Introduction

Sonchus oleraceus L. (family; Asteraceae), commonly known as Sowthistle (Arabic name; Tilfaf), is an edible leaf vegetable. It is frequently consumed in Mediterranean countries (Guil-Guerrero et al. 1998). It is cosmopolitan and its native is Europe and North Africa (Vieira and Barreto 2006). Traditionally, it used to treat central nervous system dysfunction, and mental disorders (Lane et al. 2006). Several pharmacological properties such as antioxidant, anti-diabetic and anti-inflammatory of S. oleraceus (SO) had been reported before (Schaffer et al. 2005; Teugwa et al. 2013; Li et al. 2017). Phytochemical studies of SO have revealed the presence of sesquiterpene lactones, essential oils, flavonoids, flavonols, proanthocyanidins, phenols, saponins and alkaloids (Miyase and Fukushima 1987; Guarrera et al. 2008).

Plant secondary metabolites plays a crucial role in the treatment of Alzheimer’s disease (AD) (Oken et al. 1998); the most prevalent neurodegenerative disorder. AD is defined by the neuropathological deposition of extracellular amyloid-beta (Aß) senile plaques, intracellular neurofibrillary tangles (NFTs) (Castellani et al. 2010). The protein aggregates (Aß) provoke neuronal damage and synaptic dysfunction like the cleavage of the neurotransmitter acetylcholine (Ach) into acetate and choline. Hence, the inhibition of their formation remains one of the potential therapeutic approach for the treatment of AD which can be achieved by the inhibition of acetylcholinesterase (AChE) (Whittaker 1990). Unfortunately, synthetic AChE inhibitors have side effects and can be potentially toxic to the patients causing gastrointestinal disturbances, hepatotoxicity and short bioavailability (Alva and Cummings 2008).

In fact, oxidative damage has been proposed to be a primary event in AD (Nunomura et al. 2001). This is because brain is quite vulnerable to oxidative injury as it is composed of easily oxidized lipids with a higher oxygen consumption rate (Nunomura et al. 2001). The oxidation of nucleic acid, lipid and protein was found in the neurons of AD patients in which is the common pathological feature of AD (Lovell and Markesbery 2007). Therefore, antioxidant can ameliorate these pathological conditions (Feng and Wang 2012). Further, antioxidant activity was found to be associated with AChE activity (Ferreira et al. 2006). Precedent study also revealed that antioxidants had substantial potential to reduce the symptoms and the frequency of AD (Gutzmann and Hadler 1998). For this reason, the antioxidant had been investigated with AChE activity in the aim of searching for novel AD drugs candidates from natural sources. Thus, the aim of the present work was to determine the antioxidant and AChE properties as well as the chemical constituents obtained from different solvent extractions of the aerial parts of the Algerian SO.

Materials and methods

Chemicals and reagents

In the present study, 1,1-diphenyl-2-picrylhydrazyl (DPPH), butylated hydroxyanisole (BHA), butylated hydroxyl toluene (BHT), α-tocopherol, gallic acid (GA), quercetin, trichloroacetic acid (TCA), potassium ferricyanide, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), galvinoxyl (GOX), 1,10-phenanthroline, acetylthiocholine iodide (ATChI), galantamine, 4-nitrophenyl-α-D-glucopyranoside (≥ 99%), 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB) and Folin-Ciocalteu’s reagent (FCR) were purchased from Biochem Chemopharma. Acetylcholinesterase (AChE) from electric eel (EC 3.1.1.7, Type VI-S, 827.84 U/mg) and all other chemicals and solvents unless stated were analytical grade and were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA).

Collection of plant material

The aerial parts of SO were collected in January 2018 at Boumahra ahmed commune (36° 27′ 16.8″ North, 7° 32′ 55.6″ East), City of Guelma, which is in the Northeastern of Algeria. The plant was authenticated by the botanist, Prof. Gérard De Belair (Faculty of Sciences, University of Annaba, Algeria), where a voucher specimen was deposited (LBEE.22.01.18).

