Collection of Plant
Plant material was collected in March 2019 from the Baghdad-ul-Jadeed campus of The Islamia University of Bahawalpur, Pakistan. The plant was identified and specified voucher number 314 was allotted by taxonomist Ghulam Sarwar, Assistant professor department of Botany, The Islamia University of Bahawalpur, Pakistan. The plant was dried, mangled, pulverised, and stored in glass jar.
Dried powder of plant material (2kg) was soaked in 80% hydro-alcoholic (methanol) solvent for 3days at room temperature. It was agitated regularly during maceration. After 3 days, the plant material was passed through muslin cloth and then filtered by using Whatman-1 filter paper. This procedure was repeated twice to collect all the remaining phytochemicals. All collected filtrates were dried using a rotary evaporator at 35 ºC under less pressure. The dried crude extract was weighed and stored in an airtight container for further analysis.
Chemicals and Drugs
Indomethacin from Shanxi Guangsheng pharmaceutical, carrageenan (Sigma–Aldrich, USA) provided diclofenac sodium and paracetamol from Agron Remedies Pvt. Ltd. while tramadol was purchased from Searle Pharma- ceuticals, Indomethacin (15mg/kg), diclofenac sodium (15 mg/kg), and paracetamol 50 mg/kg and different doses of HMPA extract (100, 200,400mg/kg) were prepared in double-distilled water for administration in rats and mice.
Phytochemical Screening, Flavonoids, and Total Phenolic Contents
Phytoconstituents tests were used to screen out a variety of secondary metabolites(Hegde et al., 2015).The total phenolic contents (TPC) of HMPA extract was determined using a colorimetric Folin–Ciocalteu reagent (FCR) technique with slight modifications. The10 µl of diluted FCR (10%) mixed with 100 µl of sample solution, 90 µl of 15% w/v aqueous sodium carbonate solution. This mixture was incubated for 90 minutes at 37 C and absorbance was measured at 750 nm. The results were reported as milligram gallic acid equivalents per gram of dry extract (Sajid-Ur-Rehman et al., 2021b).
An aluminum chloride colorimetric test was used to assess the total flavonoid content (TFC), and quantity of total flavonoids was reported rutin equivalents (mgRE/g extract) (Zengin et al., 2015).A calibration curve with a range of 0-100 µl (0–100 mg) was created using rutin as the standard. The 25 µl of 1% sodium nitrite solution and 100 µl of test solution was mixed and stand for 5 minutes then added 10 µ l of 1% aluminum chloride solution again stand for five minutes Afterwards 36 µl of 4% of sodium hydroxide was mixed and was diluted by using methanol (28 µl) and at 510 nm, absorbance was read. The calibration curve was used for the calculation of TFC and expressed as milligram rutin equivalent per gram of dry extract (mg of RE/g extract).
Determination of Antioxidant Potential
Antioxidant activity of HMPA extract was assessed by using two radical scavenging assays i.e., DPPH, ABTS
While reducing power was determined by using i.e., FRAP, CUPRAC assay reported protocol by (Asif et al., 2016, Khurshid et al., 2019).
Gas Chromatography-Mass Spectroscopy (GC–MS) Analysis
The phytoconstituents of HMPA extract were determined using GC MS analysis (Agilent, 6890 series Hewlett Packard, 5973) mass choosy detection systems, with HP-5MS column (250 m in diameter, 30 m in length 0.25 m in film thickness). The temperature at the inlet was 220 °C. The oven temperature steadily increased from 60 °C to 280 °C at a rate of 3 °C/min. The carrier gas was pure helium gas (99.995 percent purity) flowing at a rate of 1 mL/min in constant flow mode.1.0 L of prepared extracts diluted with individual solvents were injected in a split-less mode. The compound was identified using a NIST library search (NIST 14).
Wistar albino rats weighing 150–230 g were kept in the animal house of the Pharmacology & Physiology research laboratory at IUB Punjab, Pakistan. All animals used in the study were housed in polycarbonate cages that measured (47 ×34× 18 cm3). The typical conditions of temperature (25±2°C) and humidity (50–55%), as well as exposure to 12 hour light and dark cycle, were maintained throughout the study. The animals were fed conventional animal food and given free access to water. To reduce animal stress, acclimatized to the test environment for one week before the trial began. All experimental protocols were reviewed by the Pharmacy Animal Ethics committee with approval no PAEC22/73 in the Faculty of Pharmacy, IUB Punjab, Pakistan.
Physiological and pathological investigations were made on albino mice (25–30 g) kept under normal circumstances. Mice were arbitrarily separated into five groups of six mice and starved overnight before the experimental assay, which was supplied with only water. Animals were given HMPA extract at escalating doses of 0.5, 1, 3, 5, and 10 g/kg/oral by stomach intubation, whereas the control group was given a 10% DMSO solution in distilled water. The animals were intensively monitored for 48 hours and then for 14 days. Animal behavior such as convulsion, tremor, salivation, writhing reflex, and behavior pattern was detected, as well as usual unwanted effects such as diarrhea and weight loss (Javed et al., 2020).
