Introduction
Inflammation is a non-specific adaptive response triggered by harmful stimuli, due to endogenous or exogenous tissue attack, the purpose of which is the recovery of tissue damage.1, 2 It contributes to all pathological processes including infectious and non-infectious diseases.3 The mechanism of injury due to inflammation is partly attributed to the release of reactive oxygen species by activated neutrophils and macrophages.4, 5 Inflammation is mainly mediated by secretory phospholipase A2 (sPLA2), which plays a key role in releasing free arachidonic acid, which is the precursor of pro-inflammatory lipid mediator and ROS production. Cyclooxygenase-1/2 and lipoxygenase catalyze the synthesis of pro-inflammatory mediators such as prostaglandins, thromboxanes and leukotrienes. Moreover, the arachidonic acid cascade is considered as an important source of ROS such as O2 - and OH. The generation of free radicals, if it persists, leads to a more deleterious complication. ROS play an essential role in inflammation, which itself is a component and or manifestation of infectious and chronic diseases.6, 7 In Mali, ethnopharmacological surveys have identified that the species of Ximenia americana L., Strychnos spinosa Lam, Cola cordifolia (Cav.) R.Br., Vitellaria paradoxa C.F.Gaertn. Saba senegalensis (A. DG.) Pichon, Cochlospermum tinctorium Perrier ex A. Rich, Terminalia macroptera (Guill. & Perr.) and Leptadenia hastata Vatke are indicated in the traditional treatment of diseases with inflammatory component such as, infections, arthritis, pain, tuberculosis, hepatitis, leprosy.8, 9, 10 The mentioned plants have high levels of total polyphenols, flavonoids, and hydrolyzable and condensed tannins.11 They have already been the subject of several pharmacological studies, including antimycobacterial, hepatocurative, immunomodulatory, anti-inflammatory and antiradical properties.9, 10, 11, 12 Therefore, the evaluation of the pharmacological properties of these plants commonly used in Malian traditional medicine has attracted considerable interest due to their therapeutic effects. The study of the mechanisms by which plant extracts demonstrate their anti-inflammatory and antioxidant activities in biological systems is complex.13 Models for the preliminary evaluation of anti-inflammatory and antioxidant activities use in vitro screening assays, inhibitors of secretory phospholipase A2, 15-lipoxygenase for inhibitory activity of pro-inflammatory enzymes, and the free radical scavenging test for scavenging activity Chemical constituents that can inhibit inflammation may also serve as appropriate agents to inhibit lipid oxidation.14, 15 In this study, the objective was to evaluate the antiradical activity of the extracts and their ability to inhibit phospholipase A2 and 15-lipoxygenase. xczc.
Material and Methods
Plant collection and authentification
The leaves of Leptadenia hastata Vatke and the roots of Cochlospermum tinctorium Perrier ex A. Rich., Ximenia americana L. and Strychnos spinosa Lam. were harvested in Kati. The leaves of Cola cordifolia (Cav.) R.Br., of Saba senegalensis (A.DG.) Pichon, of Terminalia macroptera Guill.& Perr. and Vitellaria paradoxa C.F.Gaertn. were harvested in Samé. The samples were authenticated at the Department of Traditional Medicine (DMT) of the National Institute for Public Health Research in Mali and a herbarium of each plant was deposited with reference numbers 1481; 0048; 0027; 0150; 3024; 3005; 2468; 2792 respectively.
Chemicals
All solvents and reagents used were of analytical grade. sPLA2 (Type V) Inhibitor Screening Assay Kit Item No. 10004883 and Lipoxygenase Inhibitor Screening Assay Kit Item No. 760700 from Cayman Chemical Co. (MI, USA).
Preparation of aqueous and hydroethanolic extracts
After drying, the plant parts were pulverized. On each powder, aqueous decoction and 70% hydroethanolic maceration methods were used as previously described.11
ABTS (2, 2’-azinobis- [3-ethylbenzothiazoline-6-sulfonic acid] Radical Scavenging
The capacity of extracts to scavenge the ABTS radical cation was determined according to the procedure described by Boualam K.16 A stock solution of ABTS (7 mM) was mixed with 2.45 mM of potassium persulfate (K2S2O8). The mixture was incubated for 12-16 hours in the dark. The ABTS·+ solution (7 mM) was diluted with distilled water to achieve an absorbance of 0.7 ± 0.02 at 734 nm. 950 µL of this solution was added to 300 μL of extract solution at different concentrations (0.1-100 μg/mL). After incubation for 7 min at room temperature, the absorbance was measured at 734 nm. The blank was prepared by replacing the extract solution with methanol. The test was performed in triplicate and the percentage inhibition was determined by the following equation:
% Inhibition = (Ab – As) /Ab ×100
Ab: Absorbance of blank; AS: Absorbance of sample/reference compound.
