The A. angustifolia leaves were collected from the Isipingo beach suburb (29°59’44.0”S 30°56’38.0”E), located on the southern coast of KZN, South Africa, under the eThekwini Metropolitan Municipality. In contrast, the AS leaves were collected from the veld along the R34 road (29°27’29.6”S 31°04’30.9”E), under the iLembe district municipality, KZN, South Africa.

The South African National Biodiversity Institute (SANBI) verified the taxonomic identity of the plants, and the two samples were added to the KZN Herbarium with the accession numbers A. sisalana (152364) and A. angustifolia (152365).

To avoid injuring the sampler, the atopic, harmful spiky points of the leaves were removed before cutting both the A. angustifolia and the A. sisalana leaves. The cut leaves were transported to the laboratory, where side thorns on the A. angustifolia leaves were removed, and these leaves were washed with tap water to remove dirt, rinsed with distilled water, and arranged on a paper towel to remove excess water. The leaves were then cut into tiny pieces and spread in a fume hood (on a paper towel) to dry for a week. The dried Agave leaves were ground into a fine powder and put in a sterile, airtight plastic zip-lock bag before being placed in the cabinet to await the analysis’ subsequent stage.

Cold maceration was used to extract the 2 kg of finely crushed Agave leaves over 72 h at room temperature, with occasional stirring. The solvents used were non-polar hexane, polar ethyl acetate, and most polar methanol (Dharajiya et al. 2017). The plant material was sequentially extracted with solvents, with increasing polarity, ranging from hexane to methanol to obtain different solvent extracts. Two kilogram of the pulverised Agave sample was soaked in 1.5 L of hexane and covered with foil to prevent solvent evaporation, as well as ingress of foreign matter. It was ensured that the powdered material was covered by the solvent to ensure maximum extraction.

The leaves were macerated for 72 h in the fume cupboard, with daily stirring to ensure maximum extraction, filtered, rinsed with hexane, and the filtrate was put aside. The residue was soaked in ethyl acetate for 72 hours. After, it was filtered, then washed with the same solvent, and the solvent was removed with rotatory evaporator. The residue was soaked in methanol for 72 hours, after which it was filtered and rinsed with the same solvent. The solvent was removed with rotatory evaporator.

The IKA RV10 digital rotatory evaporator was used to concentrate the filtrates under reduced pressure, and the extracts were kept at 4 °C for the following experiment stage.

The Agave leaf extract underwent a preliminary phytochemical evaluation, employing standard methods to identify the presence of various phytochemicals with minor modifications (Dharajiya et al. 2017).

Sodium hydroxide test: 2 mL of 2.0% NaOH solution added to 1 mL of Agave extract produced a prominent yellow hue that disappeared when two drops of mild acetic acid were added, indicating the presence of flavonoids.

The Agave extract (0.5 g) and 10 mL of water were combined and placed in a test tube. After boiling and filtering the mixture, a small amount of 0.1% ferric chloride was added and stirred, and the colour of the mixture was examined for a blue-black or greenish-brown hue, which indicates the presence of tannins.

Ellagic test: After adding five drops of 5% FeCl₃ to 1 mL of plant extract, a yellow-green fluorescence was observed, indicating the presence of a specific phenol called resorcinol.

A total of 1 mL of chloroform, acetic anhydride, and undiluted sulphuric acid were sequentially added to 1 mL of the Agave leaf extract. The presence of terpenoids was established by the observation of a reddish-violet hue in the mixture.

A total of 3 mL of distilled water and five drops of olive oil were added to 1 mL of Agave plant extract and shaken vigorously for 2 min. The solution produced a persistent foam, which indicated the presence of saponins.

A total of 2 mL of acetic anhydride was added to 1 mL of Agave leaf chloroform extract followed by the addition of undiluted sulphuric acid. The appearance of green colouration indicated the presence of steroids.

Salkowski’s test: The presence of steroidal aglycone (a glycoside component) was indicated by the brownish-red colour when 2 mL of concentrated sulphuric acid was combined with 5 mL of the Agave extract.

