PHYTOCHEMICAL CHARACTERIZATION AND EVALUATION OF ANTIOXIDANT, ANTIMICROBIAL, ANTI-INFLAMMATORY, AND CYTOTOXIC ACTIVITIES OF SEQUENTIAL EXTRACTS FROM RIPE BANANA (MUSA PARADISIACA L.) PEEL

Classified as probably not a substantial claim.

The MTT assay assesses mitochondrial activity to indicate cell viability.

“Cytotoxic potential of the extracts was evaluated using the MTT assay, which measures mitochondrial activity as an indicator of cell viability (Abolhasanzadeh et al., 2024;Adeniyi-Akee et al., 2025;Ali et al., 2026;Zohmachhuana et al., 2022).”

• cites Abolhasanzadeh et al., 2024 sourcePartly supported

  The Materials and methods section (page unavailable) uses the MTT assay to assess MKN-45 cell viability and describes measuring formazan absorbance. It does not state that the assay measures mitochondrial activity, so that part of the attributed explanation is unsupported by our copy. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites Adeniyi-Akee et al., 2025 sourceCould not verify

  The Materials and Methods sections describe a brine shrimp lethality test and counting dead and surviving larvae to assess cytotoxicity (page unavailable). A search for MTT and mitochondrial activity found no passage explaining whether the MTT assay indicates cell viability through mitochondrial activity. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites Ali et al., 2026 sourcePartly supported

  The Materials and methods section (In vitro cell line study; page unavailable) describes MTT being metabolized to formazan and its optical density being used as an indicator of cell number. This supports the assay’s use to assess viable cells, but the paper does not specify that the activity measured is mitochondrial. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

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The dried powdered plant material and prepared extracts showed no fungal growth and were free from microbial contamination.

“The results indicated the absence of fungal growth, confirming that the plant material and extracts were free from microbial contamination and suitable for further experimental studies (Obuka et al., 2021).”

• cites Obuka et al., 2021 sourceCould not verify

  The Sample preparation and Microbiological stability tests sections (pages unavailable) describe bio-based building-material samples deliberately inoculated with fungi, not dried plant powder or prepared plant extracts. A search for powdered plant material, extracts, and microbial contamination found no passage testing whether those preparations were contamination-free. Fungal growth reported for the building materials does not establish the condition of the different preparations in the claim. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

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The sequential extracts effectively inhibited both bacterial and fungal strains in disc diffusion and MIC assays.

“Antimicrobial activity assessed by disc diffusion and MIC methods showed effective inhibition against both bacterial and fungal strains.”

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The sequential extracts showed anti-inflammatory activity in protein denaturation, HRBC membrane stabilization, and carrageenan-induced paw edema models.

“Anti-inflammatory potential was confirmed through protein denaturation, HRBC membrane stabilization assays, and carrageenan-induced paw edema model, with the methanol extract demonstrating significant activity.”

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The ethyl acetate extract showed moderate antimicrobial activity, whereas the n-hexane extract had minimal inhibition.

“Ethyl acetate extract exhibited moderate activity, while nhexane extract displayed minimal inhibition.”

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In the DPPH assay, the IC50 values were 43.5 μg/mL for n-hexane, 33.33 μg/mL for ethyl acetate, 30.8 μg/mL for methanol, and 17.7 μg/mL for ascorbic acid.

“| n-Hexane | 43.5 |”

• Also stated in: § 5. CONCLUSION · plain

  “| Ethyl Acetate | 33.33 |”

• Also stated in: § 5. CONCLUSION · plain

  “| Methanol | 30.8 |”

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In the MTT assay, the IC50 values were >200 μg/mL for n-hexane, 112.6 μg/mL for ethyl acetate, and 68.4 μg/mL for methanol.

“| n-Hexane | >200 |”

• Also stated in: § 5. CONCLUSION · plain

  “| Ethyl Acetate | 112.6 |”

• Also stated in: § 5. CONCLUSION · plain

  “| Methanol | 68.4 |”

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The ethyl acetate extract had moderate cytotoxicity, while the n-hexane extract was mostly non-toxic.

