Introduction

Ocimum basilicum L. (Basil) is a medicinal herb found in products like dietary supplements, extracts, and topical skincare formulations. The antimicrobial, anti-inflammatory, and therapeutic properties of these products are attributed to their high levels of polyphenols and flavonoids, specifically rosmarinic acid, caffeic acid, quercetin, and other antioxidants (Jayasinghe et al., 2003; Hussain et al., 2008; Romano et al., 2022). As most of these compounds are thermolabile, susceptible to oxidation, and degraded by poor processing conditions, drying applied to basil leaves makes a notable difference in the chemical quality and the subsequent potency. Though drying reduces moisture and inhibits microbial growth, it may also promote the degradation of heat-labile phenolics and other bioactive compounds depending on temperature and exposure time (Ferreira et al., 2021). The most common methods used in industry and research involve both oven drying and various types of freeze-drying, all with variable stability effects on the phenolic and flavonoid constituents. For example, in studies related to medicinal and aromatic plants, air flow, temperature, and method of drying significantly altered antioxidant capacity and phytochemical retention (Al-Hamdani et al., 2022; Hossain et al., 2010).

Although basil is one of the more studied culinary and medicinal herbs, the emphasis in much of the drying literature relates to industrial drying quality or technology optimization rather than accessible, low-cost analytical comparisons for small labs or early-stage researchers. Thus, there is a significant gap in the current literature: very few studies have assessed exactly how real-world drying conditions affect the measurement and potency of the beneficial compounds found in these herbs, such as the polyphenols and flavonoids. The present work investigates the impact of three accessible drying methods: microwave, oven, and a commonly used freeze-drying alternative method that relies on low-temperature freezer dehydration on the total phenolic and flavonoid content of basil leaves. The two standard colorimetric assays are Ferric Chloride $\left({\text{FeCl}}_3\right)$ for phenol and Aluminum Chloride $\left({\text{AlCl}}_3\right)$ for flavonoid potencies. In this research, the color of the extract samples was analyzed in terms of RGB and HEX values (six-character alphanumeric representations of color intensity), extending beyond classic absorbance measurements, providing a simple method to quantify bioactive retention across drying treatments. Basil is an appropriate model plant due to its phytochemical profile being characterized and sensitive to thermal processing conditions, thereby providing an ideal system to study how drying affects compounds related to antioxidant activity (Al-Hamdani et al., 2022; Hossain et al., 2008).

Literature Review

The bioactive polyphenols and flavonoids in plants aid specifically in antioxidant defense, antimicrobial activity, and modulation of inflammatory pathways. The current understanding of polyphenolic compounds is that the presence of multiple hydroxyl groups attached to an aromatic ring enables their function in Reactive Oxygen Species (ROS) scavenging, cell membrane stabilization, and inhibition of oxidative damage. Flavonoids are one subclass of polyphenolic compounds with additional important functions in UV protection, pigmentation, and pathogen defense (Romano et al., 2022). High final concentrations of these compounds are important in industries that utilize these plants, such as dietary supplement and topical skin care product manufacturing, for purposes of product efficacy, therapeutic potential, and also consumer confidence. Changes in drying methods, microwave-drying, oven-drying, low-temperature approaches, or otherwise, in conjunction with or alternative to freeze-drying, directly impact polyphenol and flavonoid retention, which further affects the potential chemical quality and functional benefits of the final products (Al-Hamdani et al., 2022; Ferreira et al., 2021). Identifying the drying method that preserves the greatest quantity of bioactive compounds not only optimizes batch-to-batch consistency but also enables industrial efficiency in the manufacturing process to produce large batches of these active ingredient-containing dietary supplements and topical products.