Preparation of extracts/fractions

The aerial parts of SO were air-dried under the shade area at room temperature until completely dried before crushed into a fine powder (diameter < 250 µm) using an electric mill (KWCG-102, China). Then, 200 g of SO powder was macerated in 2 L of hydro-methanolic solution (80%) for 72 h at room temperature and filtered. This combination of solution was used as it can extract the most flavonoids from the plant (Baatouche et al. 2019). The process was repeated in three times. The filtrates were then concentrated and evaporated under pressure in a rotary evaporator (R-215, Büchi Labortechnik AG, Flawil, Switzerland) at 40 °C to obtain crude extract (M.E). A part of the extract was suspended in distilled water and successively partitioned to a liquid–liquid extraction in a funnel using different solvents of increasing polarity: chloroform, ethyl acetate and n-butanol. This yielded chloroform fraction (C.F), ethyl acetate fraction (EA.F) and butanolic fraction (B.F) while the residual represented aqueous phase fraction (AP.F).

For the hot aqueous extract (HAE), 20 g of SO powder was extracted in 100 mL of distilled water for 4 h until boiling. The mixture was filtered and centrifuged at 2000 × g for 15 min. The supernatant was dried using a lyophilisation apparatus (Smach et al. 2015). HAE was kept in dry place. In the preparation of the ethanolic extract (E.E), 20 g of SO powder were mixed with 500 mL of ethanol by constantly shaking for 72 h. After that, it was filtered and concentrated in a rotary evaporator at 40 °C. It was lyophilised to give crude EE. Similarly, the macerated aqueous extracts (MAE) were prepared by mixing 50 g of SO powder with 500 mL of distilled water and constantly shaking for 72 h. Then, it was filtered and frozen before finally lyophilised. Both E.E and MAE were weighed and kept at 4 °C. The methods of extraction for E.E and MAE were modified from Dos Santos et al. (2019).

The extraction of total alkaloid was conducted according to the method reported by Dehmlow et al. (1999). 100 g of SO powder was extracted in 1 L methanol. The methanol was then removed under reduced pressure using a rotary evaporator. 4% acetic acid (500 mL) was added to the residue and it was extracted for three times in petroleum ether (100 mL each time) to remove neutral compound. 120 mL of ammonia was poured on the aqueous layer to alkalinize it until the pH reach 11. It was extracted again in 50 mL of petroleum ether for 10 times. The organic phase was evaporated to give the total alkaloid extract (TAE).

Quantification of total phenolic content

The total phenolic content (TPC) of SO samples was evaluated by using the FCR method as described by Singleton and Rossi (1965) with some modifications (Müller et al. 2010). Briefly, 10 mg of the extracts/fractions was dissolved in 10 mL of methanol using sonicator to yield a concentration of 1 mg/mL. Then, 20 µL of the extracts/fractions (1 mg/mL) were mixed with 100 µL of FCR (1:10 in distilled water) and 75 µL of sodium carbonate solution (7.5%) in the wells of 96-well microplate. After 2 h of incubation in the darkness at room temperature, the absorbance was measured using microplate reader (EnSpire® Multimode Plate Reader, PerkinElmer, Inc., Massachusetts, USA) at 765 nm against a blank. The tests were performed in triplicate. A calibration curve was generated by using the optical density (OD) of the know concentration of GA as standard. TPC was expressed as µg gallic acid equivalents/mg dried extract weight (µg GAE/mg of extract) which was based on the calibration curve of gallic acid with the linearity range from 10 to 100 µg/mL (R > 0.99).

Quantification of total flavonoid content

The total flavonoid content (TFC) of plant extracts was determined using the microplate method as described by Topçu et al. (2007). A volume of 50 µL of the extracts/fractions (1 mg/mL in methanol) was mixed with 130 µL of MeOH. This was followed by the addition of 10 µL of acetate potassium and 10 µL of aluminium nitrate. After 40 min at room temperature, the absorbance was measured at 415 nm using microplate reader (EnSpire® Multimode Plate Reader, PerkinElmer, Inc., Massachusetts, USA). All samples were performed in triplicate. TFC was expressed as µg of quercetin equivalents per milligram of dried extract weight (µg QE/mg of extract) which based on the calibration curve of quercetin with the linearity range from 25 to 200 µg/mL (R > 0.99).