“Ethyl acetate extract showed moderate activity, whereas n-hexane extract was largely non-toxic.”

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MIC values agreed with disc diffusion results, showing that lower methanol extract concentrations inhibited microbial growth.

“The MIC values corroborated the disc diffusion results, indicating that lower concentrations of methanol extract were sufficient to inhibit microbial growth.”

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DPPH, reducing power, and hydrogen peroxide scavenging all increased with concentration, and the methanol extract had the lowest IC50 values.

“The DPPH radical scavenging, reducing power, and hydrogen peroxide scavenging assays consistently displayed a concentration-dependent increase in activity, with the methanol extract exhibiting the lowest IC₅₀ values.”

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The methanol extract significantly inhibited paw oedema, particularly at 3-4 hours, suggesting suppression of prostaglandin-mediated inflammation.

“The methanol extract demonstrated significant inhibition of paw oedema, especially in the later phase (3-4 hours), indicating suppression of prostaglandin-mediated inflammation.”

• Also stated in: § Figure 13: In Vivo Anti-inflammatory Activity Profile 3.12 Cytotoxicity Assessment by MTT Assay · boosted

  “The in vivo carrageenan-induced paw edema model further validated these findings, showing significant inhibition of inflammation, particularly during the late phase associated with prostaglandin release.”

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Flavonoids were detected in the methanol and ethyl acetate extracts but not in the n-hexane extract.

“| Flavonoids | + | + | - |”

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The n-hexane extract had MIC values of 250 μg/mL against Bacillus subtilis and 125 μg/mL against Escherichia coli.

“In contrast, the n-hexane extract displayed the weakest activity, with MIC values of 250 µg/mL against Bacillus subtilis and 125 µg/mL against Escherichia coli.”

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The n-hexane extract had the lowest phenolic content (36.08 ± 1.20 mg GAE/g) and the highest IC50 (43.5 μg/mL), indicating weaker free radical scavenging.

“The nhexane extract showed the lowest phenolic content (36.08 ± 1.20 mg GAE/g) and the highest IC₅₀ value (43.5 µg/mL), suggesting weaker free radical scavenging potential.”

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The ethyl acetate extract had MIC values of 125 μg/mL against Bacillus subtilis and 62.5 μg/mL against Escherichia coli.

“The ethyl acetate extract showed moderate antimicrobial potency with MIC values of 125 µg/mL and 62.5 µg/mL against Bacillus subtilis and Escherichia coli, respectively.”

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In the H2O2 scavenging assay, the IC50 values were 41.8 μg/mL for n-hexane, 41.7 μg/mL for ethyl acetate, 35.9 μg/mL for methanol, and 17.7 μg/mL for ascorbic acid.

“| n-Hexane | 41.8 |”

• Also stated in: § 5. CONCLUSION · plain

  “| Ethyl Acetate | 41.7 |”

• Also stated in: § 5. CONCLUSION · plain

  “| Methanol | 35.9 |”

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The extract significantly inhibited Gram-positive and Gram-negative bacteria and fungi, with slightly less activity against Pseudomonas aeruginosa.

“The extract exhibited significant inhibitory effects against both Gram-positive and Gram-negative bacteria, as well as fungal strains, although slightly reduced activity was observed against Pseudomonas aeruginosa, which is known for its intrinsic resistance.”

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Both in vitro anti-inflammatory assays found the methanol extract most inhibitory, approaching diclofenac at higher concentrations, and it protected proteins and membranes similarly to NSAIDs.

“Both in vitro anti-inflammatory assays showed that the methanol extract had the highest inhibition, nearing the activity of diclofenac at higher concentrations.”

• Also stated in: § Figure 13: In Vivo Anti-inflammatory Activity Profile 3.12 Cytotoxicity Assessment by MTT Assay · boosted

  “In vitro assays, including protein denaturation inhibition and HRBC membrane stabilisation, demonstrated that the methanol extract effectively protected protein structures and cellular membranes, mimicking the action of non-steroidal anti-inflammatory drugs.”

