Tunisian Silybum species: Potential source of phytochemicals, organic acids, minerals, and proteins across various plant organs

doi:10.21203/rs.3.rs-3560005/v1

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Tunisian Silybum species: Potential source of phytochemicals, organic acids, minerals, and proteins across various plant organs

https://doi.org/10.21203/rs.3.rs-3560005/v1

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The genus Silybum comprises two closely related species, S. marianum and S. eburneum, both of which are listed among the most highly valued wild edible plants. Various local rural communities in the Mediterranean basin have incorporated these plants into their diets. In this study, we investigated the organs (stems, leaves, flowers, immature seeds, and mature seeds) of wild, potassium, edible S. marianum and S. eburneum collected from two Tunisian regions for their mineral composition, polyphenolic contents, organic acids, seed storage proteins, as well as their antioxidant potential. The Flame atomic spectroscopy revealed that, in S. marianum, stems had high sodium, while potassium and calcium were in the flower and leaf. S. eburneum had high sodium in stems and potassium and calcium in the flower, with variations in magnesium and other minerals. S. marianum shows substantial fructose variation among its organs, with the highest content in the stem (3.54 mg/g DW) and the lowest in immature seeds (0.01 mg/g DW). Conversely, S. eburneum exhibits significant heterogeneity in glucose, sucrose, and maltose levels across its organs, with maltose exclusively detected in immature seeds. A notable organ-dependent distribution of organic acids was observed for oxalic, quinic, citric, malic, succinic, lactic, formic, acetic, and propionic acids among the two species. The phenolic contents were calorimetrically evaluated and further submitted to an LC-MS analysis. Higher levels were detected in both mature and immature seeds in both species compared to the other plant parts. Also, the seeds possessed higher antioxidant activities. A total of 32 phenolics were identified with 3,4-di-O-caffeoyquinic acid, 4,5-di-O-caffeoyquinic acid, syringic acid, protocatechuic acid, naringenin, apigenin, and quercetin were detected as the dominant compounds. In both S. marianum and S. eburneum, albumins and globulins are the predominant protein fractions in both mature and immature seeds and the storage protein fraction was mostly affected by the maturity stage. This current study substantiates the versatile utility of Silybum as a sustainable resource with significant potential for its nutritional applications in the production of functional foods across diverse sectors of the food industry.

The Silybum genus is classified within the tribe Asterales of the Asteraceae family and is native to the Middle East, the Arabian Peninsula, the Indian subcontinent, North Africa, and certain regions of Europe. It has been introduced to various regions, including Japan, sub-Saharan Africa, North and South America, parts of Europe, Australia, and New Zealand [1]. The genus Silybum comprises of two closely related species: S. marianum is characterized by its variegated foliar morphology and S. eburneum exhibits a uniform green leaf [2]. Initially considered distinct species, genetic studies have since revealed that these two variants are closely related and likely represent different forms or varieties within the same species [3, 4]. S. eburneum is a non-grass herb, a wild plant characterized by its glabrous, spine-scented nature. It typically thrives as an annual or biennial species, predominantly found in marginal land, grassy banks, and river flats. This erect plant can reach heights ranging from 15 to 300 cm. It produces solitary, light purple (occasionally white) flowers, often in profusion, from the months of June through September. The leaves are uniformly green, simple, and arranged alternately along the stem. They are lanceolate in shape, featuring denticulate margins and petiolate attachments. The seeds are notably hard and come equipped with a white, silky pappus for dispersal. [4]. For S. marianum, leaves are arranged alternately and are notable for their significant size and glabrous texture, featuring spiny margins. These leaves typically measure between 50 and 60 cm in length and 20 to 30 cm in width [5]. The plant's stem exhibits a height range of 40 to 200 cm, displaying a smooth or slightly downy texture. It maintains an erect posture and undergoes branching primarily in the upper section of the plant [6]. Each stem culminates in a flower head, approximately 5 cm in diameter, displaying a distinctive red-purple hue. The inflorescences are encircled by protective spiny bracts [7]. The seeds are achenes measuring 5–8 mm in length, featuring an elongated white pappus and exhibiting a color spectrum spanning from black to brown [8]. The weight of 1000 seeds ranges from 28 to 30 g. Each flower head yields approximately 190 seeds, resulting in an average seed production of 6350 seeds per individual plant [9]. Furthermore, it is noteworthy that seeds can maintain viability in the soil for as long as nine years [10]

For centuries, various local rural communities in the Mediterranean basin have incorporated these plants into their diets. In Italy, the peeled heads and stems are consumed raw [11]. In Spain, S. marianum has a historical tradition of culinary use as a salad vegetable or as a boiled and fried dish [12], Arabs have a tradition of consuming the young fleshy stems, seeds, and sprouts [13]. In Tunisia, these plants are listed among the widely consumed plants and edible parts consist of the capitulum (flower head) and the seeds. These two components are commonly consumed either in their raw form or are utilized as essential ingredients in the traditional breakfast cereals:“Bsissa” [14]. This plant is listed among the most highly valued wild edible plants, recognized for its potential as a food ingredient with significant economic value [15] The mid-ribs of basal leaves are widely consumed, either raw or cooked, in the several Mediterranean countries [16]. The basal leaves are commonly stewed, and rarely used in their raw form as ingredients in salads [17].

The seeds of the milk thistle plant have been extensively employed in medicinal contexts for an impressive span of over two millennia, principally acclaimed for their therapeutic effectiveness in addressing liver-related maladies [18]. In their review, [19] noted that Milk Thistle received much attention as an innovative and versatile crop for agriculture in challenging environments. Additionally, the fruits of this plant are industrially utilized in the production of silymarin [20]. It is proven that the seeds of S. marianum contain a high amount of oils (enter 11.69% and 29.68%), proteins (20.35–25.25% ), carbohydrates (38.16%), fiber (27.24–29.95%) as well as numerous beneficial components such as essential amino acids, minerals, and phytochemicals with potential biological activities such as antioxidant and antimicrobial effects (El-haak et al. 2015[21, 22].

The primary aim of this study was to conduct a comparative analysis of the phytochemicals extracted from various plant organs, including leaves, stems, flowers, immature seeds, and mature seeds of S. marianum and S. eburneum, which naturally grow in Tunisia. The mineral composition was evaluated by Flame Atomic Absorption Spectroscopy (FAAS). Organic acids and free sugar contents were characterized using LC-MS analysis. The qualitative and quantitative profiles of the polyphenolic compounds were determined through spectrophotometric analysis and High-Performance Liquid Chromatography-Electrospray Ionization/Mass Spectrometry (LC-ESI/MS). Additionally, the antioxidant activity of the extracts was assessed through three in vitro methods: total antioxidant capacity, DPPH free radical scavenging activity, and reducing power assay.

