Phytochemical Profiling, Gas Chromatography–Mass Spectrometry Analysis, and In Vivo Activity of Terminalia mantaly in Rats

Summary

Here, we present a protocol that integrates phytochemical analysis, Gas Chromatography–Mass Spectrometry characterization, and validated in vivo assays to evaluate the anti-inflammatory, analgesic, and antipyretic activities of Terminalia mantaly, providing a reproducible framework for pharmacological assessment of medicinal plants.

Abstract

Synthetic drugs for inflammation, pain, and fever are effective but often cause adverse effects, increasing interest in plant-derived therapeutic alternatives. Terminalia mantaly H. Perrier, traditionally used in African medicine, has limited scientific validation. This study presents an integrated experimental protocol combining phytochemical screening, quantitative analysis of phenolic and flavonoid compounds, gas chromatography–mass spectrometry (GC-MS) characterization, and in vivo pharmacological testing to evaluate the therapeutic potential of the methanolic extract of T. mantaly. Phytochemical screening confirmed the presence of multiple bioactive classes, and GC–MS analysis identified twenty constituents, including fatty acids, phytosterols, tocopherols, and other bioactive compounds with known anti-inflammatory and antioxidant properties. In vivo pharmacological evaluation in rats demonstrated dose-dependent anti-inflammatory activity in the carrageenan-induced paw edema model, significant prolongation of the pain-withdrawal latency in the tail immersion assay, and a reduction in yeast-induced pyrexia. At higher doses, the extract showed effects comparable to standard drugs such as diclofenac and paracetamol. Acute toxicity and cytotoxicity assessments indicated a favorable safety profile within the tested dose range. Together, these findings validate the ethnomedicinal use of T. mantaly and demonstrate a reproducible experimental framework for linking phytochemical composition with pharmacological activity. The protocol provides a practical methodological reference for researchers investigating medicinal plants and supports the development of plant-derived agents for the management of inflammatory and febrile conditions.

Introduction

Inflammation, pain, and fever are interconnected pathological processes that underlie a wide range of acute and chronic diseases. Although synthetic drugs such as nonsteroidal anti-inflammatory agents, corticosteroids, and paracetamol remain the mainstay of therapy, their long-term use is often associated with gastrointestinal irritation, renal complications, hepatotoxicity, and other adverse effects¹. These limitations have prompted an increasing interest in medicinal plants as safer, more sustainable alternatives for managing inflammatory and febrile conditions. Natural products are well recognized for their rich diversity of bioactive molecules, many of which serve as leads for modern drug development. Within this context, T. mantaly, a member of the family Combretaceae, has drawn attention due to its ethnomedicinal use in African traditional systems for treating infections, gastrointestinal ailments, and inflammatory disorders^2,3. Despite its traditional relevance, systematic pharmacological studies validating its therapeutic potential remain limited. In particular, there is a scarcity of comprehensive investigations that integrate phytochemical screening, in vivo biological evaluations, and advanced chemical profiling methods, such as GC–MS. Addressing this gap is crucial for scientifically substantiating its folkloric applications and identifying potential lead compounds for pharmaceutical use⁴. In the present study, the methanolic extract of T. mantaly was subjected to detailed phytochemical and pharmacological evaluation. Preliminary analysis confirmed the presence of key metabolites, including phenolics, flavonoids, tannins, saponins, and terpenoids, all of which are known to contribute to anti-inflammatory, analgesic, and antioxidant effects⁵. Quantitative assays further revealed that the extract contained substantial levels of phenolic and flavonoid compounds, with values comparable to reference standards, suggesting a strong antioxidant potential.

