The Effect of Grinding Techniques on the Microstructural Properties of Purslane (Portulaca oleracea L.) Powder, Its Total Phenolics Before and After In Vitro Simulated Gastrointestinal Digestion, and Its Antioxidant Capacity

Abstract

Purslane (Portulaca oleracea L.) is a plant recognized as a valuable source of nutrients and bioactive compounds such as omega-3 fatty acids, antioxidants, vitamins, and minerals. This study investigates the effects of grinding techniques (knife, ball, and planetary ball mill) on the properties of purslane powder (surface microstructure, particle size distribution, and color), their influence on the phenolic content in the extracts of purslane powder before and after in vitro simulated digestion process, and the antioxidant activity of the purslane extracts. The results showed that applied grinding techniques affected the particle size distribution and surface morphology of the powder, which in turn influenced the gastrointestinal stability of the dominant phenolic compounds in purslane powder extracts. The powder obtained via ball milling, characterized by the highest proportion of fine particles (x < 100 µm), showed the highest content of total phenolics (656 mg GAE/L). Ball milling resulted in high preservation of the dominant phenolic acids in the powder extract after simulated gastric and intestinal digestion (83.55% and 69.42%) and high free radical scavenging activity (DPPH and ABTS) and ferric reducing power (FRAP). The results obtained emphasize the nutritional and biological benefits of purslane in the form of a fine powder.

1. Introduction

Purslane (Portulaca oleracea L.) is an edible halophyte plant of the purslane family (Portulacaceae) with important nutritional, medicinal, and pharmacological benefits. Some other common names are garden purslane, little hogweed, pusley, wild portulaca, pourpier in France, verdolagas in Mexico, and Ma Chi Xian in China. This plant is also characterized by its potential for phytoremediation, as it is able to remove pollutants (especially heavy metals such as cadmium, mercury, lead, and copper) from the environment and has a special photosynthetic metabolic pathway—CAM (Crassulacean acid metabolism)—which enables the plant to adapt to drought stress [1,2]. This ethnomedicinal plant is highly resistant to negative climatic factors, allowing it to grow in fields, roadsides, and infertile areas [3]. Interestingly, purslane’s tolerance for salinity increases after the first cut, and some authors consider purslane to be an optimal plant for cultivation in highly saline waters [4]. The fossilized remains of purslane date back to prehistoric times, and it has been reported to be native to Europe, Africa, North America, Australia, and Asia [5,6].

The nutritional importance of purslane is based on its high content of omega-3 fatty acids (contains five times more omega-3 fatty acids than spinach), minerals (potassium, magnesium, calcium, phosphorus, and iron), essential amino acids, vitamins (vitamin K and B vitamins—riboflavin, pyridoxine, niacin, thiamine, and panthothenic acid), and antioxidants (vitamin C, vitamin E, β-carotene, glutathione, and phenolic compounds) [5,7,8,9]. The leaves and stems of purslane are a rich source of crude protein (17.9%) and crude fiber (20.3%) [10,11]. It also contains various organic acids such as citric acid, fumaric acid, succinic acid, and oxalic acid [12]. Due to its high oxalate content, it should not be consumed excessively by individuals prone to forming kidney stones. Purslane leaves can be eaten raw, commonly served with yogurt, or cooked in a manner similar to spinach. In the Mediterranean region, purslane is often used as an ingredient in salads, soups, pesto, or smoothies. It also pairs exceptionally well with meats such as lamb, pork, and beef. Purslane seeds can be used in powder form and mixed with cereals to bake bread or cakes. Their consumption significantly lowers the serum concentration of fasting blood glucose [5,13].

