ino-5-chloro-7-phenylpyrido [3,4?d]pyridazine-1,4(2H,3H)dione) was purchased from Wako Chemicals Gmbh (Neuss, Germany).
Horseradish peroxidase (HRP) was obtained from Roche (Mannheim, Germany) and human Myeloperoxidase was from Calbiochem, EMD Millipore (Bellirica, MA USA). Caffeic acid, Chlorogenic acid (purity: 95%), Gallic acid (purity: 97%) was purchased from Sigma-Aldrich. Rutin (purity ≥ 99%), isoquercitrin (purity ≥ 99%) and, quercetin (purity ≥ 98.5%) were HPLC grade and purchased from Extrasynthese. Water was treated using a Milli-Q water ultra-purification system before use.
2.3. Microscopic Analysis
Powder observations were made using European Pharmacopeia reagent  . Observations were made with a Zeiss Primo Star 200Ò microscope and pictures were taken with Smart Phone Samsung E7.
2.4. Preparation of Extracts
Aqueous extracts were prepared by infusion of 10 g of sample powders with 150 mL of water for 10 minutes. The infusion was cooled to room temperature before filtration and the evaporation of the solvent was performed by lyophilisation (Apparatus: Christ Alpha 1 - 4 LSC®). The extracts were then weighed and kept in dark hermetic flasks at 4˚C.
2.5. Phytochemical Analysis
2.5.1. Thin Layer Chromatographic Analysis
Analytical TLC of 10 μL of solution for 10 mg×mL−1 of methanolic and dichloromethane extracts was carried out on normal phase Silica gel 60 F254 plates (Merck), using different eluents for the identification of secondary metabolites  .
2.5.2. Phenolics Contents
1) Total phenolics content
Total phenolic content of methanolic extracts (Methanol 80%) was determined according to the Folin-Ciocalteu method as described previously  . A calibration curve of gallic acid (0.025 - 0.4 mg×mL−1) was prepared, and phenolic contents were determined in triplicate from the linear regression equation of this curve. The results were expressed as milligrams of gallic acid (GA) equivalent per gram of dried matter.
2) Total flavonoids content
The flavonoid content of the extracts was determined by UV-Vis spectrophotometry. Results are expressed in mg equivalent of quercetin per g (mg QE/g) of dry vegetal material  .
3) Total anthocyanins content
The determination of the anthocyanin content of the extracts was performed by the procedure reported by Bahati et al. (2017)  .
2.6. Cell-Free, Cellular and Enzymatic Assays
The extracts and standard molecules were solubilized in water, thus their effect was compared to a control test performed with water alone.
2.6.1. Radical Scavenging Activity
ABTS and DPPH assay were performed according to the method described by Kapepula et al. (2017)  .
2.6.2. Cellular Antioxidant Activity
1) Cell culture and treatment
Human promyelocytic leukemia cells (HL-60) were obtained from the American Type Culture Collection (ATCC, USA) and cultured in appropriate medium (IMDM) obtained from Biowest (France)  .
2) Measurement of Cellular Antioxidant Activity (CAA)
The cellular antioxidant activity consists of evaluating the capacity of the extracts to modulate the production of ROS in cell models often used to study inflammatory response. The L012 probe was used to monitor the extracellular ROS production in HL-60 cells. The ROS produced by PMA-activated monocytes HL60 were measured by L012-enhanced chemiluminescence (CL). This experiment was based on this method: Two microliters of extract solutions at final concentrations of 0.5, 1, 5 and 10 μg mL−1, 5 μL of HRP (30 μg mL−1), 5 μL of CaCl2 (11 μg mL−1), 20 μL of L012 (0.25 μg mL−1) and as last 10 μL PMA (0.486 μM) were added to a volume of 158 μL cell suspension (250,000 cells). In each assay, three wells were loaded without plant extract and were taken as control for the ROS production (100% ROS induced chemiluminescence). To measure the basic ROS production of the cells at the absence of activation, three other wells without plant extract received no PMA (NA, not activated). The CL produced was measured for 30 min at 37˚C on a Fluoreskan (Fluoreskan Ascent FL, Fischer Scientific, Tournai, Belgium).
2.6.3. Inhibition of Myeloperoxidase Oxidant Activity
PBS solutions of the plant extracts to the final concentrations of 1, 10 and 20 μg mL−1 were incubated for 10 min with human MPO at a final concentration of 25 ng×mL−1. After incubation, 100 μL of the mixtures were loaded into the 96-well microtitration plates and incubated for 10 minutes at 37˚C in darkness. The MPO peroxidase activity (enzymatic activity) was detected by adding 100 μL of AmplexÒ Red solution (80 μM) solution dissolved in phosphate buffer (50 mM, at pH 7.5), H2O2 (10 μM) and 10 μL sodium nitrite (10 mM). The oxidation of AmplexÒ Red into the fluorescent adduct resorufin (λ excitation = 544 nm; λ emission = 590 nm) was monitored for 30 min at 37˚C with a fluorescent plate reader (Fluoroskan Ascent, Fisher Scientific). A control assay set as 100% MPO activity was performed with MPO in the presence of PBS instead of the plant extracts  .
