Healthy, mature specimens of Aloe vera leaves were collected from mature plants in April and May 2024 from a garden in Taif City, KSA. Fresh leaves, approximately 50–55 cm in length, with thick stems and a dark green color, were randomly selected and cut by removing the leaf rinds in the early morning for the experimental work. The leaves were transported to Taif University in polyethylene bags to prevent oxidation and microbial contamination.

After surface sterilization and washing with distilled water for 5 min to remove any sticking materials, dust, and impurities, the leaves were allowed to dry at room temperature. Using a sterile stainless steel knife, the leaves were manually cut, and the gel portion was retained after the outer green skin was peeled off. The gel was carefully collected using a sterile scalpel and weighed on an electric balance to calculate the yield as a percentage. Water extracts were then prepared for the anti-influenza determinations. First, the gel was frozen at −80°C for 1 h and then freeze-dried using ALPHA 1–4 LSC (Germany) at −50°C and a pressure of 0.010 mbar for 1 day (Qiao et al., 2020). The dried gel was ground using a non-metallic electric grinder and sieved through a300 μm mesh. The Aloe vera powder was stored in a zipper storage bag at −80°C until further use. Figure 1 shows the summary of the experimental study.

MDCK cells and influenza AP/R/8 virus were obtained from the Shanghai Institute of Biological Sciences, China. Cells (1.5 × 10⁴) were grown and incubated at 37°C for 24 h in a 96-well plate. The influenza virus solution was diluted to 4 × 10³ in DMEM containing trypsin–EDTA for the infection, with non-infected MDCK cells used as the control. The MDCK cells were rinsed two times with PBS without the AP/R/8 solution. Approximately 2 g of fresh gel was added to 10 mL of sterile water and then stirred and filtered to remove any residue. Different concentrations of Aloe vera gel, ranging from 0 mg/mL to 8 mg/mL, were prepared, and 10 μL of each concentration was added to 90 μL of AP/R/8 virus solution (TCID50) in a 96-well plate. All samples were analyzed in triplicate and kept in a carbon dioxide incubator with humidity for 48 h. The cells were washed, and cold acetone (70%) was added for 1 h at a temperature of −4°C, according to the method described by Gansukh et al. (2016). The samples were measured using a Synergy HT Microplate Reader (BioTek, USA) at 510 nm for anti-influenza activity, and the results were expressed as IC₅₀ (Al-Eisa et al., 2023a).

The gel was spread on either one or both sides of the cells in the Petri dishes and allowed to dry at ambient temperature. HUVECs were obtained from the Shanghai Institute of Biological Sciences, China. The control consisted of a healthy HUVEC line. DMEM medium was used, supplemented with FBS (10%), L-glutamine, and penicillin–streptomycin, and the cells were incubated at 37°C in a 5% CO₂ atmosphere. The cells were washed with PBS, then exposed to Tris/EDTA, and incubated at 37°C in a 5% CO₂ atmosphere. Afterward, the cells were separated, centrifuged at 1000 g for 10 min, and counted using trypan blue dye after exposure to ultraviolet C at a wavelength of 254 nm (Kostyuk et al., 2008). The initial cell count recorded 600,000 cells at 0 h. The cells were then exposed to ultraviolet C for different durations (1, 1.5, 2, and 4 h) for the control and for the gel applied to one or both sides.

The microbial population was applied in vitro using Mueller–Hinton agar as the medium. Two kinds of foodborne bacterial isolates, Bacillus subtilis and Pseudomonas aeruginosa, were obtained from the Agricultural Culture Collection, Egypt. To prepare the plates, 15 milliliters of the medium were added to sterile Petri dishes, allowed to harden for 5 min, and then uniformly swabbed with a 0.1% inoculum suspension before being allowed to dry for another 5 min. The extracts were placed onto 6 mm sterile disks at a concentration of 40 mg/disk. After placing the loaded disks on the surface of the medium and allowing the extract to diffuse for 5 min, the plates were incubated for 24 h at 37°C (Sami et al., 2021e; Sami et al., 2021c). The microbial population was detected in triplicate, and the results were expressed as the zone of inhibition in millimeters (mm).