Purslane is listed by the WHO (World Health Organization) as one of the most commonly used medicinal plants and is described as a “Global Panacea” [5]. Since ancient times, purslane has been used in folk medicine across many countries for its neuroprotective, hepatoprotective, antidiabetic, antioxidant, antifatigue, anti-inflammatory, and anticancer activities [14]. Its pharmacological benefits were described by the central figures of pharmacology, Dioscorides and Galen, as a remedy for headaches, inflammation, erysipelas, bladder problems, worms, dysentery, and hemorrhoids [15]. Among Native Americans, it is also used as a medicinal plant (they make a plant decoction or a mixture of plant juice and honey), while in traditional Chinese medicine, it is known as a “long-lived plant” [16]. The pharmacological significance of purslane is based on the presence of various classes of phytochemicals such as flavonoids, alkaloids, phenolic acids, lignans, polysaccharides, and catecholamines [2]. The antioxidant activity of purslane has been reported by many authors [17,18,19]. Purslane can influence the balance of intestinal microbiota and reduce intestinal inflammation processes, as shown in studies with broilers and mice [20,21]. According to the available data, there is no study on the gastrointestinal stability of the dominant phenolic compounds from purslane.

Grinding technology, as a fundamental process for size reduction, is widely used in the food industry. Powdered foods are easy to store and transport. Particle size is a crucial factor for the physicochemical and nutritional properties of the powder, and it improves its solubility and the extraction rate of bioactive compounds [22]. Ball milling is the most commonly reported superfine grinding device. By reducing the particle size, superfine grinding increases the surface area, resulting in powders that are easily dispersed, have excellent infusion properties, and release more efficient bioactive substances [23].

In powder form, purslane can have a positive effect in aquaculture and pharmacology as an eco-friendly substance due to its immunological and immunomodulatory potential. There are a few, very recent, studies on purslane leaf powder (PLP) and its use in aquaculture as a dietary supplement to reduce the harmful effects of cadmium [24,25]. Salman et al. [26] investigated the use of purslane powder in the preparation of traditional kishk—a dried, fermented dairy product popular in the Levant region. The study of the effects of different grinding techniques in the processing of purslane powder is of great interest for the production of powders with the highest content of bioactive compounds. When all the benefits (nutritional, medicinal, and economic) of this easy-to-grow plant are summarized, it is obvious that purslane is an optimal candidate for a natural superfood whose potential has not yet been fully exploited.

To the best of our knowledge, this is the first study to investigate the effects of different grinding techniques (knife, ball, and planetary ball mill) on the properties of purslane leaf and stem powder (surface microstructure, particle size distribution, and color). The phenolic content in the extracts obtained from the purslane powders, their in vitro gastrointestinal stability, and the antioxidant activity of the extracts via the DPPH, FRAP, and ABTS methods were also determined in this study.

2. Materials and Methods

2.1. Plant Material

Fresh plant material (leaves and stems) (Figure 1) of wild purslane (Portulaca olearacea L.) was collected in May 2024 in the area of Split (Croatia) (the GPS coordinates are 43°31′ N 16°27′ E/43.51° N 16.45° E). The botanical identification of the plant material was performed at the Faculty of Science, the University of Split, and voucher specimens were deposited at the Department of Food Technology, the Faculty of Chemistry and Technology, Split, Croatia. Prior to freeze-drying, the plant material was stored in Ziplock bags (26 × 27 cm) at a temperature of −80 °C in an automated sample cold storage system (Thermo Scientific HERAfreeze, Marietta, OH, USA). The total weight of the plant material was 80 g.

2.2. Freeze-Drying of Collected Plant Material

The plant material (80 g) was freeze-dried over a period of 24 h using a freeze-dryer (FreeZone 2.5, Labconco, Kansas City, MO, USA) at a temperature of −50 ± X °C and a pressure of 0.122 mbar. After the drying process, the plant material was weighed, divided into four equal samples (20 g), stored in sealed plastic bags, and treated with helium to avoid oxidative deterioration during storage at room temperature.

2.3. Grinding of Freeze-Dried Plant Material

The first portion of the dried plant material (20 g) was ground with a coffee grinder for 2 min with occasional breaks to avoid heating the plant material. The second portion of the dried plant material (20 g) was ground with a ball mill (Retsch, MM 400, Berlin, Germany) for 3 min at a frequency of 30 Hz. The third portion of the dried plant material (20 g) was ground with a planetary ball mill (Planetary Micro Mill Pulverisette 7 premium line, Fritsch, Berlin, Germany) for 5 min at a rotational speed of 700 rpm.

