Plant material and extraction
The plant material consisted of the aerial part of C. quadrangularis which was harvested in June in the Cameroon coastal region, more precisely in the Department of Mougo (Nkongsamba). The botanical authentication of this specimen was carried out at the national herbarium of Yaoundé in Cameroon by comparison with a sample kept under number 36966 HNC/Cam. After the harvest, the plant was dried in the shade, then it was crushed with a grinder to was obtained by decoction of 300 g of C. quadrangularis powder in 3.25 l of distilled water for 20 min then filtered using filter give a fine powder which was used to prepare the various extracts. The aqueous extract paper. The filtrate obtained was evaporated in an oven at a temperature of 40 °C and the powder obtained (17 % yield) representing the aqueous extract was transferred to a dry bottle, then kept for the various pharmacological tests. The ethanolic extract was obtained by maceration of 300 g of C. quadrangularis powder in 3.25 l of 99% ethanol for 48 h and then filtered using filter paper. The filtrate was concentrated using a rotary evaporator at 70 °C. The extract which was obtained (3 % yield) was placed in the open air for complete evaporation of the ethanol, then placed in an oven for evaporation of the water. The powder obtained was transferred to a dry vial and then stored for the pharmacological tests.
Quantitative phytochemistry
Dosage of total phenols
The oxidation of phenols, the mixture of phosphomolybdic acid (H3PMo12O40) and phosphotungstic acid (H3PW12O40) is reduced to a solution consisting of molybdenum and tungsten blues therefore the absorption varies according to the quantitative/qualitative composition of the pH and phenolic mixtures obtained by adding sodium carbonate (Luis et al. 2009). For this test, a solution of extract (20 µL, 2 mg/ml) or distilled water (white tube), Folin-Ciocalteu reagent (100 µL, diluted 10 times), sodium carbonate (80 µL, 20%) was stirred, incubated (water bath, 20°C, 30 minutes), and the absorbance was read (spectrophotometer, 765 nm). Gallic acid (0.015 to 2 mg/ml) was used to draw the standard curve which made it possible to obtain the results in milligram equivalent of gallic acid per gram of powder (Ramde-Tiendrebeogo et al. 2012).
Assay of total flavonoids
A solution of extract (100 µL, 2 mg/ml), aluminum chloride (50 µL, 1.2%) and potassium acetate (50 µL, 120 mM) was incubated (30 minutes, temperature ambient) and the optical density was read (415 nm). Quercetin (0.015 to 2 mg/ml) was used to draw the standard curve which made it possible to obtain the results in milligram equivalent of quercetin per gram of powder (Chang et al. 2002).
Quantification of total tannins
A solution of extract (100 µL, 2 mg/ml) or distilled water (white tube), Folin-Ciocalteu reagent (500 µL, diluted 10 times), sodium carbonate (1000 µL, 35%) and distilled water (8.4 mL) was stirred, incubated (30 minutes, room temperature), then the optical density read at 700 nm. Tannic acid (100, 200, 300, 400 and 500 µg/mL) was used to draw the standard curve which made it possible to obtain the results in milligram equivalent of tannic acid per gram of powder (Govindappa et al. 2011).
In vitro assays
Immunomodulatory activity
The production of ROS (intracellular/extracellular) through the chemiluminescence test, the production of cytokines through the ELISA test and cell proliferation were used to determine in vitro the immunomodulatory properties of the extracts of C. quadrangularis according to the protocols described by Mbiantcha et al. (2017) and Matah Marte et al. (2020).
Isolation of human polymorpho neutrophils (PMNs)
The method described by Zimmerman (1983) was used to isolate neutrophils. In a 30- year-old voluntary donor (having given his consent for the purposes of this study), 10 ml of blood was taken from the bend of the elbow and introduced into a tube (with heparin). At equal volume, this blood was combined with Hanks Balance Salts Solution (HBSS) (MP Biomedicals Inc., Sigma and Research Organics) and lymphocyte separation medium (LSM) (MP Biomedicals Inc., Sigma and Research Organics) and left to stand (30 minutes). The supernatant was removed, mixed with LSM (5 ml) and centrifuged (400 g, 20 minutes, room temperature). The pellet collected was mixed with distilled water (1 ml, 1 minute, lysis of the red blood cells), then 1 ml of HBSS-- (2x) (stopping of the lysis). HBSS was added again and the tube centrifuged (10 minutes, 4 °C, 300g) and the supernatant discarded. Then 1 ml of HBSS-- was then introduced into the tube and kept cold. Trypan blue exclusion (viability test), hemocytometer (cell count) were used and for each test, 1 x 106 cells/ml was the cell concentration used.
