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The experimental approaches described in this study generated reproducible in vitro and in vivo data that enabled assessment of both the phytochemical composition and pharmacological activities of the methanolic extract of T. mantaly. All experimental measurements were performed in replicates, and results are presented as mean ± standard deviation (SD). Statistical significance between groups was determined using one-way ANOVA followed by Tukey’s post hoc test, with p < 0.05 considered significant.
Estimation of total phenolic and flavonoid contents using the Folin–Ciocalteu and aluminum chloride assays produced consistent absorbance values across replicate measurements, with calibration curves showing appropriate linearity for gallic acid and rutin standards (Figure 1 and Figure 2). The total phenolic content of the extract was quantified as 1.084 ± 0.445 mg gallic acid equivalent per g extract, while total flavonoid content was 0.114 ± 0.008 mg rutin equivalents per g extract. Under optimal conditions, absorbance readings remained stable with minimal variation between replicates, indicating reliable spectrophotometric performance. In contrast, irregular absorbance values or reduced linearity of the standard curves may reflect issues related to reagent freshness, incubation duration, or spectrophotometric handling. All spectrophotometric assays were performed in triplicate to ensure measurement reliability.
GC-MS of the methanolic extract revealed chromatograms containing multiple well-resolved peaks corresponding to identifiable phytochemical constituents. The GC–MS full scan chromatogram of the methanolic extract is shown in Figure 3. GC–MS profiling revealed twenty identifiable compounds with varying relative abundances (Table 1). The major constituents included 3,5-dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one (22.44% peak area), linoleic acid (15.41%), γ-sitosterol (14.52%), and n-hexadecanoic acid (11.85%), while other compounds such as vitamin E, squalene, and stigmasterol were detected in smaller proportions. Most of these compounds have been reported to possess anti-inflammatory, antioxidant, and membrane-stabilizing properties, suggesting they may contribute to the pharmacological effects observed in in vivo assays. Successful analyses were characterized by reproducible retention times and consistent peak area distributions across runs. Reduced peak resolution, baseline drift, or retention time variability may occur due to column contamination, inappropriate temperature programming, or excessive sample loading.
Treatment with the methanolic extract produced a dose-dependent attenuation of paw edema across the observation period. At the highest dose (300 mg/kg), paw thickness was reduced to 2.72 ± 0.79 mm at 4 h, compared with 2.66 ± 0.64 mm in the diclofenac group (Figure 4 and Supplementary Table 1). Correspondingly, percentage inhibition increased over time, reaching 59.16% at 4 h for the highest extract dose, closely approaching the standard drug value of 60.06% (Supplementary Table 2). Consistent reductions across time points indicate reliable anti-inflammatory responses, whereas increased variability may arise from inconsistencies in carrageenan administration or paw thickness measurement.
The control group showed a decline in latency from 6.06 ± 1.35 s at baseline to 5.08 ± 1.38 s at 120 min, whereas extract treatment produced a dose-dependent increase, reaching 11.32 ± 0.88 s at 120 min for the 300 mg/kg dose, comparable to 11.62 ± 0.59 s in the paracetamol group (Figure 5 and Supplementary Table 3). Correspondingly, the percentage inhibition of nociception increased over time, reaching 122.83% at 120 min with the highest extract dose, compared with 128.74% with the standard drug (Supplementary Table 4). Valid experimental outcomes are indicated by stable baseline latency values and progressive increases after treatment, while erratic responses may be associated with fluctuations in water bath temperature or variation in tail immersion depth.
Administration of the methanolic extract and paracetamol led to a gradual reduction in rectal temperature over the observation period. At the highest dose, temperature decreased to 37.21 °C at 3 h, closely matching the paracetamol group value of 37.18 °C (Figure 6 and Supplementary Table 5). Rectal temperature (°C) was measured at 0, 1, 2, and 3 h following oral administration of T. mantaly methanolic extract (100, 200, and 300 mg/kg) or paracetamol (150 mg/kg) in yeast-induced febrile rats (Figure 6), and the percentage reduction relative to the febrile control group is presented in Supplementary Table 6.
Overall, the representative results shown in Figures 1–6 illustrate the reproducibility of the experimental procedures and provide practical reference points for evaluating both successful outcomes and technically sub-optimal results when applying these methods.

Figure 1. Estimation of total phenolic content of T. mantaly methanolic extract. Total phenolic content was determined using the Folin–Ciocalteu method. The graph shows the comparative absorbance response of the T. mantaly methanolic extract (TMWM) and the gallic acid standard at different concentrations measured at 765 nm. Gallic acid was used as the reference standard. Data represent mean ± SD of triplicate determinations. Please click here to view a larger version of this figure.

Figure 2. Estimation of total flavonoid content of T. mantaly methanolic extract. Total flavonoid content was determined using the aluminum chloride colorimetric method and measured at 415 nm. Rutin was used as the reference standard. Data are expressed as mean ± SD of triplicate determinations. Please click here to view a larger version of this figure.

Figure 3. Gas chromatography–mass Spectrometry chromatogram of the methanolic extract of T. mantaly. The chromatogram shows the separation of the detected constituents, with peaks corresponding to the compounds listed in Table 1. Please click here to view a larger version of this figure.