Quantification of total flavonol content

Total flavonol content (TFLC) was measured using the method of (Kumaran and Joel Karunakaran 2007). Briefly, a volume of 50 µL of the extracts/fractions was mixed with 50 µL of aluminium chloride and 150 µL of sodium acetate solution (in water). After 2.5 h of incubation, the absorbance was measured at 440 nm. All tests were carried out in triplicate. The results were expressed as µg of quercetin equivalents per milligram of dried extract weight (µg QE/mg of extract) which based on the calibration curve of quercetin with the linearity range from 25 to 200 µg/mL (R > 0.99).

DPPH radical scavenging activity

The scavenging ability of the extracts/fractions towards the DPPH radical was determined using the method of (Blois 1958) with modifications involving the use of a high-throughput microplate system. In a 96-well microplate, 160 µL of the methanolic DPPH solution was mixed with 40 µL of the samples in methanol at different concentrations (12.5–800 µg/mL). After 30 min at room temperature in the darkness, the absorbance was recorded at 517 nm against a blank using a microplate reader (EnSpire® Multimode Plate Reader, PerkinElmer, Inc., Massachusetts, USA). BHA, BHT and α-tocopherol were used as comparison while methanol was used as a control. The percentage of inhibition of radical scavenging activity was calculated using Eq. 1. The concentration of samples providing 50% inhibition (IC50 of the samples) was obtained from the plotted graph of the percentage of inhibition versus the concentrations of samples.

$${\text{Percentage of Inhibition}}\,\left( \% \right) = \frac{{A_{{{\text{control}}}} - A_{{{\text{sample}}}} }}{{A_{{{\text{control}}}} }} \times 100$$
(1)

where Acontrol is the absorbance of the control reaction and Asample is the absorbance of the test samples.

ABTS radical scavenging assay

The ABTS radical cation (ABTS•+) decolourisation assay was performed according to the method described by Re et al. (1999) with slight modifications. At first, ABTS was diluted in water to 7 mM concentration. Next, ABTS•+ solution was prepared by reacting ABTS solution with potassium persulfate (final concentration: 2.45 mM). The mixture was allowed to stand in the dark at room temperature for 16 h before use. After that, the mixture was diluted in water to get an absorbance equal to 0.700 ± 0.020 at 734 nm. Then, 40 µL of the extracts/fractions in methanol at different concentrations and 160 µL of diluted ABTS•+ solution were added to each well of microplate. After 10 min, the absorbance at 734 nm was recorded using a microplate reader (EnSpire® Multimode Plate Reader, PerkinElmer, Inc., Massachusetts, USA). The percentage of inhibition was calculated using the Eq. 1 and the IC50 values were deducted from the plotted graph. BHA and BHT were used as the positive controls.

Phenanthroline assay

The (Szydłowska-Czerniak et al. 2008) method was adopted for this assay. This assay measured the formation of tri-phenanthroline complex. In the presence of hydroxyl radical, Fe2+ was oxidized to Fe3+ and combined with 1,10-phenanthroline to form tri-phenanthroline. Otherwise, this reaction was disrupted if the antioxidant was able to scavenge the hydroxyl radical. The reaction mixture containing 10 µL of different concentration of the extracts/fractions in methanol, 30 µL of 1,10-phenanthroline solution in methanol (0.5%), 50 µL ferric chloride (FeCl3) (0.2%) and 110 µL of MeOH was incubated in the dark for 20 min at 30 °C. The absorbance of the reaction mixture was then read at 510 nm. The percentage of inhibition was calculated according to the Eq. 1 and compared with BHT as a classical metal chelator. The results were presented as IC50 value in µg/mL.

GOX free radical scavenging assay

This assay was performed as shown by Shi et al. (2001). It measured the scavenging ability of the antioxidants toward the stable GOX radicals by donating hydrogen in the hydroxyl groups to form resonance-stabilized GOX radicals. Basically, GOX solution was appeared in deep blue and decolourised to become purple over the time as its odd electron was paired off. In brief, 160 µL of GOX (0.1 mM) was mixed with 40 µL of each sample at different concentrations. The reaction was carried out at room temperature and monitored for 2 h. The decrease in GOX concentration was recorded by measuring the absorbance at 428 nm.