For the determination of moisture and total solid contents, the Aloe vera gel samples were freeze-dried at −50°C under a pressure of 0.010 mbar until a constant weight was achieved. Standard assays were performed to evaluate the proximate analysis of ash (Ignition method), protein (Kjeldahl method), fiber (acid and alkaline hydrolysis method), and fat (Soxhlet extraction) contents, according to the assays described by the Association of Official Analytical Chemists (AOAC) (AOAC, 1990). Total polysaccharides in the Aloe vera gel sample were detected according to the assay described by Sami et al. (2019). The phenol-sulfuric assay was used, with glucose as the standard, and absorbance was measured at a wavelength of 490 nm (754 PC, Shanghai, China). Approximately 1 g of the aloe vera gel was mixed with 10 mL of distilled water and shaken regularly for 2 h. The solution was filtered and analyzed using a digital pH meter (Ramdhini and Anggraini, 2024). All chemical components were detected in triplicate, and the results were calculated as percentages, except for the pH value.

Approximately 5 g of the Aloe vera gel was ashed at 410–440°C. The white-ash residue was dissolved in a mixture of HNO₃, H₂SO₄, and HClO₄ in a ratio of 11:6:3, filtered, and then adjusted to 100 mL with distilled water for mineral evaluations (Sami et al., 2021b). An atomic absorption spectrometer (Perkin Elmer, A Analyst 800) with suitable hollow cathode lamps was used separately for each element. The results were expressed as μg/100 g for Cd, Co, Cu, Ni, Pb, Mn, V, A, Cr, Fe, Se, and Zn and as mg/100 g for Mg and P.

The antioxidant activity of the Aloe vera gel was detected using the 1,1-diphenyl-2picrylhydrazyl (DPPH) method, according to its ability to scavenge radicals or donate hydrogen (Rokayya et al., 2013). Approximately 1 mg of the gel was dissolved in 1 mL of MeOH and 150 μL of a DPPH solution with a concentration of 0.1 mM. The mixture was kept in the refrigerator for 30 min, shaken thoroughly, and measured at a wavelength of ~0.800, with an absorbance of 515 nm. DPPH antioxidant activity was detected against MeOH as a standard and calculated as IC₅₀ (μg/mL).

Vitamin C (ascorbic acid) in the Aloe vera gel was detected using the colorimetric method based on the oxidation–reduction reaction of 2,6-dichlorophenol as a redox dye. L-ascorbic acid can be oxidized to L-dehydroascorbic acid, resulting in a rose-pink color in the acidic solution (Cisternas et al., 2014).

All experiments were performed in triplicate, and the results were expressed as mean ± standard deviation (SD). The means were compared using one-way ANOVA (version 8.2, SAS, Cary, NC, USA) with a significance level of a p-value of <0.05.

Herbal medicines derived from natural resources play a significant role in the development of antiviral medications. Novel influenza viruses have pathogenic effects on public health, and there is a need to identify new anti-influenza compounds. Aloe vera gel contains anthraquinone derivatives, which have the potential to exhibit antiviral activities. The control and different concentrations of the Aloe vera gel extracts were evaluated against the influenza AP/R/8 virus, as shown in Figure 2. The highest IC₅₀ value was reported for the control (untreated cells), while the lowest IC₅₀ value (4.23 mg/mL) was reported for the treated cells at a concentration of 8 mg/mL. The lower IC₅₀ value indicated enhanced antiviral activity. The results from using the MDCK cell lines are in agreement with those reported by Rezazadeh et al. (2016) who studied the anti-influenza activity in the Vero cell line. Sydiskis et al. (1991) reported the inhibitory effects of Aloe vera gel against poliovirus, herpes simplex, cytomegalovirus, varicella zoster virus, influenza, rhabdovirus septicemia, papillomavirus, immunodeficiency virus, and both RNA and DNA viruses. Majumder et al. (2019) reported the antiviral effects of Aloe vera on coronavirus and its management in in-vitro and in-vivo studies. Mulu et al. (2015) reported the significant effects of Aloe vera, which are similar to those of the lopinavir/ritonavir drug. The anthraquinone derivatives in the Aloe vera plant, such as aloe-emodin, emodin, and chrysophanol, were the main contributors to the anti-influenza activity (Gansukh et al., 2016).