Acute Anti-inflammatory Activity
Carrageenan Induced hind‑paw edema model was used with minor modification to determine in vivo anti-inflammatory potential of HMPA extract. Wistar albino rats have fasted with free access to water, and animals were divided into five groups (n=6). All groups received 0.1 mL freshly prepared 1% carrageenan injected into the right hind paw except control (Sajid-Ur-Rehman et al., 2021a). The digital Vernier caliper (Mitutoyo, Japan) was used to measure the paw size of rats. The rats were given a vehicle (distilled water 5 ml/kg, i.p), HMPA extract (100, 200, and 400 mg/kg, i.p) and standard indomethacin (15 mg/kg, i.p) half an hour earlier. The paw edema of all above-mentioned groups was measured just before and after carrageenan injection at "0 h," and subsequently at 1, 2, 3, and 4 hours. The previously reported formula was used to compute the % inhibition of paw edema (Javed et al., 2020a).
Wistar albino rats have fasted with free access to water, and animals were divided into five groups of six animals each. Before induction of pyrexia, the rectal temperature of all animals was measured using a digital clinical thermometer. All animals were injected in the back neck with a 15 % w/v of Brewer's yeast (10 ml/kg aqueous solution) to induce pyrexia. Rectal temperature was measured after 18 hours, and animals with a temperature increase of at least 0.5 °C were included in the study. The pyretic animals were treated orally with HMPA extract (100, 200, and 400 mg/kg) and standard paracetamol (PCM 50 mg/kg orally) distilled water (5 mg/kg, orally to control group. The rectal temperature of all animals was measured at the 1st, 2nd, 3rd, and up to the 6th hour following HMPA extract administration by putting a digital clinical thermometer 2 cm into the rectum (Javed et al., 2020a)
Analgesic activity of HMPA extract was measured by using tail immersion method, hot plate method, Acetic acid-induced writhing method. Wistar albino rats (100 – 190 g) were divided into six groups each group having six animals. To control group 10% DMSO (5 ml/kg) was given orally, to the standard group diclofenac sodium (15 mg/kg) and tramadol (30 mg/kg) was given orally. All animals were treated with HMPA Extract (100,200,400 mg/kg) orally.
Tail Immersion Method
The tail Immersion method was performed with minor modifications as described(Sajid-Ur-Rehman et al., 2021b). For measuring tail-flick response animals were divided into five group and each group having six animals. In this study, a thermostat was set to keep the water temperature between 50± 55°C. The rat tail (3cm) was immersed in hot water, and the reflexes of the tail retracting from the hot water were recorded before and after dose administration in rats. Following that, the experimental animals were administered HMPA extract and standard drug and recorded the reaction time again at 30, 60, 90, and 120 minutes.
Hot Plate Method
The hot-plate test was performed with minor modifications as described by (Sajid-ur-Rehman et al., 2021c).The HMPA extract and standard drug was given orally to the respective groups after one hour of dose administration the individual rat was placed on a hot plate (VELP®, Italy) set to 50–55 °C.
The response time of rats was recorded in seconds by observing licking, lifting, or jumping before and after the therapy at 30, 60, 90, and 120 minutes. To avoid paw injury, 25 seconds was set as the cut-off time
Acetic acid-induced writhing method
According to (Sajid-Ur-Rehman et al., 2021a)the acetic acid-induced writhing model was used with minor modification. For this experiment, albino rats (100–150 g) were divided into five groups (n=6). The control group got DW 5 ml/kg while the standard group received diclofenac sodium 15mg/kg whereas the treatment group got HMPA extract in various doses (100, 200, and 400 mg/kg) orally. 30 minutes after treatment 0.6 % v/v acetic acid (0.1 mL) was administered orally. The numbers of abdominal muscle contractions (writhes) were counted for 5 minutes.
Molecular docking analysis
The chemical compounds got from the results of GCMS analysis were subjected to molecular docking with some anti-inflammatory protein targets. The docking analysis used the crystal structures of the Cyclooxygenase 1 (PDB: 4O1Z) and Cyclooxygenase 2 (PDB: 5IKV) protein targets from the PDB (https://www.rcsb.org/). Polar hydrogens were inserted into the protein. Water, inhibitors, and extra chains were removed from the protein and then converted into the PDBQT format. To analyze their potential against the proteins, the ligands were taken from PubChem (https://pubchem.ncbi.nlm.nih.gov/). The ligands were input into Open Babel and used the PyRx application to reduce their energy. The compounds were then transferred to PDBQT format for further investigation. The grid box is then constructed in the desired dimensions. Finally, the Discovery studio visualized the interactions.
Result values were represented in mean ± SEM (n = 6). Two-way ANOVA followed by a test tokay’s was performed using Graph Pad Prism (San Diego, CA, USA) software. A p-value of less than 0.05 was considered statistically significant.