Animal models were used because the pharmacological responses evaluated involve integrated inflammatory, neural, and thermoregulatory pathways that cannot be reproduced in vitro. The in vivo assays provided clear pharmacological evidence of bioactivity. The anti-inflammatory (paw edema induced by carrageenan), analgesic (tail immersion), and antipyretic (pyrexia induced by yeast) properties were tested in rats, demonstrating dose-dependent anti-inflammatory activity similar to that of diclofenac sodium at higher doses. Similarly, the extract enhanced tail-immersion latencies in rats, confirming its analgesic efficacy, while yeast-induced pyrexia models showed marked antipyretic effects, closely matching those of paracetamol⁶. Chemical profiling through GC–MS analysis provided deeper insights into its bioactive composition. A total of 20 compounds were identified, including 3,5-dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one, linoleic acid, γ-sitosterol, squalene, and vitamin E. These compounds are well documented for their antioxidant, anti-inflammatory, and cardioprotective properties, supporting the pharmacological outcomes observed^7,8. Importantly, toxicity assessments revealed negligible cytotoxic effects, underscoring its safety at the tested doses. Taken together, this study addresses a critical gap by providing both phytochemical and pharmacological evidence for the medicinal value of T. mantaly. The findings not only substantiate its traditional applications but also highlight its potential as a natural source of lead compounds for developing novel therapies against inflammation and related disorders. The experimental models used in this study primarily reflect acute inflammatory and nociceptive responses and therefore provide preliminary pharmacological evidence rather than a direct indication of chronic therapeutic efficacy.

Protocol

All animal experiments were conducted in accordance with international guidelines for the care and use of laboratory animals and complied with OECD recommendations. The study protocol was approved by the Animal Care and Use Committee (IACUC) of Bahauddin Zakariya University, Multan. (Approval No: 3 /UREC/2025). All efforts were made to minimize animal suffering and reduce the number of animals used.

1. Preparation of T. mantaly whole methanolic extract (TMWM)

1. Collect fresh stems, roots, and leaves of T. mantaly, authenticate the plant material, and deposit a voucher specimen in a recognized herbarium.
   NOTE: The plant material used in this study was collected from Punjab Nursery, Mattital Road, Multan, on November 12th, 2023. The geographic coordinates of the collection site are 30.2236° N, 71.4755° E. The plant was taxonomically authenticated by taxonomist Prof.Dr. Zafar Ullah Zafar, Professor of botany, Bahauddin Zakariya University, Multan, and a voucher specimen (www.theplantlist.org/tpl1.1/record/kew-2434420) was deposited in the herbarium of Bahauddin Zakariya University, Multan. The plant was collected from its natural habitat under appropriate environmental conditions, and the collection site was documented with photographs.
2. Wash the plant material with tap water followed by distilled water to remove contaminants, then shade-dry at 25–30 °C for 7–10 days until constant weight is achieved.
3. Grind the dried material to coarse powder and weigh 500 g using an analytical balance. Transfer the powder into a clean glass container and add 2.5 L of methanol.
4. Macerate the mixture at room temperature for 72 h with intermittent shaking every 6–8 h. Filter through Whatman No.1 filter paper.
5. Concentrate the filtrate under reduced pressure using a rotary evaporator at 39 °C, then store the dried extract in airtight containers at room temperature until use.
   CAUTION: Methanol is toxic and flammable. Perform handling inside a fume hood while wearing gloves and protective eyewear. Dispose of solvent waste according to institutional chemical safety guidelines.
   ​NOTE: The dried extract can be stored in airtight containers at room temperature for several weeks before further analysis.

2. Estimation of total phenolic content (Folin–Ciocalteu method)

1. Prepare gallic acid stock solution (100 µg/mL) by dissolving 10 mg gallic acid in 100 mL methanol and prepare serial dilutions ranging from 1–10 µg/mL.
2. Dissolve 10 mg extract in 10 mL methanol to obtain a 1 mg/mL solution. Transfer 0.5 mL of each standard or sample into test tubes.
3. Add 2.5 mL Folin–Ciocalteu reagent diluted 1:1 with water, then add 2.0 mL of 7.5% sodium carbonate solution. Mix and incubate at 25 ± 2 °C for 30 min.
4. Measure absorbance at 765 nm against a reagent blank and calculate total phenolic content as mg gallic acid equivalents per g extract.