2.4. Characterization of Purslane Powder

2.4.1. Determination of Particle Size Distribution (PSD)

After grinding, the product obtained was sieved using standard sieves with a mesh size of 1000 to 63 µm and a Retch vibratory sieve shaker (Retsch AS 200 Basic, Berlin, Germany). The sieving was carried out for 10 min at an amplitude of 1.5 mm. The results shown are based on the particle size distribution (PSD) of three analyzed samples. The same figure also shows the mean diameters and standard deviations of the products calculated using Equations (1) and (2).

To gain better insight into the effect of grinding in three applied mills on product properties, it is useful to analyze two main distribution parameters in addition to the particle size distribution function: the mean diameter (x_m) and its standard deviation (S.D.) [27]. The mean diameter is calculated according to the following equation:

(1)$x_m={\displaystyle \frac{\sum x·∆Q_3\left(x\right)}{\sum ∆Q_3\left(x\right)}}$

while the standard deviation, which represents the average deviation from the mean diameter, is calculated through the following expression:

(2)$S.D.=\sqrt{{\displaystyle \frac{\sum {\left(x-x_m\right)}^2·∆Q_3\left(x\right)}{\sum ∆Q_3\left(x\right)}}}$

2.4.2. Color

The color of the purslane powder was determined using a spectrophotometer (PCE-CSM 3, PCE Instruments, Meschede, Germany). Before the measurements, the device was calibrated with a white calibration plate. The color parameters were defined according to the CIE scale: L*—lightness (brightness), a*—red component (redness), b*—yellow component (yellowness), and chroma (color saturation) and hue (angle, color tone) [28,29].

2.4.3. Powder Surface Microstructure via Scanning Electron Microscropy (SEM)

The microstructures of unground and ground purslane were determined using a Schottky field emission scanning electron microscope JEOL JSM-7610FPlus (JEOL Ltd., Tokyo, Japan). Prior to the examination, the samples were coated with a 5 nm-thick gold layer using the Quorum Q150 ES plus sputter coater (Quorum, Laughton, UK). The signal from the backscattered electrons was used for imaging, with an acceleration voltage of 1 kV and a working distance of 15 mm.

2.5. Extraction of Ground and Unground Plant Material via Hot Maceration

The extraction was carried out according to a slightly modified method by del Pilar Fernandez Poyatos [30]. First, 1 g of plant powder and freeze-dried unground purslane (control) was resuspended in 69 mL of distilled water, brought to a temperature of 70 °C, and stirred in an ultrasonic bath (room temperature and 40 kHz frequency) for 15 min. The extract was cooled to room temperature, it was centrifuged at 4000 rpm for 10 min, the supernatant was filtered using Whatman No. 1 filter paper and freeze-dried (FreeZone 2.5, Labconco, Kansas City, MO, USA) for 24 h, and the dried plant material was resuspended in distilled water to the final concentration (15 g/L).

2.6. In Vitro Gastrointestinal Digestion

Static INFOGEST in vitro gastrointestinal digestion (gastric and intestinal phases) was performed according to the method described by Brodkorb et al. [31] and Minekus et al. [32]. To simulate in vitro digestion, salivary amylase, pancreatin, and bile salts were purchased from Sigma-Aldrich (St. Louis, MO, USA), and Merck KgaA (Darmstadt, Germany). Rabbit gastric extract (RGE15) was purchased from Lipolytech (Marseille, France). All digestion experiments were performed in duplicate.

The equation for calculating the gastrointestinal stability rate (%), from the concentrations of samples before and after digestion, is as follows:

(3)$\mathrm{gastrointestinal} \mathrm{stability} \mathrm{rate} \left(\%\right)={\displaystyle \frac{{\mathrm{concentration} }_{\mathrm{after} \mathrm{digestion}}}{{\mathrm{concentration}}_{\mathrm{before} \mathrm{digestion}}}}·100$

2.7. Determination of Total Phenolic (TP)

The total phenol content in purslane powder extracts was determined spectrophotometrically (760 nm) using the Folin–Ciocalteu reagent according to the method described by Singleton and Rossi [33]. The measurement was compared with a standard calibration curve of a gallic acid solution (1–10 µg/mL), and the results were expressed in grams of gallic acid equivalents (GAE) per liter of extract. The experiment was performed in triplicate.