Ultraviolet radiation can be absorbed by nucleic acid, proteins, and cellular membranes and lead to the destruction of chemical bonds and the formation of free radicals (Lekmine et al., 2022). Continuous exposure at a higher rate or for a longer period has negative effects on oxidative free radical-driven reactions and can lead to cell death (Ntourtoglou et al., 2022). Figure 3 presents the differences in the cell counts of the HUVECs among the control, cells treated with the gel applied to one side, and cells treated with the gel applied to both sides after exposure to ultraviolet C for 0, 1, 1.5, 2, and 4 h, respectively. The initial cell counts reported 600.000 viable cells after 1 h of exposure to ultraviolet C. For the cells treated with the gel applied to one side and cells with the gel applied on both sides, 290.000 viable cells were reported. After 1.5 h, the cells treated with the gel applied to one side and cells treated with the gel applied on both sides reported 130.000 viable cells, which represented double the count compared to the control. After exposure to ultraviolet C for 2 h, all cells in the control group and the cells treated with the gel applied to one side died, while the cells treated with the gel applied on both sides reported 29.000 viable cells. Following continuous exposure to ultraviolet C for 4 h, all cells in all groups died. Tariq et al. (2019) reported the protective role of Aloe vera against ultraviolet radiation in the range of 300–400 nm. The two-sided application of the gel under UV-C exposure yielded better results than the single-sided application. This might be due to the high amount of antioxidant components and polyphenol content (Tariq et al., 2019). The results of the current study are in agreement with those of Yao et al. (2016), who found that sterols in Aloe vera gel can help prevent skin photo-aging. Another study by Pugh et al. (2001) applied gamma radiation to animals and treated them with Aloe vera, which demonstrated positive effects on wound healing.

The microbial population was detected in the Aloe vera gel extracts at concentrations of 0, 20, 40, 50, and 100 μL, showing resistance to all concentrations against the tested bacterial isolates, such as Bacillus subtilis and Pseudomonas aeruginosa, Figure 4. The biological activity was evaluated by measuring the inhibition zone (mm). The lowest inhibition activity was detected against the Gram-positive bacteria Bacillus subtilis (inhibition zone ≥5 mm), with a value of 2.20 mm at a concentration of 20 μL. The highest inhibition activity among the two foodborne bacterial isolates was detected against the Gram-negative bacteria Pseudomonas aeruginosa (inhibition zone ≥10 mm), with a value of 21.29 mm at a concentration of 100 μL. The current results are in agreement with those of Smith-Palmer et al. (1998), who reported that Gram-positive bacteria are more susceptible to inhibition by natural herbal extracts compared to Gram-negative bacteria. Khattak et al. (2020) reported strong inhibitory effects of Aloe vera gel against several isolates, including Pseudomonas aeruginosa. The high presence of phytochemical compounds in Aloe vera gel is the main reason for its healing properties as it reduces bacterial counts, inhibits bacterial efflux, neutralizes toxins, prevents biofilm formation of the bacteria on wounds, and enhances the effectiveness of certain antibiotics (Kumar et al., 2017).

The antioxidant activity, measured as radical scavenging activity, presented an IC₅₀ value of 79.11 μg/mL, as shown in Table 3. Our DPPH radical result is in agreement with that reported by Lotfizadeh et al. (2023). The antioxidant activity is associated with the ethyl acetate fraction and the phenolic nature of the Aloe vera plant (Asamenew et al., 2011). DPPH radical results can vary due to differences in solubility, reactions, and analytical conditions (Li et al., 2021).

Humans need vitamins in small quantities, with few exceptions, as they cannot synthesize most vitamins (Sami et al., 2014). The ascorbic acid content of the aloe vera gel sample was detected (0.005%), as shown in Table 3. Mitra et al. (2023) reported that Aloe vera contains a variety of vitamins, including A, C, E, B1, B2, B6, B9, and B12.