Steam distillation was performed by method (Baibuch et al., 2023) with some modifications. At 120°C and a flower-to-liquid ratio of 1:2 (w/v), rose hydrosol was subjected to reflux for 0, 5, 10, and 15 min. At the same temperature (120°C) and a reflux time of 5 min, the flower-to-liquid ratio varied as 1:1, 1:2, 1:3, and 1:4. Additionally, maintaining a reflux time of 5 min and a flower-to-liquid ratio of 1:2, the distillation temperature was set to 110, 120, 130, and 140°C.

Based on the single factor experiment of extracting rose hydrosol and taking the factors of flower-liquid ratio (A), reflux time (B), and distillation temperature (C) as random factors and the sensory score as the response value (Y), we conducted response surface analysis with three factors and three levels. The experimental design is shown in Table 1.

The study considered whether stamens were included in the optimum extraction process parameters. Four groups of control rose hydrosol products were established for sensory evaluation, as detailed in Table 2. The sensory indexes of rose hydrosol can directly influence consumer acceptance. Therefore, high-quality rose hydrosol can be guaranteed by sensory evaluation from three aspects: color, aroma, and moisturizing effect and preference level. Through this sensory index to determine the subsequent experiments, the extraction process parameters of rose hydrosol. For sensory evaluation, we selected six evaluators (the ratio of male to female was 1:1, and the average age was 22) from the laboratory. The evaluators underwent training and had no biased preferences, ensuring the reliability of the results. The sensory evaluation was carried out in a sensory evaluation laboratory at the Institute of Food Science and Technology of Shihezi University, in compliance with ISO 8589:2007 (Frangipane et al., 2024).

Samples (20 mL) were placed in a 50 mL beaker to observe the color and viscosity of rose hydrosol. Odorless absorbent paper was chosen, cut into strips measuring 8 cm in length and 4 cm in width, for odor evaluation of the samples. The sensory evaluation indexes are detailed in Table 2. Through the sensory evaluation of similar hydrosol products, four comprehensive indexes have been developed.

DPPH radical scavenging activity was determined as previously reported (Chen et al., 2020) with some modifications. Vitamin C (Vc) control solutions were prepared with distilled water at concentrations of 0.10 mg/mL, 0.40 mg/mL, 0.80 mg/mL, 1.00 mg/mL, and 2.00 mg/mL. To measure the DPPH radical scavenging activity, 1 mL of each Vc solution and 1 mL of DPPH solution at various concentrations were mixed in test tubes, reacted for 30 min at room temperature in the dark, and A₁ was measured at 517 nm. Additionally, 1 mL of ethanol was mixed with 1 mL of Vc solution to measure A₂, and 1 mL of DPPH solution was mixed with 1 mL of distilled water to measure A₃, both following the same procedure. The Vc solution was then replaced with rose hydrosol extracted under optimal conditions, and its DPPH free radical scavenging rate was determined using the same method. The DPPH radical scavenging rate of the rose hydrosol was compared with that of the different concentrations of Vc solution. Each group of samples was tested three times, and a microplate reader was used to measure the optical density of each sample. To calculate DPPH radical scavenging rate, we used the following formula:

where A₁ is the absorbance value at 517 nm (1 mL of vitamin C [Vc] solution and 1 mL of DPPH solution reacted for 30 min at room temperature in the dark); A2 is the absorbance value at 517 nm (1 mL of ethanol and 1 mL of Vc solution reacted for 30 min at room temperature in the dark); and A3 is the absorbance value at 517 nm (1 mL of DPPH solution and 1 mL of distilled water reacted for 30 min at room temperature in the dark).