2.7. Statistical Analysis
Each concentration was tested in triplicate in each assay, and at least 3 different assays were performed. All results were expressed as mean values ± standard deviation (SD). The statistical analysis was performed with GraphPad 7.0 (GraphPad Software, San Diego California, USA). Two-way analysis (ANOVA) was used; multiple comparisons of all data were performed using the “Tukey” Multiple Comparisons Test and the level of statistical significance was set at p < 0.05. The IC50 values were calculated with GraphPad Prism 7.0 under application of the function “log (inhibitor) vs. normalized response-variable slope” after converting the concentrations into their decimal logarithm.
3. Results and Discussion
3.1. Botanical Microscopic Features
Microscopical analysis of powders of leaves, flowers and seeds of C. occidentalis revealed the features below (Figures 2-4). Leaves were characterized by upper
Figure 2. Unicellular non glandular trichome (A), Glandular trichome (B), Paracytic stomata (C) and Scalariform vessels (D) from leaves of C. occidentalis at 40×.
Figure 3. Pollen grains (A), Inner epidermis (B), Fragment of fiber with unicellular non-glandular trichome (C), and Scalariform vessels (D) from flower of C. occidentalis at 40×.
Figure 4. Epicarp in surface view showing stomata (A), Endosperm layer in surface view (B), Pluricellular non glandular trichomes (C), Parenchymatous layers of the testa in surface view and non glandular trichomes (D) from seeds of C. occidentalis at 40×.
epidermis in surface view with part of the underlying palisade, cells of the spongy mesophyll containing cluster crystals of calcium oxalate, lower epidermis of the leaf in surface view showing paracytic stomata, parenchyma containing numerous calcium oxalate crystals, isolated calcium oxalate cluster crystals, covering and glandular trichomes, scalariform vessels, polyedric starch granules, lignified fibers. The diagnostic characters of flowers were: abundant covering and glandular trichomes, inner and outer epidermis of the corolla, spirally thickened vessels and associated parenchyma, abundant pollen grains. The diagnostic characters of the seeds were: pluricellular non glandular trichomes, cork, epicarp in surface view showing stomata, epidermis of the testa with underlying oil cells, part of the pericarp showing the innermost layer of the mesocarp, the endocarp, parenchymatous layers of the testa in surface view, epiderms and palisade of the cotyledons in sectional view, thicker-walled cells of the endosperm, pollen grain.
Microscopic analysis is one of the cheapest methods to correctly identify the raw materials from herbal medicines and is useful for the identification and authentification of botanicals and for detecting the adulterated and poor quality  . The knowledge of botanical microscopic details of different parts of C. occidentalis, is important for the evaluation of their quality such as raw materials for traditional medicines. Microscopy analysis allows the identification of herbal drugs and the detection of individual components of a mixture.
Phytochemical analysis revealed the presence of phenolic acids, flavonoids, iridoids, tannins and terpenes. TLC fingerprints of different parts were different and characteristic. They showed the presence of glycosylated flavonoids and phenolic acids as main compounds for flowers, leaves and seed pods. By comparison with used standards, they showed that flowers and leaves contain quercetin. Menthol is detected equally in all extracts from different parts (Figure 5).
Previous phytochemical screening revealed the presence of alkaloids, saponins, tannins, reducing sugar, phenols, anthraquinones, cardiotonic glycosides, resins, sterols in different parts of this plant. Leaves contained phlobatannins, chrysophanol, emodin, physcion, tetrahydroanthracene derivatives, germichrysone and occidentalins A and B    . Flowers contained anthraquinones, emodin, physcion and physcion-1-O-β-D-glucoside. The study of phytochemicals of C. occidentalis reveals that the nature and amount of phytochemicals vary according to climatic conditions. Stems, leaves and the root bark of the plant from Ivory Coast, Africa contained small amount of saponins, no alkaloids, sterols, triterpenes, quinines, tannins and flavonoids. However, a large amount of alkaloids were found in the stem, leaves and fruits of the species from Ethiopia  .
3.3. Antioxidant Activities
ABTS and DPPH assays showed that infusion extracts of the Cassia parts have the ability to scavenge free radicals connected with their IC50 values (Table 1).
Table 1. IC50 values (μg×mL−1) of infusion extracts of parts of C. occidentalis on ABTS and DPPH assays (Mean ± SD, n = 6) and their phenolic contents.
Figure 5: TLC chromatogram of dichloromethane extracts from C. occidentalis with oleanolic acid, menthol, thymol as standards; developed with toluene/ethyl acetate (93:7; v/v) and visualized at visible with sulfuric anisaldehyde reagent. Terpenes are detected as violet spots.
