Introduction

Background of the research

Hypoxis hemerocallidea (H. hemerocallidea) is a plant native to Southern Africa that is widely used in traditional medicine and contains a variety of bioactive secondary metabolites (Aremu et al., 2021). H. hemerocallidea, also known as "African potato," is used in traditional medicine throughout Southern Africa to treat endocrine gland dysplasia. Extracts H. hemerocallidea are also applied as a remedy for delirium, bad dreams, and impotence (Porcel and Schutta, 2015). The bioactive attributes of H. hemerocallidea is as a result of its metabolic composition. Some of the many metabolites reported in H. hemerocallidea include; rooperol (Mkhize et al., 2022; Badeggi et al., 2022) and iridoid glycoside harpagoside which have been determined to have antibacterial, anti-inflammatory, antioxidant, and anticancer activity (Mkhize et al., 2022). As a result, HIV/AIDS patients frequently consume it to boost their immunity and improve their overall well-being (Gao et al., 2022).

Due to the medicinal properties of H. hemerocallidea and similar species, researchers have continuously sought for ways to enrich these bioactive compounds through various extraction techniques. One such technique is supercritical fluid extraction (SFE), which is a popular method for extracting a range of bioactive compounds (España et al., 2020). The disadvantages of SFE extraction include the need for elevated pressure (Mkhize et al., 2022), the risk of volatile compound losses (Gao et al., 2022), and the large number of parameters to optimize. Another method that has been explored is microwave-assisted extraction (MAE). This method is desired for its robust capability to extract metabolites in a short time frame (Zia et al., 16). However, microwave-assisted extraction (MAE) has several drawbacks which include high equipment costs (Gao et al., 2022) and the requirement for large amounts of toxic organic solvents, among others (Gao et al., 2022). The QuEChERS method has been desired by researchers for its ability to be quick, easy, cheap and effective. This technique has been studied for the extraction of mycotoxins from plants (Miró-Abella et al., 2017), polyaromatic hydrocarbons in food (Słowik-Borowiec et al., 2022), pesticides and pharmaceuticals in fertilizers (Dong et al., 2023).

Though QuEChERS has its advantages, it also has a tendency of being time consuming and often suffers from poor extraction efficiency. Furthermore, the majority of the clean-up solvents used in the original QuEChERS method are toxic and require large volumes making the technique wasteful and expensive to run. A modified QuEChERS technique is therefore required to minimize the drawbacks experienced with the traditional QuEChERS method. To the best of our knowledge, this project is the first of its kind that was directed at applying the modified QuEChERS method for the enrichment of two bioactive compounds, β-sitosterol and solasodine, from H. hemerocallidea simultaneously. Optimization of extraction of β-sitosterol and solasodine was based on a chemometric study design (Box-Behnken) where parameters such as ultrasonication time, ultrasonication power, partitioning salt and centrifugation time were assessed. As a prototype, the explored method, which has strong pharmaceutical relevance, could meet the insatiable appetite for affordable medicines comprised of compounds from natural products.

Methods and materials

Chemicals and reagents

The salts Na2SO4 (anhydrous ≥ 99% purity), (NH4)2SO4 (anhydrous ≥ 99% purity) and ethanol (99% CP) were purchased from Associated Chemical Enterprises (Johannesburg, South Africa) and Sigma-Aldrich (Johannesburg, South Africa). Ultra-pure water (0.005 µS, 18 mΩ) using a Direct-Q 5UV distiller (MA, USA) was applied for the preparation of the salt solutions. H. hemerocallidea were purchased from Essentially Natural located (Cape Town, South Africa). Individual standard solutions of β-sitosterol (5000 μg mL-1, in methanol) and solasodine (5000 μg mL-1, in methanol) were purchased from Sigma Aldrich (Modderfontein, South Africa). LC-MS grade water and methanol were purchased from Sigma Aldrich (Modderfontein, South Africa).

