*3.4. Antioxidant Activity Measured by the DPPH+*• *Method*

The values of antioxidant activity in the tested herbs against the DPPH•<sup>+</sup> radical ranged from 9.5–1061.1 µM Trolox/g DM, which is shown in Table 3. The highest antioxidant activity against the DPPH•<sup>+</sup> radical was shown by garlic bulbs (933.050 µM Trolox/g DM), stinging nettle herb (1034.725 µM Trolox/g DM) and cleavers herb (1061.068 µM Trolox/g DM). The lowest antioxidant activity against the DPPH•<sup>+</sup> radical was found in the extract from psyllium seed husk, (9.475 µM Trolox/g DM); (Table 3).

#### *3.5. Antioxidant Activity Measured by the FRAP Method*

The three herbs with the highest antioxidant activity determined using the FRAP method were stinging nettle herb (1698.517 µM Trolox/g DM), chamomile flower heads (1699.766 µM Trolox/g DM) and cleavers herb (1769.766 µM Trolox/g DM), as shown in Table 3. The lowest antioxidant activity determined by the FRAP method was found in milk thistle extract (26.252 µM Trolox/g DM), which corresponded to the results obtained earlier in the study among others, total polyphenol content and antioxidant activity against the ABTS•<sup>+</sup> radical (Table 3).

#### *3.6. PCA Analysis*

The PCA analysis showed that the first component is essentially related to the antioxidant activity, as the factor describing FRAP, DPPH•<sup>+</sup> , ABTS•<sup>+</sup> and polyphenols. The second component, with slightly smaller factor loads, describes the variability of the basic composition of the tested raw materials (Figure 1A).

This analysis also confirmed a very high degree of correlation between the antioxidant activity (determined by various methods) and the content of polyphenols, and confirmed the correlation of the dry matter content with the protein content. In turn, the content of DM and CP was negatively correlated with NFE. A thesis can also be formulated about the lack of correlation between antioxidant activity, the content of polyphenols and the proximate composition of the tested raw materials.

The analysis of the factorial coordinates of the cases (Figure 1B) indicates the existence of four clusters of points (surrounded by ellipses). Raw materials in clusters in the second and third quadrants of the coordinate system are characterized by the greatest activity of the antioxidant activity.

Herbs that proved the highest antioxidant activity include: stinging nettle herb, marjoram herb, cleavers herb, common chamomile flower heads and garlic bulbs. Other raw materials are characterized by a smaller antioxidant activity and a significant differentiation of the basic composition. This is evidenced by the indicated extreme groups, i.e., two-element groups in the first and fourth quarters. The first one includes seeds of black cumin and fruits of milk thistle, whose raw materials were characterized by a low content of NFE, in contrast to the husks of psyllium seeds and black chokeberry fruits, of course, with significantly lower antioxidant activity.

*Appl. Sci.* **2023**, *13*, x FOR PEER REVIEW 10 of 15

**Figure 1.** Biplot based on first two principal component axes for nutritional value and antioxidant activity (**A**) and distribution of 27 herbs on the first two components obtained from principal com‐ ponent analysis (**B**). Abbreviation crude protein (CP), crude fibre (CF), ether extract (EE), crude ash (CA), nitrogen free extract (NFE), 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid (ABTS•+ rad‐ ical), 2,2′‐Diphenyl‐1‐picrylhydrazyl (DPPH•+), ferric ion reducing antioxidant parameter (FRAP). **Figure 1.** Biplot based on first two principal component axes for nutritional value and antioxidant activity (**A**) and distribution of 27 herbs on the first two components obtained from principal component analysis (**B**). Abbreviation crude protein (CP), crude fibre (CF), ether extract (EE), crude ash (CA), nitrogen free extract (NFE), 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS•<sup>+</sup> radical), 2,20 -Diphenyl-1-picrylhydrazyl (DPPH•<sup>+</sup> ), ferric ion reducing antioxidant parameter (FRAP).

