INTRODUCTION:

Inflammation is a non-specific response to a microcirculation tissue injury caused by physical, chemical, or biological stimuli¹. However, when there is a loss of homeostatic control over this process of defense, inflammation plays a damaging role that contributes to the appearance and worsening of diseases². Pain is a vital, physiologic, multidimensional sensory experience, which is a key early warning device, an alarm system that announces the presence of a potentially damaging stimulus³. Due to its broad pharmacological relevance, which includes anti-inflammatory, analgesic, and antipyretic actions with fewer side effects, numerous studies have recently focused on therapeutic plant-derived natural chemicals such as flavonoids, steroids, polyphenols, coumarins, terpenes^4,5.

Flavonoids are diphenyl propanoids that are widely present in plant foods and make up a major portion of the human diet⁶.

Apigenin is a flavone under the category of natural flavonoids that is abundantly present in common fruits, vegetables, nuts, onions, oranges, and tea⁷. Numerous studies have revealed that Apigenin has a wide range of pharmacological properties, including antiviral, anticancer, anti-oxidative, and anti-inflammatory actions^8,9. The present study was carried out to evaluate the analgesic and anti-inflammatory activities of Apigenin.

MATERIALS AND METHODS:

Experimental animal:

Albino Swiss mice were procured from Shree Farm, Nimgaon (MH); CPCSEA Regis. No. 1231/B/08. Then all animals were acclimatized for seven days under well-controlled conditions of temperature (22°C) and humidity (55°C) and a 12-hour light-dark cycle. The animals had free access to a standard laboratory diet, and tap water was provided ad libitum under hygienic conditions. Each experimental group had a separate set of animals, and care was taken to ensure that animals used for one response, i.e., were not employed in any other activity. Animals were habituated to laboratory conditions for at least 48–72hours before the experimental protocol to minimize, if any, nonspecific stress. The experiment protocol was approved by the Institutional Animal Ethical Committee (IAEC No. 149/1999/CPCSEA) prior to the experiments.

Materials used:

Apigenin was purchased from Natural Remedies Private Limited, Bangalore (Karnataka). Carrageenan and other chemicals like dimethylsulphoxide (DMSO), disodium hydrogen phosphate, potassium dihydrogen phosphate, and sodium chloride were procured from the Department of pharmacy, GGV, Bilaspur, C.G.

Experimental protocol:

After acclimatization, selected animals were divided into five different groups, with six animals in each group. Each group was treated with vehicle (s.c.), Carrageenan (2%, s.c.), Apigenin (25 and 50mg/kg, s.c.) and Indomethacin (25mg/kg, s.c.). An Apigenin solution was prepared in DMSO and administered subcutaneously. Carrageenan was prepared in phosphate buffered saline (PBS) and to get a final concentration of 2% Carrageenan solution, 0.01ml of this solution was then administered subcutaneously into the sub-plantar surface of the right hind paw of mice in each group except normal group.

Treatment schedule:

The day on which Carrageenan was administered subcutaneously (s.c.), 30 minutes after Carrageenan administration, Apigenin was given subcutaneously with different samples for different groups.

Estimation of paw volume:

Firstly, paw volume was recorded for both the hind left and right paw of each mouse in each group using an electronic Vernier caliper. Then, with the exception of the normal group, Carrageenan was administered in the right paw. Then at 3 hours, 6 hours, and 9 hours after injection, the state of thickening of the murine plantar hind right paw was measured using a Verniercaliper (accuracy of 0.01mm). The thickening of Paw in milliliters (ml) was calculated using the formula:

Final volume – Intial volume

% Increase = --------------------------------- x 100

Final volume

Furthermore, on the other hand, mice were injected subcutaneously into the right plantar hind paw with Apigenin, and after 30 min, 2% Carrageenan in PBS was injected sub-plantarly in a volume of 0.01ml into the right hind paw. Then at 3 hours, 6 hours, and 9 hours after Carrageenan administration, thickening of the paw was calculated using the formula:

Percentage inhibition of paw edema = (1 – …….) x 100

Where Vc represents the average increase in paw volume (average inflammation) of the Carrageenan-treated group of mice at a given time, and Vtis the average inflammation of the drug-treated (test and standard drug) mice at the same time. The percentage inhibition formula was used to determine the percentage inhibition of paw edema.