Chromatographic and spectral conditions

All mass spectral measurements were done using a UPLC triple quadrupole 9030 mass spectrometer (Shimadzu, Japan) with an electrospray interface (ESI) operating in positive mode. The mass range was 100–1000 m/z. Table 1 shows the optimized MS compound dependent parameters for solasodine and β-sitosterol. All samples were centrifuged using a bench-top NF 1200 centrifuge (Vacutec, Turkey). Samples were shaken using mini orbital shaker SSM1 (Lasec, UK). Quantification of each standard was done by selecting the most abundant pre-cursor to product ion transitions in multi reaction monitoring mode. The following parameters were used: drying gas flow = 10 L min-1 and drying gas temperature = 250 °C.

Table 1 The optimized MS compound dependent parameters for solasodine and β-sitosterol
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For chromatographic separation, a Shimadzu 9030 LC instrument consisting of an auto-sampler, thermo-stated column compartment and a binary pump (Shimadzu, Japan) was employed. Data acquisition was done using the Lab solutions software. Standards were separated using a Shimpack C18, 2.1 × 100 mm, 2.7 μm column from Shimadzu (Honeydew, South Africa). The column was maintained at 40 °C. The injection volume was 10 µL. Mobile phase A was 0.1% formic acid in ultrahigh purity water (v/v), and mobile phase B was 0.1% (v/v) formic acid in methanol. Chromatographic separation was achieved using a 7 min gradient elution method consisting of the following settings: the initial conditions were 5% solvent B at a flow rate of 0.4 mL min−1 and was maintained for the next 2 min. The conditions were changed to 95% solvent B at 3 min. This was kept constant for 1 min. The conditions were then changed to 5% solvent B at 5 min, and maintained for the next 2 min.

Preparation of standard solutions

A stock solution of solasodine and β-sitosterol with the concentration of 1000 µg L−1 was prepared in ethanol and stored at 4 °C until analysis. A series of 10 working standard solutions at the concentration levels of 2–500 µg L−1 were prepared.

Sampling and sample preparation

Grounded leaves of H. hemerocallidea were purchased from Essentially Natural located in Cape Town. The solvent, ethanol (5 mL), and 50 mg of H. hemerocallidea powder was poured into a centrifuge tube. The mixture was then sonicated for 5–30 min at an ultrasonication power ranging from 150–300 W. After ultrasonication, the sample was added into a centrifuge tube containing the partitioning salts which was (NH4)2SO4 and Na2SO4, and centrifuged for 4–20 min. Following centrifugation, 10 mg of cellulose was poured into a centrifuge tube where cellulose, a clean-up agent, was added. The mixture was then vortexed for 5 min and then centrifuged for 4–20 min. Following centrifugation the clear solution was used for analysis on the UPLC triple quadrupole mass spectrometer and the lower residue was discarded.

Results and discussion

Calibration curves and instrumental method optimization

The β-sitosterol calibration curve was obtained from standard solutions at various concentrations with a correlation coefficient of 0.9857. The regression equation was found to be y = 9.4428x + 38.921. The calibration curve for solasodine was created by performing a least-square linear regression analysis of the peak area (y) versus analyte concentration (x) also over a concentration range of 2–500 µg L-1. A linear calibration graph with a correlation coefficient of 0.9966 and the regression equation of y = 14184x + 43916 for solasodine was obtained.

Using a sensitive and robust tandem MS approach (UPLC-MS) with settings presented by Mokgehle et al. (2022) it was possible to efficiently fingerprint solasodine at m/z 157 (product ion) and β-sitosterol based at m/z 113 (product ion) as shown in Figs. 1, 2, respectively. Thereafter, based on the MRM transitions at m/z 414 → 157 and at m/z 415 → 113 of solasodine and β-sitosterol, respectively, the analytes were quantified as shown in Table 2

Fig. 1
figure 1

Molecular transition of β-sitosterol (m/z = 415) to product ion (m/z = 397) after the loss of water, and further fragmentation resulted in product ions at m/z = 285 and at m/z = 113

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Fig. 2
figure 2

Molecular transition of solasodine (m/z = 414) to product ion (m/z = 396) after the loss of water and to the product ion at m/z = 157