2.1. Plants collection

The plant material used includes the plants Silybum marianum and Silybum eburneum (leaves, stems, flowers, mature and immature seeds). S. marianum was harvested in Médenine (33°21'21.4"N 10°29'05.6"E), located in the southeast of Tunisia, while S. eburneum was harvested in Sidi Bouzid (35°04'42"N 9°20'06"E), located in the center of Tunisia. Whole plants were collected in March 2021 while the collection of mature seeds was carried out in May 2021. After harvesting, the plants were washed and separated into organs: leaves, stems, flowers, and seeds (Fig. 1).

2.2. Minerals analysis

The different plant parts of both species were finely ground into a powder. Subsequently, 0.5% nitric acid was added to each sample. The mixtures were left to stand for 48 hours in darkness with vigorous agitation. Afterward, the resulting extracts were filtered, and the concentrations of the mineral elements were determined using an atomic absorption spectrophotometer (Shimadzu AA6800) that was equipped with WIZAARD 2.30 control software. The concentration of mineral components was calculated by referencing calibration curves specific to each element [23].

2.3. Soluble sugar contents.

For the measurement of soluble sugar contents was determined as detailed in [24], a 200 mg sample of samples were ground in 1 ml of 80% ethanol, followed by incubation at 80°C for 20 minutes. Afterward, it was centrifuged at 13,000g for 20 minutes. This extraction process was repeated twice to maximize sugar molecule extraction. The resulting supernatant was then stored at 4°C until needed. Subsequently, the soluble sugar content was quantified using the high-performance liquid chromatography (HPLC) method as described by [25].The separation was conducted on an XBridge amide column (3.5 µm, 4.6 × 150 mm; Waters), and sugar standards for HPLC from Sigma-Aldrich were employed. The soluble sugar contents were expressed as milligrams per gram of dry weight (mg/g DW).

2.4. Organic acid contents

The organic acid composition in the different part of Silybum species were determined as previously detailed in [24]. In brief, a wight of 200 mg of samples were ground in 1 ml of ultrapure water and then incubated at 80°C for 1 hour. After incubation, they were subjected to centrifugation at 11,000g for 20 minutes, and the resulting supernatant was collected and stored at 4°C until needed. The analysis of these extracts was carried out using LC–MS (liquid chromatography-mass spectrometry). LC analysis was performed using a Shimadzu UFLC XR system HPLC instrument, and separation occurred on a PerkinElmer Brownlee™ Aqueous C18 type column (5 µm, 250 × 4.6 mm). The identification of organic acids was based on standards. The contents of oxalic, quinic, citric, malic, succinic, lactic, formic, acetic, and propionic acids were expressed as mg/g DW.

2.5. Storage proteins contents

The extraction of the four storage protein fractions from mature and immature seeds of S. marianum and S. eburneum was conducted using the method of [26], slightly modified as detailed in [27, 28]. The seeds were first ground and defatted. The albumin fractions were extracted using 1 mL of distilled water (pH 6.5). The globulin fractions were extracted in a Tris-HCl buffer containing 100 mM Tris-HCl and 0.5 M NaCl (pH 8.1). The prolamin fractions were extracted with a 55% (v/v) aqueous isopropanol solution, and the glutelin fraction was extracted using a 0.2 M acetic acid solution. The content of the fractions was evaluated following the procedure outlined by[29], using a standard range of Bovine Serum Albumin (B.S.A). The amount of albumin, globulin, prolamin and glutelin were measured in mg/g DW.

2.6. Secondary metabolite screening

2.6.1. Extraction of active ingredients

A wight of 10 grams of powdered leaves, stems, flowers, both mature and immature seeds samples were extracted with 100 mL pure methanol by cold maceration at 40°C for 24 h. The mixture was centrifugated at 4500 rpm for 15 min and the obtained supernatant was filtered through 0.2 µm syringe PTFE membrane filter. The solvent was evaporated under reduced pressure at 35°C using rotary vacuum evaporator. The resulting residues were stored in sterile glass bottles under refrigeration until they were utilized.

2.6.2. Total polyphenol and flavonoid contents.

The total phenolic and flavonoid contents in the different plants organs were colourimetrically evaluated using the conventional Folin-Ciocalteu and the aluminum chloride methods were respectively following the procedures outlined by [30]. For the polyphenols, to a volume of 125 µL of plant extract, 500 µL of distilled water, and 125 µL of Folin-Ciocalteu reagent Na₂CO₃ (7%) were added. To bring the total volume to 3 mL, distilled water was added, and the mixture was then incubated for 90 minutes in darkness. After incubation, the absorbance of the samples was measured at a wavelength of 760 nm. The results obtained were expressed as milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g DW). For flavonoids, 75 µL of NaNO2 (7%) and 150 µL of freshly prepared AlCl₃ (10%) were mixed with 125 µL of seed extract. After a 5-minute incubation, 500 µL of NaOH (1 M) was added to the resulting mixture and the final volume was adjusted to 3 mL with distilled water. The absorbance of the samples was measured at 510 nm. The total flavonoid content was expressed as milligrams of quercetin equivalents per gram of dry weight (mg QE/g DW).

2.6.3. Characterization of extracts by LC-ESI/MS

HPLC-MS analyses were conducted using a Shimadzu UFLC XR system, comprising an electrospray ionization source (ESI), two LC-20ADXR solvent delivery units, a SIL-20AXR autosampler, an SCL-10A system controller, a CTO-20 AC column oven, and a DGU-20AS degasser (Shimadzu, Kyoto, Japan. The negative ionization mode was employed, and the ESI conditions were set as follows: capillary voltage of -3.5 v, nebulizing gas flow rate of 1.5 l/min, dry gas flow rate of 15 l/min, DL temperature of 280°C, block source temperature of 400°C, and voltage detector of 1.35 V. Identification of compounds was accomplished by comparing their retention time and mass spectra with those of reference standards. Identification of compounds was accomplished by comparing their retention time and mass spectra with those of reference standards [31].

2.7. Antioxidant potential

2.7.1 Total antioxidant activity

The total antioxidant capacity was investigated through to the phosphomolybdenum assay as detailed by [32]. To 200 µL of sample extract, 2 mL of reagent solution composed of sulfuric acid (0.6 M), sodium phosphate (28 mM), and ammonium molybdate (4 mM) was added and the resulting mixture was incubated for 90 minutes at 95°C. The absorbance was determined at 700 nm versus a blank. Results are expressed as equivalents of gallic acid (mg GAE/g DW).