For this reason, the quantification of polyphenols and flavonoids in plant material has been accurately based on colorimetric assays utilizing their chemical reactivity. To elaborate, the FeCl₃ assay forms colored complexes with phenolic hydroxyl groups, producing a dark green or brown color proportional to total polyphenol concentration, hence reflecting the amount of antioxidant molecules available. The ${\text{AlCl}}_3$ assay reacts with flavonoid hydroxyl groups to yield a yellow complex measurable by spectrophotometric means. These reactions proceed because both metal ions form coordination complexes with particular functional groups residing within the target compounds, providing a direct and reproducible chemical result. Such colorimetric changes correlate with their strength of concentration, hence making them suitable for comparisons among various drying methods (Hossain et al., 2010; Velioglu et al., 2017; Shafiei et al., 2015). These assays, with digital color analysis such as RGB extraction and conversion to HEX codes, enable comparison of compound retention across different drying methods. By integrating the use of knowledge in polyphenol and flavonoid chemistry with validated colorimetric assays, this work establishes evidence-based guidance in selecting the best drying method for optimum retention of bioactive content (Velioglu et al., 2017). Such insights are directly relevant to supplement manufacturers, skincare formulators, and potentially drug developers who seek to ensure potency, quality, and consistency of products, with implications for industrial efficiency and overall consumer benefit. One of the main motivators for this research was the possible concern that, for products designed to rely on specific active components (e.g., catechins in green tea), using a drying method that has the potential to diminish those compounds could undo much of the work involved in isolating them, emphasizing the importance of understanding how drying affects overall retention levels.

Methods

A controlled laboratory experiment examining the polyphenol and flavonoid content of a basil sample using three different drying methods was conducted. One batch of fresh basil leaves was obtained commercially to ensure all samples originated from the same source. Leaves were washed under filtered tap water, patted dry with paper towels, and handled using nitrile gloves and safety goggles to maintain sanitation before and during processing. A total of 126 g of fresh basil was used for the entirety of this study. This material was then divided into equal amounts among three experimental groups corresponding to drying methods: microwave drying, oven drying, and a low-temperature freezer dehydration method. For each of these drying methods, the material was further divided into two subsets, one for later analysis with ${\text{FeCl}}_3$ and one for ${\text{AlCl}}_3$, yielding six experimental conditions with two replicates each (n = 2).

Microwave-dried samples were treated in low-heat 30-second bursts, repeated a total of seven times, with proper caution to avoid overheating or burning. Oven-dried samples were placed in an oven preheated to 45–50 °C for a total of five hours, with all materials facing equal heat. The low-temperature freezer dehydration group samples were laid out flat and left in the freezer for five nights, allowing gentle dehydration without significant thermal degradation before being thawed for four hours to defrost prior to crushing. Each drying method was treated to produce a consistent moisture reduction across all samples.

Each sample subset was then finely powdered in a separate mortar and pestle after it had completely dried. Then, 2 g of each ground sample was placed into a glass vial with 20 mL of 40% food-grade ethanol, labeled, and mixed by gently flipping upside down for 2 minutes. Mixtures were then allowed to rest, refrigerated, and kept away from light for a total of three nights. At the end of this time, the solid materials were completely filtered from the extracts using filter paper, and the clear extracts were collected. Each extract was divided such that 2 mL was available for both the ${\text{FeCl}}_3$ and ${\text{AlCl}}_3$ assays. Extracts were then stored in sealed, labeled containers and refrigerated in a dark location until testing.

For the ${\text{FeCl}}_3$ assay, 2 mL of each extract was combined with 0.5 mL of 1% ${\text{FeCl}}_3$ solution in an airtight, clear container, gently mixed, and left to react for 15 minutes until a colored complex proportional to total polyphenols developed. For the ${\text{AlCl}}_3$ assay, 2 mL of extract was combined with 0.5 mL of 10% ${\text{AlCl}}_3$ solution under the same conditions to quantify flavonoids. Digital images were taken directly from above under uniform lighting against a plain white background, and HEX and RGB color values were measured from these images to quantify accurate relative concentrations of the phenols and flavonoid contents. To ensure accuracy, color values were independently verified using multiple color analysis tools, which consistently reproduced the same RGB and HEX measurements for each sample, supporting the reliability of the color-based comparisons to use for this assay.