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Table 2 Lists of experiments using RSM for MS peak optimization
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The concentration of β-sitosterol and solasodine obtained as a function of temperature when parameters such as ultrasonication time, ultrasonication power, centrifugation time and partitioning salts were used is tabulated in Table 2. Generally, the concentrations of solasodine obtained are greater than those of β-sitosterol. Besides, the extraction method used to enrich the studied metabolites; this may suggest that solasodine is prominent in the leaves of H. hemerocallidea, which is a unique finding as solasodine has mainly been reported in Solanum species (Mokgehle et al., 2022; Lal et al., 2022; Begum et al., 2023). Furthermore, solutions containing (NH4)2SO4 generally extracted more solasodine than β-sitosterol in comparison to Na2SO4. This could be due to the doubly charged sulfate ions in Na2SO4 and (NH4)2SO4 forming strong hydrogen bonds with the surrounding water molecules, weakening the solasodine-water interaction and increasing the extent of solasodine precipitation (salting-out) from the hydration sphere. Besides the anionic (SO42-) effect playing a role in salting-out, another distinguishing factor in the salting-out performance of the salts may have been the cationic effect. According to the Hofmeister series Na+ repels analytes from the aqueous phase to a greater extent than NH4+, minimizing the salting-out process (Kang et al., 2020). This is due to the greater hardness and smaller size of Na+, which explains the generally higher concentration of solasodine than β-sitosterol obtained with (NH4)2SO4 compared to Na2SO4 (Kang et al., 2020) (Table 2).

MRM transitions of β-sitosterol and solasodine

Figure 1 shows a fragmentation pattern of β-sitosterol. The β-sitosterol molecular ion was identified at m/z 414, where its molecular formula was C29H50O (Azeez et al., 2018). The product ion at m/z 396 was due to the loss of a water molecule by the molecular ion. The product ion at m/z 397 was further fragmented which involved dealkylation resulting in the product ion at m/z 113 (Azeez et al., 2018).

The ion due to solasodine [M+H]+ ion was isolated at m/z 414. The protonated ion of solasodine had undergone dehydration, resulting in the fragment at m/z 396. Multiple cleavages in the second ring can explain the formation of the ion at m/z 157 as shown in Fig. 2.

Fig. 3a, b shows the mass spectrum of solasodine and β-sitosterol, respectively. The mass spectrum of solasodine contains 3 main (most intense) peaks. The loss of water was represented by the peak at m/z 396 and solasodine at m/z 414. A third peak was also found which was a fragment of m/z 414 was noted at m/z 157. A similar trend was also observed for β-sitosterol.

Fig. 3
figure 3

Product ions obtained during MRM scans for a β-sitosterol and b solasodine

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Evaluation of significant and insignificant parameters in the extraction of β- sitosterol and solasodine

Figure 4 is a Pareto chart, which displays the absolute values of the standardized effects indicating the parameter that were significant in the extraction of the analytes of interest (Kamaruddin et al., 8). The standardized effects are t-statistics that are used to test the null hypothesis that the effect is 0. The y-axis on the Pareto chart represents parameters investigated in this study, the x-axis represents F-values and the bars represents P values. The parameters investigated were ‘ultrasonication time’ as represented by parameter A, ‘ultrasonication power’ as B and ‘centrifugation time’ as C. The results of 34 experiments using a full factorial design to estimate the effects of the aforementioned parameters at two different levels for each parameter show that only the square of ultrasonication time (A2) was a significant parameter due to p < 0.05. Single parameters such as A B or C and parameters which involved interactions of more than parameter such as AB and AC were not significant (P > 0.05).

Fig. 4
figure 4

Pareto charts of β-sitosterol

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Figure 5 depicts the response surface models for AB, AC, and BC, which correspond to (NH4)2SO4 and Na2SO4, respectively. The response surface models evaluate the multivariate interaction between centrifugation time, ultrasonication time, and ultrasonication power. In this case, the dependent variable was Z (solasodine concentration), and the independent variables were x (ultrasonication time) and y (centrifugation time). Equation (1) was derived from the quadratic fit between the predicted and experimental values for extraction of solasodine and had an r2 = 0.9966.

