Reagents, Chemicals and Equipment.
Different reagents and chemicals used in this study were procured from various sources: Morphine was purchased from Sigma Aldrich (St. Louis, MO, USA); Sodium nitroprusside from Merck Ltd (Mumbai, India); Trypsin and Casein from Hi-Media Lab. Ltd. (Mumbai, India). Sulfanilamide, Sodium hydroxide, Linoleic acid, Phosphoric acid, Ascorbic acid, Sulfuric acid, Arachidonic acid and Sodium linoleate were purchased from SD Fine Chem. Ltd, (Mumbai, India); Hemoglobin, Thiobarbituric acid, Ethylenediaminetetra acetic acid, Bovine albumin and Lipopolysaccharide were from Central Drug House Pvt. Ltd., (New Delhi, India); Dimethyl sulfoxide (DMSO) and Sodium chloride from Geochim Sarl. (Western region, Cameroon); Diclofenac sodium, Ibuprofen, Vitamin C, Indomethacin and other reagents were provided from local suppliers in Pakistan.
To assess neuropathic pain, mechanical hyperalgesia (analgesimeter, UGO Basil, Italy), thermal hyperalgesia (Hotplate, Ugo Basile, Monvalle VA, Italy) and cold allodynia (cold water (4 °C)) equipment were used.
Plant Material, Extraction and Isolation.
The whole plant of Scyphocephalium
. ochocoa, was collected in Franceville, Upper Haut Ogooue Province, South-East of Gabon, then identified by a botanist from the National Herbarium of Gabon (M. Yves Issembe) by comparing it with a preserved specimen under the number So 083/UM. The bark was removed, sliced up, shade dried and crushed (Feuya Tchouya et al. 2021). Thus, 3.5 kg of powder was macerated in 7 L of a mixture of dichloromethane (DCM)/methanol (MeOH) (1:1) and kept at room temperature during 72 h. After filtration and concentration in a rotary evaporator, a new extraction of the residual powder was carried out and the various crude extracts were mixed. The final concentrate was dried (under vacuum) to give 250 g (7.14 % yield) of crude extract. It was dissolved in a MeOH/H2O mixture (1:1), then successively extracted with n-hexane and with ethyl acetate. Extraction of each mixture was carried out in vacuum to give 41.5 g of n-hexane extract (16.6 % yield), 45 g of ethyl acetate extract (18 % yield) and 95 g of aqueous residue (38 % yield). The hexane extract (30 g) was subjected to column chromatography (silica gel, n-hexane/ethyl acetate of increasing polarity), which gave 0.875 g of Scyphocephalione A (2.92 % yield) at an n-hexane/ethyl acetate polarity of 90:10 (Feuya Tchouya et al. 2021).
In vitro assays.
The evaluation of the anti-inflammatory properties of Scyphocephalione A was done following the in vitro cyclooxygenase activity and 5-Lipoxygenase activity inhibition tests as well as the in vitro protein denaturation technic. The methods described by Djuichou Nguemnang et al. (2019) and Mbiantcha et al. (2020) were used.
Preparation of lymphocyte culture.
A culture (human peripheral lymphocytes, RPMI 1640 (HIMEDIA), streptomycin, penicillin, fetal calf serum, phytohemagglutinin (HIMEDIA)), was filtered using cellulose acetate 0.2 µm pore brand Sartorios. After addition of 1 x 106 cells/ml of plasma, the culture was incubated for 72 h. After another addition of 1 μl of lipopolysaccharide and a 24 h incubation period, the culture received different concentrations (100, 200, 500 and 1000 μg/mL) of Scyphocephalione A, Diclofenac and Ibuprofen, then incubated again for 24 h. The culture was next centrifuged for 10 min at 6000 rpm, followed by removal of the supernatant and addition of 50 µl of lysis buffer. Finally, the culture was centrifuged again as previously described (Viji, and Helen 2008).
Assay of Cyclooxygenase, 5-Lipoxygenase and Protein Denaturation Inhibition.
A mixture of Tris-HCl buffer, cyclooxygenase, hemoglobin, glutathione, arachidonic acid and TCA (10 % in HCl 1N, 0.2 ml), was incubated at 37 °C for 20 min. Then after addition of TBA (0.2 ml), it was heated with boiling water for 20 minutes, cooled and then centrifuged for 3 minutes at 1000 rpm. The supernatant was used to assess cyclooxygenase activity at 632 nm (Viji, and Helen 2008).
To assess 5-lipoxygenase activity, the starting mixture (25 ml) consisting of linoleic acid (70 mg), Tween 20 (4 ml of non-oxygenated water), sodium hydroxide (0.5 N) and non-oxygenated water was divided into portions (0.5 ml each). Each portion was rinsed with nitrogen, frozen and passed into a quartz cuvette at 25 °C. A control solution, prepared from 0.2 ml of sodium linoleate, 2.75 ml of Tris buffer (pH 7.4) and 50 ml of enzyme was used. The optical density (OD) was measured at 234 nm (Viji, and Helen 2008) and the percentage (%) of inhibition was calculated as follow:
In a solution (1 ml) containing distilled water (control tube), Diclofenac or Scyphocephalione A at different concentrations (100, 200, 500 and 1000 μg/ml), the bovine serum solution (1 ml, 5%) was added. The mixture was incubated (15 min, 27 °C) and placed at 70 °C (10 min). After cooling (room temperature), the OD was measured at 660 nm (Padmanabhan and Jangle 2012) and the percentage of inhibition was calculated using the same formula presented above.