3. Estimation of total flavonoid content (Aluminum chloride method)

1. Prepare a rutin stock solution (1000 µg/mL) by dissolving rutin in methanol, then prepare serial dilutions in the range of 10–80 µg/mL.
2. Transfer 0.5 mL of the standard or extract solution into test tubes and add 1.5 mL methanol, 0.1 mL of 10% aluminum chloride (AlCl₃) solution, 0.1 mL of 1 M potassium acetate solution, and 2.8 mL distilled water.
3. Incubate at room temperature for 30 min and measure absorbance at 415 nm. Express results as mg rutin equivalents per g extract⁹.

4. Gas chromatography–mass spectrometry (GC–MS) analysis

1. Dissolve 10 mg extract in 1 mL HPLC-grade methanol and filter through a 0.22 µm syringe filter into a GC vial.
2. Perform analysis using an Agilent 7890B GC coupled with a 5977A MS equipped with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 µm). Use helium as carrier gas at 1.0 mL/min with injector temperature at 250 °C.
3. Maintain ion source temperature at 230 °C and quadrupole temperature at 150 °C. Program the oven from 60 °C (2 min hold) to 180 °C at 10 °C/min and then to 280 °C at 5 °C/min with a final hold of 10 min.
4. Inject 1 µL of the filtered extract in split mode (10:1) and record chromatograms over an m/z range of 40–600. Run a solvent blank before sample injection.
5. Identify compounds by comparing spectra with the NIST mass spectral library and confirm identities using retention time and peak area normalization.

5. Anti-inflammatory activity (carrageenan-induced paw edema)

1. Use healthy Albino Wistar rats weighing 180–220 g and acclimatize for 7 days. Fast animals overnight before the experiment with free access to water.
2. Randomly divide animals into five groups (n = 5 per group). Administer normal saline to controls; orally administer 100, 200, and 300 mg/kg to the treatment groups; and administer diclofenac sodium (40 mg/kg, subcutaneously) to the standard group.
3. After 1 h, inject 0.1 mL of freshly prepared 1% carrageenan into the subplantar region of the right hind paw.
4. Measure paw thickness at 0, 1, 2, 3, and 4 h using a digital vernier caliper and calculate the increase relative to baseline.
5. Determine the percentage inhibition of edema compared with the control group¹⁰.
   CAUTION: Carrageenan powder may cause irritation. Avoid inhalation and skin contact and dispose of biological waste according to institutional biosafety procedures.

6. Analgesic activity (tail immersion test)

1. Use acclimatized Albino Wistar rats maintained under a 12 h light/dark cycle with free access to food and water.
2. Divide animals into five groups (n = 5). Administer distilled water to controls, extract orally at 100, 200, and 300 mg/kg to treatment groups, and paracetamol (20 mg/kg) to the standard group.
3. Maintain the water bath at 55 ± 1 °C. Immerse 1–2 cm of the distal tail and record baseline withdrawal latency using a 15-s cut-off time.
4. Repeat measurements at 30, 60, 90, and 120 min after treatment, ensuring the same portion of tail is immersed each time.
5. Calculate the percentage increase in latency and express results as mean ± SD.

7. Antipyretic activity

1. Use acclimatized rats and record normal rectal temperature using a lubricated digital thermometer.
2. Prepare a 20% brewer’s yeast suspension in distilled water and inject subcutaneously at 10 mL/kg into the dorsum to induce pyrexia.
3. After 18 h, measure rectal temperature again and exclude animals that do not show at least a 0.5 °C increase.
4. Randomly divide febrile rats into five groups (n = 5 per group). Administer normal saline to the control group, plant extract orally at 100, 200, and 300 mg/kg to the treatment groups, and paracetamol (150 mg/kg) to the standard group.
5. Measure rectal temperature at 0, 1, 2, and 3 h after treatment and calculate the reduction relative to baseline¹¹.
   CAUTION: Brewer’s yeast suspensions are biological materials and should be handled using standard biosafety precautions. Dispose of unused suspension and contaminated materials as biological waste.
   ​NOTE: After yeast injection, animals may be returned to cages and maintained under standard conditions until temperature measurement.