2.8. HPLC-DAD Analysis of Phenolic Compounds

The separation, identification, and quantification of the individual phenolic acids was carried out in the hydrolyzed extracts. Hydrolysis was carried out with 1.2 mol/L hydrochloric acid for 2 h at 80 °C and 300 rpm. The hydrolyzed extracts were centrifuged, and the supernatants were analyzed on an Agilent 1100 Series device with a UV/VIS detector (Agilent Technologies, Santa Clara, CA, USA). Separation was performed on a Poroshell 120 SB-C18 non-polar column (4.6 × 75 mm, 2.7 μm particle size) using the Zorbax Rx-C18 guard column (4.6 × 12.5 mm, 5 μm particle size). The injection volume was 25 µL, the flow rate was 1.0 mL/min, and the column temperature was set to 30 °C. The mobile phases and gradient profile were as in the work of Šola et al. [34]. Protocatechuic acid and benzoic acid were analyzed at 254 nm, syringic acid at 280 nm, and chlorogenic, ferulic, and sinapic acid at 310 nm.

2.9. In Vitro Antioxidant Activity

2.9.1. DPPH^• (2,2-Diphenyl-1-picrylhydrazyl) Scavenging Activity

The DPPH radical scavenging ability of the samples was measured according to the method described by Yen and Duh [35]. The radical scavenging activity was calculated using the following equation:

(4)$\mathrm{DPPH} \mathrm{radical} \mathrm{scavenging} \%={\displaystyle \frac{A_0-A_1}{A_0}}·100$

A₀ is the absorbance of the control, and A₁ is the absorbance of the sample. Samples were prepared and measured in triplicates.

2.9.2. ABTS 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulphonic Acid) Radical Scavenging Activity

The ability to scavenge ABTS radical was measured according to the method described by Shah and Modi [36]. Trolox and ascorbic acid were used as positive controls, while the mixture of methanol and ABTS^•+ solution served as a negative control. The percentage ABTS inhibition was expressed as a percentage using the same equation as the DPPH method. Samples were prepared and measured in triplicate.

2.9.3. FRAP (Ferric Reducing/Antioxidant Power)

The reducing potential of the aqueous plant infusions was measured according to the method of Benzie and Strain [37]. In the FRAP assay, the antioxidants in the sample reduce the Fe³⁺-TPTZ complex to the ferrous form at a low pH of 3.6 resulting in an increase in absorbance at 595 nm. Ascorbic acid was used as a standard and the results were expressed in mg ascorbic acid equivalents (AAE)/mL extract. The experiment was performed in triplicate.

2.10. Statistical Analysis

The statistical analysis was performed using the Statistica 14.0 program (TIBCO Software Inc., Palo Alto, CA, USA). Comparisons of samples’ means were carried out using a one-way analysis of variance (ANOVA), followed by Duncan’s New Multiple Range Test (DNMRT). Statistical significance was set to the p ≤ 0.05 level.

3. Results and Discussion

3.1. Purslane Powder Characterization

3.1.1. Surface Microstructure

Figure 2a–e shows the surface microstructure of freeze-dried unground purslane (leaves and stems) (a,b) and powders obtained after grinding with a knife mill (c), a ball mill (d), and a planetary ball mill (e), as determined via scanning electron microscopy (SEM). The sieves that retained the largest amount of powder were selected for Figure 2.

Micrographs of the surface microstructure of the unground freeze-dried plant material (leaf and stem) show, as expected, the compact, unbroken structure of the plant material. The micrograph showing the surface microstructure of the stem is visually interesting because it shows a large number of porous, honeycomb-like structures that are certainly formed during the drying process of the plant material. The surface structure of a freeze-dried leaf is rough and uneven, with small clusters and occasional grooves. Micrographs of the surface microstructure of purslane powder show broken, coarse structures with particles of different diameters, depending on the type of grinding technique. The surface microstructure of purslane powder after the knife mill contains sharp cuts with clearly visible edges of the particles and the rather broken structure (shredded). After grinding with a ball mill, the surface microstructure is less fragmented than after grinding with a knife mill, the edges are not as clearly defined, and the plant material is lumpy with some round formations (Figure 2c). The micrograph showing the powder obtained after grinding with a planetary ball mill differs at first glance from the surface structure shown for the other two types of powder. The planetary ball mill produced a powder whose surface structure appears to consist of many fine particles with smooth edges and a very irregular shape corresponding to the bimodal particle distribution in Figure 3.