Estimation of analgesia:

Eddy’s hot plate analgesiometer:

A hot plate was used to observe the central anti-nociceptive activity¹⁰. To avoid blister formation and skin damage, the animals were placed on Eddy's hot plate analgesiometer and kept at a temperature of 55 +0.5°C for a maximum of 15 seconds per exposure. The mice were screened for an initial nociceptive effect after 3 hours, 6 hours, and 9 hours of Carrageenan administration. Furthermore, this procedure was repeated in the drug-administered group. Reaction time was recorded as the latency period when the animals licked their fore and/or hind paws and/or jumped.

RESULT:

Anti-inflammatory effect:

Carrageenan-induced paw edema in mice:

Apigenin reduced paw volume when administered subcutaneously in DMSO at a dose of 25mg/kg body weight (3 to 9 hours), while at a dose of 50mg/kg; the reduction was time-dependent and was 54.6% (3 to 9 hours) (Table). The effects of Apigenin 25 mg/kg and Apigenin 50mg/inhibitory kg on Carrageenan-induced paw edema provided evidence of their anti-inflammatory activities.

Percentage inhibition of Apigenin:

In the control group, Carrageenan 2% in PBS was administered s.c. into the sub-plantar region, indicating the formation of edema in mice. To cause inflammation in the Apigenin 25mg/kg group, 0.02ml of the drug was given subcutaneously. This treatment caused a reduction in paw volume (41.21% paw inhibition), although not as much as the Apigenin 50 mg/kg dose. In the Apigenin 50 mg/kg group, inflammation was induced, and 0.02ml of Apigenin administration similarly resulted in a reduction in paw volume (paw inhibition of 54.6%); however, this was less pronounced than with Indomethacin. In the Indomethacin group, inflammation and 25mg/kg Indomethacin induced a significant decrease in paw volume (61.4% paw inhibition) more than the Apigenin (25mg/kg) and 50mg/kg treatment groups (Table).

Table 1: Anti-inflammatory effect and percentage inhibition of different Apigenin doses on Carrageenan induced mouse paw edema

Paw edema (mm)				Inhibition (%)
Treatment	3 h	6 h	9 h	3 h	6 h	9 h
Carrageenan 2%	3.114±0.081	3.364±0.0114	3.518±0.102	-	-	-
Apigenin25 mg/Kg	2.03±0.093^**	2.15±0.177^**	2.068±0.318^***	34.81	36.08	41.21
Apigenin 50mg/Kg	1.80±0.228^**	1.73±0.285***	1.65±0.262***	41.87	48	54.6
Indomethacin 25mgKg	1.34±0.060^***	1.41±0.346***	1.35±0.616***	56.3	59.5	61.4

Values are expressed as mean SD, and the number of animals is 6.

**p 0.001 compared with the change in paw volume of a control group (2% Carrageenan).

***p 0.0001 compared with the change in paw volume of a control group (2% Carrageenan).

Estimation of analgesia:

Eddy’s hot plate analgesiometer:

A separate group of animals (n = 6) were taken and evaluated for their time of response on Eddie's hot plate (in seconds) at 3h, 6h, and 9h after s.c. administration of Carrageenan into the sub-plantar region. Again, response time on the hot plate was recorded as time latency. Administration of Carrageenan produced a significant time-dependent decrease in latency (increase in jumping and licking).

After Apigenin administration to Carrageenan-inflamed mice, the response time on a hot plate was recorded. Apigenin (25mg/kg) and Apigenin (50mg/kg) are both time-dependent, which is represented in). (Figure 1)Indomethacin at 25mg/kg also showed a comparable effect to Apigenin.

Figure 1: Effect of s.c. administration of Carrageenan at 3h, 6h, 9h and s.c. administration of Apigenin (25mg/kg), Apigenin (50mg/kg), and Indomethacin (25mg/kg) on experiment groups at 3h, 6h, 9h. (On hot plate)

Values are expressed as mean + SEM of 6 mice in each group. ***P<0.001; ###P<0.001; and @@@p<0.001 compared with control (Two way ANOVA and Bonferroni).