·OH radical scavenging activity was determined as previously reported (Ma et al., 2023) with some modifications. Vc control solutions were prepared with distilled water at concentrations of 0.10 mg/mL, 0.40 mg/mL, 0.80 mg/mL, 1.00 mg/mL, and 2.00 mg/mL. To measure the hydroxyl radical (·OH) scavenging activity, 1 mL of each Vc solution was mixed with 1 mL of FeSO₄ (10.0 mmol/L) solution, 1 mL of salicylic acid-ethanol solution (10.0 mmol/L), and 1 mL of hydrogen peroxide solution (8.0 mmol/L) in a test tube. The mixture was reacted in a constant temperature water bath at 37°C for 30 min, allowed to stand for another 30 min, and A₁ was measured at 510 nm. For the control measurements, distilled water was used instead of hydrogen peroxide solution to determine A₂, and distilled water was used instead of the Vc solution to determine A₃. The Vc solution was then replaced with rose hydrosol extracted under optimal conditions, and the hydroxyl radical scavenging rate was determined using the same method. The hydroxyl radical scavenging rates of rose hydrosol and Vc solutions at different concentrations were compared, with each group of samples being tested three times. To calculate ·OH radical scavenging activity, we used the following formula:

where, A₁ is the absorbance value at 510 nm (1 mL of Vc solution, 1 mL FeSO₄ solution, 1 mL of salicylic acid-ethanol solution and 1 mL of hydrogen peroxide solution reacted in a constant temperature water bath at 37°C for 30 min, and allowed to stand for 30 min); A₂ is the absorbance value at 510 nm (hydrogen peroxide solution was replaced by distilled water solution); and A₃ is the absorbance value at 510 nm (The Vc solution was replaced by distilled water solution).

⋅O₂⁻ radical scavenging activity was determined as previously reported (Bai et al., 2021) with some modifications. Control solutions of Vc were prepared in distilled water at concentrations of 0.10 mg/mL, 0.40 mg/mL, 0.80 mg/mL, 1.00 mg/mL, and 2.00 mg/mL. For the experimental procedure, 2 mL of each Vc solution with varying concentrations was mixed sequentially with 4.5 mL of Tris–HCl buffer (pH = 8.2, 50.0 mmol/L) and 0.5 mL of pyrogallol (25.0 mmol/L), thoroughly shaken, and incubated in a water bath at 25°C for 6 min. Following incubation, 1 mL of HCl (10.0 mmol/L) was added, mixed well, and A₁ was measured at 325 nm. Control measurements were conducted using distilled water instead of pyrogallol to determine A₂ and using distilled water instead of Vc solution to determine A₃, following the same protocol. Subsequently, the Vc solution was substituted with rose hydrosol extracted under optimal conditions, and the scavenging rate of ⋅O²⁻ free radicals was determined using the same method. The scavenging rates of ·O₂⁻ free radicals for rose hydrosol and Vc solutions at different concentrations were compared, with each sample group repeated three times. To calculate ·O₂⁻ radical scavenging activity, we used the following formula:

where A₁ is absorbance value at 325 nm (2 mL of Vc solution, 4.5 mL Tris–HCl buffer, and 0.5 mL pyrogallol reacted in a water bath at 25°C for 6 min and mixed with 1 mL HCl); A₂ is absorbance value at 325 nm (pyrogallol was replaced by distilled water); and A₃ is absorbance value at 325 nm (Vc solution was replaced by distilled water).

Polyphenol content was determined as previously reported (Blicharski and Oniszczuk, 2017) with some modifications. Standard solutions of gallic acid were prepared at concentrations of 0.01 mg/mL, 0.02 mg/mL, 0.03 mg/mL, 0.04 mg/mL, and 0.05 mg/mL. The polyphenol content in rose hydrosol was determined using Folin phenol colorimetry with gallic acid as the standard. Specifically, 1 mL of gallic acid solutions at the five concentrations was mixed with 5 mL of 10% Folin phenol reagent, allowed to stand for 7 min, followed by the addition of 4 mL of 7.5 g/100 mL sodium carbonate solution. The reaction proceeded at room temperature in the dark for 1 h, and absorbance was measured at 765 nm. A standard curve was constructed using distilled water as the blank control to establish the regression equation relating gallic acid concentration (y in mg/mL) to absorbance (x). The polyphenol content in rose hydrosol was determined by diluting 1 mL of the hydrosol to 100 mL with a volumetric flask and measuring absorbance using the same method as described above. The content of polyphenol in rose hydrosol was calculated according to the content of total phenol (mg/g) in each gram of rose (calculated by GAE).

where C is the content of polyphenols in the determination solution, mg/mL; V is the volume of the solution, mL; N is the dilution factor; and M is the weight of rose petals, g.