2.7.2. DPPH anti-radical activity

The antiradical potential effect of the different plant parts was evaluated against the DPPH free radical according to [33]. A volume of 50 µL of a range of concentration was mixed with 1.95 mL of 0.025 g/L DPPH solution. The obtained mixture was vigorously mixed and left to incubate for 30 min in the dark at room temperature. The reaction was monitored at 515 nm. The percentage of DPPH radical scavenging inhibition was measured as outlined below:

[(AC-AE)/AC] × 100.

Where AC refers to the control absorbance value while AE refers to the extract sample. Results are expressed as Trolox equivalent (mg TR/g DW).

2.7.3. Reducing power potential

The reducing power in the different plant parts was estimated using the method detailed in [34]. In brief, 2.5 mL of phosphate buffer (0.2 M, pH = 6.6) and 2.5 mL of potassium ferricyanide (1%) were added to tubes that contained 1 mL of extracts. The tubes were incubated for 20 minutes at 50°C, followed by the addition of 2.5 mL of 10% TCA. After the centrifugation at 13000 rpm for 10 min, 2.5 mL of the resulting supernatant was diluted with an equal volume of distilled water and mixed with 0.5 mL of FeCl₃ solution (0.1%), and the absorbance was read measured at 700 nm. The reducing power in the extracts is evaluated as the EC₅₀ values (mg/mL) which correspond to the concentration at which ferrous ions were chelated by 50%.

2.8. Statistical Analysis

The statistical analysis was carried out using IBM SPSS statistical software (version 20.0, IBM Corp., Armonk, NY, USA). The data were presented as means ± standard deviation (SD) from three replicates and subjected to analysis of variance (ANOVA). To compare the means, Duncan's post-hoc tests were performed. Students-T test was conducted to determine the significant difference effect of species, organs, and their combined effects. Linear regression analysis was employed to determine the EC50 and IC50 values for the RPA and DPPH assays. The significance level was set at 5% to determine the differences between means.

3.1. Mineral Composition

The mineral analysis conducted on two plant species, S. marianum and S. eburneum, has revealed intriguing insights into the distribution of essential elements across various plant parts (Table 1). In S. marianum, sodium content was highest in the stem (19.8 mg/100g), while potassium (27.2 mg/100g) and calcium (166.2 mg/100g) were most abundant in the flower and leaf, respectively. Magnesium, on the other hand, showed a relatively lower but varied presence in different parts, with the leaf containing 5.06 mg/100g. In S. eburneum, the stem exhibited the highest sodium and potassium levels (58.33 mg/g and 103.3 mg/g, respectively), while the flower contained the most calcium (230.1 mg/100g). Similarly, magnesium content displayed variations across plant parts. Copper, iron, and manganese levels varied significantly between the two species and across plant parts, with copper ranging from 0.04 to 0.1 mg/100g, iron from 0.17 to 0.35 mg/100g, and manganese from 0.04 to 0.08 mg/100g. These findings shed light on the mineral composition of these plants and can have implications for agricultural and nutritional studies. In his study, [16] the mineral content Mid-ribs of basal leaves of S. marianum varies significantly, with wide-ranging concentrations: Sodium (Na) spans from 24.7 to 128 mg/g, K from 432 to 1300 mg/100g, Ca from 42 to 171 mg/100g, Mg from 10.3 to 22.6 mg/100g, Cu from 0.01 to 0.17 mg/g, Fe from 0.47 to 0.55 mg/100g, Mn from 0.03 to 0.21 mg/100g, and Zn from 0.21 to 0.35 mg/100g. Additionally, [35] suggested that the stems of S. marianum also could be a potential source of minerals by their highest concentration of Si and Al.

3.2. Free sugar composition in S. marianum and S. eburneum organs.

Table 2 summarizes the carbohydrates composition in S. marianum and S. eburneum across various organ types. The investigation elucidates critical components within these plants, encompassing fructose, glucose, sucrose, and maltose. This profiling reveals noteworthy differentials in the chemical constituents of the examined organs. Specifically, S. marianum exhibits substantial variation in fructose content across its stem segments, while SE demonstrates considerable heterogeneity in glucose, sucrose, and maltose levels in its corresponding organs. S. marianum exhibits substantial variation in fructose content across its organs, with values ranging from 0.01 mg/g DW in immature seeds to 3.54 mg/g DW in stem. In contrast, S. eburneum demonstrates considerable heterogeneity in glucose, sucrose, and maltose levels in its corresponding organs. For instance, S. eburneum 's leaves display a glucose content of 3.83 mg/g DW and sucrose content of 3.31 mg/g DW, while stem exhibits markedly lower glucose (0.81 mg/g DW) and sucrose (1.62 mg/g DW) values. Maltose was only detected in the immature seeds of S. eburneum. Significant differences emerge clearly for fructose and maltose. However, glucose primarily exhibits significance related to species, while its interactions with organs do not reach conventional significance levels. For sucrose, significance is noted in species effect, but the main effect of organs and species-organ interaction are not statistically significant (Table 7). These results are higher in comparison with [22], where the contents of fructose, glucose, sucrose contents in the defatted S. marianum seeds were 0.1139, 0.168 and 1.6433 mg/g respectively. The free sugars in the aerial parts of S. marianum are widely discussed. [36] and [37] reported glucose, galactose and mannose, rhamnose, xylose and arabinose. Additionally, mannitol, sucrose, fructose, raffinose, arabinose and galactose were detected in the stem of the plant [35]. The organ-specific disparities in sugar content observed in our study are in alignment with the findings of [38], who reported varying concentrations of fructose, glucose, and myo-inositol in different parts of the flowers, leaves, and seeds.