DISCUSSION:

According to the results of this study, Apigenin (25 mg/kg, s.c.) causes paw edema, one of the most practical ways to screen anti-inflammatory drugs, and has a strong anti-inflammatory effect on Carrageenan¹¹. The development of Carrageenan-induced paw edema is bi-phasic, with the first phase being linked to the production of histamine, serotonin, and kinins and the second phase being linked to the release of prostaglandins and bradykinins whereas the kinins and prostaglandins mediate the more prolonged delayed onset responses called second phase^12-14. Both Apigenin (25mg/kg) and Apigenin (50mg/kg) significantly inhibited Carrageenan-induced paw edema, and this effect was dose and time-dependent. This anti-inflammatory effect of Apigenin may be due to the presence of flavonoids that possess anti-inflammatory and analgesic activity. Flavonoids are known to inhibit the enzyme prostaglandin synthetase, more specifically the endoperoxides, and are reported to produce anti-inflammatory effects^15,16. Because prostaglandins are involved in pain perception, Apigenin's analgesic activity could be due to inhibition of their synthesis. The presence of the flavonoids identified might be responsible for the anti-inflammatory and analgesic activities of Apigenin¹⁷.

This mechanism leads to the possibility of more than one mechanism being involved¹⁸. One possible interpretation of the results presented here is that Apigenin may be involved in the anti-inflammatory effect on Carrageenan-induced paw edema.

Prostaglandins have been long recognized as a major intermediary of inflammation. They're arachidonic acid metabolites synthesized by COX- 1 and COX- 2^19,20. Results indicate that Apigenin caused a statistically significant reduction in PGE2 content. This finding underlines the anti-inflammatory effect of Apigenin. It was also determined how apigenin affected the release of TNF in the inflammatory exudates in addition to the prostaglandin E2 (PGE2) evaluation. TNF is a pleiotropic cytokine involved in both acute and chronic inflammation²¹. More importantly; TNF induces the synthesis of PGE2. Apigenin's ability to reduce TNF release is consistent with the current study's PGE2 findings. As a result, an alternative explanation for the resulting decrease in PGE2 levels in inflammatory exudates could be interference with TNF release. Furthermore, in the same animal model, Apigenin also managed to increase the total antioxidant capacity in the inflammatory exudates. This increase in the total antioxidant capacity was also in reverse dose order, with the highest capacity obtained with the lowest dose tested.

In Carrageenan-induced paw edema (2%) in mice; paw volume was increased compared to normal mice, which was determined by a Vernier caliper. Administration of Apigenin at a dose of 25mg/kg, s.c., produced a significant effect on Carrageenan-induced paw edema, which was represented by percentage inhibition (41.2%). Whereas when a higher dose of Apigenin (50 mg/kg, s.c.) was administered, similar effects were observed, but the percentage inhibition (51% of the lower dose) was higher. This dose- and time-dependent effect of Apigenin is due to the presence of flavonoids and a reduction in oxidative stress.

In another study, the analgesic activity of Apigenin was determined. Carrageenan-induced inflammation showed an increase in time latency when subjected to a hot plate and tail flick. In this study, the s.c. injection of Apigenin (25mg/kg) increased the time latency period produced by Carrageenan injection (2%, s.c.). Similar to Apigenin (25mg/kg), Apigenin (50mg/kg) likewise considerably lengthened the delay. The results of the apigenin test on a hot plate and the tail flick test also show that it has anti-inflammatory effects and lessens the pain brought on by the injection of Carrageenan.

CONCLUSION:

In this study, Apigenin was taken based on the literature survey. Apigenin possesses anti-inflammatory and analgesic properties by inhibiting NF-B activation and the LPS-induced inflammatory response through multiple mechanisms in macrophages. The results provided important scientific evidence for the potential application of Apigenin as a therapeutic agent for inflammatory diseases.

Inflammation was induced in the animals by s.c. administration of Carrageenan to the sub-plantar surface of the right hind paw. The drug-treated group was compared to the control group for changes in paw volume and pain perception. After obtaining the results, statistical analysis showed that the 50 mg/kg Apigenin significantly decreased the paw volume and increased the duration of the response on a hot plate.

Thus, it can be summarized by stating that 50 mg/kg of Apigenin gives a promising effect on the treatment of inflammation.

ACKNOWLEDGMENTS:

We are thankful to Department of Pharmacy, Guru Ghasidas Vishvavidyalaya (GGV), Bilaspur, (C.G.) for support.

CONFLICTS OF INTEREST:

The authors declare no conflicts of interest.

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Received on 28.04.2023 Modified on 22.08.2023

Research J. Pharm. and Tech 2024; 17(4):1527-1530.

DOI: 10.52711/0974-360X.2024.00241