The materials and the techniques used in each procedure were the same for all experiments, so that a valid comparison among groups would be possible. Safety precautions included wearing gloves and goggles when handling the ethanol and the ${\text{FeCl}}_3$ and ${\text{AlCl}}_3$ solutions. This design allowed all processing and analysis to maintain experimental consistency and reproducibility.

Descriptive statistics for all treatment groups were calculated, and mean HEX and RGB values were compared using a one-way ANOVA to test the differences between the chosen drying methods. The level of statistical significance was identified using the p-value. Comparisons of HEX and RGB values across the experimental conditions were performed to assess both polyphenol and flavonoid content intensity.

Additionally, all used literature was selected based on relevance to drying methods, basil plants, nutrient management, phenols and flavonoids, supplement and topical manufacturing, and medicinal or nutraceutical plant uses. Only reputable peer-reviewed research, clinical trials, and review articles published in English from 2000 to 2025 were chosen for this study.

Results

Before the addition of ${\text{FeCl}}_3$ and ${\text{AlCl}}_3$, all basil ethanol extracts displayed a similar pale yellow-green baseline coloration with minimal variation between drying methods (mean HEX ≈ #B0BF93; RGB ≈ 176, 191, 147), indicating comparable extraction efficiency prior chemical reaction. The color development revealed distinct variability between the analyzed basil samples, which had undergone the different drying processes compared, for ${\text{FeCl}}_3$ and ${\text{AlCl}}_3$ assays. The low-temperature freezer dehydration method provided colors that were the darkest and most saturated, while medium levels of color development were obtained for microwave-dried samples, and light-colored, brownish-gray specimens were the result for oven-dried ones. In the case of ${\text{FeCl}}_3$, the of the color evolution resulted in olive-green hues for freezer-dried samples (HEX = #556B2F, RGB = 85, 107, 47), light green hues for microwaved samples (HEX = #6B8E23, RGB = 107, 142, 35), and green-brown hues for oven-dried samples (HEX = #8B7D6B, RGB = 139, 125, 107). In ${\text{AlCl}}_3$ assays, the intensity of the color evolution resulted in the highest intensity of yellow-green hues for freezer-dried samples (HEX = #9ACD32, RGB = 154, 205, 50), medium intensity for microwaved samples (HEX = #BDB76B, RGB = 189, 183, 107), and very low intensity for oven-dried samples in terms of visual color spectrum (HEX = #D2CFC4, RGB = 210, 207, 196). Based on one-way ANOVA of the associated HEX and RGB unit values of color evolution of the respective compounds for both assays, there was a statistically significant effect of drying processes on the ${\text{FeCl}}_3$ (polyphenol) and ${\text{AlCl}}_3$ (flavonoids) color evolution with a significance level of p < 0.05, supporting the hypothesis that drying method affects phenolic and flavonoid retention.

Discussion

These differences in the retention of compounds based on drying techniques strongly suggest the vulnerability of basil’s bioactive compounds to heat. Specifically, the low-temperature freezer dehydration method, no heat, was the most effective in the retention of polyphenolic and flavonoid compounds, suggesting that reduced heat exposure likely limits thermal degradation of these compounds. Conversely, the use of microwave and oven drying techniques involving higher temperatures likely contributes to partial degradation of these compounds, implying that oven drying results in the greatest loss.

The above research illustrates that the use of traditional colorimetric tests, in conjunction with computer-based color analysis (HEX and RGB indices), constitutes a feasible and replicable indicator of the retention of the bioactives. The results have immediate application to the formulation of medical herbs, vitamin supplements, and topical creams in that they provide a specific indication of the importance of making knowledgeable decisions regarding the drying process to enhance the efficacy and medical value of plant-based products. The currently modest scale of investigation, with straightforward protocols, represents a model that can help illustrate the importance of the drying process regarding the retention of certain bioactives. However, it is crucial to note that the small sample size (n = 2 per treatment group) of this study limits statistical power, and the ANOVA results should be interpreted as exploratory rather than definitive.