3.1.2. Particle Size Distribution (PSD)

The results of the particle size distributions of the powders after grinding in three different mills are shown in Figure 3 as mass distribution functions, i.e., as the mass percentage of the particles in a certain size interval (ΔQ₃ (x)/Δx). It can be seen from the results that the product from the ball mill has the highest proportion of fine particles (x < 100 µm), followed by the knife mill. In addition, the products from these two mills have a negligible proportion of particles in the size range larger than x ≥ 250 µm. On the other hand, the product from the planetary ball mill has the lowest proportion of fine particles and the highest proportion of coarse particles (200 µm < x < 500 µm). The powders obtained in the ball mill and the planetary ball mill are characterized by bimodal functions, whereby this property is more pronounced in the product from the planetary ball mill. Both planetary and ball mills are based on the same comminution principle, namely the impact and friction principle. While the impact breaks the large particles into coarse fragments, the friction leads to the formation of fine particles. In this study, the presence of fines, which is a consequence of friction, i.e., abrasion, indicates that this mechanism is predominant in the ball mill. The bimodal size distribution with the second mode in the coarse fraction indicates that the coarse particles were not adequately ground in the planetary ball mill. Other authors also reported a bimodal distribution of the particles in the powder obtained with a planetary ball mill after a short grinding time and came to the conclusion that the bimodal distribution only changes to a unimodal distribution after a long grinding time (>60 min) [38,39].

The calculated values in Figure 3 show that the smallest average particle size is achieved in the ball mill and the coarsest in the planar mill. The higher values of the standard deviations in the planetary ball mill and the ball mill respectively indicate the presence of particles in the coarser fraction, which is due to incomplete, i.e., insufficient grinding.

3.1.3. Color of Purslane Powders

Table 1 shows the color parameters of the purslane powders obtained. The L* values of the color parameter range from 53.65 (PPK) to 39.50 (PPP), while PPB has an L* value of 61.68. L* represents the brightness of the color, which can be characterized by values from 0 to 100, whereby a value of 100 representing white (light) and a value of 0 for black (dark). The results presented show that the PPB sample was significantly darker than the PPP sample, while the PPK sample did not differ significantly from the other samples. Positive values of the a* color coordinate for all three samples indicate that the samples have a pronounced slightly red color component, with the PPP sample having a significantly higher intensity of red pigments than the other two samples, while positive values of the b* color component indicate the presence of yellow color in the samples, and notably higher intensity of yellow pigments had PPK and PPB samples. Markedly higher color saturation also had PPB (Chroma = 26.77) and PPK (Chroma = 25.91 ± 0.04), which indicates that the grinding process on a ball mill had the least influence on the color of the sample. The hue values of all samples, although similar in range from 85.80 to 87.18, significantly differed between all three samples, with the PPB sample having the highest value, followed by the PPK sample, and the PPP sample had the lowest value. Based on the hue values, we conclude that the values are in the first quadrant, which means that yellow and green tones dominate in all samples. The obtained results of the characterization of purslane powder after different grinding methods are difficult to compare with the findings of similar studies, as no similar study was found in the available literature.