AChE inhibition assay

The inhibitory activity of AChE was investigated using a modified version of (Ellman et al. 1961) in a 96-well microplate. 150 µL of Tris buffer (0.1 M, pH 8.0), 10 µL of the extracts/fractions in ethanol with different concentrations (3.125 – 200 µg/mL) and 20 µL of AChE enzyme solution (5.32 × 10–3 U) were mixed and incubated for 15 min at 25 °C. Subsequently, the reaction was started by adding 10 µL of DTNB (0.5 mM) and 10 µL of ATChI substrate solution (0.71 mM). The absorbance of the mixture was measured spectrophotometrically at 412 nm in every 5 min for 15 min. A blank containing Tris buffer and ethanol instead of enzyme solution was used. A control mixture was also prepared by replacing ethanol instead of extract and was considered as 100% enzyme activity. The percentage of inhibition was calculated as in Eq. 2. The concentration of extracts/fractions providing 50% of inhibition (IC50) was determined by plotting the percentage of inhibition versus the concentrations of extracts/fractions.

$${\text{Percentage of inhibition}}\left( \% \right) = \frac{{\left( {E - S} \right)}}{E} \times 100$$
(2)

where E is the activity of the enzyme without the sample (100% enzyme activity) and S is the activity of the enzyme in the presence of the sample.

LCMS–QTOF analysis

The determination of the phytochemical constituents of the active extracts/fractions was carried out using LCMS-QTOF (LC 1200 Series, Agilent Technologies, Palo Alto, California, USA) equipped with a standard autosampler. The chromatography column was ZORBAX Eclipse Plus C18 (EPC-18) column (2.1 × 100 mm, 1.8 µm) from Agilent Technologies. Firstly, the samples were prepared by dissolving the extracts/fractions (4 mg) in methanol (HPLC grade). The samples were filtered by passing through a Millipore nylon filter disk (0.22 µm) using an injector to remove any particulates before they were injected into the LCMS system. The injection volume was 2 µL and the column temperature was set at 40 °C. The samples were run using a gradient elution programme at a flow rate of 0.25 mL/min. The mobile phases that were used consisted of ultra-purified water plus 0.1% formic acid (A) and 5% acetonitrile (B). The solvent gradient was applied as follows: 0 min, 5% B; 36 min, 95% B; 41 min, 95% B; 41.1 min, 5% B; 48 min, 5% B. The LCMS system was coupled to a Quadrupole-Time-of-Flight (QTOF) (Agilent Technologies, Palo Alto, California, USA) and a detector was equipped with an electrospray ionization source (ESI) operating in the positive ion mode. The optimum values of the ESI–MS parameters were: nitrogen in which was used as drying gas at a temperature of 325 °C, drying gas flow at 10 L/min; nebulising gas pressure at 30 psi; capillary voltage potential at 4000 V and finally, fragmentor voltage of 175 V was chosen and applied to the samples. The detection was carried out within a mass range of 100–1100 m/z. For analysis of the samples, the accurate mass data of the molecular ions were processed using Agilent Mass Hunter Qualitative Analysis B.05.00 software (Agilent Technologies, Palo Alto, California, USA). All products were recognized by interpreting their MS data and comparing them with the available bibliography.

Statistical analysis

All the experimental data were displayed as mean ± standard deviation (SD) of three trials. The one-way analysis of variance (ANOVA) followed by Tukey, Least Significance Difference (LSD) post-hoc tests, and the homogeneity test of variances by the Levene test were conducted using IBM SPSS Statistics V20 software (IBM Corporation, New York, USA). The level of significance was set at p < 0.05 value.

Results and discussion

Yield of extractions

The extraction yields of different extracts/fractions of SO were reported in Table 1. Based on the results, AP.F produced the highest yield of extraction (41.73%) while the lowest amount of yield was C.F (0.14%). According to Dhanani et al. (2017), the amount of yield indicated the effects of the extraction conditions. Thus, the variations in the extraction yields was due to the differences in the polarity of the solvents that were used as well as the method of extractions which directly impacted the biological activities of the final extracts (Hayouni et al. 2007; Pellegrini et al. 2007). As the most polar solvent, water was the most efficient solvent in extracting the phytochemical constituents from the plant. In fact, the combination of water and organic solvent (methanol) which also had high polarity further enhanced the efficiency of the extraction. This was supported by the previous study that showed high polar solvent resulted greater percentage of yield as compared to less polar solvent (Abubakar et al. 2017). The study by Jacotet-Navarro et al. (2018) also found that hydro-alcoholic mixtures gave maximum yield of extraction.