3.3. Organic acid contents in S. marianum and S. eburneum

The organic acid contents in different organs of S. marianum and S. eburneum are summarized in Table 3. While oxalic acid and quinic acid were not detected in the parts of S. marianum, their contents differed significantly from S. eburneum, with higher levels found in the leaves (0.031 and 0.7 mg/g DW, respectively). Among S. marianum organs, citric acid is only detected in the leaves at the concentration of 6.50 mg/g DW and varied from 0.064 to 0.27 for the immature seeds and stems of S. eburneum. Malic acid levels vary in response to plant part and species, with amounts ranging from 0.28 (mature seeds) to 15.03 mg/g DW (leaves) for S. marianum and from 0.13 to 0.56 in the mature seeds and flower of S. eburneum. Succinc acid shows variability across plant parts. The concentrations in leaves are higher in both species, measuring 1.01 mg/g DW for S. marianum and 1.23 mg/g DW for S. eburneum. However, succinic acid is not detected in the immature seeds of either species, nor in the mature seeds of the first species and the stems of the second species. Lactic acid is present only in the seeds of S. eburneum, measuring 0.18 and 0.21 in the mature and immature seeds respectively. While the concentrations were in the range of 0.63—3.72 in the leaf and immature seeds for S. marianum. Formic acid exhibits varying concentrations across plant parts, with values ranging from 0.03 to 0.47 mg/g DW in SM and from 0.12 to 0.4 mg/g DW in SE. Higher amounts of acetic acid were detected in the leaves of both plants, measuring 37.22 and 38.7 mg/g DW for SM and SE, respectively compared to other plant parts. Propionic acid is detectable in all plant parts of SM, with values ranging from 0.22 mg/g DW to 0.45 mg/g DW. In the plant organic acids are involved in several fundamental pathways including intermediate or end products in catabolic and metabolic pathway [39].Some of them, like malic, citric and oxalic acids, seems to be related to processes operating within the rhizosphere such as nutrient acquisition, metal detoxification, mitigation of anaerobic stress in root systems and mineral weathering [40]. The quinic, malic, shikinic, citric and fumaric acids were previously described in the aerial parts of S. marianum with a total value of 53 mg/g dw [41]. Organic acids are classified as weak acids on a chemical level, exhibiting only partial dissociation. They widely used in food preservation, spanning centuries. Recently, organic acids such as formic acid, butyric acid, propionic acid, acetic acid and formic acid, citric acid, malic acid, and lactic acid have been reported for their potential antibacterial, immune potentiating properties [42]. In their study, [43] observed that among several edible plants, S. marianum exhibited the highest values for total organic acids, with the highest content of oxalic acid (662.03—464.50 mg/100 g) and fumaric acid (2.96—26.29 mg/100 g). Malic and citric acids were only detected in one population, with levels of 1.69 and 1.49 mg/100 g, respectively. Our findings align with those of [41] who suggest that Silybum species could be considered for incorporation into food formulations as acidulants, owing to the abundant presence of these organic acids in various parts of the plants.

3.4. Storage protein contents in the mature and immature seeds S. marianum and S. eburneum

In both S. marianum and S. eburneum, albumins and globulins are the predominant protein fractions in both mature and immature seeds (Table 4). For S. marianum, in mature seeds, albumins account 28.382 mg/g, and globulins for about 32.182 mg/g. However, in mature S. eburneum seeds, albumins comprise about 21.846 mg/g, and globulins about 16.221 mg/g. Furthermore, there are notable differences in the levels of prolamins and glutelins between the two plants, particularly in mature seeds. In mature SM seeds, Prolamins are measuring about 2.172 mg/g, and glutelins about 1.042 mg/g, while in mature SE seeds, prolamins are at 4.408 mg/g, and glutelins around 3.127 mg/g. The seeds of the plant receive significant attention because the unique property of this plant is the accumulation of silymarin in the pericarp and seed coat. This compound is well-known for its detoxifying effect and its ability to stabilize liver functions. As a result, the plant has been widely cultivated for pharmaceutical purposes in several countries. The seeds contain a high amounts of total protein, measuring 16.5% [44] and 19.1 g/100 g [13]. According to [45] the seeds proteins characterized by elevated levels of glutamic acid and essential amino acids, ranging from 32.33 to 38.24 g per 100 g of protein, which the recommended FAO/WHO requirements for infants aged 2 to 5 years. However, there is a deficiency in lysine, methionine, and cysteine, which necessitates supplementation from alternative protein sources, such as those found in milk. In their study [46], it was noted that albumin was the predominant fraction, followed by globulins, with smaller amounts of glutelins and prolamins. Also, the molecular mass of the obtained fraction varied from 16 to 122 kDa. No allergic reactions to milk thistle proteins have been reported. [47] demonstrated that incorporating defatted milk thistle seed flour at a 3% level in wheat bread shows promise for improving bread characteristics and potentially reducing liver damage in male rats. This suggests that defatted milk thistle seeds, in flour form, could be effectively used in functional food production.

3.5. Phytochemical characterization of S. marianum and S. eburneum

Spectroscopy analysis

The results obtained by the Folin Ciocalteu and the chloride ammonium assays revealed that total polyphenol and flavonoid contents varied considerably across different plant parts for S. marianum and S. eburneum (Table 5). For both species, the highest polyphenols amounts are observed in the mature seed, with S. marianum showing substantially higher value (161.364 mg GAE/g DW) compared to S. eburneum (57.876 mg GAE/g DW). Conversely, S. eburneum exhibits significantly higher content in the flower and stem (24.631 and 4.702 mg GAE/g DW) compared to S. marianum (5.858 and 0.735 mg GAE/g DW). The total flavonoid contents varied significantly among both species. Our findings indicate that S. marianum displays a substantially higher content in their organs compared to S. eburneum. For S. marianum, the content varied from 2.581 to 41.886 mg QE/g DW for the stems and mature seeds respectively. These contents were in the range of 3.576—17.554 mg QE/g DW for the leaf and mature seeds extracts respectively. Statistical analysis indicates strong statistical evidence for the significant effects of species, organs, and their interaction on the measured phytochemical contents (Table 7). The Asteraceae family is widely recognized for containing well-known species such as chamomile (Matricaria recutita L.), dandelion (Taraxacum officinale Web.), marigold (Calendula officinalis), and yarrow (Achillea millefolium L.). These plants are notable for their richness in polyphenols and are extensively utilized for their various beneficial effects [48]. While milk thistle S. marianum has been extensively characterized worldwide in terms of phytochemicals biological activities, our knowledge regarding S. eburneum is quite scarce in comparison. The total polyphenol contents in the Algerian S. marianum revealed that the highest value was recorded in the seed extract (127.39 mg GAE /g DW), followed by flowers (42.22 mg GAE /g DW), leaves (22.25 mg GAE g DW), and twigs (9.05 mg GAE/g DW). While the flower and seed parts possessed higher flavonoid (34.06 and 19.41 mg EQ/g DW) compared to the other plant parts [49]. In the seeds of S. marainum, the polyphenol and flavonoid contents were found to be 29 mg GAE/g DW and 3.39 mg EC/g DW respectively [50]. The study conducted by [51] found that polyphenols and flavonoid contents in S. marianum seeds were 245.183 mg GAE /g DW and 88.151 mg quercetin/g DW respectively. Additionally, the study performed by [52] explained that the polyphenols in milk thistle genotypes were in the range 206—360 mg GAE per 100 g achenes, 30—70 mg rutin equivalent per 100 g. Moreover, [53] Noted that the total polyphenols in the seed varied from 24.17 to 35.07 mg GAE/g while the flavonoids varied from 16.01 to 29.09 mg QE/g. It is well-known that the extraction of phytochemicals from S. marianum is highly influenced by both the type of solvent and the extraction method as evidenced by [54]. By comparing the oil characteristics of milk thistle seeds at three maturation stages (immature, intermediate, and mature), [55] noted that the maturity stage significantly influenced both the policosanol profile and the biological activities of milk thistle oil. The oil extracted from immature seeds had the highest total policosanol content and exhibited the greatest DPPH and ABTS radical scavenging activity.