3.2. Gastrointestinal Stability of Phenolic Compounds from Purslane

According to the available data, there is no previous study on the gastrointestinal stability of the dominant phenolic compounds from purslane extract using an in vitro digestion method. In this study, a static INFOGEST in vitro digestion method mimicking gastric and small intestinal phases of digestion is used. This method is now recognized as a standardized and practical static digestion method developed within the COST INFOGEST network. It uses constant ratios of meal to digestive fluids and a constant pH value for each step of the simulated digestion [31]. Figure 4 shows the results of total phenols in unground freeze-dried purslane and purslane powder before and after simulated gastric and intestinal digestion. The results show that the highest content of total phenolics before and after in vitro simulated digestion was determined in the PPB sample (655.82 mgGAE/L and 475.26 mgGAE/L), while the lowest content of total phenolics was determined in the PPP sample (395.43 mg GAE/L and 316.56 mg GAE/L). The extracts of PPK and PPB had higher total phenolics than the unground sample (control) (average 140%). The gastrointestinal stability of total phenols was higher in all samples after the gastric phase than after the intestinal digestion phase. In general, the stability of phenolic compounds decreased by 20% after the intestinal digestion phase. The reduction in the content of phenolic compounds after the in vitro simulated intestinal digestion phase is observed by many authors and could be related to their low alkaline stability [40,41,42]. The range of total phenols in purslane powder extracts is in agreement with other authors [43,44].

Table 2 shows the gastrointestinal stability of the dominant phenolic compounds from unground freeze-dried purslane and purslane powders obtained via different grinding techniques. The dominant phenolic compounds identified in the extracts of unground purslane and purslane powders were phenolic acids, mainly protocatechuic, chlorogenic, and ferulic acids. PPK and PBB showed a higher content of chlorogenic acid than the unground sample (control), while all extracts from purslane powders had a higher content of ferulic acid compared to the unground sample. Compared to the other powders, the ferulic acid content in the undigested extract of purslane powder obtained with a ball planetary mill was more than 2 times higher than in the other samples (Table 2). This shows that the grinding technique can alter the phenolic profile of plant powders. There are not many studies on the effects of different grinding techniques on phenolic composition. However, Liu et al. [45] showed that the grinding method can affect the phenolic composition and antioxidant capacity of Tartary buckwheat flour. The presence of chlorogenic acid as dominant and the presence of ferulic acid in purslane was demonstrated by other authors [46,47]. Protocatehuic acid was found to be the dominant phenolic compound in the methanolic extracts of purslane seeds [48]. The results from Table 2 show a high stability of phenolic acids from purslane powder extracts after the gastric digestion phase (average: 83.55%), and a lower stability after the intestinal digestion phase (average: 69.42%). Furthermore, the grinding techniques could influence the gastrointestinal stability of phenolic acids, but there are no similar studies to compare the results obtained. Other authors also report a high stability of chlorogenic acid under gastric conditions and a low stability under intestinal conditions [49]. Studies with rats showed that chlorogenic acid in its intact form can be absorbed in the stomach [49].

Particle size had a significant effect on the gastrointestinal stability and antioxidant activity of the phenolic compounds and is consistent with the results of this study. Particles of smaller size have a larger specific surface area, S (S ∝ 1/x_m). It is the larger specific surface area that ensures a higher rate of mass transfer of beneficial components from the plant material. Therefore, superfine powder provides higher phenolic stability, bioacessibility, and antioxidant activity [50,51].

3.3. Antioxidant Activity of Extracts from Unground and Ground Purslane

The antioxidant activity of the extracts of unground and ground purslane was evaluated using three antioxidant methods (DPPH, ABTS, and FRAP), as shown in Figure 5, Figure 6 and Figure 7. All methods measure the ability of a compound to scavenge free radical. As expected, the extracts of all samples showed high antioxidant activity in all methods, and this is consistent with the results of other authors [17,43]. However, there are no studies on the influence of grinding techniques on the antioxidant activity of purslane. The influence of particle size on extraction yield and antioxidant activity is shown in studies with green tea powder or Vaccinium bracteatum Thunb leaf powder [52,53]. As shown in Figure 5, the PPK and PPB samples showed higher DPPH radical scavenging activity compared to the extract of unground purslane (control). The PPP sample showed the lowest DPPH radical scavenging activity.

Figure 6 shows the ABTS radical scavenging activity of the tested samples. The ABTS radical scavenging method is considered suitable for testing phenolic compounds [52]. The results show a dose-dependent radical scavenging ability. The PPB sample showed a significantly higher radical scavenging potential than all other tested samples. Protocatehuic acid, as the dominant phenolic acid in purslane, is widely distributed in plants, and its radical scavenging ability has been demonstrated through various methods [54]. Some authors reported that the reduction in particle size of the powder of macroalgae increases the ABTS potential [55].