Table 1 The yield of extraction and antioxidant potential of the extracts/fractions of SO in different assays
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However, there was a great reduction in the percentage of yield when different methods of extraction were used. The hot extraction (10.92%) and maceration method (6.50%) in aqueous condition caused the decrease in the percentage of yield by 4 and 7 times as compared to liquid–liquid extraction. In the case of different solvent extractions, ethanol yielded higher mass of extraction (33.10%) than water (6.50%). It was the second highest amount of yield. This is due to the plant material contains high levels of polar compounds that are more soluble in ethanol rather than in water (Thakur & Arya 2012; Piechocka et al. 2020). Therefore, it can conclude that the use of organic solvent especially ethanol in solid–liquid extraction possessed the greatest performance in getting better yield for this plant.

Total phenolic, flavonoid and flavonol contents

The results of total phenolic, flavonoid and flavonol contents were presented in Figs. 1, 2 and 3 respectively. EA.F contained the highest amount of phenolic compounds (259.20 ± 23.51 µg GAE/mg) and followed by B.F (167.53 ± 4.57 µg GAE/mg). The lowest phenolic content was obtained from C.F (23.31 ± 2.21 µg GAE/mg) while the rest were between 28.51 µg GAE/mg and 89.98 µg GAE/mg. Similarly, EA.F and B.F exhibited the top amount of total flavonoid with the value of 128.33 ± 1.00 µg QE/mg and 112.85 ± 7.82 µg QE/mg respectively. The other samples demonstrated the value of total flavonoid content between 15.88 µg QE/mg and 54.45 µg QE/mg with the lowest amount was also in C.F. For the total flavonol content, the highest values were still in EA.F (233.73 ± 0.88 µg QE/mg) and B.F (221.38 ± 3.41 µg QE/mg) but MAE comprised the least amount of flavonol (137.21 µg QE/mg). Meanwhile, the range of flavonol content in the other samples were between 141.25 μg QE/mg and 187.54 µg QE/mg.

Fig. 1
figure 1

Total phenolic content of different extracts/fractions from the aerial parts of SO. GAE: Gallic acid equivalent, DW: Dry weight of the samples, M.E: methanolic extract, C.F: chloroformic fraction, EA.F: ethyl acetate fraction, B.F: butanolic fraction, AP.F: aqueous phase fraction, MAE: macerated aqueous extract, HAE: hot aqueous extract, E.E: ethanolic extract and TAE: total alkaloid extract. a−e: Results with different superscript letters were significantly different

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Fig. 2
figure 2

Total flavonoid content of different extracts/fractions from the aerial parts of SO. QE: Quercetin equivalent, DW: Dry weight of the samples, M.E: methanolic extract, C.F: chloroformic fraction, EA.F: ethyl acetate fraction, B.F: butanolic fraction, AP.F: aqueous phase fraction, MAE: macerated aqueous extract, HAE: hot aqueous extract, E.E: ethanolic extract and TAE: total alkaloid extract. a−f: Results with different superscript letters were significantly different

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Fig. 3
figure 3

Total flavonols content of different extracts/fractions from the aerial parts of SO. QE: Quercetin equivalent, DW: Dry weight of the samples, M.E: methanolic extract, C.F: chloroformic fraction, EA.F: ethyl acetate fraction, B.F: butanolic fraction, AP.F: aqueous phase fraction, MAE: macerated aqueous extract, HAE: hot aqueous extract, E.E: ethanolic extract and TAE: total alkaloid extract. a−f: Results with different superscript letters were significantly different

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Our findings showed that EA.F and B.F had the highest total phenolic, total flavonoid and total flavonol content in despite of low extraction yield. In a study carried out by Teugwa et al. (2013), total phenolic content of the hydroethanolic and methanolic extracts of the whole plant of SO were measured at 616.89 ± 19.20 and 182.25 ± 16.76 µg catechine equivalent/g of dry extract respectively which were higher than the current results. In another research, high amounts of TPC and TFC had been obtained from the aerial parts of SO grown in China when methanol was used (Xia et al. 2011). Consistently, the methanolic extract of SO which was grown in Korea had the highest value of TPC, and the lowest values of TPC were obtained in ethanolic and water extracts (Yin et al. 2007). This was also not in line with our results. The differences may be due to geographical distribution and various environmental conditions, which can affect the synthesis of the phytochemicals in the plant (Hossain and Shah 2015).

