solvents and reagents used were of analytical grade. COX (ovine) Colorimetric Inhibitor Screening Assay Kit Item No. 760111 was from Cayman Chemical Co.,MI, (USA),  Lipid Rich Bovine Serum Albumin  from  AlbuMax  II,  (New  Zealand);  Trichloroacetic acid from PROLABO, (France) and Potassium ferricyanide K₃Fe (CN)₆; Hydrogen peroxide; Iron chloride from PROLABO, (Belgium).

Plants materials and extraction

The roots of Cochlospermum tinctorium Perrier ex A. Rich., Ximenia americana L., the leaves of Saba senegalensis (A.DG.) Pichon, of Terminalia macroptera Guill & Perr, and Vitellaria paradoxa C.F.Gaertn were harvested. These specimens have been certified by the Department of Traditional Medicine (DMT) of the National Institute of Public Health of Mali and the herbarium of each plant is kept with reference number 0048, 0027, 3005, 2468 and 2792, respectively. On each harvested plant organ, two extraction methods, aqueous decoction and 70% hydroethanolic maceration were used as described previously (Ballo et al., 2022).

In vitro anti-inflammatory activity

Cyclooxygenase (COX-1 and COX-2) inhibition test

COX-1 (ovine) and COX-2 (recombinant ovine) extract inhibition test was performed using COX (ovine) Colorimetric Inhibitor Screening Assay Kit item No. 760111, that measures cyclooxygenase peroxidase activity by monitoring the appearance of oxidized N,N,N',N'-tetra-methyl-p-phenylenediamine (TMPD) at 590 nm as described previously (Ilic et al., 2014). These tests were performed according to the manufacturer's instructions. Briefly, it consisted of the addition of assay buffer (150 µl), hemin (10 µl) and enzyme (10 µl) (either COX-1 or COX-2); and 10 µl of extract, reference drug into the inhibitor wells.10 µl of ethanol (used as diluent for the extracts) were used for the 100% initial activity and background wells, which contained only test buffer (150 µl) and hemin (10 µl). The plate was incubated for five minutes at 25°C. 20 µl of the colorimetric substrate solution were added, followed by 20 µl of arachidonic acid to all wells. The plate was then incubated for another two minutes at 25°C and the absorbance was read at 590 nm. Test products, extracts, diclofenac and celecoxib were used so that their final well concentrations ranged from 0.45 to 113.64 μg/ml. The test was carried out in duplicate. The inhibitory capacity was determined according to the following formula:

% Inhibition = [(AEA - AIA) / AEA] × 100.

AEA: Activity enzyme test absorbance - background wells absorbance; AIA: Activity inhibition test Absorbance - background wells absorbance. The results were expressed in concentrations that inhibit 50% (IC₅₀) of the enzyme activity.

Inhibition of protein denaturation

In vitro anti-inflammatory activity of crude extracts, expressed as protein denaturation was performed according to previously used methods (Deshpande et al., 2009; Ghosh et al., 2015; Rahman et al., 2015). The test solutions consisted of mixtures of different concentrations (100, 250 and 500 μg/ml) of extracts (50 µl) or standard drug sodium diclofenac (50 µl) with 0.5% w/v bovine albumin serum (BSA) (450 µl).  A mixture of distilled water (450 µl) and different extracts (50 µl) with various concentrations (100, 250 and 500 μg/ml) served as product control solution. BSA 0.5% w/v (450 µl) aqueous solution and distilled water (50 µl) was used as a control solution. The preparations were incubated at 37°C for 20 min, followed by a temperature increase to 57°C for 3 min. After cooling, 2.5 ml of saline buffer phosphate solution (pH 6.3) was added  to  all  solutions. The  absorbance  was  read  by  UV-visible spectrophotometer at 660 nm. The control represented 100% protein denaturation. The percentage inhibition of protein denaturation was calculated using the following formula:

Percentage inhibition = 100 - [{(optical density of test solutions - optical density of product control) / optical density of test control} × 100].

In vitro antioxidant activity

Ferric reducing antioxidant power

The ferric reducing antioxidant power was assessed according to an experimental protocol previously used by the authors (Abu-Serie et al., 2018; El-Haci and Bekkara, 2016). Briefly, 500 µl of extracts at different concentrations (0.1 to 200 µg/ml) is mixed with 1.25 ml of 0.2 M phosphate buffer solution (pH 6.6) and 1.25 ml of a 1% potassium ferricyanide (K₃Fe (CN) ₆) solution. The preparation was incubated at 50°C for 20 min, and cooled down to room temperature. 1.25 ml 10% trichloroacetic acid was then added. Afterward, the tubes were centrifuged at 3000 rpm for 10 min. Finally, 2 ml of the supernatant were added to 2 ml of distilled water and 400 μl of a 0.1% freshly prepared iron chloride (FeCl₃) solution were added to the mixture. The absorbance was read against a blank at 700 nm. Ascorbic acid was used as a positive control and was treated under the same experimental conditions as the extracts. An increase in absorbance indicates greater reducing power. The results were expressed as IC₅₀ values which translate into the concentration of antioxidant used to obtain an absorbance of 0.5.