Figure 7 shows the results of the ferric reducing capacity of unground and ground purslane extracts. FRAP is a good method to measure the reducing capacity of water-soluble compounds such as phenols, and the FRAP values correlate positively with the content of total phenols, which is also confirmed through the results obtained [56]. The PPB sample had a significantly higher percentage of FRAP inhibition than all other samples and dose-dependent FRAP values, while the PPP sample, at the same p value ≤ 0.05, had the lowest FRAP values (Figure 7). Shu et al. [50] reported on the influence of particle size on FRAP values and concluded that a reduction in particle size increases FRAP values, which is consistent with the results obtained in this study. The mechanical force used during grinding breaks open the cell wall of the plant material, which increases the release and accessibility of biologically active compounds, such as phenols [26]. In addition, by increasing the surface area and reducing the particle size, the release of bioactive compounds from the plant material is improved. For this reason, the PPB sample showed higher antioxidant activity in all methods compared to the other samples tested.

4. Conclusions

Purslane is an edible wild plant with great nutritional and medicinal potential. The results of this study show that the grinding technique can improve this potential by producing a fine powder from purslane. The use of a ball mill resulted in a powder with a higher proportion of fine particles (x < 100 µm) compared to other grinding techniques (knife and planetary ball). Extracts from purslane powder after ball grinding had the highest content of total phenols compared to extracts from freeze-dried, unground purslane (control), and extracts from powder after knife and planetary ball grinding. The gastrointestinal stability of the phenols was the highest in extracts from purslane powder after ball grinding following simulated digestion in the stomach and intestine. HPLC-DAD analysis revealed that protocatehuic acid, chlorogenic acid, and ferulic acid are the dominant phenolic compounds in purslane extracts. However, their content depends on the grinding techniques. Ferulic acid was only dominant in the extract of purslane powder after planetary ball grinding. The extract of purslane powder after ball grinding had the highest antioxidant activity in all tested methods (DPPH, ABTS, and FRAP), which correlates positively with the particle size. Many studies have already proven that purslane can be used as a natural superfood or powerful dietary supplement in human nutrition and aquaculture, but the results of this study suggest its potential use in the form of very fine powder.

Author Contributions

Conceptualization, T.B.; methodology, T.B., M.B., I.Š., D.Đ. and M.Ć.; formal analysis, A.B., D.R., T.B., I.Š., M.B. and M.Ć.; investigation, T.B., D.R., M.B., I.Š. and D.Đ.; resources, T.B. and I.Š.; data curation, M.Ć., T.B., M.B. and I.Š.; writing—review and editing, T.B., M.Ć., A.B., I.Š. and M.B.; supervision, T.B. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

This research was partially supported under the project “Functional integration of the University of Split Faculty of Science/Faculty of Chemistry and Technology/Faculty of Maritime Studies through the development of scientific research infrastructure in the building of the three faculties” (KK. 01.1.1.02.0018).

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Figures and Tables

Figure 1 Scheme of purslane plant material processing.

Figure 2 Micrographs of the surface microstructure of freeze-dried, unground purslane (a) leaf (FPL) and (b) stem (FPS), (c,d) purslane powder after grinding with a knife mill (PPK) on 90 and 710 µm sieves, (e,f) purslane powder after grinding with ball mill (PPB) on 63 and 250 µm sieves, and (g) purslane powder after grinding with a planetary ball mill (PPP) on µm 250 sieve scanned with SEM.

Figure 3 Comparison of particle size distributions, mean particle diameters, and standard deviations of freeze-dried purslane powder obtained after grinding with applied mills. PPK = purslane powder after grinding with a knife mill; PPB = purslane powder after grinding with a ball mill; PPP = purslane powder after grinding with a planetary ball mill.

Figure 4 Total phenolic content of unground and ground purslane before and after in vitro digestion. The values represent the means ± standard deviations of three biological replicates and three technical replicates. Different small letters indicate significant differences between the samples in the same digestion phase, while different capital letters indicate significant differences among one sample after different digestion phases (one-way ANOVA, Duncan test, p ≤ 0.05). Control = freeze-dried, unground purslane; PPK = purslane powder after grinding with a knife mill; PPB = purslane powder after grinding with a ball mill; PPP = purslane powder after grinding with a planetary ball mill.