Generally, those intermediate polar solvents such as ethyl acetate and butanol were found to be more potent in extracting phenolic compounds (Ajayi et al. 2017). In a study on Armoracia rusticana, it was revealed that ethyl acetate extraction possessed high TPC as ethyl acetate is the best solvent for selectively extracting polyphenols (Thavamoney et al. 2018). Moreover, most of the flavonoids are known to be less polar or semi-polar in nature. Because of that, a research on Monotheca buxifolia suggested that hexane is a poorer solvent for flavonoids recovery compared to those more polar solvents such as ethyl acetate and butanol (Jan et al. 2013). Furthermore, the ethyl acetate and the n-butanol fractions were reported to be more effective in extracting phenol compounds from crude extract according to the previous research. It could be suggested that phenol compounds mostly occupy the ethyl acetate fraction as compared to the other fractions, resulting low extraction yield and high TPC (Nakamura et al. 2016). This explained the highest value of TPC, TFC and TFLC in EA.F and B.F as compared to other solvents. Thus, EA.F and B.F has a better extraction potency in extraction of phenolics than other organic solvents like chloroform.

Antioxidant capacity

Polyphenolic compounds are the major constituents that present in the plant and mostly possess as potent antioxidants (Elufioye et al. 2019). Hence, the antioxidant activities of the plant samples were evaluated via four complementary techniques instead of a single method which were DPPH, ABTS, galvinoxyl and phenanthroline assays. This is because to give better conclusive results of the antioxidant properties of the extracts (Sacchetti et al. 2005; Houghton et al. 2007). Among them, DPPH assay is the most used method in evaluating antioxidant capacity by evaluating the radical scavenging activity. The antioxidant potential of the different samples was reported in Table 1 and the results were expressed as IC50 and A0.5 values.

Generally, the extracts/fractions of SO displayed the scavenging abilities in concentration-dependent manner. Among the extracts/fractions, EA.F and B.F showed the highest DPPH and galvinoxyl scavenging activities and had the strongest ability of inhibiting the hydroxyl radical as measured in phenanthroline assay. However, EA.F was more efficient in scavenging the free radical of DPPH and GOX than the B.F. Interestingly, EA.F exhibited almost the same capability in scavenging DPPH as α-tocopherol and BHT. The results also revealed EA.F and B.F fractions that possessed the highest TPC value favoured to show stronger DPPH radical scavenging activity. Similarly, the study by Nakamura et al. (2016) showed that EA.F gave the highest DPPH activity. In contrast, the DPPH activity of methanol extract of S. asper from Pakistan was higher than in EA.F (Rahmat et al. 2012). According to Thavamoney et al. (2018), the radical scavenging activity was greatly affected by the presence of the hydroxyl group in the C-3 position. Therefore, stronger DPPH fractions might contain a lot of phenolic compounds that are structurally effective for scavenging the DPPH radicals (Nakamura et al. 2016).

In ABTS•+ assay, TAE was the most powerful in the reduction of ABTS•+ radical as compared to other extracts/fractions. Moreover, it was comparable to the control BHT and BHA where no significant difference was observed. Otherwise, C.F was found to be the weakest in ABTS•+ scavenging activity. On the other hand, EA.F and B.F showed the highest antioxidant activities among all fractions in GOR and phenanthroline tests which were firstly reported in this plant. This were in accordance with their high phenolic, flavonoid and flavonol contents. TAE showed strong ABTS•+ scavenging activity due to the presence of reducing molecules that may inhibit the potassium persulfate activity and hence reduced the production of ABTS•+. Wang et al. (1999) found that some compounds which had ABTS•+ scavenging activity did not showed DPPH· scavenging activity. The non-active phenolic compounds, which were not detected in the DPPH radical scavenging assay, reacted in the ABTS radical assay, and therefore lowered its values.