LC-MS analysis of polyphenolic compounds

The different organs of different parts of the S. marianum and S. eburneum plant, including its leaves, stems, flowers, mature seeds, and immature seeds were further submitted to LC-ESI/MS analysis (Table 6) for a qualitative and quantitative investigation of phenolic compounds. A total of twenty-two phenolics have been tentatively identified including 16 phenolic acids and 16 flavonoids. For S. marianum, among the detected phenolic acids, 3,4-di-O-caffeoyquinic acid is found in substantial quantities in stem (3402.4 µg/g DE), leaf (11405.2 µg/g DE), and flower (20538 µg/g DE), and parts, with the highest concentration observed in the flower. However, this compound was not detected in either immature or mature seeds. Protocatechuic acid is significantly higher than in and flower (4133.5 µg/g DE) and leaf (326.4 µg/g DE) while 4,5-di-O-caffeoyquinic acid is higher in leaf (2269.1 µg/g DE) and stems (503.2 µg/g DE) parts. Quinic acid is most prevalent in mature seeds (10404.5 µg/g DE) and immature seeds (6389.3) compared to flower (357.9 µg/g DE) stem (104) and leaf (72.1 µg/g DE) parts. Additionally, o-coumaric acid is prevalent in the flowers and stems and not detected in the other plant parts. Trans cinnamic acid is highly abundant in mature seeds (7319.9 µg/g DE) and present in the stem (482.1 µg/g DE). Apigenin main constituent detected in the flower. Indeed, quercetin possessed higher levels, and it was found in the range of 3.4 to 1814 for leaf and the immature seeds, followed by naringenin that varied from 24.01 µg/g DE to 1694.4 µg/g DE for the stems and immature seeds, respectively. Quercetrin and quercetin-3-o-galactoside are prevalent in the flower and mature seeds. For S. eburneum parts, the identified polyphenolic compounds are lower than that detected in the S. marianum organs. In the leaf part, Chlorogenic acid was the main compounds (82.4 µg/g DE), followed by apegenin-7-o-glucoside (63.6 µg/g DE). In the stems, higher content of syringic acid (168.1 µg/g DE), apegenin-7-o-glucoside (139.9 µg/g DE), and apigenin (27.3 µg/g DE). In the flower, 13 compounds were characterized mostly predominated by Kaempferol (1281.3 µg/g DE), apigenin (877.5 µg/g DE), naringenin (160.1 µg/g DE), syringic acid (110.02 µg/g DE) and protocatechuic acid (56.7 02 µg/g DE), quinic acid (34.2 µg/g DE), and chlorogenic acid (33.9 µg/g DE).

In their analysis of phenolics in 15 genotypes of S. marianum, [52] found varying concentrations of phenolic acids in the extracts, with notable amounts of chlorogenic acid (148—361.6 mg/kg), caffeic acid (2.2—33.6 mg/kg), and ferulic acid (9.7—26.5 mg/kg). Apigenin (2–11.9 mg/kg) and luteolin (3.5–79.7 mg/kg) were identified as the most abundant flavonoids, while luteolin 7-O-glucoside and quercetin were absent in all genotypes studied. In addition, (Mhamdi et al.2016) identified same phenolic compounds, in S. marianum seeds, like isosilybin A (21.9%), silybin B (17.67%), isosilybin B (12.8%), silybin A (12.2%), silychristin (7.9%) and silydianin (7.5%). Also, the authors reported higher contents of silybin that varied from 3086 to 9499 mg/kg.

3.6. The antioxidant activities in the different parts of S. marianum and S. eburneum

The antioxidant potential in different parts of the S. marianum and S. eburneum plant, including its leaves, stems, flowers, mature seeds, and immature seeds, were determined with a focus on three key components: total antioxidant capacity (TAC), free radical DPPH scavenging activity, and reducing power assay (Table 5). In S. marianum extracts, both mature and immature seeds possessed higher TAC activities (31.14 and 24.66 mg GAE/g DW). Also, the flowers show higher activity (28.64 mg GAE/g DW) compared to leaves (19.9 mg GAE/g DW) and stems (8.06 mg GAE/g DW). While in S. eburneum, the flower part had the highest activity (22.85 mg GAE/g DW), which was 5.78 folds higher than that of the stems, 4.91 folds higher than that of immature seeds, and about 3.8 folds higher than that of both leaves and mature seeds. Additionally, both mature and immature seeds of the two species exhibit higher DPPH radical scavenging activities and reducing power assays compared to the other plants parts. Statistical analysis indicates strong statistical evidence for the significant effects of species, organs, and their interaction on total antioxidant capacity, DPPH, and FRAP assays (Table 7). Our study is supported by several previous studies that have shown that the seeds exhibit higher antioxidant activity. For instance, [56] reported that the seeds of the Pakistan milk thistle possessed the greatest antioxidant potential compared to the stems, leaves, and roots. Also, in the Algerian S. marianum, the seeds were found to possess the highest total antioxidant capacity, while the leaf extract exhibited the lowest activity. Additionally, the seeds demonstrated the most significant DPPH radical activity, with twigs showing the lowest potential [49]. As evidenced by [53] the seeds of S. marianum possessed high DPPH radical scavenging activities that varied from 18.9 to 25.01% and potential FRAP that was in the range 9.73—17.69 acid ascorbic equivalents. The seeds extract showed high preventive effect against the protein and damage activated by oxidative Fenton reaction, high lipid peroxidation. Also, the seed possessed important DPPH, reducing power [57].

In this study, the mineral, organic acid, free sugar, proteins, phytochemicals as well as the antioxidant potentials from stems, leaves, flowers, immature seeds, and mature seeds of wild edible S. marianum and S. eburneum were investigated. The plants organs showed higher content of minerals including sodium, potassium, calcium, and magnesium. Additionally, S. marianum shows substantial fructose variation among its organs, with the highest content in the stem and the lowest in immature seeds. Conversely, S. eburneum exhibits significant heterogeneity in glucose, sucrose, and maltose levels across its organs. A notable organ-dependent distribution of organic acids was observed for oxalic, quinic, citric, malic, succinic, lactic, formic, acetic, and propionic acids among the two species. The colorimetric analysis revealed higher levels of polyphenol and flavonoid contents in both mature and immature seeds in both species compared to the other plant parts. A total of 32 polyphenolic compounds were identified through LC-ESI/MS, with 3,4-di-O-caffeoyquinic acid, 4,5-di-O-caffeoyquinic acid, syringic acid, protocatechuic acid, naringenin, apigenin, and quercetin were detected as the dominant compounds. In both S. marianum and S. eburneum, albumins and globulins are the predominant protein fractions in both mature and immature seeds and the storage protein fraction was mostly affected by the maturity stage. This current study substantiates the versatile utility of Silybum as a sustainable resource with significant potential for its nutritional applications in the production of functional foods across diverse sectors of the food industry.

