All chemicals and reagents used in this study, unless otherwise stated, were of analytical and HPLC grades, sourced from Merck and Sigma Aldrich.

Aloe barbadensis leaves were collected between May 2021 and June 2021 from plants in their natural habitats in Kisumu, Elgeyo-Marakwet and Baringo Counties, representing the hot, moderately hot and cold regions of Kenya, respectively (Table 1). They were identified and authenticated by a taxonomist at the Department of Biological Sciences, Moi University, Kenya where voucher samples (CKN 010521, CKN 020521, CKN 030521) were deposited. Voucher samples were also deposited at the Herbarium of University of Eldoret, Eldoret, Kenya for future reference.

Extraction was done according to the method used by Sánchez-Machado et al. [22]. Fresh latex, fresh gel, dry latex and dry gel were extracted and stored separately. The leaf latex of A. barbadensis was obtained by cutting the leaves transversally near the base using a knife and arranging them concentrically. They were drained for 30 minutes into a clean beaker. Then the collected latex was left in open air for 3 days to allow complete evaporation of water [22] [27].

The resulting leaves were washed under running tap water and then distilled water. They were surface sterilized with 10% sodium hypochlorite to prevent microbial contamination. Fresh gel from the leaf skin was removed and the inner and middle layers were scraped [28] and homogenized in a blender. For dry samples, fresh gel was dried at 40˚C for 24 hours in an oven while the fresh latex was dried at room temperature (25˚C) for 24 hours. The dried samples from both gel and latex were separately extracted. The crude extracts thus obtained were kept at 4˚C in a refrigerator until commencement of analysis [22].

Measured 5 mg of dry latex were dissolved in PBS at pH 3 and topped up to the 5 mL mark. A total of 50 mg of dry gel was prepared by dissolution in PBS to make a 10 mL solution. All samples were sonicated on a digital Ultrasonic Cleaner (LMUC Series, Model-USBT-6-Liters) for 10 minutes and filtered through 0.45 μm pore size nylon membrane filters [22] [29]. The extraction process was replicated twice. All the extracts obtained were freeze dried using a FD5-serries freeze dryer (MRC Scientific Instruments).

Isolation of aloin using precipitation method gave very low yields. Thus, Preparative Thin Layer Chromatography (PTLC) method by Geremedhin et al. [27] was used with some modifications. Calcium sulphate (15%) mixed with silica (85%) were slowly dissolved in distilled water. The mixture was then poured onto a glass plate and spread with a metric ruler. The plate was put in a shaker to perfect the preparation then it was sun dried naturally and later activated in the oven at 120˚C for 2 hours.

Isolates were obtained from the latex extracts using PTLC prepared by coating the glass plate with silica gel type G (Size: 10 - 40, Sigma-Aldrich, USA) at 0.5 mm thickness in the laboratory of Pharmacy Department at Mbarara University of Science and Technology, Uganda. The dry latex was dissolved in methanol and applied directly as a band to the coated PTLC (20 cm × 20 cm) over one side of the plate. Ethyl acetate and methanol mixture (7:3) were used as a solvent system to develop the chromatogram. The isolates were checked for their purity using silica gel over TLC plate (0.25 mm thickness).

The chromatographic zones were visualized using ultraviolet light at 254 nm and 366 nm. After visualization, the chromatographic zones were coded in ascending order of retention factor (Rf) values. Then, each band was carefully scrapped off separately from the plate and dissolved in methanol and chloroform (1:1), filtered and concentrated [27].

A portion of the powder (0.1 g) was subjected to silica chromatography column using 75% water acetic acid: 25% ethyl acetate (v/v) to afford clean aloin.

Aloin in the extracts was quantified using HPLC. The analysis was performed on an UFLC Shimadzu HPLC system (Prominence Model, Tokyo, Japan) at the Analytical and Pharmaceutical Laboratory of Mbarara University of Science and Technology, Uganda. The HPLC system comprised of an LC-20AD pump, a Phenomenex Luna C18 column (250 × 4.6 mm × 5 µm), temperature-controlled sample trays, an online degasser (DGU-20A5R) and UV detector.

Aloin quantification was carried out using the external standard method as described by Meng et al. [30] with some modifications. A stock solution of 1 mg/mL of aloin standard was prepared by weighing 1 mg and dissolving in 1 mL of HPLC grade methanol. From this, working solutions of 0.5, 5, 10, 20, 50, 100 and 150 µg/mL of aloin were prepared. Triplicate injections of 10 μL of each concentration were run to establish the calibration curve and ensure the linearity.

A reversed-phase HPLC assay was carried out using a binary isocratic elution with a flow rate of 0.8 mL/min at a column temperature of 25˚C, a mobile phase of acetonitrile/0.1% acetic acid (1:3) and a detection wavelength of 254 nm. The acquisition time for aloin injection was 15 minutes. Data were processed with LC-Solution Software. Solvents and water were filtered through a 0.45 μm nylon membrane with the aid of Buckner, enhanced by ILMVAC GmbH vacuum pump.

For the preparation of the calibration curves, standard stock solutions were prepared in methanol, filtered and appropriately diluted to obtain the desired concentrations. The calibration graphs were plotted as the linear regression of the peak areas versus concentrations. To determine the aloin contents, both gel and latex extracts (1 mg/mL in PBS) were analyzed using HPLC-UV. The amount of aloin was calculated from the calibration curve.

Quantitative data obtained from triplicate analyses were expressed as means ± standard deviations of replicates. To identify any significant differences between means, One-Way Analysis of Variance (ANOVA) was performed with Tukey post hoc test at p < 0.05. The analyses were performed using Graph Pad Prism for windows (v9.0, Graph Pad Software, California, USA).

Linearity of the standard curves was estimated as the determination coefficient (r²) from plots of the integrated peak heights versus the concentrations of the standard (Figure 3). All the standard curves produced throughout the course of the study appeared linear upon visual inspection. The coefficients of determination (r²) were ≥99.9%, therefore considered linear and acceptable for quantifying aloin in the extracts. Linear regression does not give equal weight to the calibration points especially at low levels when a seven-point calibration curve is used but the r² for four-point calibration curve was ≥99.9%.

Precision of the method was established in terms of the Relative Standard Deviation (RSD) which was found to be between 0.393% and 0.76% for repeatability. On the other hand, reproducibility of total aloin in the samples was established through analysis of the same samples for 3 days, and the RSD values for the samples were found to be less than 0.392 for the seven chromatograms.

The aloin contents of dry gel and latex samples were determined in different preparations of PBS extracts. Though the latex from the three regions were able to separate along with the aloin standard when dissolved in the same solvent system, the corresponding gels regions did not show any separations (Figure 4, Table 2).

As seen in Table 3, latex samples quantified had higher aloin amounts than the gels, with the highest content being 237.971 ± 5.281 mg aloin/g DW for dry latex from Elgeyo-Marakwet followed by those from Baringo (198.409 ± 2.000 mg aloin/g DW) and then Kisumu (40.760 ± 0.088 mg aloin/g DW). For gels, the aloin content followed the order Kisumu > Baringo > Elgeyo-Marakwet.