Fig. 5
figure 5

RSM for extraction of solasodine using (a, c, e) (NH4)2SO4 and (b, d, f) Na2SO4

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Quadratic fit equation for extraction of solasodine:

$$ z \, \left( {x,y} \right) \, = \, 22.27x - \, 27.65y \, - 0.44xy \, + \, 0.25x^{2} + \, 0.45y^{2} + \, 2269 $$
(1)

The response surface models (RSM) shown in Figure 5 are color coded. The blue region on the RSM for solasodine extraction indicates low extraction concentrations of solasodine, whereas the green region indicates high extraction of the same analyte (Fig. 5a–e). The graphs in Fig. 5a–c are RSM for solasodine extraction, where (NH4)2SO4 exhibited low solasodine extraction, as most of the graph is shaded in blue, whereas in Fig. 5e solasodine extraction improved with increasing centrifugation time and decreasing ultrasonication power when the same salt was used. It was also observed, particularly for Na2SO4, that the ultrasonication time of 15–20 min resulted in better extraction of solasodine and concurs with the significant effect (p < 0.05) of the squared of ultrasonication time in Fig. 4.

Figure 6 depicts the responses to the multivariate interactions for the extraction of β-sitosterol. Equation (2) was derived from the quadratic fit between the predicted and experimental values for extraction of β-sitosterol and had an r2 = 0.9857.

Fig. 6
figure 6

RSM for extraction of β-sitosterol using (a, c, e) (NH4)2SO4 and (b, d, f) Na2SO4

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Quadratic fit equation for extraction of β-sitosterol:

$$ z \, \left( {x,y} \right) \, = \, 5.91x \, {-} \, 29.17y \, - 0.44xy \, + \, 0.25x^{2} + \, 0.45y^{2} + \, 2483 $$
(2)

From the RSM obtained for β-sitosterol extraction (Fig. 6a, c, e), it was observed that NH4+ ions from (NH4)2SO4 had a higher salting-out ability for β-sitosterol than Na+ ions derived from Na2SO4. This is also observed by the color coded regions of higher extraction as shown by the light blue (Fig. 6a, c), green and yellow (Fig. 6e) colors. This also indicated that (NH4)2SO4 was the more effective partitioning salt. Besides the influence the Hofmeister series in extraction of β-sitosterol, there could be other influential factors such as the nature of interaction between the solute and the hydration sphere. Solasodine has the capability to form (O–H…H–O) interactions with the hydration sphere, while β-sitosterol can form both (O–H…H–O) and (N–H…N–H) interactions (Arumuganainar et al., 2023). Therefore, this effect could have to some extent limited the salting-out process of Na2SO4.

Extraction optima for modified QuECHERS

Optimums obtained for extraction of β-sitosterol and solasodine

The best conditions for the optimal extraction of solasodine and β-sitosterol are summarized Tables 3, 4, respectively. The desirability factor for optimal extraction of both analytes was 1.00 which indicated that the data was reliable. Both analytes required the same conditions for optimal extraction; this could be to their similar structures.

Table 3 Optimum conditions for extraction of solasodine
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Table 4 Optimum conditions for extraction of β-sitosterol
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Conclusion

The effect of ultrasonication time, ultrasonication power, and centrifugation time, on the modified QuEChERS extraction of solasodine and β-sitosterol from H. hemerocallidea was investigated in this study. The salting-out effect of the sulfate ion resulted in the increased precipitation of solasodine compared to β-sitosterol and explained the higher concentrations of solasodine extracted compared to β-sitosterol. The cationic effect in salting-out was another distinguishing factor in the generally superior extraction performance of (NH4)2SO4 over Na2SO4 for solasodine and β-sitosterol. The optimal extraction conditions for both analytes were an ultrasonication time of 30 minutes, ultrasonication power of 300 W, and centrifugation time of 12 minutes, with a desirability of 1. The square of ultrasonication time (A2) was the determined to be a significant parameter (p < 0.05) in extraction of the studied analytes. The extraction of solasodine and β-sitosterol from H. hemerocallidea could be improved by using miniaturized methods or even greener solvents such as deep eutectic solvents (DES). This modified QuEChERS which only used two green solvents, water and ethanol, with (NH4)2SO4 as a salting-out agent, is a promising technique that can be applied to separate other potential nutraceutical compounds from other natural products.