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The study uses chloroform and ethyl acetate for Soxhlet extraction of Tamarindus indica seeds and peel, yet it does not justify the choice of these two particular solvents despite their vastly different polarity. Why did the authors not include a more polar solvent such as methanol or aqueous ethanol, which are commonly used in phytochemical extraction and may better represent a broader spectrum of bioactive compounds?
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Section 2.1: The seeds and peel were extracted separately using chloroform and ethyl acetate via Soxhlet apparatus. These specific chloroform and ethyl acetate extracts were then used throughout the paper for all biological testing.
Section 2.3.3: The protocol states: “100 mg of each seeds and peel extracts (wt) (chloroform and ethyl acetate) were taken separately and soaked in approximately 9 ml of ethanol for a day and filtered…” This step is critically problematic.
The method for total terpenoid content begins by re-dissolving or re-extracting the initial chloroform/ethyl acetate dried extract in ethanol. Terpenoid solubility and yield can vary drastically between solvents like chloroform, ethyl acetate, and ethanol. Therefore, the terpenoid value subsequently obtained (after ethanol soaking and petroleum ether fractionation) does not accurately represent the terpenoid content present in the original chloroform or ethyl acetate extract that was actually tested in the bioassays.
This creates a fundamental flaw in correlating the reported phytochemical content (Fig. 1, Results 3.2) with the observed biological activities. If the quantitative data does not reflect the chemical composition of the extract tested, any conclusion about which compound or extract is responsible for an effect is compromised.
– Could the authors clarify the intended protocol in Section 2.3.3? Was the goal to quantify terpenoids in the chloroform/ethyl acetate extracts, or was this a separate ethanol-based extraction from the original powder meant to estimate total terpenoids in the plant material independent of the main study extracts?
– If it was the former, has the team validated that this ethanol-soaking step provides an accurate quantitative measure of the terpenoids originally present in the non-polar (chloroform) or mid-polar (ethyl acetate) extracts? A different quantification method (e.g., direct gravimetric analysis after solvent removal or a colorimetric assay compatible with the original solvent) would be more appropriate.
– Similarly, for consistency, were the phenolic and flavonoid assays (Sections 2.3.1 & 2.3.2) performed directly on the chloroform/ethyl acetate extracts, or were those extracts also subjected to a secondary solvent step before analysis?
Viewing 2 replies - 1 through 2 (of 2 total)
As a fellow researcher who has carefully read your study, I appreciate the effort in investigating the bioactivities of Tamarindus indica seed and peel extracts. However, I would like to raise a few critical points for clarification and improvement.
Firstly, I note that another reader has already questioned the rationale behind the choice of chloroform and ethyl acetate as extraction solvents, especially in the absence of more polar alternatives like methanol or aqueous ethanol, which are commonly used for a broader recovery of bioactive compounds. I fully agree with that concern and emphasize the need for justification, especially given the phytochemical diversity known in tamarind.
Secondly, I observed a methodological inconsistency that requires explanation. In your phytochemical screening (Table 1), the ethyl acetate extract of the peel was reported to contain only terpenoids, with phenols and flavonoids absent. However, your GC-MS analysis (Table 6) clearly identified isovanillic acid, a phenolic compound, in the same extract. This discrepancy raises concerns about the accuracy or sensitivity of the qualitative phytochemical tests employed. Could the authors clarify this inconsistency, and whether quantitative methods were cross-validated?
Lastly, the study would have benefitted from a broader solvent gradient or even a polarity-based fractionation approach. This would allow a more comprehensive assessment of the phytochemical landscape and corresponding bioactivities. Limiting the analysis to two mid-to-low polarity solvents potentially underrepresents the pharmacological potential of T. indica constituents.