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The methanolic extract had MIC values of 62.5 μg/mL against Bacillus subtilis and 31.25 μg/mL against Escherichia coli.

“The methanolic extract exhibited the strongest antibacterial activity, demonstrating the lowest MIC values of 62.5 µg/mL against Bacillus subtilis and 31.25 µg/mL against Escherichia coli.”

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In the carrageenan-induced paw edema model at 4 h, the methanol extract (200 mg/kg) produced 30.4% inhibition, ethyl acetate (200 mg/kg) 17.8%, and n-hexane (200 mg/kg) 9.8%.

“| Methanol Extract (200 | 30.4% |”

• Also stated in: § 5. CONCLUSION · plain

  “| Ethyl Acetate (200 mg/kg) | 17.8% |”

• Also stated in: § 5. CONCLUSION · plain

  “| n-Hexane (200 mg/kg) | 9.8% |”

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The methanol extract showed significant cytotoxicity with a lower IC50, suggesting potential anticancer properties.

“The methanol extract exhibited significant cytotoxic activity with a lower IC₅₀ value, indicating potential anticancer properties.”

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The methanol extract was the richest in phytochemicals, having the greatest amounts of phenolics, flavonoids, and anthocyanins.

“Methanol extract is phytochemically richest”

• Also stated in: § Figure 13: In Vivo Anti-inflammatory Activity Profile 3.12 Cytotoxicity Assessment by MTT Assay · plain

  “The methanol extract exhibited the highest yield and a markedly richer phytochemical profile, particularly in phenolics, flavonoids, tannins, and glycosides.”

• Also stated in: § 5. CONCLUSION · plain

  “Among the evaluated extracts, the methanol fraction exhibited the highest levels of phenolics, flavonoids, and anthocyanins, translating into superior antioxidant, antimicrobial, antiinflammatory, and cytotoxic activities.”

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The methanol extract had significant free radical scavenging, microbial inhibition, anti-inflammatory activity in vitro and in vivo, and notable cytotoxicity.

“The methanol extract, in particular, showed significant free radical scavenging ability, effective microbial inhibition, substantial anti-inflammatory activity in both in vitro and in vivo models, and notable cytotoxic potential.”

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In the ripe banana peel extracts, anthocyanins were mainly found in the methanol fraction, with a small amount in the ethyl acetate fraction and none in the n-hexane fraction.

“Anthocyanins were predominantly present in the methanol extract, with minor presence in the ethyl acetate extract and absence in the n-hexane extract.”

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The methanol extract had significantly more total phenolics and flavonoids, which led to better antioxidant performance in all tests.

“Quantitative phytochemical analysis showed significantly higher levels of total phenolics and flavonoids in the methanol extract, which directly led to better antioxidant performance across all tests.”

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The total phenolic content values were 36.08 ± 1.20 mg GAE/g for n-hexane, 43.09 ± 1.40 mg GAE/g for ethyl acetate, and 43.68 ± 1.50 mg GAE/g for methanol.

“| n-Hexane | 36.08 ±1.20 | 43.5 | | |”

• Also stated in: § 5. CONCLUSION · plain

  “| Ethyl | 43.09 ± 1.40 | 33.33 | | |”

• Also stated in: § 5. CONCLUSION · plain

  “| Methanol | 43.68 ± 1.50 | 30.8 | | |”

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The methanol extract had the strongest antimicrobial activity against all tested strains, particularly Gram-positive bacteria.

“The methanol extract showed the highest antimicrobial activity across all tested strains, especially against Gram-positive bacteria.”

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The ethyl acetate and methanol extracts had higher TPC values (43.09 ± 1.40 and 43.68 ± 1.50 mg GAE/g) and lower IC50 values, indicating stronger antioxidant activity than the n-hexane extract.

“The ethyl acetate and methanolic extracts, which possessed higher TPC values of 43.09 ± 1.40 mg GAE/g and 43.68 ± 1.50 mg GAE/g respectively, exhibited lower IC₅₀ values, indicating stronger antioxidant activity compared to the n-hexane extract.”