All extracts showed a weaker capacity to scavenge DPPH radical. DPPH assay only detect hydrophilic antioxidants in the contrary of ABTS assay which detect hydrophilic and lipophilic antioxidants. Phenolic compounds could be major contributors to antioxidant activity of infusions of Cassia parts. The total phenolic contents varied significantly (p < 0.05) between the parts of Cassia. Results showed that flowers was the part with the highest amount of total phenolic content. Leaves had the highest amount of flavonoids (Table 1). Previous studies reported the antioxidant activity of different parts of C. occidentalis. Singh et al. (2017) showed that methanolic extract from seeds of this plant had moderate DPPH radical scavenging activity  . Methanol extract from leaves was proved to be the most effective among the organic extracts for scavenging hydrogen peroxide radical  .
Beside conventional cell-free antioxidant assays, it can be pertinent to evaluate the antioxidant and anticatalytic potentials of plant extracts in cellular models such as those involved in ROS production and inflammatory responses. The addition of increasing concentrations of the infusion extracts from different parts of C. occidentalis (0.5, 1, 5 and 10 μg×mL−1), and quercetin (10−6 to 10−4 M) resulted in a dose-dependent decrease of HL-60 ROS production in comparison to the control test performed with water (Figure 6). With this cellular model involved in inflammation, our results showed that the activities of aqueous extracts are significantly higher (p < 0.05) in the following order: Leaves > Flowers > Seeds > Seed Pods.
These results suggest that the compounds of infusion extracts are good extracellular ROS scavengers. Antioxidants especially phenolic acids and flavonoids have ability to scavenge biological free radicals. The aqueous extracts used in local medicine preserved an antioxidant capacity. Interestingly, leaves and flowers exhibited a higher cellular antioxidant activity.
3.4. Inhibition of Peroxidasic Activity
Peroxidases generally use H2O2 as one of the substrates and participate in oxidizing drug and xenobiotic detoxification, innate immunity, hormone biosynthesis and the pathogenesis of inflammatory diseases   . In MPO direct technique, all infusion extracts of parts of Cassia exhibited a dose-dependent inhibitory effect on MPO activity in the range concentrations of 1 to 20 µg mL−1 (Figure 7). The leave and flowers extracts showed higher inhibition effect as compared to other plant parts. Seeds showed weak activity at tested concentrations and seedpods were practically not active. This inhibitory effect was significantly higher
Figure 6. Modulatory effects of quercetin and extracts from parts of Cassia occidentalis on chemiluminescence response (CL) by PMA stimulated HL60 monocytes (Means ± SD, n = 6). The CL intensity results from the reaction between L012 and the ROS produced by the non-activated (NA) and activated HL60 (A). The CL response of stimulated HL60 in the presence of water used to solubilize the extracts was defined as 100%. P-values (**p < 0.01) calculated by two-way ANOVA followed by Sidak Multiple Comparisons Test indicated a significant effect of the extracts vs. water control.
Figure 7. Effects of gallic acid and infusion extracts of parts of Cassia on MPO activity. The percentage of inhibition was calculated for each sample concentration versus the corresponding control (MPO + Water), taken as 100% (mean ± SD, n = 6). Samples vs. Control Water: ns: no significance. P-values (****p < 0.0001) calculated by two-way ANOVA indicated a significant effect vs. water control set as 100% response. ns = not significant vs. Water control. There are statistical differences between extracts of Flowers and Leaves and control.
(p < 0.001) and showed a better interaction of molecules of Cassia extracts with the enzyme.
Phenolic compounds such as flavonoids found in different parts were reported to be excellent ROS scavengers and inhibitors of MPO    . MPO and its metabolites are biomarkers for infectious diseases and a wide array of non-infectious and neurodegenerative disorders  . The inhibitors of MPO activity are promising therapeutic agents  . Altogether the results showed that the extracts tested showed the highest acellular and cellular antioxidant activity, also the highest inhibition on MPO activity related to their phytochemicals. Cassia occidentalis is known such as a plant with many applications in traditional medicine. Pharmacological properties such as anti-inflammatory, anticarcinogenic, antimutagenic, antiplasmodial, antirheumatic and hepatoprotective were reported on this species  . Oxidative damage and inflammatory process play a crucial role in the diseases treated by herbal formulations from Cassia   . Obtained results support the potential therapeutic interest of all aerial parts of Cassia and could justify their use in traditional medicine and local nutraceutical resources.
The microscopic analysis of powders from flowers, leaves and seeds of C. occidentalis allowed the identification of specific microscopical features. TLC analysis indicated that phenolic compounds and terpenes are major secondary metabolites in this plant. Aqueous extracts from all parts exhibited good antioxidant activity with flowers and leaves as the most active, which is consistent with the higher phenolic content. The leaves and flowers extracts showed higher inhibition effect on MPO as compared to other plant parts. However, the in vitro activities should be complemented in the future with in vivo evaluation which could be promoted as medicine and as nutraceutical resource with high antioxidant capacity.
The authors are thankful to Laboratory of Pharmacognosy and the Centre for Oxygen Research and Development (C.O.R.D.) of University of Liège (Belgium) and Académie de recherche et d’enseignement supérieur (ARES) for their support in this research project.
*These authors contributed equally to this article.
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