Selection of experimental animals

This study employed healthy Wistar albino rats of both sexes weighing 140-160 grams obtained from the laboratory. All participants were subjected to rigorous monitoring for infection, and any rats exhibiting symptoms of the condition were excluded from participation in the study. Compliance with animal welfare legislation was observed throughout the animal experiment.

Preparation of experimental animals

The rats were randomly assigned to five groups, each containing six animals, and were treated as given in Table 2.

Carrageenan-induced rat paw edema method

The anti-inflammatory effects of NUM were assessed in Wistar albino rats using the carrageenan-induced rat paw edema model as outlined by Winter et al., 1963. The rats were fasted overnight, and the test drug was administered orally via gastric gavage tubes and suspended in a vehicle. One hour post administration of the test drugs, all groups received a sub plantar injection of 0.1 ml of 1% carrageenan in the right hind paw. Paw thickness was measured using a plethysmograph at baseline and at hourly intervals for up to 6 hours following carrageenan injection. The percentage inhibition of paw thickness in the treated groups was determined by comparing their mean paw thickness to that of the control group (Winter & Risely, 1963).

% Inhibition = 100 (1-Vt/Vc)

(V - Control mean paw thickness, Vt - Test mean paw thickness).

Selection of Experimental Animals

This study employed healthy Swiss albino mice of both sexes, weighing 25–40 grams, which were obtained from the laboratory. All participants were subjected to rigorous monitoring for infection, and any mice exhibiting symptoms of the condition were excluded from participation in the study. Compliance with animal welfare legislation was observed throughout the animal experiment.

Preparation of experimental animals

Swiss albino mice were randomly assigned to five groups, each consisting of six animals, and were treated as given in Table 3:

Eddy’s hot plate method

The hot plate test was employed to measure response latency time following the procedure described by (Eddy & Leimbach, 1953). Mice that exhibited an initial nociceptive response within 15 seconds were selected for the study. The cut-off time for hot plate latencies was set at 15 seconds. The overnight-fasted mice were given the test drugs orally via gastric gavage tubes, with the drugs suspended in a vehicle. The standard drug was administered intraperitoneally. The animals were placed on a hot metal plate maintained at 55°C and surrounded by a Plexiglas cylinder (26 cm in height, 19 cm in diameter). The time elapsed between placing the animal on the hot plate and the occurrence of hind paw licking, shaking, or jumping off the surface was recorded as the response latency in seconds. The specificity and sensitivity of the test were enhanced by recording the reaction time of the first observed behavior, whether it was paw licking or jumping. Responses were measured at 0, 60, 90, and 120 minutes (Eddy & Leimbach, 1953).

The percentage protection against thermal pain stimulus was calculated using the following formula:

% Protection = Test Mean – Control Mean/Test Mean X 100

Selection of Experimental Animals

The animal source for the study was Kerala Veterinary and Animal Science University, Kerala, Mannuthy, utilizing Wistar albino rats (one male) aged 6-8 weeks with a body weight of 200-220 grams. The animals were acclimatized for 7 days prior to dosing, with veterinary examinations conducted at the time of purchase and at the end of the acclimatization period. The housing conditions included a temperature of 24 ± 2°C, relative humidity between 30% and 70%, and a 12:12-hour dark and light cycle. Each animal was identified by individual marking, animal number, and cage number using picric acid. The diet consisted of rat chow pellets and RO water, and the animals were housed in stainless steel cages with dry grass bedding.

Acetylcholine-induced ileum formation in rats

Overnight-fasted adult male Wistar albino rats weighing between 200 and 220 g were used in this study following the methods outlined by (Carvalho, Almeida, Melo, Cavalcanti, & Marçal, 2009). Under mild pentobarbital anaesthesia, the rats were euthanized by cervical dislocation. The abdomen was opened, and two pieces (each 2 cm long) of the ileum were dissected from a segment 10-15 cm proximal to the ileocecal junction. Both isolated ileum segments were mounted for tension recording and allowed to equilibrate for 1 hour in separate 50 ml organ baths containing aerated normal Tyrode solution (composition in mM: NaCl, KCl, CaCl₂, MgCl₂). H₂O, NaHCO₃, NaH₂PO₄, and glucose; _pH 7.4). The tissues were maintained at 37°C and bubbled with air.

The response of the first ileum was recorded using a frontal lever with various concentrations of NUM (ranging from 0.12 µg to 32 µg). The response of the second ileum was recorded using a frontal lever with various concentrations of acetylcholine until submaximal responses were obtained. The isolated ileum was then incubated for 45 minutes with NUM (1. 2 µg/ml). Following this incubation period, the response of the ileum to various concentrations of acetylcholine was recorded again in the presence of NUM (Carvalho et al., 2009).

Anti-Inflammatory Activity

The carrageenan-induced paw oedema in Wistar albino rats is the most commonly used method for performing acute anti-inflammatory screening pharmacology studies. Following the initiation of inflammation in the rat paw, the inflammatory process begins and continues. The first phase (0-2 hours) of inflammation is mediated primarily by histamine and serotonin, as well as an increase in prostaglandin production. The second phase of inflammation (2-3 hours in duration) is sustained by bradykinin-mediated release and the release of leukotriene and polymorph nuclear cells. NUM was administered orally at 6, 12, and 24 mg/kg at low, middle, and high doses, respectively. No inhibition of edema was observed in the low-dose groups at 6 hours. In the moderate-dose and high-dose groups, moderately significant changes were noted. In the mid-dose group, at 2 hours after test drug administration, moderately significant changes in edema reduction were noted but were less pronounced than those in the high-dose group. At 2 hours after drug administration, more significant changes were noted in the high-dose group than in the standard drug group, which was treated with 5 mg/kg indomethacin. The highest rate of inhibition was observed in the high-dose group (24 mg/kg) at 2 hours. The results are depicted in Figure 3.

Anti-Nociceptive activity

Central pain at the supraspinal and spinal levels was evaluated by the eddy’s hot plate thermal method. NUM was administered orally at 12, 24, and 48 mg/kg at low, moderate, and high doses, respectively. According to Eddy’s hot plate method, NUM increases the thermal stress tolerance of animals. At a low dose, the thermal stress tolerance was less significant at +90 minutes. In the mid-dose group, the difference was moderately significant at +60 minutes, whereas in the high-dose group, it was highly significant at +60 minutes and equal to that of the standard drug. This shows that after oral administration of the drug, the analgesic effect occurs after one hour, whereas for the standard drug administered iv, the analgesic effect occurs after 30 minutes. The results are depicted in Figure 4.

Anti-Spasmodic Activity

In terms of antispasmodic activity, when acetylcholine was administered to the ileum of rats, it produced a certain amount of normal contraction and a normal tissue response. This sample was used as a control. When 1.2 µg/ml NUM was administered at various concentrations, such as 0.2, 0.4, 0.6, 0.8, and 0.16 ml, contractions and tissue responses increased rather than decreased compared to those in the control group. This shows that the test drug NUM has no significant antispasmodic effect on the smooth muscles of the rat ileum.

Therefore, the test drug Nava Uppu mezhugu has significant antinociceptive (analgesic) activity and acute anti-inflammatory activity. The results are depicted in Figure 5.

The toxicity and safety profile of the test drug was determined previously by (Meenaloshini & Master of Thesis. National Institute of Siddha, Chennai - 47., 2019) as a master’s thesis but has not been published thus far (Meenaloshini & Master of Thesis. National Institute of Siddha, Chennai - 47., 2019).