IC50 was estimated using graph of free radical scavenging against the extract and standard concentrations.
Enzyme inhibitory activity
Phospholipase A2 inhibition assay
Type V sPLA2 (phospholipase A2) has been shown to be involved in eicosanoids formation in inflammatory cells, such as macrophages and mast cells.17 Cayman’s sPLA2 Inhibitor Assay allows the Screening Type V sPLA2 inhibitors. Thus, the assay of this enzyme was performed using the method described by Cayman Chemical Co. (MI, USA) in catalog No. 10004883. For this purpose, the extracts dissolved in ethanol were used so that their final concentrations in the wells ranged from 3.03 to 151.52 μg/mL. The assay utilizes the 1.2-dithio analog of diheptanoyl phosphatidylcholine as a substrate. Upon hydrolysis by sPLA2, free thiols are detected using DTNB (5,5 dithio bis-(2-nitrobenzoic acid). Briefly, add 10 µL of sPLA2 to all wells except the background wells and 10 µL of ethanol or Thioetheramide-PC or extracts to the 100% initial activity wells, positive control inhibitor wells and inhibitor wells respectively. Then dispense 200 µL of Diheptanoyl Thio-PC solution followed by 10 µL of DTNB into all wells. The plate was carefully shaken for 10 seconds and then incubated for 15 minutes at 25°C. The test was performed in duplicate and 96-well microplates were used. The absorbance was read at the wavelength of 405 nm. The percentage inhibition of sPLA2 was calculated by the formula:
% Inhibition = [(AEA - AIA) / AEA] X 100
15-Lipoxygenase inhibition assay
The lipoxygenase inhibition test was performed using the Lipoxygenase Inhibitor Screening Assay (LISA) kit, Cayman Chemical Co. (MI, USA) in catalog No.760700 according to a previously reported method.13 A solution of 15-lipoxygenase (90 μL), extract (10 μL) and assay buffer (100 μL) were placed in the test wells and incubated for five minutes at 25°C. The reaction was initiated by adding arachidonic acid solution (10 μL) as substrate to each test well. All test wells were covered and placed on a shaker for 10 minutes. Chromogen (100 μL) was added to each well to stop enzymatic catalysis and prevent further development of the reaction and placed the plate on a shaker for five minutes. The levels of hydroperoxides (HP) produced were measured at 490 nm absorbance. Concentrations ranging from 4.76 to 238.09 μg/mL of each extait as well as nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor standard, were tested for lipoxygenase inhibitory activity. The whole test was performed in duplicate. The inhibitory capacity was determined according to the following formula:% Inhibition = [(AEA - AIA) / AEA] X 100AEA : Activity enzyme test absorbance - background wells absorbance.AIA : Activity inhibition test Absorbance - background wells absorbance.
IC50 value (concentration at which there was 50% inhibition) was deduced from graph representing the percentage inhibition of the enzyme activity.
Statistical analysis and expression of results
The results were expressed as mean ± SD. The statistical analysis was carried out using One-way ANOVA followed by the Tukey test about Graph Pad Prism® version 5.03. Differences were considered statistically significant, very significant and highly significant when p <0.05 (*), <0.00l (**), <0.000l (***) respectively.
Table 1
Effects (IC50 ± S.D. μg/mL) of the aqueous and hydroethanolic extracts on scavenging of ABTS radical.
Table 2
Inhibitory effects (IC50 ± S.D. μg/mL) of aqueous and hydroethanolic extracts on sPLA2.
Table 3
Inhibitory effects (IC50 ± S.D. μg/mL) of aqueous and hydroethanolic extracts on 15-LOX.
Results and Discussion
A total of 16 extracts of 8 plants, 8 obtained by hydroethanolic maceration and 8 by aqueous decoction were screened for ABTS antiradical activity, type V sPLA2 inhibition and 15-lipoxigenase inhibition.