A few drops of Wagner’s reagent were added to 2 mL of Agave extract and 1.5% hydrochloric acid. The formation of a brownish precipitate demonstrated the presence of alkaloids.

The Agave extract samples were reconstituted in dimethyl sulphate (DMSO) to yield a final concentration of 100 mg/mL. Sonication was used for the samples with solubility challenges, and the extracts were kept at 4 ^oC after being sonicated. The prepared DPPH (120 µL, made by dissolving 0.1 mM in ethanol) and 120 µL of Tris-HCl buffer (50 mM, pH 7.4) were put into a 96-well plate containing a five-microliter sample. The plate was then incubated in the dark for 20 min at room temperature. Using a BioTek^® PowerWave XS spectrophotometer (Winooski, VT, USA), the absorbance was measured at 513 nm, and the % radical scavenging equation was computed as follows:

The buffer was swapped for the 5 µL sample that served as the control. Two final concentrations of 250 µg/mL and 500 µg/mL of plant extracts were tested.

The FRAP method was used to evaluate the possible antioxidant activity of the Agave extracts. The following adjustments were made to the procedure that was published by Benzie and Strain (1996) to measure the ferric-reducing ability of the extracts and antioxidant controls. Dimethyl sulphate was used to generate stock solutions of the compounds (100 mg/mL) and the positive control, trolox (10 mM). Twenty millilitres of sodium acetate buffer (300 mM), 2 mL of freshly prepared TPTZ solution (10 mM TPTZ and 40 mM HCl dissolved at 50 ºC in a water bath), 2 mL of freshly prepared FeCl₃ solution (20 mM ferric chloride in distilled water), and 2 mL of distilled water comprised the FRAP reagent. A 96-well plate containing 50 µL of the samples and 200 µL of FRAP reagent was incubated for 30 min at 37 °C. Using a BioTek^® PowerWave XS spectrophotometer (Winooski, VT, USA), the absorbance was measured at 593 nm. The results were expressed as (Fe₂SO₄ [µmol/L]/mg sample), which was calculated using a ferrous sulphate (Fe₂SO₄) (1.25 µmol/L – 200 µmol/L) standard curve.

This study followed all ethical standards for research without direct contact with human or animal subjects.

The results from the phytochemical screening of the Agave plant leaves, presented in Table 1, showed that both Agave species leaf extracts contained phytoconstituents with therapeutic benefits.

The qualitative analysis of A. angustifolia revealed that the plant contained terpenes or terpenoids, aglycones (a steroidal portion of glycosides), flavonoids, phenols, and saponins, while steroids and alkaloids were not detected in the extracts. The A. angustifolia hexane extract showed a strong content of terpenoids and a weak presence of flavonoids, tannins, phenolic compounds, saponins, and glycosides, while steroid and alkaloid compounds were not detected. A moderate content of flavonoid compounds was observed in the A. angustifolia ethyl acetate extract, while saponin and glycoside compounds were weakly present, and the compounds of tannins, phenolics, terpenoids, steroids, and alkaloids were not detected. The A. angustifolia methanolic extract revealed a strong presence of flavonoids, moderate saponins, and a weak availability of tannins, terpenoids, and glycosides, while phenolics steroids, and alkaloids were not detected. The hexane extract of A. sisalana indicated moderate content of tannins, phenolics, and steroids, and a faint presence of flavonoids, terpenoids, saponins and glycosides, but alkaloid compounds were not detected. On the other hand, AS ethyl acetate extract showed a high presence of flavonoid and alkaloids, while phenolics were moderately present, and tannins, saponins and glycosides were slightly detected. Flavonoids and alkaloids were strongly detected in the AS methanolic extract, while saponins were moderately detected. Phenolics, glycosides and terpenoids were slightly present, and tannins and steroids were not detected in the methanolic extract of A. sisalana.