Flavonoid content was determined as previously reported (Blicharski and Oniszczuk, 2017) with some modifications. A standard solution of rutin was prepared by weighing 25 mg of rutin standard and dissolving it in a 100 mL volumetric flask, adjusting the volume with ethanol to obtain a concentration of 0.25 mg/mL rutin. The flavonoid content in rose hydrosol was determined using the sodium nitrite-aluminum nitrate-sodium hydroxide color development method. Various volumes (0, 1.0, 3.0, 5.0, 7.0, 9.0 mL) of the rutin standard solution were transferred into 25 mL volumetric flasks, diluted to 15 mL with anhydrous ethanol, and then treated sequentially with 1 mL of 5% sodium nitrite solution for 5 min, followed by 1 mL of 10% aluminum nitrate solution for another 5 min, and finally with 5 mL of 10% sodium hydroxide solution before adjusting the volume to 25 mL with anhydrous ethanol. A standard curve was constructed using distilled water as a blank control to establish the regression equation correlating rutin concentration (y in mg/mL) to absorbance (x). The flavonoid content in rose hydrosol was calculated as rutin equivalent (RE) in mg/mL. To analyze the hydrosol, 1 mL was diluted 100 times in a 100 mL volumetric flask. The content of total flavonoids in rose hydrosol was calculated according to the content of total flavonoids (mg/g) per each gram of roses (calculated as RE).

where C is the content of flavonoids in the solution, mg/mL; V is the volume of the solutions, mL; N is the dilution factor; M is the weight of rose petals, g.

DPPH is a singlet electron with strong oxidation ability. When it is dissolved in water, it is blue-purple, and the solution has the maximum absorption at 517 nm. When antioxidant is added to the solution, the antioxidant can pair with DPPH, and the stronger the pairing ability is, the smaller the absorbance will be (Chen et al., 2020). Therefore, the ability of antioxidant to scavenge DPPH can be judged by measuring the absorbance. Figure 4A showed that the DPPH scavenging rate increased with increasing Vc concentration (0.1–1 mg/mL). The scavenging rate of rose hydrosol to DPPH was 26.13%, which was similar to that of 0.1 mg/mL Vc. This indicated that rose hydrosol possessed a notable ability to scavenge DPPH and had great potential for use as a natural antioxidant. Antioxidants in food have an important influence on the scavenging ability of hydroxyl radicals (Hassan et al., 2024). From Figure 4B, when the concentration of Vc was in the range of 0.1–1.6 mg/mL, the scavenging rate of p-OH radical was increasing with the increase of Vc concentration, and it increased slowly, basically floating around 60%. The scavenging rate of rose hydrosol on ·OH was 55.56%, which showed that rose hydrosol had antioxidant activity in vitro. By detecting the hydroxyl radical scavenging ability of different antioxidants, we can evaluate their antioxidant effects and provide basis for the screening and development of antioxidants (Hassan et al., 2024). As shown in Figure 4C, the scavenging ability of Vc for ⋅O²⁻ increased with Vc concentration. The scavenging rate of rose hydrosol for superoxide radicals was 10.76%, demonstrating a weaker, yet notable, antioxidant activity. This finding is consistent with previous studies (Gateva et al., 2024).

Polyphenols and flavonoids are a large class of components with antioxidant effects. Mainly due to the fact that they contain multiple phenolic hydroxyl structures. Phenolic hydroxyl groups have strong redox ability and can react with free radicals, thus reducing the damage of free radicals to biomolecules (Hassan et al., 2024). From the gallic acid standard curve (Figure 5), we obtained a linear regression equation y = 0.05915x - 0.0023 (R² = 0.9996). The polyphenol content in rose hydrosol was 14.9 ± 2.5 GAE mg/g; however, the long transport times of raw materials might reduce the polyphenol content. The polyphenols in rose hydrosol had antioxidant activity, consistent with a previous study (Alonso et al., 2022). From the rutin standard curve (Figure 5), we obtained a linear regression equation y = 0.10097x - 0.00149 (R² = 0.9995), and the content of flavonoids in rose hydrosol was 19.3 ± 1.3 RE mg/g, which was consistent with a previous study (Alonso et al., 2022). The content of flavonoids may be reduced due to the long transport time of raw materials, the temperature difference between the origin of the roses and the laboratory, and the extended duration of the experiment.