Hydrogen peroxide scavenging activity

The hydrogen peroxide (H₂O₂) scavenging activity of the extract was determined according to previously described methods (Bhatti et al., 2015; Nabavi et al., 2008). Briefly, 2 ml of extract at concentrations ranging from 0.1 to 100 μg/ml was mixed with 0.6 ml of 40 mM H₂0₂ solution. Ascorbic acid was used as positive control.

Statistical analysis

Results are expressed as mean ± SD. Multiple groups were compared using one-way ANOVA test followed by the Tukey test. Differences were considered statistically significant when p <0.05 (*), very significant when p <0.00l (**) and highly significant when p <0.000l (***). 

In vitro anti-inflammatory activity

Using different biochemical in vitro methods, we investigated the anti-inflammatory properties of the selected plant extracts.

Cyclooxygenase (COX-1 and COX-2) inhibitory activity

Prostaglandins   are   inflammatory   mediators  produced from arachidonic acid by the action of cyclooxygenase enzymes. The constitutive form (COX-1) is expressed throughout the body and performs a number of homeostatic functions such as maintaining normal gastric mucosa and influencing renal blood flow and aggregation (Parente and Perretti, 2003). In contrast, the inducible form (COX-2) is expressed in response to inflammatory and physiological stimuli, and is involved in the production of prostaglandins that mediate pain and support the inflammatory process (Lamon et al., 2010). The inhibitory effect of the selected plant extracts on constitutive COX-1, along with their inhibitory effects on the inducible COX-2 was examined. The results of COX-1 and COX-2 inhibition were expressed as IC₅₀ values (Figures 1 and 2), which is very important to determine the relationship between concentration and effect of a product. The literature shows a insufficiency of data expressed in IC50 values. From these results, a number of interesting observations can be made (Elgorashi and McGaw, 2019). First, a general observation is that the inhibitory effect of hydroethanolic extracts was greater than that of aqueous extracts, and this difference could be observed both in the cases of COX-1 and COX-2 inhibition. Indeed, all IC₅₀ values of hydroethanolic extracts are significantly to highly significantly higher than those of aqueous extracts, both for COX-1 and COX-2 inhibitory effect (p<0.05 to p<0.0001). The only exception was observed in the case of extracts of C. tinctorium for which the IC₅₀ value of the hydroethanolic extract is not significantly different than the IC₅₀ value of the aqueous extract (Figure 1, p>0.05). Secondly, the investigated plant extracts appeared to be more active in inhibiting COX-2 activity than COX-1. Thus, except hydroethanolic extract of V. paradoxa, all hydroethanolic extracts exhibited an IC₅₀ value lower than 30 ug/ml (though those values remain ~4 times less active than the reference drug celecoxib) for COX-2 activity inhibition, the most active extract in that regard being hydroethanolic extract of X. americana. All extracts, except hydroethanolic extract of C. tinctorium, V. paradoxa and S. senegalensis had IC₅₀ values higher than 30 ug/ml on COX-1 activity. Subject to further studies this particular difference could be a point of interest in the context of selectivity research for existing anti-inflammatory drugs. Indeed, non-selective AINS are generally known to have important gastrointestinal and renal adverse effects, while studies on selective COX-1 inhibitors have shown that COX-1 inhibition is responsible for these adverse effects (Parente and Perretti, 2003). Comparatively, COX-2 inhibitors appear to have more selective therapeutic effects on pain, inflammation, arthritis (Gautam et al., 2010). In this regard, the extraction products of aqueous decoction of C. tinctorium, both extracts of X. americana and T. macroptera exhibit a striking profile, not only for their significant potential anti-inflammatory properties but also  and  more  importantly  because   of   the   apparent selectivity for COX-2 enzyme that they show. Moreover, comparative chemical analysis of the extracts, including between genera and species of plants, as well as between types of extracts, could give more information on the chemical aspect of the inhibitory action of the extracts. As is shown in Figures 1 and 2, striking differences in COX inhibition activity are apparent, not only between the plant species but also between the extracts for the same plant, at least for number of them. Thus, in the case of C. tinctorium, aqueous and hydroethanolic extracts appear to have the same  degree of COX-1 inhibition (p>0.05) while the hydroethanolic extracts showed 2.5 times more activity than the aqueous extracts on COX-2 inhibition (Figure 2). Studies have shown that the aqueous extract of Cochlospermum tinctorium contain high levels of polysaccharides, while the hydroethanolic fraction appears to be richer in polyphenols. A quiet opposite profile is apparent for X. americana and T. macroptera, to a lesser extend however (Ballo et al., 2021; Nergard et al., 2005).

Among the present investigated plant extracts, 3 extracts appeared to be of significant interest in terms of therapeutic perspective for inflammatory conditions. Nevertheless, subject to thorough investigation, these products represent interesting potential alternatives for anti-inflammatory/analgesic medicines with less adverse effects.