8. Acute toxicity studies

1. Conduct the study according to Organization for Economic Co-operation and Development (OECD) guideline 423 using healthy Albino Wistar rats of either sex weighing 180–220 g.
2. Fast animals overnight before dosing and randomly divide them into groups (n = 5 per group).
3. Administer a single oral dose of 5 g/kg body weight of the extract to the treatment animals and distilled water (1 mL/kg) to the control animals.
4. Observe animals continuously for the first 4 h after dosing, periodically during the first 24 h, and daily for 14 days.
   NOTE: Observation criteria include changes in skin and fur condition, eyes and mucous membranes, respiratory pattern, salivation, tremors, convulsions, locomotor activity, and general behavioral responses.
5. Record clinical signs of toxicity, mortality, and body weight on Day 0, Day 7, and Day 14.
6. Assess safety based on survival rate and observed behavioral or physical changes¹².
   ​NOTE: Behavioral scoring includes monitoring of posture, gait, activity level, grooming behavior, response to external stimuli, and signs of lethargy or hyperactivity.

9. Statistical analysis

1. Compile all experimental data and express results as mean ± standard deviation with n = 5 animals per group.
2. Enter the data into GraphPad Prism software in column format.
3. Assess data normality using the Shapiro–Wilk test before applying comparative analysis.
4. Perform statistical analysis using GraphPad Prism software by selecting: Analyze → One-way ANOVA → Tukey multiple comparisons.
5. Perform one-way analysis of variance followed by Tukey’s post hoc multiple comparison test to determine statistical significance between groups.
6. Consider differences statistically significant at p < 0.05.

Results

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.

Discussion

In this study, a combination of phytochemical characterization and established in vivo pharmacological models was used to evaluate the anti-inflammatory, analgesic, and antipyretic potential of the methanolic extract of T. mantaly. The experimental models employed are widely used in natural product research and provide a practical framework for preliminary assessment of biological activity.

The reproducible retention times of the compounds support the extract's chemical complexity...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Albino Wistar rats	Institutional animal house	N/A	Experimental animals
Aluminum chloride	Sigma-Aldrich	237051	Flavonoid assay
Brewer’s yeast	Sigma-Aldrich	YSC2	Pyrexia induction
Carrageenan	Sigma-Aldrich	C1013	Paw edema induction
Centrifuge	Eppendorf	5810R	Sample processing
Diclofenac sodium	Sigma-Aldrich	D6899	Anti-inflammatory standard
Digital rectal thermometer	Omron	MC-246	Rectal temperature measurement
Digital vernier caliper	Mitutoyo	500-196-30	Paw thickness measurement
Distilled water	In-house	N/A	Solvent
Folin–Ciocalteu reagent	Sigma-Aldrich	F9252	Phenolic assay
Gallic acid	Sigma-Aldrich	G7384	Phenolic standard
GC–MS system	Agilent Technologies	7890B–5977A	Phytochemical profiling
GraphPad Prism	GraphPad Software	Version 8.0	Statistical analysis software
Methanol (HPLC grade)	Merck	106009	Extraction solvent
Oral gavage needle	Instech	FTP-20-38	Oral administration
Paracetamol	Sigma-Aldrich	P0300000	Analgesic and antipyretic standard
Potassium acetate	Merck	104820	Flavonoid assay
Rotary evaporator	Büchi	R-200	Extract concentration
Rutin	Sigma-Aldrich	R5143	Flavonoid standard
Sodium carbonate	Merck	106392	Phenolic assay
UV–Visible spectrophotometer	Shimadzu	UV-1800	Absorbance measurement
Water bath (thermostatic)	Memmert	WNB14	Tail immersion test