Peritoneal macrophages isolation from mice
Mice (Roswell Park Memorial Institute medium (NMRI)) weighing 22 to 24 g, having received Fetal bovine serum (FBS) (1 ml, i.p.), after 72 hours and sacrifice of the animals, the RPMI (Roswell Park Memorial Institute medium, MP Biomedicals Inc., Sigma) 10% (10 ml), was introduced into the peritoneal cavity which was massaged for 2 minutes. Using a syringe, the macrophage-rich RPMI was extracted from the peritoneum and centrifuged at 400g at 4 °C for 20 minutes. In the pellet, the incomplete RPMI was introduced and centrifuged again at 4 °C (10 minutes, 300 g), then addition of incomplete RPMI/HBSS (1 ml). The same methods of viability, counting and the same number of cells per ml for each test were used (Mesaik et al. 2006; Mahomoodally et al. 2007).
Chemiluminescence assay
The chemiluminescence method in 96-well plates described by Mesaik et al. (2006) and Mahomoodally et al. (2007) was used. C. quadrangularis extracts (25 µl, 3.125 to 100 μl/ml), ibuprofen (25 µl, 3.125 to 100 μl/ml), whole blood (25 µl) or neutrophils (25 µl, 1 x 106 cells/ml) or macrophages (25 µl, 1 x 106 cells/ml) were mixed in the test wells. The control wells did not receive the plant extracts. Plates were introduced into a thermostatically controlled chamber of the luminometer (20 minutes, 37 °C), then the Zymosan/Phorbol 12-myristate 13- acetate (PMA) (Fluka, European Cell Culture Collection) mixture (25 μl) and/or the luminol/lucigenine mixture (25 μl, 7 × 105 M) were added to each well and the plate was read in RLU (relative light units) and the percent inhibition was calculated:
Cytokines, T-Cell proliferation and MTT assay
Cell Culture
The human monocytic cell line THP-1 (ATCC® TIB-202) (American Type Culture Collection (Manassas, VA, USA)), grown in the Roswell Park Memorial Institute (RPMI-1640) containing glucose (4.5 g/l), inactivated fetal calf serum (10%), L-glutamine (1%), nonessential amino acids (1%), streptomycin (10 μg/ml) and 100 U/ml penicillin.
Immunoassay for Cytokines
THP-1 cells (2.5 x 105 cells/ml) (American Type Culture Collection (Manassas, VA, USA)) were mixed with vitamin D3 (0.1 μM) and PMA (100 ng/ml) to be differentiated into macrophages for 24 hours. Each well containing the cells received the extracts (2, 10 and 50 μg/ml) and Lipopolysaccharide (LPS, 2 μg/ml). TNF-α, IL-6, IL-1β and IL-10 were quantified using commercial kits (R&D Systems, Minneapolis, USA) to the ELISA test (Enzyme- linked immunosorbent assay). After 4 hours, optical density (450 nm) was read by a microplate reader (Model 680; BioRad Laboratories, Mississauga, ON, Canada), then TNF-α, IL-6, IL-1β and IL-10 quantity was calculated using a standard curve constructed (serial dilutions of cytokine standards) supplied with the kit (Mahajna et al. 2015).
T-Cell proliferation assay
The cell proliferation test in 96-well white plates (Rita et al., 2014). C. quadrangularis extracts (50 μl; 1, 10 and 100 μg/ml), prednisolone (50 μl; 1, 10 and 100 μg/ml), T lymphocytes (50 µl, 2 × 106 cells/ml) and phytohaemagglutinin-L (PHAL) (50 µl, 7.5 µg/ml) were mixed. Negative (T lymphocytes and RPMI (150 μl)) and positive (T lymphocytes, PHAL and RPMI (100 μl)) controls were also prepared. The plate was incubated (5% CO2, 37 °C, 72 hours), then addition of 0.5 μCi/well (methyl 7 3H) of thymidine (25 μl), further incubation for 18 hours and cell harvesting (filter in fiberglass). The counts per minute (CPM) were use to determined the percent inhibition (%):
MTT Assay
For this test carried out in 96-well plates, seeded THP-1 (20,000 cells), differentiated into macrophages (0.1 μM of vitamin D3 and 100 ng/ml of PMA) for 24 hours, then addition of extracts of C. quadrangularis (1 – 100 µg/ml) and incubated (24 hours, 37 °C). The culture medium was then removed, the cells washed (PBS), addition of 100 µl (500 µg/ml) of 3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and RPMI, incubated (4 hours, 37°C), removal of the medium, addition of isopropanol (100 μl, 0.08N) and further incubation (15 minutes, 37 °C). An ELISA plate reader set to 570 nanometers was used to read the absorbance (Mahajna et al. 2015).
