Introduction

Inflammation is a natural biological response to injury or infection, playing a critical role in the body's healing process. However, chronic inflammation can lead to a host of diseases, including arthritis, cardiovascular disorders, and even cancer. Traditionally, synthetic anti-inflammatory drugs, such as non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids, have been the cornerstone of inflammation management. While these drugs are effective, their long-term use is often associated with significant side effects, including gastrointestinal bleeding, cardiovascular risks, and renal impairment. These limitations have spurred a growing interest in natural alternatives, particularly plant-based anti-inflammatory agents, which offer a safer and potentially more sustainable approach to managing inflammation. Plants have been used for centuries in traditional medicine systems across the world to treat inflammatory conditions. Numerous studies have identified a wide array of bioactive compounds in plants, such as flavonoids, alkaloids, terpenoids, and polyphenols, which exhibit potent anti-inflammatory properties. These natural compounds work through multiple mechanisms, including the inhibition of pro-inflammatory enzymes, scavenging of free radicals, and modulation of immune responses. Unlike synthetic drugs, which often target a single pathway, plant-based anti-inflammatory agents tend to exert their effects through a multi-targeted approach, potentially leading to fewer side effects and a broader therapeutic impact. One such Agad is Champak Agad. Acharya Vagbhat has mentioned Champak Agad in Keetlootadivishpratishedham chapter of Uttarsthan. This Agad is indicated to be useful in all Loota and Keeta Visha and can treat symptoms of insect bites.^[1] Most of symptoms of Insect bites are manifested on skin like Pain, inflammation, burning sensation, redness, and secondary infections due to breaking of skin barrier. The benefits of natural anti-inflammatory plants extend beyond their efficacy. They are often more accessible, cost-effective, and culturally acceptable, particularly in regions where traditional medicine is deeply rooted. Moreover, growing body of scientific evidence supporting their therapeutic potential has led to increased integration of plant-based remedies into modern healthcare practices.

4. Analytical Study

Extraction Process

• Extraction involves the separation of medicinally active portions of plant or animal tissues from the inactive or inert components by using selective solvents in standard extraction procedures.
• It is the method of removing active constituents from a solid or liquid using a liquid solvent.
• The purpose of this extraction procedure for crude drugs is to attain the therapeutically desired portion.
• There are various types of extraction procedures like maceration, infusion, percolation, etc.
• For the present study Soxhlet Extraction method was chosen.

5. Soxhlet Extraction Method

For the aim of extraction, distilled water and ethanol were used as solvents hence, two extracts of the drug were obtained.

• Aqueous extract
• Alcohol extract - ethanolic

Materials Required

• Round bottom flask (500 ml)
• Screw cap tube
• Precision electronic scales
• Tarred Petri dish
• Solvents: 95% ethanol and distilled water
• Soxhlet chamber/Thimble (500 ml)
• Heating mental
• Condenser along with 2 tubes
• Cotton
• Water bath
• Glass beads
• Filter paper

For Ethanolic Extraction

100g of drug was taken and put in Soxhlet chamber, which was then filled with 100ml of ethanol. Another 250ml of ethanol was added to round-bottom flask along with some glass beads, and entire setup was connected to a heating mantle.

Figure 1.b: Soxhlet heat extraction

Figure 1.c: Water Bath

Figure 1.d: Final prepared Ethanolic extract and Aqueous extract

• Negative control samples contained the same amount of egg albumin and PBS with 2.0 ml of distilled water.
• The mixture was incubated at 37± 2°C for 15 min and then heated at 70°C for 5 min to induce denaturation.
• After cooling, the absorbance was recorded at 660 nm using the vehicle as a blank. The percentage inhibition of protein denaturation was calculated using the formula

(%) Percentage inhibition = A_c - A₁/A_c × 100

Where A₁ is the absorbance of the test sample and A is the absorbance of the control

Figure 2.a: Fresh egg albumin

Observations and Results

Table 1: Anti-inflammatory activity of Diclofenac (standard drug) at different concentrations.

Table 2: Anti-inflammatory activity of Champak Agad extracts at different concentrations

The present study demonstrated concentration-dependent inhibition of egg albumin protein denaturation by extracts of Champak Agad at concentrations of 25 mg/ml, 50 mg/ml, 100 mg/ml, 150 mg/ml, and 200 mg/ml. Diclofenac sodium, used as a standard drug at a range between 25mg/ml and 100 mg/ml, also showed concentration-dependent inhibition of protein denaturation, being used as a reference.

The maximum inhibition of 25% at the concentration of 25 mg/ml was observed with aqueous extract, while the ethanol extract achieved 22.41%. The inhibition increased further to 42.5% and 38.79% at a concentration of 50 mg/ml in water and ethanol extract respectively.

In this case, a more pronounced effect was observed compared with lower concentrations. The inhibition rate with aqueous extract at a concentration of 100mg/ml reached 67.5%, while for the ethanol extract it gave 53.8%. At 150mg/ml, the inhibition reached 77.5% with the aqueous extract and 66.55% with the ethanolic extract.

At the highest concentration of 200 mg/ml, the aqueous extract showed the highest inhibition (85%), while the ethanolic extract followed closely with 82.5%.

Similarly, berberine acts through its signaling pathways to modulate inflammation in many systems by upregulating COX-2 after pro-inflammatory cytokines, with certain pathways eliciting their behaviour.^[6] Manjishtha (Rubia cordifolia) causes significant inhibition of the production of pro-inflammatory cytokines, with the consequent amelioration of knee swelling/thickening inflammation during models of rheumatoid arthritis.^[7] Tagar extracts have been shown to produce a dose-dependent reduction in carrageenan-induced paw edema; additionally, Tagar supports acute inflammatory response.^[8] Nagkesar (Mesua ferrea) modulates NF-κB signalling to exhibit anti-inflammatory properties and reduce the levels of key cytokines such as IL-1 and TNF-α.^[9] Pattang (Caesalpinia sappan) suppresses the expression of IL-1β and TNF-α, inhibiting NO production via iNOS downregulation, and reduces COX-2 activity through NF-κB signal pathway inhibition.^[10]

The Tikta Rasa and Ushna Virya characteristics of these ingredients enhance digestion, metabolism, and thus nutrient absorption, boosting modulated immunity. Proper digestion aids in the availability of antioxidants and nutrients that relieve oxidative stress and inflammation.^[11] Modern immune research backs metabolic reprogramming that allows immune cells to redirect themselves to an anti-inflammatory state^[12] and hence supports the anti-inflammatory actions of Champak Agad. The concentration-dependent inhibition of protein denaturation by Champak Agad is consistent with the combined effective action of its constituents. These results bridge the world of ancient Ayurvedic wisdom with contemporary evidence as they vouch for the therapeutic scope of Champak Agad as a natural anti-inflammatory drug.

Conclusion

The present study demonstrates that Champak Agad extracts exhibit significant anti-inflammatory activity through concentration-dependent inhibition of protein denaturation. The ethanolic and aqueous extracts were found to inhibit denaturation with increasing rates when higher concentrations were used, where the aqueous extract always outperformed the ethanolic extract consistently. The maximum inhibition of the aqueous extract was achieved at the highest concentration tested of 200 mg/ml, confirming its superior effectiveness.