CRediT authorship contribution statement

Samah Maaloul: Methodology, Data Curation Writing - Original Draft. Maher Mahmoudi: Conceptualization, Investigation, Writing - Original Draft, Writing - review & editing. Fayçal Boughalleb: Data Curation, Methodology, Formal analysis. Talel Bouhamda: Methodology. Hedi Mighri: Methodology, Gérard Lizard: Conceptualization, Formal analysis, Data Curation. Raoudha Abdellaoui: Conceptualization, Investigation, Supervision,

Acknowledgment

The authors would like to acknowledge the technical staff of the Arid Regions Institute, Medenine (IRA), for their help with the conduct of these experiments.

Ethics approval and consent to participate.

Not applicable

Consent for publication

Not applicable

Declarations of interest

The authors declare that they have no conflict of interests.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Table 1: Mineral composition (mg/100g DW) in S. marianum and S. eburneum plants

		Na	K	Ca	Mg	Cu	Fe	Mn
S. marianum	Leaf	18.73 ±1.67^B	39.6 ±5.1^B	154.4 ±5^C	5.06±0.25^B	0.04 ±0.004^B	0.35 ±0.02^A	0.07 ±0.002^A
	Stem	19.8±2.71^A	44.2 ±6.7^A	100.5 ±6.7^E	3.3 ±0.18^E	0.07 ±0.01^C	0.2 ±0.03^B	0.048 ±0.003^D
	Flower	9.93 ±0.77^C	27.2 ±0.71^C	166.2 ±0.8^B	3.9 ±0.24^D	0.08 ±0.01^B	0.25 ±0.01^B	0.052 ±0.003^C
	Mature Seed	6.86 ±1.26^D	8.06 ±0.8^E	114 ±25.80^D	5.1 ±0.61^A	0.1 ±0.01^A	0.2±0.03^B	0.054 ±0.007^C
	Immature Seed	6.8 ±0.283^D	11.4 ±0.28^D	326.8 ±7.4^A	4.46±0.2^C	0.1±0.01^A	0.2 ±0.02^B	0.06 ±0.008^B
S. eburneum	Leaf	50.94 ±2.65^c	56.26±9.02^c	224.6 ±24.4^a	24.2 ±4.4^a	0.05 ±0.01^c	0.3 ±0.07^a	0.06 ±0.003^b
	Stem	58.33 ±14.5^b	103.3 ±18.3^a	178.8 ±14.9^b	21 ±2.14^b	0.02 ±0.001^d	0.17 ±0.016^b	0.05 ±0.01^b
	Flower	64.9 ±2.7^a	93.64±4.67^b	230.1 ±17.7^a	13.4 ±1.5^d	0.10 ±0.021^a	0.3 ±0.06^a	0.06 ±0.01^b
	Mature Seed	41.38 ±7.1^e	54.7 ±6.6^c	106.8 ±23^c	16.06 ±0.57^c	0.09 ±0.02^b	0.25 ±0.05^a	0.08 ±0.02^a
	Immature Seed	48.180±10.7^d	46.1 ±7.2^d	116.3 ±38.5^d	16.6 ±1.5^c	0.09 ±0.02^b	0.3 ±0.01^a	0.04±0.02^c

Values are expressed as mean ± SD. Same letters in the same column show no significant difference between plant organs.

Table 2: Content of organic carbohydrates (mg/g DW) in the organs of S. marianum and S. eburneum

		Fructose	Glucose	Sucrose	Maltose
S. marianum	Leaf	0.313 ± 0.02^C	12.05 ±2.9^B	0.14 ±0.01^C	nd
	Stem	3.54±0.29^A	17.65±14.8^A	0.21±0.01^B	nd
	Flower	1.14±0.1^B	4.24±0.255^C	0.24±0.13^B	nd
	Mature Seed	0.01±0.005^D	0.49±0.08^D	0.61±0.01^A	nd
	Immature Seed	0.017±0.007^D	nd	0.1±0.02^C	nd
S. eburneum	Leaf	4.5 ±1.07^c	3.83 ±0.78^a	3.31 ±2.27^a	nd
	Stem	5.4±1.07^a	0.81±0.57^c	1.62±0.34^c	nd
	Flower	4.8±0.2^b	1.6±0.27^b	1.07±0.57^c	nd
	Mature Seed	0.32±0.07^e	3.6±0.63^a	2.67±0.24^b	nd
	Immature Seed	0.57±0.007d	0.15±0.02^d	3.36±0.35^a	2.26±0.12

Values are expressed as mean ± SD. nd: not detected. Same letters in the same column show no significant difference between plant organs.

Table 3: Content of organic acids (mg/g DW) in S. marianum and S. eburneum parts

		Oxalic acid	Quinic acid	Citric acid	Malic acid	Succinc acid	Lactic acid	Formic acid	Acetic acid	Propionic acid
S. marianum	Leaf	nd	nd	6.5±1.27	15.03 ±1.3^A	1.01±0.057^A	0.63±0.02^A	0.18 ±0.03^C	37.22 ±1.9^A	0.45 ±0.02^A
	Stem	nd	nd	nd	5.21±0.48^C	0.08±0.03^C	0.21±0.07^C	0.037±0.01^D	1.8±0.5^C	0.22±0.001^C
	Flower	nd	nd	nd	11.05±2.7^B	0.16±0.09^B	0.61±0.32^A	0.17±0.007^C	2.12±0.3^B	0.36±0.07^B
	Mature Seed	nd	nd	nd	0.28±0.017^E	nd	0.26±0.01^C	0.47±0.007^A	0.41±0.01^D	0.23±0.017^C
	Immature Seed	nd	nd	nd	1.74±0.05^D	nd	3.72±0.09^B	0.28±0.04^B	0.5±0.03^D	0.22±0.015^C
S. eburneum	Leaf	0.031 ±0.013^a	0.7 ±0.26^b	0.25 ±0.1^a	0.45 ±0.06^b	1.23±0.14a	nd	0.2 ±0.02^c	38.7±3.1^a	0.44 ±0.08^b
	Stem	0.003±0.002^c	1.1±0.146^a	0.27±0.09^a	0.33±0.04^c	nd	nd	0.12±0.01^d	0.21±0.032^e	Nd
	Flower	nd	1.13±0.091^a	0.161±0.036^b	0.56±0.165^a	0.87±0.26^b	nd	0.2±0.02^c	6.4±1.2^b	0.46±0.064^b
	Mature Seed	0.013±0.001^b	0.2±0.013^b	0.073±0.027^c	0.134±0.005^e	0.5±0.044^c	0.18±0.02^b	0.32±0.05^b	0.57±0.03^d	Nd
	Immature Seed	0.024±0.008^a	nd	0.064±0.01^c	0.214±0.026^d	nd	0.21±0.06^a	0.4±0.02^a	1.4±0.2^c	1±0.14^c

Values are expressed as mean ± SD. nd: not detected. Same letters in the same column show no significant difference between plant organs.