Figure 5 DPPH radical scavenging activity of extracts from unground and ground purslane. Values represent the means ± standard deviations of three biological replicates and three technical replicates. Different letters indicate significant differences between the samples for each concentration separately (one-way ANOVA, Duncan test, p ≤ 0.05). Control = freeze-dried, unground purslane; PPK = purslane powder after grinding with a knife mill; PPB = purslane powder after grinding with a ball mill; PPP = purslane powder after grinding with a planetary ball mill.

Figure 6 ABTS radical scavenging activity of extracts from unground and ground purslane. Values represent the means ± standard deviations of three biological replicates and three technical replicates. Different letters indicate significant differences between the samples for each concentration separately (one-way ANOVA, Duncan test, p ≤ 0.05). Control = freeze-dried, unground purslane; PPK = purslane powder after grinding with a knife mill; PPB = purslane powder after grinding with a ball mill; PPP = purslane powder after grinding with a planetary ball mill.

Figure 7 Free radical scavenging capacity (FRAP) extracts from unground and ground purslane. Values represent the means ± standard deviations of three biological replicates and three technical replicates. Different letters indicate significant differences between the samples for each concentration separately (one-way ANOVA, Duncan test, p ≤ 0.05). Control = freeze-dried, unground purslane; PPK = purslane powder after grinding with a knife mill; PPB = purslane powder after grinding with a ball mill; PPP = purslane powder after grinding with a planetary ball mill.

Table 1

Color parameters of obtained purslane powders.

	Color Parameters
	L*	a*	b*	Chroma	Hue
PPK	53.64 ± 0.09 ab	1.4 ± 0.01 b	25.87 ± 0.04 a	25.91 ± 0.04 a	86.9 ± 0.03 b
PPB	61.68 ± 2.22 a	1.32 ± 0.13 b	26.73 ± 0.77 a	26.76 ± 0.78 a	87.18 ± 0.19 a
PPP	39.50 ± 17.34 b	1.67 ± 0.02 a	22.77 ± 0.03 b	22.83 ± 0.03 b	85.80 ± 0.05 c

Values represent the means ± standard deviations of three biological replicates and three technical replicates. Different letters indicate significant differences among the samples in a column (one-way ANOVA, Duncan test, p ≤ 0.05). PPK = purslane powder after grinding with a knife mill; PPB = purslane powder after grinding with a ball mill; PPP = purslane powder after grinding with a planetary ball mill

Table 2

Gastrointestinal stability of dominant phenolic compounds from unground and ground purslane.

Phenolic Compounds	Control			PPK			PPB			PPP
	UN (µg/g)	GA (%)	IN (%)	UN (µg/g)	GA (%)	IN (%)	UN (µg/g)	GA (%)	IN (%)	UN (µg/g)	GA (%)	IN (%)
Benzoic	20.90	100	34.54	16.03	74.85	100	14.06	99.43	100	10.12	72.13	76.18
Protocatehuic	2010.03	88.60	41.29	2318.08	74.50	61.10	2059.76	69.47	29.08	804.40	65.67	57.34
Chlorogenic	380.20	100	80.33	467.55	65.12	68.71	365.89	85.44	72.27	389.15	95.31	73.53
Syringic	11.57	71.39	93.17	40.57	45.00	14.61	8.77	57.24	91.67	7.68	93.88	32.42
Ferulic	242.17	86.83	86.83	287.80	63.66	76.17	253.05	89.73	73.34	666.61	88.36	64.24
Synapic	8.93	56.77	56.77	8.00	77.37	46.12	9.20	100	49.89	6.81	63.14	56.24

The gastrointestinal stability (%) of phenolic acids was calculated according to the formula described in Section 2.6. UN = undigested; GA = gastric; IN = intestinal; control = freeze-dried, unground purslane; PPK = purslane powder after grinding with a knife mill; PPB = purslane powder after grinding with a ball mill; PPP = purslane powder after grinding with a planetary ball mill.

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