In vivo assays
To performed all tests, adult male and female mice (Mus musculus, BALB/c) and rats (Wistar) of 3 months old and, weighing on average 27 ± 1 (mice for acute pain assay) and 170 ± 2 g (rat for neuropathic pain assay) were used. They were raised in the International Centre for Chemical and Biological Sciences (ICCBS), University of Karachi, Pakistan under natural conditions (21 ± 2 °C temperature, 12 h/12 h cycle) with access to food and water. The experiments were conducted in agreement with the Institutional Committee for the Use, Care and Standards of Animals (IACUC) of ICCBS following protocol n° 1209004, accepted by the committee of ethics of ICCBS (Mbiantcha et al. 2017).
In the acute pain assays, 18 adults’ mice were used for each method. They were partitioned into 3 groups of 6 animals each and treated as follows:
- A negative control group (Vehicle), receiving DMSO 2.5%, p.o.;
- A positive control group (Indo), receiving Indomethacin (10 mg/kg, p.o.);
- A test group (Scyp A), receiving Scyphocephalione A, 50 mg/kg, p.o.).
For the neuropathic pain assays, 24 adult rats were used. There were distributed in 4 groups of 6 animals each and processed as follows:
- A neutral control group (Normal control), without Vincristine sulphate administration;
- A negative control group (Vincristine), receiving Vincristine sulphate (100 μg/kg, i.p.) + vehicle (2.5% DMSO, p.o.);
- A positive control group (Morphine), receiving Morphine (10 mg/kg, p.o.) + Vincristine sulphate (100 μg/kg, i.p.);
- A test group (Scyp A), receiving Scyphocephalione A (50 mg/kg, p.o.) + Vincristine sulphate (100 μg/kg, i.p.).
For both in vivo assays the volume of administration was 10 ml/kg. The experiment was carried out for 15 days. On the 15th day after the last test, all the animals were anesthetized by injection of thiopental (50 mg/kg, i.p.), then the rib cage of each rat was carefully opened and the blood was drawn by catheterization of the abdominal artery and introduced into a 1st tube containing EDTA for the determination of the haematological parameters, then into a second tube without EDTA for the determination of biochemical parameters.
Acetic acid-induced acute pain assay
The test was performed by administrating to each mouse 10 ml/kg (i.p) of acetic acid, 1 h after the different treatments. Then, mice were placed in sets of two in a glass cage. Five minutes after, the number of writhing (stretching) was recorded for a period of 30 min (Vogel, and Vogel 1997). The percentage of inhibition (% I) was determined according to the formula below:
Formalin-induced acute pain assay
The test was conducted as follow: 1 h after administration of the different substances, each mouse received an intra-plantar injection of 2.5 % formalin (100 µl), then each animal was individually placed in a glass cage. The paw licking time (pain materialization) was determined using a chronometer between 0 and 5 min (neurogenic pain), then between 15 and 30 min (inflammatory pain) (Gaertner et al. 1999). The percentage of inhibition (% I) was determined according to the formula below:
Vincristine-induced neuropathic pain assay
The experiment was conducted following the method described by Chiba et al. (2017). Briefly, apart from the animals of the normal control group, all the other rats received Vincristine sulphate (100 μg/kg, i.p.) in two series to induce neuropathic pain: from the 1st to the 5th day, then from the 8th to the 12th day. The treatments with Scyphocephalione A and Morphine were performed one day before induction and continued daily until day 15.
Behavioral assessment of neuropathic pain
To assess the effect of Scyphocephalione A on neuropathic pain, mechanical hyperalgesia thermal hyperalgesia and cold allodynia trials were used.
For the mechanical hyperalgesia trial, the hind paw of the rat, placed in a pressure applicator, was stimulated by increasing pressure (cut-off of 250 g) until paw withdrawal. The nociceptive threshold value was considered as the force (g) obtained (Hajimashhadi et al. 2017) and was evaluated on days 0, 1, 3, 5, 7, 9, 11, 13 and 15. The percentage (%) of pain sensitivity latency was determined.
In the thermal hyperalgesia trial, animals were gently placed over the heating plate (50 ± 0.5 °C), the paw withdrawal latency was recorded in seconds and a cut-off latency of 20 seconds has also been observed (Bhardwaj et al. 2016). The paw withdrawal latency was evaluated on days 0, 1, 3, 5, 7, 9, 11, 13 and 15. The percentage (%) of paw withdrawal latency was determined.
Regarding the cold allodynia trial, the tail of each animal was soaked in cold water (4 °C), the time of inactivity of an animal to withdraw its tail was determined with a cut-off of 20 seconds in case of lack of reaction (Mbiantcha et al. 2017). The tail withdrawal latency was evaluated on days 0, 2, 4, 6, 8, 10, 12, 14 and 15. The percentage (%) of tail withdrawal latency was determined.
Haematological and Biochemical estimates
Blood collected into tube containing EDTA was used to evaluate haematological parameter such as haemoglobin, red blood cell (RBC), white blood cell (WBC) and Platelet count. Blood collected into tubes without EDTA was centrifuged at 4900 rpm (5 min), then the serum was taken for the assay of alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), creatinine (Bhardwaj et al. 2016; Mbiantcha et al. 2017), glutathione (GSH) (Ellman 1959), catalase (Sinha 1972), superoxide dismutase (SOD) (Misra, and Fridovich 1972), NO and malondialdehyde (MDA) (Wilbur et al. 1949).
For in vitro assays, samples were ran out in triplicate and the results were presented as percentage inhibition; while for the in vivo assays, the results were analyzed by one-way ANOVA followed by Tukey or Bonferroni post-tests. Data were expressed as mean ± SEM. A p-value < 0.05 was considered significant (GraphPad Prism, version 5.03).