Table 4: Storage protein contents (mg/g DM) in mature and immature seeds of S. marianum and S. eburneum

		Albumins	Globulins	Prolamins	Glutelins
S. marianum	Mature seeds	28.38±2.74^A	32.18±0.62^A	2.17±0.424^A	1.04±0.345^A
S. marianum	Immature seeds	15.30±1.66^B	13.39±2.19^B	1.88±0.564^B	0.52±0.130^B
S. eburneum	Mature seeds	21.84±1.78^b	16.22±1.47^a	4.40±0.263^a	3.12±0.586^a
S. eburneum	Immature seeds	24.06±2.78^a	8.23±0.20^b	2.36±0.498^b	3.12±0.284^a

Values are expressed as mean ± SD. Same letters in the same column show no significant difference between plant organs.

Table 5: Phytochemicals and antioxidant activities in the different parts of S. marianum and S. eburneum.

		Phytochemicals		Antioxidant activity
		Polyphenols (mg GAE/g DE)	Flavonoids (mg QE/g DE)	TAC (mg GAE/g DE)	DPPH (EC₅₀µg/mL) (mg TR/g DW)	FRAP ( EC₅₀µg/mL) (mg TR/g DW)
S. marianum	Leaf	1.7±0.14^D	7.34±0.97^D	19.9±1.99^D	0.77±0.08^D	1.42±0.07^D
	Stem	0.73±0.08^E	2.85±0.30^E	8.06±0.67^E	0.45±0.02^E	1.27±0.22^D
	Flower	5.85±0.71^C	12.26±0.50^C	28.64±2.37^B	1.48±0.21^C	3.89±0.20^C
	Mature Seed	161.36±13.91^A	41.88±0.43^A	24.66±0.40^C	3.36±0.004^B	44.91±1.12^A
	Immature Seed	55.86±8.27^B	23.88±1.11^B	31.14±0.49^A	4.43±0.01^A	31.416±3.13^B
S. eburneum	Leaf	1.4±0.20^e	3.57±0.21^e	5.84±0.32^b	0.59±0.06^c	1.6±0.28^e
	Stem	4.7±1.61^d	7.86±2.45^d	3.94±2.01^d	1.51±0.43^b	3.25±0.58^d
	Flower	24.63±6.02^c	16.9±1.99^b	22.58±1.08^a	2.46±0.34^a	8.11±0.85^c
	Mature Seed	57.87±4.55^a	17.55±1.46^a	5.91±0.41^b	2.14±0.01^a	16.04±0.52^a
	Immature Seed	39.91±5.20^b	13.21±0.26^c	4.59±0.35^c	2.45±0.06^a	9.73±0.63^b

GAE: gallic acid equivalent; QE: gallic acid equivalent; Values are expressed as mean ± SD. Same letters in the same column show no significant difference between plant organs.

Table 6: Polyphenolic compounds (µg/g DW) identified by LC-ESI/MS analysis in S. marianum and S. eburneum.

		*S. marianum*					*S. eburneum*
		Leaf	Stem	Flower	Mature seeds	Immature seeds	Leaf	Stem	Flower	Mature seeds	Immature seeds
1	Gallic acid	ND	ND	ND	ND	ND	ND	ND	2.9±0.7^b	0.7^c	3.3±0.7^a
2	protocatechuic acid	326.4^B	ND	4133.5^A	ND	ND	ND	20.3±15.9^b	56.7±16.6^a	4.6±0.2^c	3.6±0.7^d
3	3,4-di-O-caffeoyquinic acid	11405.2±135.1^C	3402.4±10.5^B	20538±3109^A	ND	ND	ND	ND	ND	ND	ND
4	4,5-di-O-caffeoyquinic acid	2269.1±58^A	503.2^B	ND	ND	ND	ND	ND	ND	ND	ND
5	quinic acid	72.1±5.1^E	104.0±3.9^D	357.9±49.9^C	10404.5^A	6389.3^B	4.6±0.8^e	16.01±5.9^d	34.2±4.9^c	64.6±15^b	107.5±24.5^a
6	1,3-di-O-caffeoyquinic acid	3.5A	2.2±1.8^B	ND	ND	ND	2.6±0.2^b	ND	7.4±1.3^a	ND	ND
7	Salviolinic acid	19.2±0.4	ND	ND	ND	ND	ND	ND	ND	ND	ND
8	Chlorogenic acid	ND	ND	ND	ND	ND	82.4±18.8^a	ND	33.9^b	ND	ND
9	trans ferulic acid	5.9^C	ND	58.3^B	1103.9^A	ND	0.2±0.01^b	0.7±0.3^a	0.8±0.1^a	ND	ND
10	syringic acid	103.6±22.8^B	ND	1374.3±189^A	ND	ND	13.2±4.7^c	168.1±28.9^b	110.02±10.7^a	ND	6.02±1.3^d
11	p-coumaric acid	207.8^B	ND	ND	ND	2663.9^A	0.5±0.04^d	0.2±0.1^e	3.2±0.3^a	2.1±0.04^b	1.7±0.1^c
12	Rosmarinic acid	ND	114.2±61.6	ND	ND	ND	ND	ND	ND	ND	ND
13	o-coumaric acid	ND	214.4±134.6^A	29.4^C	1175.2^B	ND	ND	ND	ND	ND	ND
14	Caffeic acid	ND	27.6	ND	ND	ND	ND	ND	ND	8.2±0.6^a	0.8±0.4^b
15	Salviolinic acid	ND	ND	6.2	ND	ND	ND	ND	ND	ND	ND
16	trans cinnamic acid	482.1^B	ND	ND	7319.9^A	ND	ND	ND	ND	2.1±0.3^b	7.5±0.8^a
17	Luteolin	3.8^D	ND	24.8±2.7^C	118.3^B	126.8^A	2.1±0.2^b	ND	68.7±12^a	ND	ND
18	Cirsilineol	128.04^B	181.4^A	ND	ND	ND	4.7	ND	ND	ND	ND
19	Rutin	5.4^C	1.7D	14.6±2.2^B	ND	509.1^A	ND	ND	ND	ND	ND
20	Luteolin-7-o-glucoside	4.9^A	4.7^A	ND	ND	ND	16.9±7.1^b	20.9±5.9^a	ND	1.2±0.2^c	ND
21	Epicatechin	ND	157.04^B	216.4±58^A	ND	ND	ND	ND	ND	ND	ND
22	Acacetin	ND	ND	32.8±22.7^A	ND	ND	ND	ND	ND	ND	ND
23	Catechin (+)	ND	ND	6.8	ND	ND	ND	ND	ND	ND	ND
24	Cirsiliol	ND	ND	ND	ND	ND	0.4±0.1^d	18.2±10.1^b	ND	10.8±0.5^c	77.8±3.7^a
25	Quercetin-3-o-galactoside	18.4±2.1^D	10.4±6.3^E	101.9±11.9^C	194.1^B	454.03^A	6.1±0.9^c	4.4±1.9^b	ND	ND	454.03^a
26	Naringin	28.9±4.8^B	4.8±2.3^C	50.7±11.1^A	ND	ND	ND	34.1±10.8	ND	ND	ND
27	Quercetrin	16.9±2.7^C	3.2±0.4^D	266.2±20.6^A	ND	226.8^B	10.4±0.7^c	21.9±5.3^b	140.7±40.4^a	ND	ND
28	Apegenin-7-o-glucoside	3.8±1.6^C	1.3^D	ND	8.9^B	30.5^A	63.6±0.9^b	139.9±54.1^a	ND	3.3±0.2^c	2.5±0.1^d
29	Kaempferol	5.01±1.2^D	ND	19.8±14.5^C	152.6^B	198.1^A	17.3±0.5^b	ND	1281.3±82.2^a	ND	ND
30	quercetin	3.4^E	104.7±139.6^C	5.3±3.1^D	1470.9^B	1814.9^A	ND	1.4^b	ND	ND	3.9±0.1^a
31	Naringenin	141.2±19.7^D	24.01^E	469.9±140.5^C	3091.5^A	1694.4^B	11.7±1.2^c	9.8±4.5^e	160.1±17.3^a	26.5±3.6^b	10.1±2.9^d
32	Apigenin	322.1±124.7^C	114.2±73.4^D	7077.5±16.1^A	ND	936.5^B	11.1±1^c	27.3±7.1^b	877.5±158.6^a	0.4±0.1^e	1.4±0.05^d
	*Detected compounds*	22	18	19	10	11	16	14	13	11	13