Anti-infammatory activity
The in vitro evaluation of the anti-inflammatory properties of the aqueous and ethanolic extracts of C. quadrangularis in this study was made on the enzymes cyclooxygenase (COX1 and COX2), 5-lipoxygenase (5-LOX) and on the denaturation of proteins according to the protocols described by Mbiantcha et al. (2020; 2022) were used.
Lymphocyte isolation
A mixture of 3 ml of Ficoll solution and 3 ml of human blood was centrifuged (400 g, 30 minutes) at room temperature, then the lymphocytes were removed by aspiration and the trypan blue method made it possible to check cell viability, then by microscopic observation the lymphocytes were confirmed by their reduced cytoplasm and large nucleus. A suspension of 1 x 106 cells/ml was cultured in tubes containing filled with 1 ml of fetal bovine serum, 1 ml of RPMI medium, 1 ml (100 μg/ml) of antibiotic (streptomycin and penicillin), 1 ml phytohemaglutinin, filtered (cellulose acetate filter, 0.2 µm) and incubated (37 °C, 72 hours); then addition of lipopolysaccharides (1 µl, 1 µg/ml) to each new incubation tube (24 h, 37 °C) (Viji and Helen, 2008). The cyclooxygenase inhibition test was performed following the protocol described by Viji and Helen (2008).
The tubes containing 1 ml of the different extracts (62.5, 125, 250 and 500 µg/ml) or ibuprofen (62.5, 125, 250 and 500 µg/ml) or 0.9% NaCl were incubated (24 hours at 37 °C), centrifuged (400 g, 10 minutes), then cell lysis buffer (50 µL) was introduced into each tube after removal of the supernatant and further centrifugation (400 g, 10 minutes). Subsequently, Tris-HCl buffer (2.75 ml, 100 mM, pH= 7.4), human hemoglobin (5 mM), glutathione (5 mM), cyclooxygenase (10 µl) and arachidonic acid (200 µM) were added, the tubes were incubated (37 °C, 20 minutes), 0.2 ml of thiobarbituric acid (TBA) and 0.2 ml of trichloroacetic acid (TCA, 10% in HCl) were also added, then the tubes were heated (boiling water bath, 70°C, 20 minutes), cooled and centrifuged (1000 rpm, 3 minutes). The optical density of the supernatant (variation of arachidonic acid) was read at 632 nm.
As for the 5-lipoxygenase activity inhibition test, the tubes were again prepared as before (cyclooxygenase test), then a solution (2 ml) containing phosphate buffer (2.75 ml; pH 7.4 ; 50 mM), 5-lipoxygenase (50 µl) and sodium linoleate (0.2 ml) were added and incubated (25 °C, 30 minutes). The increase in absorbance (234 nm) characteristic of the formation of hydroperoxy-linoleic acid materialized the activity of 5-lipoxygenase (Viji and Helen 2008).
For protein denaturation, 1 ml of distilled water or Diclofenac (62.5, 125, 250 and 500 μg/ml) or different extracts (62.5, 125, 250 and 500 μg/ml) was added 1 ml of bovine serum (5 %), incubated (15 minutes, 27 °C), placed for 10 minutes at 70 °C; then the optical density (OD) at 660 nm was read and the percentage inhibition was determined (Padmanabhan and Jangle 2012).
Antioxidant activity
The in vitro evaluation of the antioxidant properties of extracts of C. quadrangularis in this study was made on the DPPH radical scavenging tests, the ABTS•+ discoloration test and the nitric oxide scavenging test according to the protocols described by Mbiantcha et al. (2020) were used.
DPPH radical scavenging activity: Three (3) ml of a working solution was obtained by diluting DPPH (24 mg/100 ml methanol) (MP Biomedicals Inc., Sigma and Research Organics) with methanol for an absorbance of 0.98 ± 0.02 at 517 nm, was mixed with each extract (100 μl, 1 mg/ml) or standard or methanol (100 μl). Absorbance (OD) (517 nm) was measured for 30 min (Brand Williams et al. 1995). The following formula was used to calculate antioxidant activity:
ABTS•+ Discoloration Test: Ten (10) ml of a solution of ABTS (7 mmol/l, 9.5 ml), potassium persulfate (100 mmol/l, 245 μl) and distilled water was diluted with 0 .1 mol/l potassium phosphate buffer (pH 7.4) to an absorbance of 0.70 ± 0.02 (734 nm, dark, room temperature, 18 hours). The absorbance of the mixture of 10 µL of each extract (62.5, 125, 250 and 500 μg/ml) and 2.99 ml of the ABTS solution was read at 734 nm (Re et al. 1999) and allowed the percentage of antioxidant activity to be calculated using the formula previously described.