The difference in antioxidant activity might be due to the composition of phenolic compound in the samples that contained a high number of hydroxyl groups. This groups were responsible for a high antioxidant activity (Arabshahi-D et al. 2007). Besides that, the method and conditions of extraction (temperature and time) also affected these activities (Robards. 2003). The difference in the stoichiometry of reactions between the antioxidant compounds in the samples and the various radicals might also be inferred as a reason for the difference in their scavenging potential (Wang et al. 1999). Factors like stereo selectivity of the radicals or the solubility of the samples in different testing systems had been reported to affect the capacity of samples to react and quench different radicals (Wang et al. 1999).

AChE activity

Different samples of SO were tested to determine their ability as AChE inhibitors and the results were depicted in Table 2. M.E exhibited the most potent inhibitory activity of AChE, followed by C.F, EA.F, B.F, E.E, HAE, TAE and MAE. It was important to note that in spite of the lowest content of phenolic, flavonoid and flavonol compounds in C.F; this fraction was good in inhibiting AChE. On the other hand, literature data of the inhibitory effect of SO against AChE were insufficient. In this work, M.E exhibited the lowest IC50 (27.07 µg/mL) in inhibiting AChE activity, which was more powerful than the methanolic extract of S. asper (IC50: 65 µg/mL) from Pakistan (Khan et al. 2012). Previous study also showed that methanolic extract gave good inhibitory activity against AChE, followed by ethyl acetate fraction, suggesting that the active constituents could be a cocktail of polar compounds in inhibiting AChE (Elufioye et al. 2019). However, in another research, hexanic extract was the most effective extraction against AChE (Ayaz et al. 2014). These suggested that the organic solvents were able to effectively extract the molecules which inhibited the enzyme at the best.

Table 2 The inhibitory activity of AChE in different extracts/fractions of SO
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Phytochemical compounds in the plant extracts/fractions

In order to evaluate the compounds that were responsible for the various bioactivities as examined above, the samples with the best antioxidant activity (EA.F and B.F) and enzyme inhibition (M.E) were subjected to compound analysis using LC–MS/QTOF. The identification of the compounds was presented in Table 3 and illustrated in Fig. 4. Their retention time and mass spectrometry obtained by MS/QTOF were compared with the reported data in the literature. M.E, EA.F and B.F contained a total of 10, 17, and 12 compounds respectively. Among them, 14 compounds were phenolic acid derivatives while the rest was composed of sesquiterpene lactones and phenylpropanoid derivatives. Quininic acid was the main compound in the M.E while gallic acid was the main compound in EA.F and B.F. Quininic acid was detected at (1.07 min) in the ESI-QTOF positive ion mode. It gave [M–H]+ ion at m/z 204.0669 in accordance with the molecular formula C11H9NO3. Gallic acid was found in the EA.F and B.F at the same retention times (4.86 min) and it gave [M–H]+ ion at m/z 171.0285 with the molecular formula C7H6O5 respectively.

Table 3 Identification of compounds in three active extracts/fractions of SO using LC–MS/QTOF
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Fig. 4
figure 4

Base peak chromatograms data detected in the aerial parts of SO by LC–MS/QTOF in positive ionization mode. The compounds in the three samples: (a) M.E, (b) EA.F and (c) B.F were characterized, numbered and presented as in Table 3. 1: Quininic acid; 2: Aesculin; 3: 3-(acetyl-oxy)-1-methoxy-1-(3,4,5-trimethoxyphenyl) propane; 4: Corchoionoside C; 5: Sonchuside H; 6: Loliolid; 7: Melampolide; 8: 15-O-β-D-glucopyranosyl-11β,13-dihydro urospermal A; 9: Tanacetin; 10: Macrocliniside A; 11: 2-(3,4-dihydroxyphenyl)-2-{[2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3,4-dihydro-2H-chromen-3-yl]oxy}-3,4,5,7-chromanetetrol; 12: Gallic acid; 13: Luteolin 7- β -D-glucosiduronic acid; 14: Sinapinic acid; 15: Catechol; 16: Apigenin-7-O-ß-D-glucuronide methyl ester; 17: Apigenin; 18: 15-hydroxy-4β,15,11β,13-tetrahydroreynosin; 19: 3-hydroxy-1-(4-hydroxy-3,5-di methoxyphenyl)-1-methoxypropane; 20: D-(-)-mannitol; 21: Ethyl linoleate; 22: Quinic acid; 23: Vitamin C; 24: 3-hydroxyflavone