Figure 4. Anti-inflammatory activity of T. mantaly methanolic extract in carrageenan-induced paw edema. Paw thickness (mm) was measured at 0, 1, 2, 3, and 4 h following oral administration of T. mantaly methanolic extract (100, 200, and 300 mg/kg) or diclofenac sodium (40 mg/kg) in Albino Wistar rats. Data are expressed as mean ± SD (n = 5). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (*p < 0.05, **p < 0.01 vs control). Please click here to view a larger version of this figure.

Figure 5. Analgesic activity of T. mantaly methanolic extract assessed by the tail immersion test. Tail withdrawal latency (seconds) was recorded at 0, 30, 60, 90, and 120 min following oral administration of T. mantaly methanolic extract (100, 200, and 300 mg/kg) or paracetamol (20 mg/kg). Data are expressed as mean ± SD (n = 5). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Please click here to view a larger version of this figure.

Figure 6. Antipyretic activity of T. mantaly methanolic extract in yeast-induced pyrexia. Rectal temperature (°C) as measured at 0, 1, 2, and 3 h following oral administration of T. mantaly methanolic extract (100, 200, and 300 mg/kg) or paracetamol (150 mg/kg) in yeast-induced febrile rats. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. Data are expressed as mean ± SD (n = 5). Please click here to view a larger version of this figure.
| Sr. No. | R.T | Peak Area | Compound Name | Mol.Formula | Mol. Weight
(g/mol) | Chemical Class |
| 1 | 2.389 | 22.44 | 3,5-Dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one | C6H8O4 | 144.12 | Pyranones |
| 2 | 3.157 | 13.73 | 5-Hydroxymethylfurfural | C6H6O3 | 126.11 | Furan |
| 3 | 10.15 | 1.49 | Methyl palmitate | C17H34O2 | 270.45 | Fatty acid methyl ester |
| 4 | 10.94 | 11.85 | n-Hexadecanoicacid | C16H32O2 | 256.42 | Fatty acid |
| 5 | 12.11 | 0.25 | 9,12-Octadecadienoic acid (Z,Z)-, methyl ester | C19H34O2 | 294.47 | Fatty acid |
| 6 | 12.20 | 0.75 | trans-13-Octadecenoic acid, methyl ester | C19H36O2 | 296.49 | Fatty acid methyl ester |
| 7 | 12.64 | 1.36 | Heptadecanoic acid, 16-methyl-, methyl ester | C19H38O2 | 284.50 | Fatty acid |
| 8 | 13.58 | 15.41 | Linoleic acid | C18H32O2 | 280.40 | Fatty acid |
| 9 | 16.45 | 0.01 | Stearic acid | C18H36O2 | 284.48 | Fatty acid |
| 10 | 16.84 | 0.08 | Heneicosanoicacid | C21H42O2 | 326.60 | Fatty acid |
| 11 | 19.69 | 0.98 | 6H-Thieno[2,3-b]pyrrole-5-carboxylic acid | C7H5NO2S | 167.18 | Heterocyclic carboxylic acids |
| 12 | 22.45 | 1.77 | Squalene | C30H50 | 410.70 | Triterpene |
| 13 | 22.82 | 0.20 | α-TocospiroB | C29H50O4 | 462.70 | Benzopyranoids |
| 14 | 24.85 | 2.15 | VitaminE | C29H50O2 | 430.70 | Tocopherols |
| 15 | 25.49 | 0.66 | Campesterol | C28H48O | 400.70 | Phytosterols |
| 16 | 25.66 | 1.60 | Stigmasterol | C29H48O | 412.70 | Phytosterols |
| 17 | 26.07 | 14.52 | γ-Sitosterol | C29H50O | 414.7 | Phytosterols |
| 18 | 26.30 | 0.40 | 2(1H)Naphthalenone,exahydro-4,8a-dimethyl-6-(1-methyl ethenyl)- | C15H22O | 218.33 | Sesquiterpenoid ketone |
| 19 | 26.612 | 0.82 | α-Amyrin | C30H50O | 426.70 | Pentacyclictriterpenoid |
| 20 | 27.48 | 3.07 | 2-Ethylacridine | C15H13N | 207.27 | Acridines |
Table 1. GC–MS profile of T. mantaly methanolic extract. Identified compounds with corresponding retention time (RT), peak area (%), molecular formula, molecular weight, and chemical class based on GC–MS analysis.
Supplementary Table 1. Anti-inflammatory activity of T. mantaly methanolic extract (paw thickness). Paw thickness (mm) measured at different time points following carrageenan injection. Data are expressed as mean ± SD (n = 5). *p < 0.05, **p < 0.01 compared with control using one-way ANOVA.Please click here to download this file.
Supplementary Table 2. Percentage reduction of paw edema. Percentage inhibition of carrageenan-induced paw edema calculated relative to control animals at corresponding time points.Please click here to download this file.
Supplementary Table 3. Analgesic activity of T. mantaly methanolic extract (tail immersion test). Tail withdrawal latency expressed as mean ± SD (n = 5). Statistical significance was determined using one-way ANOVA.Please click here to download this file.
Supplementary Table 4. Percentage inhibition of analgesic activity. Percentage increase in tail withdrawal latency calculated relative to baseline values following treatment.Please click here to download this file.
Supplementary Table 5. Antipyretic activity of T. mantaly methanolic extract. Rectal temperature (°C) measured at different time points in yeast-induced febrile rats. Values represent mean ± SD (n = 5).Please click here to download this file.
Supplementary Table 6. Percentage inhibition of antipyretic activity. Percentage reduction in rectal temperature calculated relative to febrile control animals.Please click here to download this file.