ABTS Radical Scavenging activity
On each sample of the 8 plants in the study, two different extraction methods were performed. Eight (8) extract were obtained by hydroethanolic maceration and eight (8) by aqueous decoction. One of the most commonly used antioxidant methods is ABTS·+. It is characterized by excellent reproducibility under certain assay conditions. The ABTS+ radical must be generated by enzymes or chemical reactions. ABTS+ can be dissolved in aqueous and organic media, in which the antioxidant activity can be measured, due to the hydrophilic and lipophilic nature of the compounds in the samples.18 Other authors also report that antioxidant substances can prevent and delay lipid oxidation by acting on the reduction of ROS and the production of other free radicals.19 The antiradical activity of the extracts varied between 7.29 ± 0.61 and 43.31 ± 0.25 µg/mL and 2.69 ± 0.25 µg/mL for gallic acid. Recurrently, the hydroethanolic extracts gave the best antiradical activities. The hydroethanol extract of T. macroptera revealed the strongest reducing power with an IC50= 7.29 ± 0.61 µg/mL. Compared to the other extracts, no significant difference was observed with the hydroethanolic extract of C. tinctorium (IC50= 7.93 ± 0.48 µg/mL), X. americana (IC50= 8.14 ± 0.39 µg/mL) and with the aqueous extract of T. macroptera (IC50= 9.23 ± 0.77 µg/mL). A significant difference (p < 0.0001) was noted with both extracts of L. hastata, C. cordifolia, S. spinosa and S. senegalensis (Table 1). Previously, similar results showed, strong antiradical activity with T. macroptera, X. americana, C. tinctorium and S. senegalensis were reported.20, 21, 22, 23
Phospholipase A2 inhibition
This study demonstrated an inhibitory effect of these Malian plants on sPLA2, 15-LOX. The inhibitors of these enzymes could become key compounds in the development of new anti-inflammatory drugs. Table 2 shows the inhibitory effect of hydroethanolic and aqueous extracts on phospholipase A2. The effect on phospholipase A2 enzymatic activity of the hydroethanolic extracts was greater than that of the aqueous extracts. Molander et al.15 and Ouedraogo et al.24 found similar results with higher percent inhibition for hydroethanolic extracts. Hydroethanolic extracts of C. tinctorium, T. macroptera, X. americana and Thioetheramide-PC (reference compound) inhibited with inhibitory concentration 50 (IC50) of 21.43 ± 0.66; 23.72 ± 0.71; 24.49 ± 0.39 and 15.23 ± 0.4 µg/mL respectively. These results confirm the inhibitory capacity of these plants on sPLA2 demonstrated by Molander et al.15 and Bernard et al.25 Previous studies have reported high levels of polyphenols and flavonoids in these plants. These phytoconstituents are known for their anti-inflammatory properties.11, 26, 27. The hydroethanolic extracts of C. tinctorium showed significant inhibitory activity (p < 0.0001) compared to the aqueous extract of X. americana and both extracts of V. paradoxa and S. senegalensis11, 28, 26 The hydroethanolic extracts of C. tinctorium showed significant inhibitory activity (p < 0.0001) compared to the aqueous extract of X. americana and both extracts of V. paradoxa and S. senegalensis (Table 2). The non-activity of the extracts of L. hastata and S. spinosa was observed.
15-lipoxigenase inhibition
The best inhibitory activity on 15-lipoxigenase was obtained with aqueous and hydroethanolic extracts of X. americana with IC50 of 18.35 ± 1.26; 18.47 ± 1.12 µg/mL respectively. Nordihydroguaiaretic acid (reference compound) had an IC50 of 1.86 µg/mL. No significant difference was observed with the aqueous and hydroethanolic extracts of T. macroptera with IC50 of 19.45 ± 0.25; 19.3 ± 0.50 µg/mL respectively. Previous studies demonstrated strong inhibition of 15-LOX (IC50= 23.2 ± 0.5 µg/mL) by leaves of T. macroptera.29 A significant difference (p < 0.0001) was observed when to compared the extracts of X. americana to with the both extracts of V. paradoxa and S. senegalensis and hydroethanolic extracts of S. spinosa and C. tinctorium. However, the two extracts of L. hastata and the aqueous extracts of C. cordifolia, S. spinosa and C. tinctorium were not active on 15-lipoxigenase (Table 3). Isa et al.27 showed the inhibitory activity of Leptadenia hastata extracts different from our extracts on 5- lipoxygenase. This proves that the activity depends on the compounds dissolved in the extract. Previous studies have reported anti-inflammatory activity of these plants in vivo and/or in vitro.22, 29, 30, 31, 32 The non-inhibitory activity of plants on sPLA2 and 15-LOX may be explained by traditional uses that may include some inappropriate applications or, the presence of several biological targets could also be responsible for the anti-inflammatory properties of these plants.25
Conclusion
The present study showed that extracts of leaves of T. macroptera and roots of X. americana have antioxidant activity and effectively inhibit sPLA2, and 15-LOX pro-inflammatory enzymes in vitro. While in vivo studies are required, T. macroptera and X. americana may be a potential resource of sPLA2 and 15-LO that may be useful for the prevention or treatment of inflammation, and our results may therefore justify its use against diseases with an inflammatory component by traditional healers in Mali.
Statement of Ethical Approval
Our study protocol was approved by the Ethical Committee at the University of Sciences, Techniques and Technologie of Bamako with a protocol approuval # Reg. No.2021/234/USTTB.
Acknowledgment
The authors acknowledge the teams of the Laboratory of Drug Development, Joseph Ki-Zerbo University, Burkina Faso and immunogenetic laboratory of Malaria Research and Training Center, University of Sciences, Techniques and Technologies of Bamako, Mali and the department of traditional medicine, Bamako, Mali for supporting this study.