Nitric oxide (NO) scavenging test: a mixture of 0.5 ml of sodium nitroprusside (pH7.4, 5 mmol/l) and each extract (62.5, 125, 250 and 500 µg/ml ) or ascorbic acid or methanol was incubated (25 °C, 180 minutes), then 1.5 mL of Griess' reagent (phosphoric acid (2%), sulfanilamide (1 %), N-(1 %) dihydrochloride-naphthyl) ethylenediamine (0.1 %)) was added, then a new incubation (30 minutes) was made. The percentage of activity was determined from the absorbance (Abs) read at 546 nm (Benzie and Strain 1999).
In vivo assays
Neuropathic Pain, Animal Treatment, Induction and Behavioral Assessment
Fifty-four (54) Wistar rats (male and female, 160 - 200 g, 3-4 months) and reared under 19-23 °C, 12 hours light at the Research Unit of Animal Physiology and Phytopharmacology (Department of Animal Biology, Faculty of Science, University of Dschang). These animals were treated as follows : group 1 (n=6, normal control) received orally distilled water, group 2 (n = 6, negative control) received orally distilled water , group 3 (n = 6, positive control) received orally pregabalin (50 mg/kg), groups 4, 5 and 6 (n = 6, tests) treated orally with the aqueous extract of C. quadrangularis at respective doses of 90, 180 and 360 mg/kg, groups 7, 8 and 9 (n = 6, tests) treated orally with the ethanolic extract of C. quadrangularis at respective doses of 90, 180 and 360 mg/kg. All treatments were administered on the first day before the start of induction. After treatments, an intraperitoneal injection of vincristine sulphate (100 μg/kg) was administered to the rats (except the animals in group 1) for two series of five days (1st, 2nd, 3rd, 4th, 5th day, then 8th, 9th, 10th, 11th, 12th day) of successive work with 2 days off. After the start of induction, the different treatments were administered daily until the 15th day (Chiba et al. 2017; Mbiantcha et al. 2018).
For behavioral tests, mechanical hyperalgesia was induced by placing the left hind paw of each rat on an analgesimeter (UGO BASILE) on days 0, 1, 3, 5, 7, 9, 11, 13 and 15. The pain was caused by increasing the pressure (cut-of 250 g) until the paw was withdrawn. The value of the nociceptive threshold was considered as the force (g) obtained (Mbiantcha et al. 2018). While mechanical allodynia was performed using Von Frey filaments. The animals were first placed individually in cages for acclimatization over a period of 30 minutes before taking the values. Subsequently, these animals gradually received the Von Frey filaments in order of masses perpendicular to their right plantar arches. Pressure was exerted for 5 seconds until the filament bent. One test consisted of applying the same filament three times in a row for a period of 1 minute. The animals’ response was considered a painful reaction if it lifted its paw or leg and/or moved its body; licked the stimulated paw or spread the toes. The mass of the filament to which the animal responded was considered as the threshold intensity.
Sample collections and assessment of proinflammatory cytokines and Growth Factors levels
On day 15, the animals are anesthetized (thiopental, 50 mg/kg, i.p.), rib cage opened, then the blood collected (catheterization of the abdominal artery) is introduced into a dry tube and centrifuged (3000 rpm, 15 minutes), the serum collected was used to determine the amounts of IL-1β, TNFα, IL-6 and IL10. Then, the sciatic nerve of the animals is also removed, crushed, centrifuged (room temperature, 3000 rpm, 15 min, 4°C, Eppendorf 5804 R, Hamburg), the supernatant allowed the analysis of IL-1β, TNFα, IL-6, NGF and IGF) (Fatani et al. 2015).
Statistical analysis.
The individual values of each test were used to calculate the Mean and it was used to present the results of this study. Some results are expressed with standard deviation (SD) or standard error of the mean (SEM). One-way analysis of variance (Tukey's test, biochemical assays and in vitro tests) and two-way analysis of variance (Bonferroni's test, behavioral tests) were used to compare group tests with controls (normal and/or negative). At p<0.05, the differences were considered significant.