#### **4. Discussion**

#### *4.1. Proximate Composition*

The oldest method of herb preservation is drying, which is associated with the loss of water and inactivation of enzymes when begun immediately after harvest. Properly dried herbs do not ferment and do not turn moldy. Moreover, the levels of their active substances do not change over a long time.

The available literature contains little information about the proximate composition of many of the tested plants presented in this study. What is more, there is little information concerning proximate composition of morphological parts of herbs and medical plants.

It should be emphasized that some of the plants we analyzed are a rich source of CP (fenugreek), CF (stinging nettle), EE (black cumin) and NFE (psyllium). However, taking into account their consumption in the average daily diet, which is small and results from the use of these plants as an addition to dishes, their nutritional significance is minimal. Albeit they can be considered for use as raw materials for the production of food for special nutritional purposes.

Concerning protein content, Sadowska et al. [18] reported that whole plant of dried thyme contained 6.48 g of protein per DM; these results are lower than ours.

Some authors reported in their researches that black cumin contains about 32–40% of oil, 16–19.9% of protein, 1.79–3.74% of minerals, 5.5% of fiber [28]. These results are lower than we presented in our study (about 42% of fat, 21% of protein, almost 4% of ash, and 8% of fiber). On the other hand, Boskabady and Shirmohammadi [29] showed that this plant contains 26.7% of protein, 28.5% of fat, 8.4% of crude fiber, and 4.8% of total ash.

Sójka et al. [30] examined basic composition of chokeberry pomace fractions achieved as a result of industrial-scale processing of fruit into juice. They showed that this valuable raw material is rich in fat (13.9%), and proteins (24%). Chokeberry fruit are good sources of dietary fiber, i.e., at the level of 5.6% of fresh mass [31]. The lower content was determined in the present study (6.6%).

Sulieman et al. [32] showed that the contents of fiber, ash, protein, fat in fenugreek seed were 6.50%, 3.20%, 28.55% and 4%, respectively. Other authors analyzed chemical composition of different varieties of fenugreek and the ash content was in the range 3–7%, protein content 23.1–26.8%, fat content 8.8–21.0% and fiber content 5.1–7.1% [33]. These results are similar to our results, except fat content. In our study, the content of fat is much lower (4.5%).

Pi ˛atkowska et al. [34] determined proximate composition of common dandelion. The amount of protein was 15.25%, the crude fiber 13.09%, fat 6.81% and ash 9.11%. These results are similar to ours, except ash content. The content of ash was established as 24.5%.

#### *4.2. Total Polyphenols Content*

The tested herbs were characterized by a varied content of total polyphenols. Antioxidants, especially polyphenolic compounds (secondary metabolites), are produced by the protective systems of various plants in response to the destructive effects of free radicals. Higher plants produce secondary metabolites that protect them against environmental stress, pathogens or herbivores [35]. Depending on the number of aromatic rings and the way they are bound, they are divided into the following classes: flavonoids, phenolic acids, stilbenes and lignans [35–41]. In this study, selected dried herbs and plant materials were compared, from which various morphological parts of the plant were collected. These were plants commonly found in Polish meadows and gardens, easily available and cheap to obtain. The total content of polyphenolic compounds is presented in Table 3. Herbs with the highest content of these components in the conducted research turned out to be marjoram herb, flower heads of chamomile and cleavers herb. It is important to note that in many studies, the total polyphenol content is expressed as gallic acid, and in our research we used chlorogenic acid as the standard. Certainly, this affects the differences in the level of total polyphenols in the discussed material and may affect the interpretation of the results.

Our results are different from data reported by Biezanowska-Kope´c and Pi ˛atkowska [ ˙ 38]. These authors showed lower content of total polyphenols (mg CGA/100 g DM) in leaves of lemon balm, marjoram, and thyme compared to our study. Similarly, the results of Tsivelika et al. [39] in common chamomile are lower than ours, but these results are calculated per mg of gallic acid/g DM). Common cleavers in the study by Mili´c et al. [40] was characterized by a higher content of total polyphenols. This plant is unknown, and any research on the content of polyphenolic compounds may turn out to be important in the context of further study of the common cleavers profile.