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In the zone of inhibition assay, significant differences were observed between n-hexane and ethyl acetate (p=0.0015), n-hexane and methanol (p=0.0001), n-hexane and ciprofloxacin (p<0.0001), ethyl acetate and methanol (p=0.0023), ethyl acetate and ciprofloxacin (p<0.0001), and methanol and ciprofloxacin (p=0.0004).

“Significant differences were observed between nhexane and ethyl acetate extracts (**p = 0.0015), nhexane and methanol extracts (***p = 0.0001), nhexane and ciprofloxacin (****p < 0.0001), ethyl acetate and methanol extracts (**p = 0.0023), ethyl acetate and ciprofloxacin (****p < 0.0001), and methanol extract and ciprofloxacin (***p = 0.0004).”

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The methanol extract exhibited the highest yield and a markedly richer phytochemical profile, particularly in phenolics and flavonoids.

“The methanol extract exhibited the highest yield and a markedly richer phytochemical profile, particularly in phenolics, flavonoids,”

Classified as probably not a substantial claim.

Standard microbial strains representing Gram-positive and Gram-negative bacteria and fungal organisms were used.

“Standard microbial strains were selected to represent both Gram-positive and Gram-negative bacteria and fungal organisms.”

Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

Ascorbic acid was used as the standard antioxidant in the DPPH and hydrogen peroxide scavenging assays.

“Ascorbic acid was used as the standard antioxidant.”

• Also stated in: § 2.9.3 Hydrogen Peroxide (H₂O₂) Scavenging Assay · plain

  “Ascorbic acid was used as the reference standard.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Total flavonoid content experiments were conducted in triplicate.

“All experiments were conducted in triplicate to ensure reproducibility.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Positive and negative controls were used in the antimicrobial disc diffusion assay.

“Standard antibiotics (e.g., ciprofloxacin for bacteria) were used as positive controls, while solvent-treated discs served as negative controls.”

Classified as probably not a substantial claim.

Only healthy, disease-free plant material was selected for the study.

“Only healthy, disease-free plant material was selected to ensure the integrity of phytochemical and biological evaluations.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Total phenolic content measurements were performed in triplicate.

“All measurements were performed in triplicate, and results were expressed as mean ± standard deviation.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Diclofenac sodium was used as the standard reference drug in the protein denaturation assay.

“Diclofenac sodium was used as the standard reference drug.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

All animal experiments were conducted in accordance with ethical guidelines and with approval from the Institutional Animal Ethics Committee.

“All animal experiments were conducted in accordance with ethical guidelines and with approval from the Institutional Animal Ethics Committee (IAEC).”

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The extract's activity was lower than diclofenac's but still pharmacologically meaningful, suggesting potential as a natural anti-inflammatory agent with a possibly safer profile.

“Although the activity was lower than that of diclofenac, the extract exhibited a meaningful pharmacological response, indicating its potential as a natural anti-inflammatory agent with a possibly safer profile.”

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These compounds act as hydrogen donors and electron transfer agents, stabilising reactive oxygen species and preventing oxidative damage.

“Mechanistically, these compounds function as hydrogen donors and electron transfer agents, stabilising reactive oxygen species and preventing oxidative damage.”

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The ethyl acetate extracts had moderate flavonoid levels, suggesting intermediate activity.

“Ethyl acetate extracts also showed moderate flavonoid presence, indicating potential intermediate activity.”

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The methanol extract's high flavonoid content suggests strong free radical scavenging and anti-inflammatory potential.

“The high flavonoid content of the methanol extract indicates strong free radicalscavenging potential and anti-inflammatory activity.”

• AssumptionRests on an assumption: Phenolics are major contributors to antioxidant and anti-inflammatory effects.

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The extract did not exceed standard drugs, but its multi-targeted effects and natural origin make it promising for further study.

“Although the extract did not surpass the activity of standard drugs, its multi-targeted biological effects and natural origin make it a promising candidate for further investigation.”

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The DPPH activity pattern correlates with total phenolic and flavonoid content, supporting phenolics as the main contributors to radical scavenging.

