Specimens of all three target plant species were collected during their active flowering phase in the floristic region of Kartli in 2020–2021. Voucher specimens (P. woronowii – ТВPH-21166, P. macrocalyx – ТВPH-21329, P. saguramica – ТВPH-21749) are deposited in the herbarium of the Iovel Kutateladze Institute of Pharmacochemistry at TSMU.8 The plant materials were air-dried, ground into powder, and extracted three times with 80% ethanol. The resulting extracts were concentrated and subjected to freeze-drying. Further chromatographic separation was performed using a Diaion HP-20 column, eluted with a water-methanol gradient ranging from 0% to 100% methanol.9 This process yielded four fractions from each species: P.m1, P.m2, P.m3, P.m4 from P. macrocalyx; P.w1, P.w2, P.w3, P.w4 from P. woronowii; and P.s1, P.s2, P.s3, P.s4 from P. saguramica. Before in vivo testing, all fractions were dissolved in distilled water.

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Experimental animals
Outbred albino CD1 mice weighing 28±2 g (n=40) were used in the study. The animals were obtained from the animal facility of the I. Kutateladze Institute of Pharmacochemistry, Tbilisi State Medical University (TSMU), and acclimatized for one week in the Department of Preclinical Pharmacological Research under standard laboratory conditions (temperature: 20±2°C; humidity: 55–65%; 12/12-hour light/dark cycle). Animals were provided granulated food (4 g/animal/day) and water ad libitum. All procedures complied with the EU Directive 2010/63 and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health10,11 and were approved by the TSMU Ethics Committee on Animal Research (Approval No. AP-56-2022).

Assessment of analgesic activity: hot plate assay

Fractions were dissolved in distilled water and administered intraperitoneally to mice (n=6 per group) at a dose of 50 mg/kg. Control animals (n=6) received 0.4 mL of normal saline via the same route. Analgesic activity was evaluated using the hot plate assay. Mice were individually placed in a transparent cylindrical chamber with a metal surface maintained at 50±2°C. The latency to the first nociceptive response (hind paw licking or jumping) was recorded at baseline (before injection), 30 minutes, and 60 minutes post-administration. Animals with baseline latencies exceeding 15 seconds were excluded. The analgesic effect was calculated using the formula:

E (%) = ((Tn – T₀) / T₀) × 100,

where T₀ is the baseline latency, and Tn is the latency at 30 or 60 minutes post-injection.12

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Assessment of anti-inflammatory activity: carrageenan-induced paw edema assay

The same group distribution and dosing regimen as described above were applied. Acute inflammation was induced by subplantar injection of 50 μL of 1% carrageenan solution in normal saline into the right hind paw of each mouse. One hour before carrageenan injection, animals received intraperitoneal injections of either 0.5 mL of normal saline (control) or 0.5 mL of the test compound solution (50 mg/kg). Paw thickness was measured with a digital micrometer immediately before carrageenan injection (baseline) and 2 hours afterward. Anti-inflammatory efficacy was calculated as the percentage inhibition of inflammation using the formula:

E (%) = [1 – (ΔTₑₓₚ / ΔTₒₙ)] × 100,

where ΔTcₒₙ and ΔTₑₓₚ represent the mean increase in paw thickness (in conventional units) in control and experimental groups, respectively.13​