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The results were consistent with previous studies on the leaves and aerial parts of SO and other species of the same genus (Abhijeet et al. 2018; Li and Yang 2018). Aesculin and quininic acid was found in the crude extract, which were comparable with the results cited by Xu et al. (2008). Sonchusides A-D and macrocliniside A had been previously isolated and identified in the whole methanolic extract from SO (Miyase and Fukushima 1987). Meanwhile, 15-O-β-D-glucopyranosyl-11β,13-dihydro urospermal A had not only characterised in the whole plant of S. asper and roots of SO in Egypt (Helal et al. 2000; Elkhayat 2009), but it was also found in SO plant originated from Algerian as observed in this study.

Flavonoids are another important group of phenolic compounds that are widely found in Sowthistle (Abhijeet et al. 2018). Among these, gallic acid, catechol and apigenin-7-O-ß-d-glucuronide methyl ester were cited in the literature (Li and Yang 2018) and all of them were found and identified in the EA.F and B.F. Other flavonoids that were found in the fractions were luteolin 7-β-d-glucosiduronic acid, apigenin, sinapinic acid, 3-hydroxyflavone and ascorbic acid. Besides, 15-O-β-glucopyranosyl-11β,13-dihydrourospermal A, corchoionoside C and sonchuside H had been found in the M.E and EA.F. They were isolated previously for the first time from SO, S. erzincanicus and S. arvensis (Bondarenko et al. 1978; Elkhayat 2009; Ozgen et al. 2010). Interestingly, the EA.F which contained the highest amount of phytochemical contents (phenols, flavonoids and flavonols) exhibited the highest number of compounds as compared to the B.F and M.E.

From the pharmacological point of view, the presence of loliolid, quinic acid, luteolin 7-β-D-glucosiduronic acid and sinapinic acid especially in the potent EA.F are very important since they are well-known to have strong antioxidant activities and may have been responsible for the observed activity (Inbathamizh and Padmini 2013; Nićiforović and Abramovič 2014; Malgorzata et al. 2015; Ma et al. 2018). In addition, apigenin-7-O-ß-d-glucuronide methyl ester, 3-hydroxyflavone and ascorbic acid were only identified in the B.F which they had effective scavenging activity against DPPH radical (Nayak et al. 2014; Wąsik and Antkiewicz-Michaluk 2017; Kamalakararao et al. 2018). The sesquiterpene lactone melampolide (costunolide) was only present in the M.E and is known for its neuroprotective effect (Ham et al. 2012). The EA.F and B.F also showed an efficient AChE inhibitory activity that might be due to gallic acid, catechol, apigenin, sinapic acid and ascorbic acid as reported by the other studies (Nićiforović and Abramovič 2014; Badhani et al. 2015; Wąsik and Antkiewicz-Michaluk 2017; Wang et al. 2018). Thus, this study showed good antioxidant and AChE properties of the polar fractions which are good sources of phenolic compounds and can be used in the management of AD.

Conclusion

To the best of our knowledge, this was the first report that directly compared the biological activities, as well as the phytoconstituents of seven different extraction solvents of SO. This study showed that the EA.F and the B.F demonstrated the highest activity in DPPH, galvinoxyl and phenanthroline scavenging assays as well as total phenolic, flavonoid and flavonol contents. The TAE showed high ABTS activity and the M.E exhibited the highest activity in inhibiting AChE. Coupling with LC–MS/QTOF allowed us to identify 24 compounds in the three active extracts; mainly phenolics, flavonoids and sesquiterpene lactones which were responsible for their biological activities. This study gave initial insight of the effective extraction for a promising treatment of AD.