The Effect of Extraction Conditions on the Yield of Tannins and Phenolics from Acorn Shell and Cupule

IntroductionAcorn, a traditional component of the human diet, is a rich source of bioactive compounds, especially polyphenols and tannins, which possess antioxidant, antibacterial, and antiviral properties. These compounds have extensive applications in the food, pharmaceutical, and chemical industries. However, their yield and purity depend highly on extraction conditions, including solvent type and polarity, temperature, extraction time, particle size, and solid-to-solvent ratio. Solvent extraction is the most widely used among various extraction techniques due to its simplicity and efficiency. Given the limited data on tannins from native acorns in Chaharmahal and Bakhtiari Province, this study aims to evaluate the effect of solvents with varying polarity and pH on tannin extraction. The purity of extracted tannins was also determined using HPLC. The findings of this research could serve as a basis for optimizing the extraction of valuable compounds from acorn and facilitating the industrial utilization of these native resources.Materials and MethodsTo extract tannins from acorn shell, cupule, and shell-cupule mix, the samples were ground and passed through a 40-mesh sieve. Extraction was carried out using three types of solvents: distilled water, 40% ethanol solution, and 0.22% sodium hydroxide solution. The process was performed at two temperatures (60°C and 90°C) with a solid-to-liquid ratio of 1:10, for one hour under continuous stirring. The resulting extracts were filtered and then dried at 50°C. To determine the actual extraction yield in alkaline-treated samples, the amount of sodium hydroxide was quantified by titration with HCl, and its mass was subtracted from the dry extract weight to obtain the real extraction yield. Total phenolic content was measured using the Folin–Ciocalteu method by determining absorbance at 760 nm. Tannin content was estimated by subtracting non-tannin phenolics from total phenolics using PVPP as an adsorbent. In this method, PVPP was added to the extract, and after centrifugation, non-tannin phenolics were separated, allowing tannin content to be calculated by difference. HPLC analysis was performed using a C18 column and a detector set at 280 nm to confirm the presence of tannins. Tannic acid was used as the standard, and separation was achieved using a gradient of formic acid and acetonitrile. Statistical analyses were conducted using SPSS, and graphs were plotted using Excel.Results and DiscussionThe effects of solvent type and extraction temperature were examined for tannin extraction. The results indicated that increasing the temperature from 60°C to 90°C enhanced extraction yields for both distilled water and 0.22% sodium hydroxide solution. However, the effect of temperature increase was significantly more pronounced under alkaline conditions. Higher temperatures improve solvent penetration, reduce viscosity, and accelerate the diffusion of bioactive compounds, which markedly boosts tannin extraction in combination with an alkaline solvent. Regarding solvent type, ethanol demonstrated superior performance in tannin extraction. Due to its dual nature (polar and non-polar), ethanol efficiently extracted a broad spectrum of phenolic compounds. The average tannin yield using ethanol was around 51.41%, which surpassed other treatments, including alkaline extraction at high temperature (48.06%). This highlights ethanol’s strong capacity to extract condensed tannins, which are abundant in acorns. Moreover, ethanol tended to extract fewer impurities, such as carbohydrates, than polar solvents like sodium hydroxide, thereby increasing the purity of the tannin extracts. Further analysis of the total phenolic content revealed that the source material (cupule or shell) and solvent type significantly influenced the extraction yield. The highest total phenolic content (61.22%) was obtained from the cupule using sodium hydroxide, significantly exceeding other materials and treatments. This confirms the cupule’s richness in phenolic compounds, though given its lower proportion in the acorn structure, using a blend of cupule and shell is more practical. Additionally, ethanol and sodium hydroxide achieved higher yields than water, underscoring the beneficial role of alkaline and semi-polar solvents in phenolic compound release. Further specific analysis of tannin extraction also showed that ethanol-based treatments yielded the highest results. The use of 40% ethanol nearly doubled the tannin yield compared to aqueous and alkaline treatments. This high efficiency is attributed to the better solubility of tannins in polar-organic solvents like ethanol. On the other hand, extraction with sodium hydroxide at lower temperatures (60°C) resulted in lower yields, emphasizing the combined effect of temperature and solvent on extraction efficiency. HPLC analysis for tannic acid quantification confirmed that the tannin content of the shell-cupule mix and cupule alone was 48.06% and 61.23%, respectively. These findings emphasize the practical value of using a shell-cupule mixture as an industrial tannin source, as it not only contains a substantial amount of tannin but also offers better availability and a higher weight proportion compared to the cupule alone.ConclusionThis study highlights the significant influence of extraction conditions, particularly solvent type and temperature, on the extraction yield of tannins and phenolic compounds from acorn resources. Alkaline extraction at elevated temperatures was more effective for tannin extraction from the shell-cupule mix, while ethanol exhibited high efficiency due to its semi-polar nature. Although the acorn cupule is richer in total phenolics, the higher abundance of the cupule-shell blend in the fruit makes it a more practical target for industrial tannin production.