“This trend directly correlates with total phenolic and flavonoid content, confirming that phenolic compounds are the primary contributors to radical scavenging activity.”

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The methanol extract's strong inhibition suggests it stabilises proteins and membranes, likely through phenolic inhibition of inflammatory mediators.

“This indicates strong stabilisation of proteins and biological membranes, probably due to phenolic-mediated inhibition of inflammatory mediators.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Healthy, disease-free plant material was chosen to preserve the integrity of the phytochemical and biological evaluations.

“Only healthy, disease-free plant material was selected to ensure the integrity of phytochemical and biological evaluations.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The lower activity against Pseudomonas aeruginosa is expected because of its intrinsic resistance.

“The relatively lower activity against Pseudomonas aeruginosa is expected due to its intrinsic resistance mechanisms.”

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The drying and storage conditions were sufficient, and the extracts remained microbiologically stable, which ensured the reliability of later biological assays.

“This confirms that the drying process and storage conditions were adequate and that the extracts were microbiologically stable, ensuring the reliability of subsequent biological assays.”

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The methanol extract showed significant activity, nearing diclofenac's efficacy at higher concentrations, though it did not exceed the standard.

“Although the extracts did not surpass the standard, the methanolic extract demonstrated significant activity, approaching the efficacy of diclofenac at higher concentrations.”

Probably not a substantial claim: may not pass S3 (load-bearing).

A limitation is the study's use of crude extracts.

“However, the study is limited by its extract-based approach, and further work involving the isolation, purification, and structural characterisation of individual bioactive compounds is necessary to fully understand the mechanisms of action.”

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The DPPH activity trend correlates with total phenolic and flavonoid content, confirming that phenolic compounds are the main contributors to radical scavenging.

“This trend directly correlates with total phenolic and flavonoid content, confirming that phenolic compounds are the primary contributors to radical scavenging activity.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The plant was authenticated by the Botanical Survey of India with number CNH/Tech.II/2023/06 dated 24.02.2023.

“The plant specimen was taxonomically authenticated by the Botanical Survey of India, Howrah, West Bengal, and assigned the authentication number CNH/Tech.II/2023/06 dated 24.02.2023.”

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The extract likely contains bioactive compounds that reduce cell viability, possibly via apoptosis or oxidative stress.

“This suggests that the extract contains bioactive compounds capable of decreasing cell viability, potentially through the induction of apoptosis or oxidative stress pathways.”

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The antimicrobial action may involve phenolic disruption of microbial membranes, enzyme inhibition, and interference with nucleic acid synthesis.

“The antimicrobial mechanism can be attributed to phenolic-induced disruption of microbial cell membranes, enzyme inhibition, and interference with nucleic acid synthesis.”

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The selective cytotoxicity, with n-hexane mostly non-toxic, highlights polar phytoconstituents' role in biological activity.

“The selective cytotoxicity pattern, where the n-hexane extract remained mostly non-toxic, further highlights the role of polar phytoconstituents in biological activity.”

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The methanol extract was less potent than diclofenac, but its activity is pharmacologically relevant and supports therapeutic potential.

“Although not as potent as diclofenac, its activity is pharmacologically relevant and supports its therapeutic potential.”

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The polar methanol extract likely contains more bioactive constituents, such as phenolics and flavonoids, contributing to its stronger anti-inflammatory activity.

“This pattern suggests that the polar methanolic extract contains a higher concentration of bioactive constituents, such as phenolic and flavonoid compounds, which contribute to its enhanced anti-inflammatory activity.”

• AssumptionRests on an assumption: Secondary metabolites dissolve according to polarity, so polar solvents extract antioxidant-active compounds preferentially.

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A key strength is the consistent trend across all parameters: methanol was most potent, then ethyl acetate, then n-hexane.

“A key strength of the study is the consistent trend observed across all experimental parameters, with the methanol extract emerging as the most potent fraction, followed by ethyl acetate and n-hexane extracts.”

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Regression analysis yielded high R2 values (>0.98), confirming reliable dose-response behaviour.

“The high R² values (>0.98) observed in regression analysis confirm the reliability of dose-response behaviour.”