Values are expressed as mean ± SD. nd: not detected. Same letters in the same row show no significant difference between plant organs.

Table 7. P-values of the statistical analyses (ANOVA) related to the phytochemical analysis in the

	*P-Values*
	Species	Organs	Specie × Organs
Antioxidant
Total Polyphenols	< 0.0001	< 0.0001	< 0.0001
Total flavonoids	< 0.0001	< 0.0001	< 0.0001
Total antioxidant	< 0.0001	< 0.0001	< 0.0001
DPPH	0.001	< 0.0001	< 0.0001
FRAP	< 0.0001	< 0.0001	< 0.0001
Sugars
Fructose	< 0.0001	< 0.0001	< 0.0001
Glucose	0.034	0.059	0.059
Sucrose	< 0.0001	0.176	0.132
Maltose	< 0.0001	< 0.0001	< 0.0001
Organic acids
Oxalic acid	< 0.0001	0.001	0.001
Quinic acid	< 0.0001	< 0.0001	< 0.0001
Citric acid	< 0.0001	< 0.0001	< 0.0001
Malic acid	< 0.0001	< 0.0001	< 0.0001
Succinic acid	< 0.0001	< 0.0001	< 0.0001
Lactic acid	< 0.0001	< 0.0001	< 0.0001
Formica cid	0.005	< 0.0001	< 0.0001
Acetic acid	0.075	< 0.0001	0.039
Propionic acid	0.005	< 0.0001	< 0.0001
Minerals
Na	< 0.0001	0.015	0.141
K	< 0.0001	< 0.0001	0.002
Ca	0.912	< 0.0001	< 0.0001
Mg	< 0.0001	0.002	0.002
Cu	.	.	.
Fe	.	.	.
Mn
Polyphenolic compounds
Gallic acid	0.323	0.416	0.486
protocatechuic acid	0.055	0.675	0.606
30.4-di-O-caffeoyquinic acid	< 0.0001	< 0.0001	< 0.0001
40.5-di-O-caffeoyquinic acid	< 0.0001	< 0.0001	< 0.0001
quinic acid	< 0.0001	< 0.0001	< 0.0001
10.3-di-O-caffeoyquinic acid	0.029	< 0.0001	0.050
Salviolinic acid	< 0.0001	< 0.0001	< 0.0001
Chlorogenic acid	0.059	0.015	0.015
trans ferulic acid	< 0.0001	< 0.0001	< 0.0001
syringic acid	0.151	0.604	0.611
p-coumaric acid	0.106	0.656	0.688
Rosmarinic acid	< 0.0001	< 0.0001	< 0.0001
o-coumaric acid	0.162	0.587	0.623
Caffeic acid	< 0.0001	< 0.0001	< 0.0001
Salviolinic acid	< 0.0001	< 0.0001	< 0.0001
trans cinnamic acid	0.532	0.118	0.161
Luteolin	0.185	0.551	0.570
Cirsilineol	0.173	0.570	0.569
Rutin	0.284	0.453	0.453
Luteolin-7-o-glucoside	0.305	0.446	0.444
Epicatechin	0.453	0.353	0.479
Acacetin	0.299	0.450	0.450
Catechin (+)	0.004	< 0.0001	< 0.0001
Cirsiliol	0.191	0.490	0.490
Quercetin-3-o-galactoside	0.979	0.315	0.101
Naringin	0.046	0.092	0.092
Quercetrin	0.021	0.002	0.002
Apegenin-7-o-glucoside	0.329	0.431	0.431
Kaempferol	0.329	0.431	0.431
quercetin	< 0.0001	< 0.0001	< 0.0001
Naringenin	< 0.0001	< 0.0001	< 0.0001
Apigenin	< 0.0001	< 0.0001	< 0.0001

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Tunisian Silybum species: Potential source of phytochemicals, organic acids, minerals, and proteins across various plant organs

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Plants collection

2.2. Minerals analysis

2.3. Soluble sugar contents.

2.4. Organic acid contents

2.5. Storage proteins contents

2.6. Secondary metabolite screening

2.6.1. Extraction of active ingredients

2.6.2. Total polyphenol and flavonoid contents.

2.6.3. Characterization of extracts by LC-ESI/MS

2.7. Antioxidant potential

2.7.1 Total antioxidant activity

2.7.2. DPPH anti-radical activity

2.7.3. Reducing power potential

2.8. Statistical Analysis

3. Results and discussion

4. Conclusion

Declarations

References

Tables

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