Alkaloids and terpenoids are known to protect plants from entrants and herbivores as well as to repel pathogens (López-Romero et al. 2018) and the saponins have antimicrobial and anti-inflammatory qualities (Yu et al. 2022). The methanol extract of A. angustifolia, ethyl acetate and methanol extracts of A. sisalana are laden with flavonoids, which characteristically are high in antioxidant concentrations that neutralise reactive oxidative stresses caused by ultraviolet B (UVB) rays and preserve the integrity of overall skin quality and appearance. The UVB radiations destroy filaggrin, which maintains the protective functions of skin barriers. Studies have established that plant extracts loaded with flavonoids and alkaloids protect the skin from UVB radiation damage (Chen et al. 2022). The hexane extract of A. angustifolia contained a high amount of terpenoids, which are known to demonstrate good antimicrobial, anti-allergic, and anti-inflammatory activities (Masyita et al. 2022). The availability of these phytochemicals in the Agave plants indicates the potential use of the plant extracts for antimicrobial, anti-fungal and anti-inflammatory properties.

The findings from this study agreed with a study by Hernandez-Valle et al. (2014), which established the extraction of steroidal saponins from A. angustifolia leaves.

Stępniowska et al. (2021) reported that alkaloids are used in cosmetics as antioxidants, skin-lightening or even-tone agents, antimicrobials, anti-cellulite, anti-ageing, calming, and anti-inflammatory substances. This suggests that the ethyl acetate and the methanol extracts from A. sisalana have a potential for use in cosmetic products claiming antimicrobial, anti-cellulite, anti-ageing, calming, and anti-inflammatory gentle soothing properties.

According to Kumar et al. (2020), plant phenolics offer protection against environmental stressors such as UV radiation, pathogenic infections, and predators. This indicates the potential use of A. sisalana in cosmetic products claiming UV protection. Goge et al. (2023) reported that tannins have therapeutic benefits because they facilitate the quick healing of wounds, and plants with high tannin content have strong antibacterial properties.

The antioxidant activity of the Agave leaves (Figure 2) represented the antioxidant activity of the six Agave extracts (hexane, ethyl acetate and methanol extract from A. angustifolia and A. sisalana), with the DPPH assay reported as a percentage of DPPH scavenged. Trolox served as a favourable reference point. The error bars show the standard deviation of quadruplicate values obtained in a single experiment. The A. angustifolia exhibited negligible DPPH activity in the methanol extract, but more than 50% DPPH scavenging activity in the hexane and ethyl acetate extracts. These results suggest that A. angustifolia possesses non-polar and slightly polar antioxidant compounds. At the maximum treatment concentrations, the ethyl acetate and methanol extracts of A. sisalana demonstrated more than 50% DPPH scavenging activity, while the hexane extract exhibited negligible DPPH activity. These results indicated that only polar antioxidant phytochemicals were detected in the A. sisalana plant. The ethyl acetate solvent extracted the antioxidant phytocompounds from both Agave plants. Determining the antioxidant activity using the FRAP method (Figure 3) indicated that all plant extracts demonstrated minimal antioxidant activity at the highest treatment concentration. Both plants had more activity in the methanol extract, with the lowest activity demonstrated by the hexane extract, which had higher values in A. angustifolia than in A. sisalana. The ethyl acetate extract showed higher activity for A. sisalana than for A. angustifolia. All the outcomes were noticed at the highest treatment concentration of 2 mg/mL. Although the DPPH method claimed more potent antioxidant activity, the results from the FRAP and DPPH methods showed a similar trend. These results corresponded with Munteanu and Apetrei (2021), who wrote that, although the kinetics and stages of the reactions differ, the single electron transfer (FRAP) and hydrogen atom transfer (DPPH) reactions produce identical outcomes because they are dependent on the solvent system, the antioxidant compound’s solubility, and its structure and characteristics. These findings are consistent with Araldi’s work (Araldi et al. 2018), which found that A. sisalana exhibited antioxidant activity. When comparing the two methods, the DPPH is the recommended approach for the antioxidant assay of the Agave plants.