Inhibition of protein denaturation

The effect of the extracts on the inhibition of protein denaturation is shown in Table 1. All extracts at all dose levels (100, 200 and 500 µg/ml) showed significant inhibition of protein denaturation. At 100 µg/ml, the reference drug diclofenac (51.32 ± 1.22%) was more effective (p < 0.0001) compared to all extracts. At such concentration, aqueous extract of C. tinctorium (42.07 ± 1.16%) and hydroethanolic extract of X. americana (41.33 ± 0.96%) were the most effective extracts against protein denaturation, when compared to the other extracts, and their effect was similar to that of diclofenac (p>0.05). The hydroethanolic extract of X. americana was the most effective (66.24 ± 0.55%) at 200 µg/ml and had no significant difference with diclofenac (67.34 ± 0.93%), all other extracts were very less active. At the highest concentration (500 µg/ml), the hydroethanolic extracts of C. tinctorium, and X. americana showed the highest activity against protein denaturation with a percent inhibition of 86.61 ± 1.22 and 84.5 ± 0.56% respectively and their effect was similar to that of the reference drug diclofenac (83.57 ± 1.61; p<0.001). No significant difference was observed between these two extracts (p>0.05) (Table 1). Moreover, the results are confirmed by the inhibitory activity of X. americana (77.51 ± 2.25%) against protein denaturation demonstrated by other authors (Shettar et al., 2015). Moreover, a plant of the same family as Terminalia macroptera Guill. & Perr., Terminalia glaucescens Planch. ex Benth, its anti-inflammatory activity has been proved by inhibition of protein denaturation (Das et al., 2020). The pathophysiology of the autoimmune disease rheumatoid arthritis involves protein denaturation and thus the production of autoantigens in vivo. Denaturation probably leads to an alteration of electrostatic, hydrogen, hydrophobic and disulphide properties (Deshpande et al., 2009; Ghosh et al., 2015). BSA denaturation was strongly inhibited by hydroethanolic extract of C. tinctorium and X. americana suggesting that both extracts could prevent protein denaturation in rheumatoid arthritis and thus be a potential anti-arthritic agent. 

 Ferric reducing antioxidant power (FRAP)

In this study, the results show that the reductive power of extracts and vitamin C increased with concentration in a very strongly linear manner (Figure 3a and b). The most important effects were observed with vitamin C (R² = 0.997; IC₅₀ = 8.42 µg/ml), hydroethanolic extracts of X. americana (R² = 0.996; IC₅₀ = 29 µg/ml), T. macropteria (R² = 0.990; IC₅₀ = 35.46 µg/ml). In addition, the hydroethanolic extract of X. americana was more than twice higher than hydroethanolic extracts and three times higher than aqueous extracts of C. tinctorium and V. paradoxa (Table 2). Previous studies confirm the results. Thus, a higher activity of the aqueous extract of Terminalia macroptera and X. americana have been reported (Shettar et al., 2015; Sobeh et al., 2017; Sombie et al., 2018). In this study, the hydroethanolic extract (IC₅₀ = 35.61 µg/ml) S. senegalensis showed good antioxidant activity and the aqueous extract (IC₅₀ = 69.57 µg/ml) had moderate activity. Belemlilga et al. (2019)obtained a contrary result in their study, but the DCM fraction of the hydroethanolic extract presented the highest reducing power with a concentration of 88.88 mmol TE/g. Studies have shown that the polyphenolic compounds in the extracts may be good electron and hydrogen atom donors, and thus able to stop radical chain reactions by converting free radicals into more stable products (Amarowicz et al., 2004; Shettar et al., 2015).

Hydrogen peroxide scavenging activity

Hydrogen peroxide is naturally produced in living organisms, it is not very reactive, but it causes cytotoxicity by the synthesis of hydroxyl radicals in the cell system. Therefore, H₂O₂ scavenging can contribute to the preservation of human health (Upadhyaya et al., 2007;  Jayakumar  et  al.,  2016).  The  H₂O₂  scavenging capacity of the extracts is presented in Table 2. The IC₅₀ value of H₂O₂ scavenging activity for extracts ranged from 39.31 ± 3.6 to 77.37 ± 2.16 µg/ml. However, the H₂O₂ scavenging effect of the best extracts and the standard decreased in the following order: vitamin C > aqueous extract of C. tinctorium > aqueous extract of T. macropteria > aqueous extract of X. americana (Table 2).  Kiessoun et al. (2018) in his study on the polyphenol rich fraction of Ximenia americana roots had a higher IC50 = 130 µg/ml compared to our results.  

The study clearly demonstrates that hydroethanolic extracts of X. americana, T. macroptera and C. tinctorium are potent inhibitors of cyclooxygenase-2 with low IC50 values. At the concentration of 500 µg/ml, hydroethanolic extracts of C. tinctorium and X. americana inhibited BSA denaturation more than 80%. They also have high antioxidant activity. Our data suggest that these extracts have significant anti-inflammatory activity. COX-1 and COX-2 inhibition, protein denaturation and antioxidant activity were performed to provide scientific data for in in vivo studies.

The authors have not declared any conflict of interests.